Improvements in or relating to a method and a device for dispensing an entity

Abstract

A cartridge comprising a microfluidic chip with space for micro-entity or micro-droplet 66 manipulation and an outlet, a conduit connected to the outlet and providing fluid communication between the chip and a nozzle, a dispense reservoir for storing wash fluid 60 and a controller to control the flow of wash fluid from the reservoir to contact a microdroplet at the nozzle and eject the microdroplet out of the cartridge. A method of dispensing a microdroplet from a cartridge, the cartridge comprising a chip having a microfluidic space for micro-entity manipulation and an outlet, a dispense reservoir for storing a wash fluid, and a conduit in fluid communication with the chip and terminating in a nozzle, the method comprising the steps of discharging the microdroplet out of the outlet of the chip and along the conduit towards the nozzle and ejecting a volume of wash fluid from the dispense reservoir to move the microdroplet out of the cartridge. The dispense fluid may flow into the conduit and contact microdroplet in the conduit such that the wash fluid is ejected from the nozzle. Alternatively, the wash fluid maybe ejected from the dispenser reservoir to a tip which is substantially perpendicular to the nozzle (fig 4). [Use fig 3b]

Description

The present invention relates to improvements in or relating to a method of dispensing a micro-entity and in particular, a method of dispensing a micro-entity from a cartridge. The present invention also relates to a cartridge for dispensing a micro-entity. It is commonly known in the art that a cartridge comprising a microfluidic chip, such as an EWOD or oEWOD chip, can be used for manipulating microdroplets or magnetic beads. During and / or after droplet manipulation using the microfluidic chip, many of the prospective workflows on microfluidic systems require recovery of material such as cells, beads; chemical, biological, biochemical or genetic material out of the cartridge and into conventional liquid handling vessels such as 96-, 384- or 1536-well plates or microtubes. Droplets that are dispensed out of the cartridge can be further assayed. These assays in general include, but are not limited to, PCR amplifications, DNA sequencing, RNA sequencing and cell expansion. In particular, recovery of droplets for genetic assays is often required since such assays commonly involve extreme temperature cycles that, if conducted in the cartridge, would kill any cells retained on the cartridge. In some instances, the EWOD or oEWOD chip has a critical requirement to eject droplets either one at a time or many at once in a large pool out of the chip and return the material contained therein back to the user. Droplets are then moved from the chip along a pipeline and out of the cartridge through the dispense nozzle. However, there are several deficiencies with existing dispense systems, particularly arising from the interaction of the carrier phase, the droplets and the droplet material with the end of the nozzle. Rather than forming a uniform pendant drip on the end of the nozzle, in some cases the carrier phase would wet the exterior of the nozzle and ascend the tube under capillary action. In many cases, droplets contained in the carrier phase would be carried up the nozzle and remain trapped at the nozzle even when the carrier phase eventually dripped off the end. Other related failure modes of current dispensing systems are also present including the droplets remaining trapped at the very end of the tip, the droplets wetting the surface of the tip and the drip formation being intermittent and difficult to synchronise. The consequences mean that the system could not supply material back to the user, hence leading to waste and inefficient processes. Therefore, there is a requirement to minimise the amount of carrier phase that is emitted from the nozzle, as in some cases, it interferes with the efficiency of downstream assays that the user may run on the material in the droplet. Moreover, there is a requirement to minimise the cycle-time between droplets, so that the dispensing of droplets is fast and efficient. It is also beneficial to ensure that the dispense is highly reliable such that every droplet ejected from the chip arrives in the receiving vessel and does not get retained in the fluidic network. In addition, it is desirable to ensure that the dispensing process does not impart high shear stress to the material in the droplet. Furthermore, it is important to ensure that droplets are received in the output vessel(s) in the same order in which they are ejected from the chip, such that droplets do not exchange positions during the dispense sequence and droplets and their respective contents can be identified and / or tracked. Thus, it is against this background that the present invention has arisen. According to an aspect of the present invention, there is provided a method of dispensing a micro-entity from a cartridge, the cartridge comprising: a chip having a microfluidic space for micro-entity manipulation and an outlet; a dispense reservoir for storing a wash fluid; and a conduit in fluid communication with the chip and terminating in a nozzle, the method comprising the steps of: discharging the micro-entity out of the outlet of the chip and along the conduit towards the nozzle; ejecting a volume of wash fluid from the dispense reservoir to move the micro-entity out of the cartridge. The wash fluid released from the dispense reservoir is able to contact and move the micro-entity out of the nozzle and into a receptacle e.g. a well plate. Thus, the wash fluid has the function of removing the micro-entity and / or any other material e.g. a carrier fluid that remains trapped at the tip of the nozzle. The quantity and timing of ejecting the wash fluid from the reservoir can be controlled so as to adjust in response to the nature of the micro-entity that is ejected from the tip of the nozzle. For example, a longer wash can be used in the case that a larger volume of emulsion is ejected, or that more microdroplets are ejected in each cycle. Therefore, providing the wash fluid at the tip of the nozzle can enable fast and efficient dispensing of micro-entities out of the cartridge. Moreover, the wash fluid does not impart high shear stress to the micro-entities and thus reduces any damage to the micro-entity during the dispensing process. Furthermore, the performance of ejecting micro-entities out of the cartridge can degrade over time since the dripping performance of the nozzle deteriorates as the carrier fluid, micro-entities and / or other material builds up around the tip of the nozzle. Hence, providing the wash fluid at a certain flow rate through the nozzle can remove materials around the tip of the nozzle and hence, this can prolong the performance of the dispensing process. In some embodiments, the step of ejecting the volume of wash fluid may be initiated by pressurising the dispense reservoir and then opening a valve. The valve is configured to control the fluid flow of the wash fluid from the dispense reservoir along the conduit and through the nozzle. The valve may be a one-way valve which acts to prevent the wash fluid from re-entering into the dispense reservoir. In some embodiments, the step of ejecting the volume of wash fluid may be initiated by activating an electrospray nozzle. In this case, the wash fluid may be in the form of an aerosol or droplet form. The wash fluid may be ejected through the nozzle. In some embodiments, upon the opening of the valve, the wash fluid may be ejected from the dispense reservoir through the chip, such as the EWOD or oEWOD chip, out through the outlet of the chip, along the conduit and through the nozzle. In some embodiments, the wash fluid can be ejected from the dispense reservoir to flow across the tip of the nozzle. The flow across the tip can be substantially perpendicular to the nozzle. In this case, the wash fluid is a liquid form. Alternatively or additionally, the wash fluid may be in a gaseous, aerosol or droplet format. In this case, the wash fluid may be sprayed across the tip of the nozzle such that it can be in contact with the micro-entity. The contact between the wash fluid and the micro-entity causes the micro-entity to exit out of the nozzle. In some embodiments, the wash fluid can be ejected through the nozzle and / or ejected to flow across the tip of the nozzle simultaneously or in a sequential manner. In some embodiments, the wash fluid may be ejected through the nozzle via a syringe. The wash fluid may be a gas and / or it may be a liquid. The wash fluid may be, but is not limited to, air, inert gas, nitrogen, water, surfactant, oil, aqueous media such as cell media, buffer such as lysis buffer, growth media and / or PCR reagents. The oil may be a fluorinated oil, a vegetable oil, a paraffin oil or a mineral oil. The wash fluid may be supplemented with detergents, surfactants and / or salts as appropriate. Alternatively, or additionally, the wash fluid may be a solvent such as an alcohol. The method as disclosed herein may further comprise the step of supplying a carrier fluid into the chip. The carrier fluid may be oil. The carrier fluid may be 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 oil, a vegetable oil or fat; or any other suitable electrically insulating liquid. In some embodiments, the method may further comprise the steps of detecting and / or analysing the micro-entities within the conduit. At least a portion of the conduit may be transparent to enable detection and / or analysis of the micro-entities within the conduit. Detecting the micro-entities within the conduit enables the identification of a microentity of interest as it passes along the conduit and through the nozzle. This may inform the user to direct the micro-entity of interest to a specific location as the micro-entity is dispensed out of the cartridge. For example, the micro-entity that has been detected may be dispensed out of the cartridge and into a well-plate for further biological and / or chemical workflows. In another example, the micro-entity detected within the conduit may not be retained by the user. In this case, the micro-entity may be directed to a waste receptacle or a waste channel. In some embodiments, the step of opening the valve may be initiated by the provision of a pressure exceeding a predetermined threshold value to the valve. The pressure required to open the valve will typically exceed 10mbar. In some embodiments, the pre-determined threshold value for the valve is between 50 mbar and 100 mbar. For example, it could be 60, 70, 80, 90 or 100 mbar. The maximum pressure for opening the valve may be in the region of 300mbar. 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. 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 mitochondria. 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. 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. The cell may be isolated from a patient or individual. The cartridge 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). 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. 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. 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. 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. 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. 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. The cell may be genetically engineered prior to encapsulation into the microdroplet. The cell may be genetically engineered after encapsulation in the microdroplet. 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. In some embodiments, micro-entity or micro-entities can be a biological and / or chemical micro-entity. The biological and / or chemical micro-entity may be any one or more of the following: 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 a cartridge comprising: a chip comprising a microfluidic space for micro-entity manipulation and an outlet; a conduit providing fluid communication between the chip and a nozzle through which micro-entities can exit the cartridge; a dispense reservoir for storing a wash fluid; a fluid flow controller configured to control the flow of wash fluid from the dispense reservoir to contact the micro-entity at the nozzle and move said micro-entity out of the cartridge. The cartridge may further comprise a connector. The connector comprises a plurality of inputs one of which is in fluid communication with the outlet of the chip and the other is in fluid communication with the dispense reservoir. The connector also comprises an output in fluid communication with the nozzle. In some embodiments, the nozzle may comprise a fluted or tapered tip, or it could be shallow tapered, steep tapered and / or have an open-sided U-shape. The formation of drips at the tip of the nozzle is governed by the rate of liquid flow into the nozzle, as well as the dynamics of drip relaxation, evaporation from the proximity of the tip and the physical properties of the nozzle. This includes the size, shape and / or material of the nozzle. The shape of the tip of the nozzle can be optimised to support drip formation e.g. providing a small drip volume with a consistent drip-to-drip behaviour. Furthermore, the shape of the tip can be particularly useful in aiding the removal of micro-entities that are trapped at the tip of the nozzle. Various configurations of the nozzle tip can be provided and this would be known and / or appreciated by the skilled person in the art. In some embodiments, the nozzle may comprise a layer of coating. The coating can be omniphobic, oleophobic or hydrophobic. The coating can be PTFE, Teflon, PEEK, Polyimide, Steel, Aluminium, Glass, fused silica, although it will be understood that other suitable materials are available. It may be a composite of the aforementioned materials. Additionally or alternatively, the coating can be PMMA, soot / carbon particles, fluorocarbon silane and / or a nanotextured omniphobic surface. In some embodiments, it may be a composite of steel internally lined with PTFE, or it may be a composite of fused silica externally jacketed with polyimide. In some embodiments, it may be a glass or fused silica nozzle coated with a hydrophobic material such as a fluorosilane. In some embodiments, the coating material may be oleophillic. Any of the materials may be coated with an anti-fouling material and / or a hydrophobic material. The hydrophobic material may prevent the wetting of droplets onto the interior or exterior of the nozzle. A coating may be used to modify the dripping behaviour of the tip of the nozzle. Advantageously a tube formed of fused silica allows for the detachment of smaller drips, ensuring that the minimum possible volume is ejected from the cartridge and maximising the throughput of droplets dispensed. In some embodiments, at least a part of the conduit comprises a layer of coating. The coating layer can be particularly useful in corners or junctions of the conduit as it can help reduce the micro-entities getting trapped in corners. Additionally or alternatively, the layer of coating may prevent adhesion of the micro-entities to the walls of the conduit and / or at the nozzle tip. The cartridge may further comprise an aqueous reservoir for supplying aqueous media into the chip and / or into the droplet generator e.g. an emulsifier. In some embodiments, the cartridge may further comprise a pneumatic manifold connected to an external pressure source. In some embodiments, the pressure source may be provided within a cartridge. The pressure source can be a pump. A single pressure source can be provided in fluid communication between a plurality of reservoirs located within the cartridge e.g. a single pressure source is provided to pressurise the aqueous and the dispense reservoirs. Providing a single pressure source can help reduce workflow and manufacturing complexities whilst also minimising resource costs. In some embodiments, the outlet of the chip may comprise a dispense loop. The dispense loop can be connected to ports on the rotary valve that allow the microentities e.g. a microdroplet to be injected into the dispense loop and then, through a repositioning of the rotary valve, ejected from the dispense loop into the conduit towards the nozzle. A mixing junction, such as a T-junction, may be provided as part of the conduit. The mixing junction can be used to introduce additional materials along the conduit. In addition, the mixing junction can be configured to prevent the trapping of microentities, such as an emulsion within the conduit. In order to avoid trapping of microentities, the mixing junction is configured to be devoid of steps, ledges and ridges. Furthermore, the orientation of the junction is such that movement of the micro-entities through the junction is not influenced by gravity or buoyancy. In some embodiments, an emulsifier configured to generate microdroplets dispersed in a carrier fluid into the chip can be provided within the cartridge. The emulsifier may be, but is not limited to, a step emulsifier, a T-junction emulsifier, a cross flow emulsifier or a membrane emulsifier or any other device(s) for imparting shear to fluids. Such emulsifiers are configured to generate droplets of varying size, composition and density. They may generate emulsions with varying ratios of continuous and dispersed phases. Any of these parameters may influence the susceptibility of the droplets to merging, disintegration, electrolysis or other deleterious effects as they enter the device. Advantageously, the present invention allows for a wide range of emulsions and different emulsifier types to be used to supply the device. Step emulsifiers have the advantage that the flow velocity of the dispersed or continuous phases does not directly influence the size of the microdroplets produced and therefore a monodisperse stream of microdroplets can be created with a varying flow velocity. If step emulsifiers are used, there may be multiple nozzles provided at the outlet of each emulsifier. Increasing the number of nozzles provided increases the size of the emulsifier, but also increases the throughput. Furthermore, in the event that one of the nozzles at the outlet of the emulsifier is blocked, another nozzle can be used to continue producing microdroplets. The nozzle can be configured to drip an aqueous fluid into a carrier phase of oil. The, or each, nozzle is located at the intersection of a droplet buffer zone and a series of channels connecting from the inlet to the droplet buffer zone. In some embodiments, at least a portion of the conduit is transparent. The conduit may comprise a detector for detection and / or analysis of the micro-entities within the conduit. The detector could be an optical detector or an electrical detector. Examples of an optical detector can be, but not limited to, a light source, lens arrangement and photodiode or a phototransistor, or lens and camera. Examples of an electrical sensor or detector can be, but not limited to, a capacitance detector or an impedance detector. According to another aspect of the present invention, there is provided use of the cartridge according to any one of the previous aspects. According to an aspect of the present invention, there is provided a species screened by the cartridge, apparatus or method as disclosed herein. According to an aspect of the present invention, there is provided a species selected by the cartridge, apparatus or method as disclosed herein. According to an aspect of the present invention, there is provided a species isolated by the cartridge, apparatus or method as disclosed herein. According to an aspect of the present invention, there is provided a species made by the cartridge, apparatus or method as disclosed herein. For example, the present invention may provide an agonist / antagonist to a species as identified by the screening, selection and / or isolation method disclosed herein. The present invention may provide an agonist / antagonist to a species as identified by the screening, selection and / or isolation method disclosed herein, for use in therapy. The species may be chemical, biochemical, or biological in nature. The micro-entity is a species of micro-dimension. According to an aspect of the present invention, there is provided a use of the cartridge, apparatus, method or species as disclosed herein. According to an aspect of the present invention, there is provided a use of the cartridge, apparatus, method or species as disclosed herein in therapy. The present invention may provide for a use of the cartridge, apparatus, method or species as disclosed herein in making a product. The product made may be chemical, biochemical, or biological in nature. The use may be peptide synthesis. The use may be synthetic biology. The use may be cell line engineering or development. The use may be cell therapy. The use may be drug discovery. The use may be antibody discovery. According to an aspect of the present invention, there is provided a use of the cartridge, apparatus, method or species as disclosed herein in analysis. The analysis may be physical, chemical, or biological. The use may be sub-cellular imaging. The use may be high content imaging. The use may be diagnostics. The use may be a biological assay. The biological assay may be high throughput screening. The biological assay may be ELISA. The use may be cell secretion. The use may be QC safety profiling. 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 cartridge for dispensing a micro-entity according to the present invention; Figure 2 illustrates a workflow for ejecting a wash fluid; Figures 3A to 3F provide a flow schematic of dispensing a micro-entity out of the cartridge; Figure 4 shows an alternative configuration of the flow channels and conduits; and Figures 5A and 5B show one possible fluid flow arising from the configuration of conduits shown in Figures 3A to 3F. Referring to Figure 1, there is provided a cartridge 10 having at least one inlet port 12 for introducing a sample into the cartridge 10. The sample can be a fluid sample, such as a liquid sample, and the sample may contain at least one micro-entity such as a biological and / or chemical entity. The sample may be an aqueous media, a buffer solution, a suspension or a particulate. In use, the sample is loaded into the cartridge 10 via through one or more inlet ports 12. In some instances, there can be different samples containing different cell types being loaded into different inlet ports 12. The sample can be loaded into the cartridge 10 via through the inlet ports 12 at the start of any experiments. In some cases, a further sample may be loaded into the cartridge 10 via through the inlet ports 12 during an experiment. As shown in Figure 1, each of the inlet ports 12 is provided with a lid 13. Once the sample is loaded into one of the inlet ports 12, the user can close the lid 13 to protect the sample and seal the aqueous reservoir 28 such that it can hold pneumatic pressure. The cartridge 10 comprises a chip 16 such as an EWOD or oEWOD chip for manipulation of a micro-entity. For example, a microdroplet can be manipulated using EWOD or oEWOD forces within the chip. Manipulation of a micro-entity such as a microdroplet may include, but not limited to, sorting, merging, transporting and / or splitting. Alternatively, the chip 16 can be an optical tweezer device, or an optoelectronic 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. The chip 16 comprises a microfluidic space for micro-entity manipulation and an outlet for removing the micro-entity out of the chip 16 As shown in Figure 1, a conduit 14 is provided to be in fluid communication between the chip 16 and a nozzle 18 through which micro-entities can exit the cartridge 10. In this context, a conduit 14 is a channel for carrying a fluid such as a liquid or a gas. The conduit 14 may be one or more flow channels that can be combined or split at a junction. The conduit may be a plurality of micro-channels that can be combined together to form a larger flow channel. The conduit 14 can be made of any materials. For example, the conduit may be made from, but is not limited to, polytetrafluoroethylene (PTFE), polyaryletherketone (PAEK) and / or polyetherketone (PEEK). In some instances, the material of the conduit 14 can be provided so as to minimise adhesion of the carrier fluid and / or the micro-entities to the walls of the conduit 14. At least a part of the conduit 14 may be coated with an omniphobic, oleophobic and / or hydrophobic coating. The coatings may reduce micro-entities adhesions to the walls of the conduit 14. Additionally or alternatively, the coating may reduce micro-entities or other materials getting trapped within the conduit e.g. in corners or bends of the conduit 14. A dispense reservoir 20 can be provided within the cartridge 10 for storing a wash fluid. The cartridge 10 may also comprise a fluid flow controller 22 configured to control the flow of the wash fluid from the dispense reservoir 20 to contact the micro-entity at the nozzle 18 and move said micro-entity out of the cartridge 10. A pneumatic manifold 24 is provided within the cartridge 10. The pneumatic manifold 24 can be connected to an external pressure source (not shown in the accompanying drawings). In use, the pneumatic manifold 24 can apply pressure to the cartridge 10 to move the fluid sample along the channels within the cartridge 10. The pneumatic manifold or pressure source 24 can be a pump configured to apply positive pressure to the cartridge 10. Alternatively, the pump may be configured to provide a vacuum within the cartridge 10. As shown in Figure 1, the pneumatic manifold 24 comprises a cluster of O-rings 25, which functions as a seal to ensure that no air, gas or liquid can escape the cartridge 10. The pneumatic manifold 24 may provide a connection to an external instrument that has a series of air outlets. The pneumatic manifold 24 may be in fluid communication to one or more reservoirs provided within the cartridge e.g. oil reservoirs 21, 23, waste reservoir 31 aqueous reservoir 28, and / or dispense reservoir 20. The connection between the pressure source and / or pneumatic manifold 24 to more than one reservoir can ensure that the reservoirs are pressurised simultaneously or in a sequential manner. As illustrated in Figure 1, the dispense reservoir 20 comprises an inlet 26 which can be connected to the aqueous reservoir 28 via a second conduit or channel 29. The aqueous reservoir 28 comprises a plurality of inlet ports 12 for introducing a sample e.g. aqueous media into the aqueous reservoir 28. Each inlet port 12 has a lid 13. The second conduit 29 comprises a filter 27 near the inlet 26 leading into the dispense reservoir 20. The connection between the aqueous reservoir 28 and the dispense reservoir 20 is configured to be pressurised using a single pressure source. The fluid flow controller is a valve 22, which can be a one-way (non-return) valve. The purpose of the one-way valve is to enable the fluid(s) to flow in one direction only. The valve can be a check valve. The material of the check valve can be perfluoroelastomer and / or PEEK. In normal use, the check valve 22 remains closed preventing the wash fluid from flowing along the conduit 14. The check valve 22 is configured to open when pressure exceeds a pre-determined threshold value to the check valve 22. Upon the opening of the valve 22, a volume of wash fluid is ejected from the outlet 30 of the dispense reservoir 20 to flow along the conduit 14. The check valve 22 may be a oneway check valve which prevents the wash fluid from returning back into the dispense reservoir 20. A connector 32 such as a Y-interconnector is provided along the conduit 14 as shown in Figure 1. The Y-interconnector 32 has two inputs 34, 36 and an output 38. The first input 34 of the connector 32 may be configured to receive the wash fluid from the dispense reservoir 20. The second input 36 of the connector 32 may be connected to the outlet of the chip 16, such as an EWOD or oEWOD chip. The output 38 of the connector 32 is connected to the nozzle 18 where the micro-entities are dispensed out of the cartridge 10. Referring to Figure 1, there is shown a dispense path 40 comprising a dispense loop 42 connected to at least one of the rotary valve (not shown in the accompanying drawings). This allows the micro-entities, which may comprise microdroplets, to be injected into a dispense or buffer loop 42 and then, through a repositioning of the rotary valve, the fluid containing microdroplets are ejected from the buffer loop 42 into the conduit 14 and travel towards the dispense nozzle 18. The dispensed sample may comprise one or more microdroplets in a carrier fluid. The dispense nozzle 18 can be configured to control the flow rate of the sample fluid out of the cartridge 10. Between the nozzle 18 and the chip 16, there is provided an optical inspection region 44. The inspection region 44 is suitable for imaging the contents of the fluids with a camera. Once the micro-entities, such as a microdroplet, are removed from the dispense loop 42, the micro-entities travel along the conduit 14 where they can be detected through imaging or any other detection method before being ejected through the nozzle 18. When the micro-entities such as microdroplets are ejected from the cartridge 10 and out of the nozzle 18, the microdroplets travel in a small volume of carrier phase that surrounds the droplet. The droplets are dispensed out of the nozzle 18 into a receptacle (not shown in the accompanying drawings) such as a well plate for further analysis. During use, the cartridge can be pressurised such that the wash fluid from the dispense reservoir 20 can flow along the conduit 14 until it contacts the micro-entities e.g. a microdroplet and moves the micro-entities through the nozzle 18 and out of the cartridge 10. Additionally or alternatively, the wash fluid may be able to flow through the chip 10 along the conduit 14 to contact and move the micro-entities out of the cartridge 10. In some instances, the wash fluid flowing through the nozzle 18 can be air, or it can be a liquid. Additionally or alternatively, the wash fluid may be provided via a separate wash fluid channel that flows in parallel with the conduit 14. The wash fluid may then be introduced into the conduit via at a junction between the wash fluid channel and the conduit. Additionally or alternatively, the wash fluid may be provided in a form of a droplet form, which can be provided by the use of an electrospray. In this case, the wash fluid may flow across the tip of the nozzle 18 to contact the microentities at the nozzle and thereby remove the micro-entities from the tip of the nozzle. Additionally or alternatively, the wash fluid may be in liquid form and can be provided to flow across the tip of the nozzle. A rail 46 located on the cartridge 10, as shown in Figure 1, mates with a groove in an instrument (not shown in the accompanying drawings) to ensure correct alignment of the cartridge 10 within the instrument. In addition, one or more alignment features 48 are provided on the surface of the cartridge 10 to ensure correct alignment of the chip 16 in the cartridge 10. Referring to Figure 2, there is provided a schematic 50 showing the dispensing process as disclosed herein. A single pressure source can be shared between the wash fluid reservoir containing the wash fluid and any other reservoir provided on the cartridge. The check valve 52 will open at high pressure that exceeds a predetermined threshold but then remain closed when the pressure within the cartridge drops below the pre-determined threshold value to the valve. As such, the wash fluid will only flow under the high pressure conditions within the cartridge. Typically, the other reservoirs on the cartridge are sealed by means of a conventional on-off valve 55 when the aqueous wash fluid is flowing. Advantageously this allows operation of the wash fluid reservoir without use of an extra pressure source on the running cartridge. As shown in Figure 2, a pressure source or controller 56 is connected to the first reservoir 54 via a channel 51. The first reservoir 54 and second reservoir 58 are connected together via the second conduit 29. As illustrated in Figure 2, the air pressure can be trapped at the top of the first reservoir 54 containing an aqueous solution 53. This enables the pressure controller 56 to pressurise the second reservoir 58 containing the wash fluid 60, for example an aqueous media. The media will flow when the pressure of the first reservoir 54 is greater than the pre-determ ined threshold value to the check valves’ 52 cracking pressure. In some instances, the predetermined threshold value is approximately 70mbar. The aqueous solution 53 provided in the first reservoir 54 can be supplied to the droplet generator, such as an emulsifier e.g. a step emulsifier as illustrated in Figure 2. Filters 57 are provided between the pressure controller 56 and the reservoirs 54, 58 and between the two reservoirs 54, 58. The filter 57 provided between the first and second reservoirs 54, 58 is configured to prevent cross contamination between the first and second reservoirs 54, 58. The filter 57 provided adjacent the pressure controller 56 prevents particulate contents of either of the reservoirs from impacting the functionality of the pressure controller 56. The check valve’s cracking pressure can be selected to be higher than the nominal operational pressure during droplet generation. By way of example only, the nominal operation pressure is less than 70mbar, preferably 60mbar. Once the check valve 52 is open, the wash fluid 60 flows along the conduit 14 into the Y-interconnector 32. The wash fluid 60 may be mixed with the fluid flow 62 containing the micro-entities e.g. a microdroplet from the chip. The fluid flow 62 from the chip enters into the Y-interconnector 32 via a further channel 42. After exiting the Y-interconnector 32, the wash fluid 60 contacts the micro-entities and moves it along the conduit 15 through the nozzle 64 to exit the cartridge into a receptacle such as a well plate. Referring to Figures 3A to 3F, there is provided a schematic showing the sequential flows of the wash fluid 60 and the fluid flow 62 containing micro-entities and carrier fluid from the chip. The carrier fluid may be HFE. Wash fluid 60 from the reservoir 58 flows through the check valve 52, along conduit 14 and into the Y-interconnector 32. The fluid flow 62 containing microdroplets 66 flows through a flow isolation region 69 of the chip 16 and subsequently through the valve 55, along channel 42 and into the Y-interconnector 32. The fluids 60, 62 are mixed in the Y-interconnector 32 and the resulting fluid or emulsion travels along conduit 15 to the nozzle 64 through which the fluid or emulsion exits the cartridge 10. In Figure 3A, conduit 14 and channel 42 are primed with a buffer solution which flows through the Y-interconnector 32 along the conduit 15 and out of the nozzle 64 in order to remove any unwanted materials or bubbles within the system before an experiment. As shown in Figure 3B, a microdroplet 66 is dispensed from the oEWOD area through an outlet 71 of the chip 16 in the direction of arrow C via the outlet of a flow isolation region69 and then into the conduit 42. The flow isolation region 69 is also provided with a flow of oil from an oil reservoir (not illustrated). The flow of oil enters the flow isolation reqion 69 in flow direction R through inlet 72 The valve 55 provided in the conduit 42 can be used to control the rate of flow of fluid 62 containing the microdroplets 66. Providing the valve 55 is open, as illustrated in Figure 3C, the microdroplet 66 travels along the conduit 42 towards the Y-interconnector 32. The junction i.e. Y-interconnector 32 is provided within the conduit 14 to enable the wash fluid 60 to combine and mix with the fluid flow 62 containing the microdroplet 66, shown in Figure 3D. As the microdroplets 66 travels along the conduit 15, a detector 68 e.g. a camera is provided to take images of the microdroplet 66 for further analysis. The wash fluid 60, which may be a media fluid, drives the microdroplet 66 in a carrier fluid towards the nozzle, as shown in Figure 3E. The wash fluid 60 provided downstream of the Y-interconnector 32 continues to drive the microdroplets 66 in a carrier fluid through the nozzle and out of the cartridge, as illustrated in Figure 3F. The dispensed pathway as illustrated in Figure 3 provides an optimised pathway, which permits the large mass of aqueous fluids, including micro-entities, to be ejected substantially intact through the minimisation of vias and / or corners. This minimises the risk of micro-entities, carrier fluids and / or other materials getting trapped in corners and thus, reduces the risk of causing blockages and loss of micro-entities during the dispense process. The total volume of the carrier fluid e.g. HFE that is dispensed out of the cartridge into a receptacle is the volume of carrier fluid contained by the channels in the chip, the conduit to the Y-interconnector and the conduit to the tip of the nozzle. Figure 4 shows an alternative configuration of the flow channels and conduits. In the example shown in Figure 4 a wash fluid conduit 41 provides a flow of wash fluid 60 across the outlet or nozzle 64 of the dispense conduit 15, thereby washing the microdroplet 66 and any surrounding dispense fluid 62 from the end of the dispense outlet conduit 15 and into a receptacle positioned below. The wash fluid conduit 41 is substantially non-parallel to the nozzle 64 through which the microdroplet will be dispensed. In the illustrated example the wash fluid conduit 41 is perpendicular to the nozzle 64. Importantly, the wash fluid 60 wets the exterior of the dispense conduit 15 and dislodges any droplets of dispense fluid 62 which may or may not include microdroplets 66 that are persisting on the outer surface of the conduit. Drips comprising wash fluid, dispense fluid or a mixture of the two fall from the outlet of the conduit 15 to the receptacle. Figures 5A and 5B show one possible fluid flow arising from the configuration of conduits shown in Figures 3A to 3F. A flow of wash fluid 60 is provided through the dispense outlet conduit 15, thereby washing the droplet 66 and surrounding dispense fluid 62 from the end of the conduit and into a receptacle positioned below. Critically the wash fluid 60 wets the nozzle 64 from the inside to the outside of the nozzle tip and this wetting and subsequent drip formation dislodges any droplets that are persisting on the outer surface. In one mechanism of action, both the dispense fluid 62 and the wash fluid 60 may rise up the exterior of the nozzle tip during dripping, through capillary action. This action may position the droplet and dispense fluid 62 high up the nozzle 64 away from the drip formation region at or adjacent the tip, hence causing the droplet to persist. However, the subsequent flow of wash fluid 60, which also rises up the exterior of the nozzle 64, will have the advantageous effect of rinsing the droplet from the nozzle 64. Figure 5B shows the flow of wash fluid 60 through the tip of the nozzle 64 dislodging dispense fluid and droplets through an alternative mechanism of action. As the wash fluid 60 flows from the conduit 15, instead of wetting the exterior of the conduit 15, instead it has the effect of pulling the preceding oil droplet downwards against the capillary rise force, and so dislodging it and any microdroplets 66 contained within. Drips comprising wash fluid 60, dispense fluid 62 or a mixture of the two, together with any entrained microdroplets 66, fall from the outlet of the conduit to the receptacle. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. “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. 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. It will further be appreciated by those skilled in the art that although the invention has 5 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

1. A method of dispensing a micro-entity from a cartridge, the cartridge comprising: a chip having a microfluidic space for micro-entity manipulation and an outlet; a dispense reservoir for storing a wash fluid; and a conduit in fluid communication with the chip and terminating in a nozzle, the method comprising the steps of:discharging the micro-entity out of the outlet of the chip and along the conduit towards the nozzle;ejecting a volume of wash fluid from the dispense reservoir to move the microentity out of the cartridge.

2. The method according to claim 1, wherein the step of ejecting the volume of wash fluid is initiated by pressurising the dispense reservoir and then opening a valve.

3. The method according to any one of the preceding claims, wherein the step of ejecting the volume of wash fluid is initiated by activating an electrospray nozzle.

4. The method according to any one of the preceding claims, wherein the wash fluid is ejected through the nozzle.

5. The method according to any one of the preceding claims, wherein the wash fluid is ejected from the dispense reservoir to flow across the tip of the nozzle.

6. The method according to claim 5, wherein the flow across the tip is substantially perpendicular to the nozzle.

7. The method according to any one of the preceding claims, wherein the wash fluid is air, water and / or surfactant.

8. The method according to any one of the preceding claims, further comprising the step of supplying a carrier fluid into the chip.

9. The method according to claim 8, wherein the carrier fluid is oil.

10. The method according to any one of the preceding claims, further comprisingthe steps of detecting and / or analysing the micro-entities within the conduit.

11. The method according to any one of claims 2 to 10, wherein the step of opening the valve is initiated by the provision of a pressure exceeding a predetermined threshold value to the valve.

12. The method according to any one of the preceding claims, wherein the microentity is a microdroplet.

13. A cartridge comprising:a chip comprising a microfluidic space for micro-entity manipulation and an outlet;a conduit providing fluid communication between the chip and a nozzle through which a micro-entity can exit the cartridge;a dispense reservoir for storing a wash fluid;a fluid flow controller configured to control the flow of wash fluid from the dispense reservoir to contact the micro-entity at the nozzle and move said micro-entity out of the cartridge.

14. The cartridge according to claim 13, wherein the cartridge further comprises a connector having inputs in fluid communication with the outlet of the chip and the dispense reservoir and an output in fluid communication with the nozzle.

15. The cartridge according to claim 13 to 14, wherein the nozzle comprises a fluted or tapered tip.

16. The cartridge according to claims 13 to 15, wherein at least a part of the conduit comprises a layer of coating.

17. The cartridge according to claim 16, wherein the nozzle comprises a layer of coating.

18. The cartridge according to claim 16 to 17, wherein the coating is omniphobic, oleophobic or hydrophobic.

19. The cartridge according to claims 13 to 18, further comprising an aqueous reservoir for supplying aqueous media into the chip.

20. The cartridge according to claims 13 to 19, further comprising a pneumaticmanifold.

21. The cartridge according to claims 13 to 20, further comprising an emulsifier configured to generate microdroplets dispersed in a carrier fluid into the chip.

22. The cartridge according to claims 13 to 21, wherein the conduit is transparent.

523. The cartridge according to claims 13 to 22, wherein the conduit further comprising a detector.

24. The cartridge according to any one of claims 13 to 23, further comprising a io valve configured to be opened to eject the volume of wash fluid.

25. Use of the cartridge according to claims 13 to 24.