Improvements in or relating to cartridges

Abstract

A cassette is provided. The cartridge comprises: at least one inlet port for introducing a sample into the cartridge; at least one emulsifier configured to generate micro-droplets; the chip comprises a first composite wall and a second composite wall, and a microfluid space for micro-droplet manipulation is arranged between the first composite wall and the second composite wall; an emulsion processing channel configured to provide fluid communication between the emulsifier and the chip; and an aqueous treatment channel for introducing an aqueous medium to the emulsifier.

Description

Technical Field

[0001] The present invention relates to improvements in or related to cartridges, and particularly to a cartridge including an emulsifier for generating micro-droplets and a chip for micro-droplet manipulation. Background Art

[0002] It is known that a cartridge can be provided to accommodate a microfluidic chip, and particularly an EWOD or oEWOD chip. Electrowetting on dielectric (EWOD) is a well-known effect in which an electric field applied between a liquid and a substrate reduces the contact angle with the surface compared to the natural state. The effect of electrowetting can be used to manipulate micro-droplets by applying a series of spatially varying electric fields on the substrate to modify the surface wettability.

[0003] A variant of this method uses optically mediated electrowetting forces to power a device for manipulating micro-droplets. In such an optically mediated electrowetting (oEWOD) device, micro-droplets are transferred through a microfluidic space defined by the containing walls, for example, between a pair of parallel plates having a microfluidic space therebetween. At least one of the containing walls includes a location hereinafter referred to as a "virtual" electrowetting electrode, which is generated by selectively irradiating a region of a semiconductor layer buried therein. By selectively irradiating the layer with light from a separate light source controlled by an optical component, a virtual path of the virtual electrowetting electrode location can be instantaneously generated, causing the micro-droplets to move along the virtual path.

[0004] The cartridge also houses an emulsifier for generating micro-droplets, which can be fed into the chip through a network of microfluidic channels. Efficiently and effectively transporting micro-droplets into the chip and providing sufficient fluid to the emulsifier to generate droplets on the cartridge are key to micro-droplet manipulation using EWOD, oEWOD, or other suitable micro-droplet manipulation techniques. In particular, for cases where an oEWOD chip is required to process a very large number of micro-droplets in parallel, it is very important that the emulsion is delivered to the oEWOD chip in a monodisperse and evenly distributed state.

[0005] Therefore, there is a need to provide a compact cartridge that houses an emulsifier and an EWOD or oEWOD chip to enable efficient generation and manipulation of multiple micro-droplets. In addition, the design of the cartridge channels in the present invention is optimized such that the dispersibility and integrity of fluids (especially biological fluids containing cells) are retained. It is also desirable to provide efficient transport of fluids and / or biological components around the cartridge.

[0006] The present invention is generated in this context. Summary of the Invention

[0007] According to one aspect of the present invention, there is provided a cartridge, the cartridge comprising: at least one inlet port for introducing a sample into the cartridge; at least one emulsifier configured to generate microdroplets; a chip including a first composite wall and a second composite wall having a microfluidic space therebetween for microdroplet manipulation; an emulsion processing channel configured to provide fluid communication between the emulsifier and the chip; and an aqueous processing channel for introducing an aqueous medium into the emulsifier.

[0008] Microdroplet manipulation can be configured to be performed by EWOD or oEWOD.

[0009] The cartridge according to the present invention provides a device for transporting biological / chemical entities (such as beads or cells) from an emulsifier to a device for interrogation without damaging the cells.

[0010] The emulsifier can be, but is not limited to, a stepped emulsifier, a T-junction emulsifier, a cross-flow emulsifier, or a membrane emulsifier or any other (one or more) devices for applying shear to a fluid. Such emulsifiers can be configured to generate droplets of different sizes, compositions, and densities. They can generate emulsions having different ratios of continuous phase and dispersed phase. Any one of these parameters can affect the sensitivity of the droplets to coalescence, decomposition, electrolysis, or other harmful effects when the droplets enter the device. Advantageously, the present invention allows the use of a wide range of emulsions and different types of emulsifiers to supply the device.

[0011] The stepped emulsifier has the advantage that the flow rate of the dispersed phase or the continuous phase does not directly affect the size of the generated microdroplets, and thus a monodisperse microdroplet stream can be created with varying flow rates.

[0012] If a stepped emulsifier is used, a plurality of nozzles can be provided at the outlet of each emulsifier. Increasing the number of nozzles provided increases the size of the emulsifier but also increases the throughput. In addition, in the case where one of the nozzles at the outlet of the emulsifier is blocked, another nozzle can be used to continue generating microdroplets. The nozzle can be configured to drip an aqueous fluid into an oil carrier phase. The nozzle 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.

[0013] Thus, the choice of the number of nozzles in each emulsifier and the number of overall emulsifiers is a balance between the substrate area / volume requirements (real estate requirements) of the cartridge and the micro-droplet throughput requirements of the chip. The cartridge is not configured to store a large volume of micro-droplets, and thus the rate of micro-droplet generation in the emulsifier must substantially match the downstream processing of micro-droplets in the chip. The chip can be an oEWOD or EWOD chip. In some embodiments, there can be between 8 and 16 nozzles in each emulsifier, and 8 emulsifiers in each cartridge.

[0014] The cross-section of the nozzle can be rectangular or can be a trapezoidal profile with two drafted side walls. The profile has a nozzle height h, which largely determines the size of the droplets detaching from the nozzle and the characteristic width w. The width is always greater than the height. In the case of the trapezoidal profile, the side walls have a draft angle that will be in the range of 0.1 degrees to 10 degrees, most typically in the range of 5 degrees to 10 degrees.

[0015] The nozzle can be set at an acute angle relative to the channel of the emulsifier. The aqueous fluid from the nozzle flows out through the stepped wall. The stepped wall has a draft angle, and the emulsifier operates effectively at this draft angle. This is an unexpected result because the prior art has stepped walls with a 0-degree vertical drop. Emulsifiers are known in the art where a slope is provided such that the nozzle depth increases along the length of the flow path. This slope of the nozzle depth is in contrast to what is seen in the device of the present invention, where the draft angle is present on the front surface of the nozzle and the nozzle depth itself is uniform throughout the nozzle structure.

[0016] The choice of nozzle depth within the channels of a stepped emulsifier determines the size of the microdroplets. This is advantageous because a stepped emulsifier within a single cassette can provide multiple different nozzle depths and thus multiple different microdroplet sizes. For example, a nozzle depth of 20 microns can generate a range of microdroplet sizes with diameters between 60 μm and 120 μm. The droplet diameter can be further varied depending on the nature of the fluid being run and the addition or removal of additives such as surfactants, density modifiers, viscosity modifiers. In particular, the droplet diameter can vary according to the composition of the aqueous fluid, which can be a cell medium of various formulations such as buffer solutions, protein mixtures, vitamin nutrients, and salts. The volume of the droplets can range from 80 pL to 800 pL; in particular, for smaller-sized droplets, in the range of 87 pL to 143 pL; in particular, for medium-sized droplets, in the range of 220 pL to 320 pL; and in particular, for larger-sized droplets, in the range of 380 pL to 800 pL. Droplets can also be formed with volumes greater than 800 pL. The supply of a wide range of droplet volumes by the cassette is particularly useful for bead-based immunoassays.

[0017] The microdroplets generated by the nozzle can have a diameter that is approximately four times the nozzle depth. The term "depth" as used herein is not intended to be limited to the vertical length of the nozzle, but this is one possible configuration. The flow through the emulsifier is substantially unaffected by gravity and thus the emulsifier can be operated in any orientation.

[0018] The emulsion treatment channel can have, but is not limited to, a linear geometry such as square, rectangular, or diamond-shaped. The diamond shape is particularly suitable for certain manufacturing techniques, including injection molding, where the required draft angle is present for all cross-sections. Alternatively, the emulsion treatment channel can have a substantially circular cross-section. The diameter, major axis, or width of the emulsion treatment channel can be less than 500 microns, and in some embodiments, less than 200 microns. In some embodiments, the diameter, major axis, or width of the emulsion treatment channel can be from 200 microns to 500 microns, such as between 200 microns and 300 microns.

[0019] Depending on the choice of molding technique used, the channel dimensions and shape of the emulsifier can be more or less constrained. In some embodiments that employ soft lithography as the molding technique, the channels can have vertical sidewalls, and for some types of masters, it can be challenging to fabricate sidewalls with any draft angle.

[0020] In some embodiments employing hot embossing techniques, sidewall draft can be optionally applied depending on the choice of the master manufacturer. For injection molding processes, the design of the mold, the operating parameters, and the choice of molding material will all affect the range of draft angles that can be achieved. For all the factors mentioned herein, it is desirable to design an emulsifier that is very robust to variations in draft angle. The emulsifier design of the present invention has been demonstrated by the inventors to produce single, dispersed droplets in the presence of a draft angle. The droplet breakup mechanism for this structure is the formation of an elongated neck of fluid within the narrowest region of the nozzle, where these necks are geometrically prone to break beyond a certain length due to the geometric mismatch between the neck and the droplet extruded on the other side of the front surface of the nozzle. Increasing the draft angle on the front surface of the nozzle does not change the droplet volume at which this breakup condition occurs, but only causes the neck to extend further beyond the nozzle at the break point.

[0021] One aspect of the present invention relates to maintaining the integrity of the emulsion such that the emulsion can move through the cartridge without interference. This is achieved through a careful selection of the channel geometry and routing. This includes a configuration that is substantially free of corners, which should be understood to mean a radius of curvature not exceeding 4 mm. Additionally, the radius of curvature should be chosen such that deposition does not occur substantially at the operating speed. For example, if the radius is greater than 4 mm, the flow rate is less than 10 ul / minute. Overall, laminar flow should be maintained.

[0022] In some embodiments, the cartridge may further include one or more through-holes connecting the emulsion processing channels. In some embodiments, the through-holes have a hydraulic diameter that is substantially the same as that of the emulsion processing channels.

[0023] The emulsion is prone to interference when traveling through the microfluidic channels. In particular, the droplets within the channels may undergo coalescence, breakup under shear, maturation, and changes in their filling rate. Each of these interferences may be caused by turns and corners in the emulsion processing channels and the transfer through the through-holes connecting the layers in the cartridge structure. They may also be caused by transfer over rough or hydrophilic surfaces. Advantageously, the cartridge design allows for the use of substantially straight channels for emulsion processing, where the emulsion can flow without disruptive corners. When there are corners in the channel, the corners are oriented such that the radius of curvature is maximized. In cases where through-holes are required in the channel, the number of through-holes is minimized and chosen such that the hydraulic diameter of the through-hole matches the hydraulic diameter of the channel into which the through-hole is inserted.

[0024] In some embodiments, the through-holes can be tapered to provide a gradually changing hydraulic diameter. In some embodiments, the channels leading to the through-holes can be tapered to provide a gradually changing hydraulic diameter.

[0025] In some embodiments, the aqueous treatment channel can be substantially cornerless and have smooth sidewalls sized between 100 μm and 400 μm. If the channel is straight, the size of the sidewalls can be the height or width of the channel. For example, if the channel has a square cross-section, the height and width can be the same. Alternatively, the size of the sidewalls can be the diameter of a channel with a circular cross-section, or the major or minor axis of a channel with an elliptical cross-section. It is obvious that the channel elongation length perpendicular to the channel size will exceed that channel size. In other words, the length of the channel is much larger than the height, width, or diameter of the channel.

[0026] In some embodiments, the aqueous treatment channel can be configured to maintain the integrity of the contents of the aqueous treatment channel during use. The aqueous treatment channel can be used to introduce particles suspended in an aqueous medium into a cartridge. Additionally or alternatively, the aqueous treatment channel can process a mixture or solution of fluid components. The aqueous treatment channel can be particularly useful for introducing biological and / or chemical entities, such as cells, beads, and / or reagents, into an emulsifier.

[0027] The surface of the aqueous treatment channel can be formed and / or treated such that laminar flow passes through the channel. This is particularly useful for reducing air bubbles within the channel. Additionally or alternatively, the aqueous treatment channel can be coated with a coating configured to prevent biological and / or chemical entities from adhering to the walls of the aqueous treatment channel. Further, the coating can also reduce shear in the fluid flow. Thus, the geometry of the aqueous treatment channel and / or the treatment of the surface of the aqueous treatment channel provides suitable conditions for processing cells. The coating can also reduce roughness and defects in the aqueous treatment channel, such as those caused by machining or molding with a machined mold. Such defects can also be removed or reduced by processes such as polishing or annealing.

[0028] The choice of materials used to form the channel and the channel walls has a crucial impact on the integrity of emulsions and biological particle suspensions as they travel through the channel. The materials used to form the channel can reduce the wetting of droplets on the channel walls. Additionally, the materials used can also reduce or eliminate the adhesion of biological particles to the channel walls. Further, certain materials will produce smoother, higher-fidelity channels and more accurately reproduce the physical structures molded therein.

[0029] In some embodiments, the channels are formed of Cyclic Olefin Copolymer (COC). In some embodiments, the channels are formed of Cyclic Olefin Polymer (COP). In other embodiments, the channels are formed of any one of Polycarbonate (PC), Polydimethlysiloxsane (PDMS), silicone rubber, and thermoplastic elastomer. Any of the above materials can be extended with pigments, which improve the visual appearance of the materials. In some embodiments, the pigments can increase the optical absorption of the materials, making the materials suitable for bonding by laser welding.

[0030] There are a variety of techniques that can be used for the assembly and construction of the cartridge. The base of the cartridge can be manufactured by 3D printing, micro-milling, hot embossing, or injection molding to form a plate with channel structures on one or both sides of the plate. The injection molding process can also introduce through-holes that insert into the channels, or alternatively, these through-holes can be created by post-molding processes such as drilling, laser ablation, micro-milling, abrasion, etching, stamping, or hot tool cutting.

[0031] To seal the channels within the plate, a top cover layer and an optional bottom cover layer are provided. These cover layers can be formed of a pressure-sensitive adhesive backing film, and after being cut to shape and perforated, they can be directly applied to the cartridge substrate. Alternatively, the cut and perforated film can also be laser welded to the cartridge base plate. Alternatively, the cover layer can also be formed of foil, film, composite material, plastic block, ceramic, or other suitable materials (e.g., FR4, metal, or glass).

[0032] The gasket can be overmolded into the base plate of the cartridge. The gasket is particularly used to seal the interfaces between the sub-components of the cartridge, such as the interface between the base plate and the emulsifier, or the interface between the base plate and the chip. Alternatively, adhesives or contact fits can also be used to fix the gasket in place, or to a retaining structure formed in the cartridge or sub-components.

[0033] Additional structures such as lids, caps, lid retainers, glass chips, and emulsifiers can be attached to the cartridge by a variety of techniques. The emulsifiers can be attached in place using threaded retaining screws, adhesives, or by laser welding. The oEWOD chips can be bonded in place using retaining clamps and / or screws, laser welding, or adhesives. Similarly, the lid retaining structure can be bonded in place using laser welding, retaining clamps and / or screws, or by adhesives. Any additional structure can be manufactured using injection molding, embossing, laser cutting, etching, micro-milling, 3D printing, or any other suitable manufacturing technique.

[0034] Additional structures can be added inside or around the cassette to provide reinforcement and protection. Such structures can include ridges, beams, rods, or bars made of materials such as steel, aluminum, fiberglass composites, ceramics, or plastics, which can prevent the cassette from bending.

[0035] The electrical connection between the chip, the unique identifier chip, and the exposed terminals on the cassette can be achieved through wires, printed circuit boards, or flexible circuit boards. Advantageously, using a flexible circuit board allows for complex electrical connection routing around the cassette without the need for a wire harness.

[0036] When pumping an aqueous dispersion of particles through a channel, the dispersion can become non-uniform. The particles can sediment due to gravity, flow focusing, and shear in the fluid flow. The particles can adhere to the channel walls either permanently or temporarily. These effects are known to occur in microfluidic channels that contain biological material particles such as cells and microbeads. The cassette is designed such that those channels that connect the input aqueous reservoir and the emulsifier (i.e., the aqueous processing channels) are substantially straight and have smooth sidewalls; any bends within the channel are routed to maximize the radius of curvature of the channel. When through-holes are required in the routing, the number of through-holes is minimized, and the hydraulic diameter of the through-holes is selected to match the hydraulic diameter of the channels to which the through-holes connect. The through-holes can be designed such that the through-holes have a tapered diameter at the proximal and / or distal ends to achieve a smooth transition of fluid flow between the channels interconnected by the through-holes.

[0037] In some embodiments, each emulsifier is a stepped emulsifier having a plurality of nozzles, and each nozzle is connected by a branched channel having a curved geometry.

[0038] The curved geometry of each branched channel is advantageous because it reduces the accumulation of cells and / or beads that may be trapped at the corners of the channel. Additionally, the curved geometry of the channel has fewer sharp turns and thus reduces the level of disturbance experienced by the cells and beads as they travel through the branched channel.

[0039] Furthermore, the curved geometry of the branched channel can help to minimize the distance and thus reduce the time required for microdroplets to travel through the emulsifier. This is advantageous because it reduces the risk of microdroplets accumulating within the branched channel and thus reduces the risk of multiple microdroplets merging with each other within the branched channel.

[0040] In some embodiments, the nozzle height can be in the range of 6 μm to 30 μm, and the stepped variation of the channels of the emulsifier can be between 20 μm and 200 μm.

[0041] In some embodiments, each stepped emulsifier further includes support columns located near the outlet of the emulsifier. The support columns provided at the outlet of each stepped emulsifier can prevent overcrowding of the microdroplets. This in turn minimizes the risk of damaging the microdroplets and / or reduces the risk of the microdroplets merging with each other.

[0042] The cartridge may further include a shield. The shield can protect the cartridge and the contents of the cartridge from being exposed to environmental conditions. The shield can be integral with the cartridge. The shield can be particularly advantageous because it protects the cartridge from exposure to the environment while also controlling and / or maintaining the conditions inside the cartridge covered by the shield. By way of example only, the shield can provide a thermal seal and / or a light seal for the cartridge.

[0043] In addition, providing a shield for most cartridge features can significantly improve the safety and durability of the cartridge. For example, providing a shield on the cartridge can assist user interaction by keeping the user's fingers away from the internal structure of the cartridge, thereby benefiting both the cartridge contents and the user.

[0044] The shield can be made of a non-conductive material such as plastic. This can help protect both the user and the overall system from unwanted exposure to electric current, thereby maintaining the integrity of the sample, the system, and the user. In addition, the shield can provide structural integrity to the cartridge by making it more rigid and thus more robust.

[0045] The shield can accommodate ducts, structures, and orifices that allow temperature-controlled air to enter the internal structure of the cartridge, making the resulting structure particularly suitable for culturing cells such as mammalian cells, bacteria, or yeast. Ducts and orifices arranged in this way can direct warm or cold air around the cartridge.

[0046] In some embodiments, the cartridge may further include one or more lids configured to cover each inlet port. In some embodiments, at least four inlet ports are provided for introducing samples into the cartridge. Providing multiple inlet ports on the cartridge can be useful when different samples need to be loaded into the cartridge for different experiments. By way of example only, a user can introduce samples containing different cells into two or more inlets. The user can then cover each inlet port with their respective corresponding lid.

[0047] In some embodiments, the cartridge may further include at least one reservoir. In some embodiments, the number of reservoirs matches the number of stepped emulsifiers such that each stepped emulsifier has a dedicated reservoir. This ensures that there is no cross - contamination between the stepped emulsifiers and also allows different aqueous fluids to be provided to each stepped emulsifier as needed. Additionally, there is at least one oil reservoir. In some embodiments, a single oil reservoir is sufficient to supply oil to all the stepped emulsifiers provided on the cartridge.

[0048] In some embodiments, the cartridge may further include at least one outlet port for dispensing the sample out of the cartridge. In some embodiments, the outlet port includes a dispensing nozzle. A detection module such as a camera may be provided at the outlet of the port to image the content of the sample flowing out of the dispensing nozzle.

[0049] The formation of droplets at the outlet of the dispensing nozzle is controlled by the flow rate of the liquid flowing into the nozzle, as well as the dynamics of droplet relaxation, evaporation near the tip, and the physical characteristics of the nozzle. This includes the size, shape, and material of the nozzle.

[0050] The nozzle can be made of PTFE, Teflon, PEEK, polyimide, steel, aluminum, glass, fused silica, but it should be understood that other suitable materials are also available. It can be a composite material of the above - mentioned materials. In some embodiments, it can be a composite of steel lined with PTFE on the inside, or it can be a composite of fused silica coated with polyimide on the outside. In some embodiments, it can be a glass or fused silica nozzle coated with a hydrophobic material such as fluorosilane. In some embodiments, the coating material can be lipophilic.

[0051] Any material can be coated with an anti - fouling material and / or a hydrophobic material. The hydrophobic material can prevent droplets from wetting the inside or outside of the dispensing tube. Coatings can be used to modify the dripping behavior at the tip.

[0052] Advantageously, a tube formed of fused silica allows the detachment of smaller droplets, thus ensuring the ejection of the smallest possible volume from the chip and maximizing the throughput of the dispensed droplets.

[0053] In some embodiments, the cartridge may further include an optical inspection area, which can be provided between the outlet port and the chip. In some embodiments, the inspection area is suitable for imaging with a camera.

[0054] In some embodiments, the dispensing path may include a buffer loop connected to a port on a rotary valve, the port allowing droplets to be injected into the buffer loop and then, by repositioning the rotary valve, the droplets are ejected from the buffer loop into the dispensing nozzle.

[0055] In some embodiments, an accompanying instrument is also provided that controls and monitors the functions of the cartridge through a variety of mechanisms. For some embodiments, these mechanisms can include pneumatic pressure, optical readout and manipulation, electrical control, and motion control. Typically, the instrument is a laboratory device designed for long-term reuse, while the cartridge is a consumable component designed for single use or with a limited service life. In some embodiments, the cartridge can be partially reusable or fully reusable.

[0056] In some embodiments, the cartridge can further include alignment features disposed on the surface of the cartridge for ensuring correct alignment of the chip within the cartridge. The alignment features disposed on the cartridge can assist the user in correctly positioning the cartridge within the instrument.

[0057] In some embodiments, for this purpose, the cartridge can be positioned within an optical device that houses one or more light sources. For example, alignment features can be used to position the cartridge relative to a first light source (e.g., a light source disposed above the cartridge). The first light source can be used to generate a sprite pattern for oEWOD.

[0058] The cartridge can also be correctly positioned by the alignment features so as to use a second light source to inspect the contents of the microdroplets. The second light source can be disposed perpendicular to the first light source.

[0059] The alignment features can be provided in the form of one or more orifices and / or one or more pins located on the surface of the cartridge; one or more holes within the body of the cartridge, and structures disposed on the edges of the cartridge and / or the shroud. In some embodiments, tracks on the distal edge of the cartridge mate with grooves in the instrument.

[0060] In some embodiments, at least one of the reservoirs is an input oil reservoir, and the cartridge further includes a rotary valve configured to connect the input oil reservoir to the stepped emulsifier.

[0061] In some embodiments, the cartridge can further include a second rotary valve configured to control the output from the chip to the outlet port of the cartridge. The second rotary valve can be used to convey microdroplets from the oEWOD chip to a dispensing nozzle located at the outlet port of the cartridge. The second rotary valve can be configured to control the dispensing of microdroplets to one or more waste reservoirs on the cartridge.

[0062] In some embodiments, the cartridge may further include a pneumatic manifold configured to introduce pressure into the cartridge. The manifold allows pressurization of fluid reservoirs embedded within the cartridge. The pressure source may be provided by an instrument pressure supply that can be automatically connected to the pneumatic manifold. The pneumatic source may be in the form of a pump configured to apply positive pressure to the cartridge. Additionally or alternatively, the pump may be configured to provide a vacuum within the cartridge. These pumps may be disposed within the cartridge structure or, conversely, these pumps may be disposed within an associated instrument. The advantage of applying pneumatic pressure from the instrument is that such a configuration can ensure that the instrument never comes into contact with biological fluids or other reagents that may contaminate the instrument. Additionally, placing pumps within a disposable cartridge structure would increase the cost and complexity of the cartridge.

[0063] In some embodiments, one or more microporous filters may be provided on the cartridge to reduce or eliminate cross - contamination between the instrument and the cartridge. Such filters may be formed of a breathable structure that prevents the entry of cells, viruses, or other biological particles while allowing air to pass through. Such filters may also prevent the entry of liquids. The size and area of the filter must be chosen such that the filter does not impede air flow for pressurizing the reservoir.

[0064] In some embodiments, the cartridge may further include a unique identifier. The unique identifier on the cartridge may be in the form of a series of numerical markings, barcodes, ID, EEPROM or other forms of non - volatile computer memory, RFID tags, transponders, or sequence codes. In some embodiments, the identifier may be used to store information that can then be retrieved to inform the user of certain workflows, capacity limitations, reusability of the cartridge; expiration date of the cartridge. It may also include various other data related to a specific experimental workflow or other information associated with the cartridge. The unique identifier may be particularly suitable for machine reading, for example, by a barcode scanner, RF scanner, or electrical connection to terminals provided on the cartridge.

[0065] In some embodiments, the cartridge is a consumable component suitable for single - use; in some cases, the cartridge is suitable for use 2, 3, 4, 5, or 6 times, but is suitable for a limited number of uses, such as fewer than 10 or fewer than 20 reuses. By updating the memory on the cartridge, the number of uses and usage history of each cartridge can be recorded. Alternatively, the number of uses and usage history can be recorded by the instrument in order to store the usage history for each unique cartridge. The storage of cartridge usage can be shared with other instruments or a central server connected to a usage network.

[0066] In some embodiments, the instrument or associated control software may limit the number of times the cartridge can be reused or limit the exact sequence of workflows permitted on the cartridge based on the usage history of the cartridge.

[0067] In some embodiments, multiple stepped emulsifiers can be oriented such that their inlets and outlets are parallel to each other. In some embodiments, up to eight stepped emulsifiers can be provided on the surface of the cartridge to generate a continuous flow of uniform and monodisperse microdroplets. The parallel configuration of the inlets and outlets is to ensure that all input paths have similar lengths and numbers of corners, thereby maintaining the integrity of the emulsion and keeping the emulsion integrity consistent between the paths. This minimizes the amount of variation that the software has to handle when loading droplets onto the chip. For example, when the emulsion enters the chip in a very high-density state, it may be more difficult for the image recognition algorithm to identify individual droplets, and / or it may be more difficult to pick up droplets using an optoelectrowetting pattern, and / or the emulsion may be more likely to wet the chip surface. Conversely, if the emulsion enters the chip in a very low-density state, the rate of loading droplets into the chip may be very slow. Therefore, the density of the emulsion provided into the chip through the emulsion processing channels must be controlled.

[0068] As another example, if the emulsion is introduced into the chip in a polydisperse state, a large number of droplets may need to be excluded from the input because they are not suitable for loading into the chip. In the case of very large droplets existing in the polydisperse emulsion, it may not be possible to process the over-sized droplets within the optoelectrowetting chip. In the case of very small droplets existing in the polydisperse emulsion, they may not be subject to the electrowetting force within the chip and thus cannot be controlled.

[0069] In some embodiments, the cartridge may further include at least one waste container. The waste container can be configured to store unwanted liquid within the cartridge so that the liquid can be removed from the chip by the user at a convenient time. Additionally, it can prevent the fluid from spraying out of the cartridge immediately and, if needed, can enable the fluid to be recycled. Further, storing the unwanted liquid in the waste container can help minimize or prevent cross-contamination of the fluid within the cartridge.

[0070] In some embodiments, emulsifiers (such as stepped emulsifiers) can be injection molded. The stepped emulsifiers can be injection molded, which can be cost-effective. Additionally, the stepped emulsifiers can be arranged to generate a large number of regular microdroplets. The generated microdroplets have a high degree of integrity so as to be used for oEWOD or EWOD manipulation.

[0071] Furthermore, manufacturing stepped emulsifiers through injection molding techniques allows for design flexibility. For example, during the injection molding process, different heights and depths can be achieved within the channels of each emulsifier.

[0072] The emulsifier can be manufactured by a hot embossing process or a soft lithography process, which has the advantage that the sidewalls of the emulsifier can be vertical rather than tapered.

[0073] Additionally or alternatively, the emulsifier can be formed by etching (including reactive ion etching). This is advantageous because it provides a smooth surface. Further, fabricating the emulsifier by etching can be highly accurate and cost-effective and can provide vertical sidewalls. Additionally or alternatively, the emulsifier can be formed by ablation or micro-machining because these techniques are suitable for producing components with a different range of critical structural dimensions (e.g., the nozzle height dimension in the emulsifier). In some embodiments, different regions of the emulsifier are formed using different fabrication techniques.

[0074] According to one aspect of the invention, there is provided the use of a cassette according to any aspect of the invention.

[0075] According to one aspect of the invention, there is provided an entity or species screened by the apparatus, device, cassette or method disclosed herein.

[0076] According to one aspect of the invention, there is provided an entity or species selected by the apparatus, device, cassette or method disclosed herein.

[0077] According to one aspect of the invention, there is provided an entity or species separated by the apparatus, device, cassette or method disclosed herein.

[0078] According to one aspect of the invention, there is provided an entity or species made by the apparatus, device, cassette or method disclosed herein.

[0079] The entity or species can be chemical, biochemical or biological in nature.

[0080] For example, the invention can provide an agonist / antagonist to an entity identified by the screening, selection and / or separation methods disclosed herein. The invention can provide an agonist / antagonist to an entity identified by the screening, selection and / or separation methods disclosed herein for therapeutic use. The entity can be chemical, biochemical or biological in nature.

[0081] According to one aspect of the invention, there is provided the use of the apparatus, device, cassette, method, biological and / or chemical entity or species disclosed herein.

[0082] According to one aspect of the invention, there is provided the use of the apparatus, device, cassette, method, biological and / or chemical entity or species disclosed herein in therapy.

[0083] The invention can provide the use of the apparatus, device, cassette, method, biological and / or chemical entity or species disclosed herein in the manufacture of a product. The manufactured product can be chemical, biochemical or biological in nature.

[0084] The use can be peptide synthesis. The use can be synthetic biology. The use can be cell line engineering or development. The use can be cell therapy. The use can be drug discovery. The use can be antibody discovery.

[0085] According to one aspect of the present invention, there is provided the use of the devices, apparatuses, cartridges, methods or species disclosed herein in an analysis.

[0086] The analysis can be physical, chemical or biological.

[0087] The use can be subcellular imaging. The use can be high content imaging.

[0088] The use can be diagnosis.

[0089] The use can be a bioassay. The bioassay can be high-throughput screening. The bioassay can be ELISA.

[0090] The use can be cell secretion.

[0091] The use can be QC safety assessment. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] The present invention will now be further and more particularly described by way of example only and with reference to the accompanying drawings, in which:

[0093] Figure 1 A cartridge according to the present invention is shown;

[0094] Figure 2 A shield provided on the cartridge according to Figure 1 is shown;

[0095] Figure 3 A plurality of emulsifiers provided within the cartridge are shown;

[0096] Figure 4A An alternative view of the emulsifier provided within the cartridge is provided;

[0097] Figure 4B A plurality of nozzles within one emulsifier are shown;

[0098] Figure 5 and Figure 6 The nozzle of the emulsifying machine is shown;

[0099] Figure 7 A view of the support column provided near the outlet of the emulsifier is provided;

[0100] Figure 8 An illustration of the alignment feature provided on the surface of the cartridge is provided;

[0101] Figure 9 One or more pins located on the surface of the cartridge are shown;

[0102] Figure 10 A through-hole structure connected between two layers of the microfluidic channel structure is shown;

[0103] Figure 11 A shield with an intake duct provided on the cartridge is shown;

[0104] Figure 12A and Figure 12B A pneumatic gasket of the cartridge according to the present invention is shown;

[0105] Figure 13A 、 Figure 13B and Figure 13C Mechanical components within the cartridge are shown;

[0106] Figure 14A and Figure 14B A slide valve is shown. Detailed Description of the Invention

[0107] Referring to Figure 1 , a cartridge 10 is provided having at least one inlet port 12 for introducing a sample into the cartridge 10. As Figure 1 shown, the cartridge has up to eight inlet ports 12 for introducing a sample into the cartridge 10. The sample can be a fluid sample (such as a liquid sample), and the sample can contain at least one biological and / or chemical entity. The sample can be an aqueous medium, a buffer solution, a suspension, or particles.

[0108] The biological and / or chemical entity can be a cell, a biomolecule, a protein, or a polypeptide, such as a hormone, an enzyme, a cell signaling molecule, a signal transduction molecule, an immunoglobulin, etc. The biological and / or chemical entity can be a nucleic acid, such as RNA, DNA, or a hybrid thereof. The biological and / or chemical entity can be a chemical substance, such as an insecticide, a toxin, an antibiotic, a fuel, a pharmaceutical drug, a vaccine, an antiviral drug, etc. The biological and / or chemical entity can be a carbohydrate, an antibody or a fragment thereof, a microbead, a particle, a compound, and / or a drug.

[0109] Other biological and / or chemical entities can also be a virus, an irritant, a cytokine, a nutrient, and / or a dissolved gas. The cells can be of the same type, such as if they are B cells or T cells (lymphocytes). It may be necessary to analyze a co-culture, where the cells within the droplet are of different types, such as a combination of reporter cells and primary cells, or a culture combining epithelial cells of different phenotypes to form a tissue-like structure.

[0110] The cell can be natural. The cell can be artificial. The cell can be a microcell. The cell can be a biological cell. The biological entity can be one or more parts of the cell, such as the nucleus and / or mitochondria.

[0111] The cell can be taken from any suitable source, such as a cell sample from a human or an animal, a plant, or a microorganism.

[0112] The cell can be a cell from a human or an animal, optionally from a mammal. The cell can be a plant cell, an insect cell, a fungal cell, a bacterial cell, or an amoeba cell. The cell can be a cell fusion body, such as a hybridoma.

[0113] The cell can be taken from a cell culture, such as a culture of stem cells, pluripotent cells, genetically engineered cells, etc.

[0114] If the cell is derived from a sample / biological sample, the cell can be any human, animal, environmental (natural, artificial, or modified), or food sample containing at least one cell type. The sample / biological sample can be selected from: feces, peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper's fluid or pre-ejaculatory fluid, female semen, sweat, fecal matter, hair, tears, cyst fluid, pleural fluid and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menstruation, pus, sebum, vomit, vaginal discharge, breast secretion, mucosal secretion, fecal water, pancreatic juice, sinus cavity lavage fluid, broncho-pulmonary aspirate, blastocoel fluid, and cord blood. Alternatively, the sample can be from a tissue sample.

[0115] The cell can be isolated from a patient or an individual. The cassette of the present invention as described herein can be used to screen such cells and return them to the patient (autologous cell transplantation). Cells can be isolated from one individual and selected for administration to a patient (allogeneic cell transplantation).

[0116] For some embodiments, the panel of microdroplets containing at least one cell contains the same type of cell, such as lymphocytes, such as T cells. Thus, the cells can be preselected before being incorporated into the microdroplets. However, some contamination can occur with any biological cell, and different types of cells may also be included in the microdroplets, such as B cells may be included in the microdroplets when T cells are the desired type.

[0117] For some embodiments, different populations of cell types may be included in the panel of microdroplets, such as if an environmental sample is being screened for the presence of unknown bacterial cells.

[0118] The cell can be a human or mammalian cell. The cell can be any suitable type from any tissue type, such as from an organ or tissue of the body.

[0119] The cell can be an immune system cell. Such cells include monocytes, macrophages, osteoclasts, neutrophils (polymorphonuclear leukocytes), dendritic cells, microglia, 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.

[0120] The cell can be a CHO cell, or it can be a Jurkat cell. In some examples, Chinese hamster ovary (CHO) cells are engineered to produce an immunotherapeutic drug (e.g., TCR), and then emulsified into microdroplets and loaded onto a microfluidic platform. Empty or multiply occupied microdroplets will be discarded. The remaining microdroplets containing single CHO cells are incubated on the chip to facilitate the production of the immunotherapeutic drug. Then the droplets containing CHO are split to obtain multi-dose drugs generated by each cell. T cells and target tumor cells are emulsified and loaded into an array on the microfluidic platform, respectively, so as to adjust the cell occupancy of each microdroplet as needed. Then the T cell and tumor cell arrays are combined. A second merging operation is used to add a dose of the immunotherapeutic drug, tracking which CHO cell each dose comes from. The resulting assay is incubated, and then the T cell killing behavior is monitored using a detection system by detecting caspase3 / 7 fluorescence (a fluorescent marker of apoptosis). CHO cells that generate an effective dose of the test drug can be dispensed from the device into a well plate.

[0121] The cell can be a pluripotent cell or a stem cell isolated or prepared by culture techniques. Pluripotent stem cells can be reprogrammed into mature cell types.

[0122] The cell can be genetically engineered before being encapsulated into a microdroplet. The cell can be genetically engineered after being encapsulated in a microdroplet.

[0123] Genetic engineering of the cell can be carried out by any suitable method, including transduction (viral gene transfer), gene editing (using nucleases such as zinc finger nucleases, TALENs, CRISPR / Cas9 bases, and prime editing), non-viral gene delivery (such as nanoparticle delivery), gene knockdown, gene knock-in, and genetic manipulation using RNA, such as gene silencing or activation or optogenetics. Genetic engineering generally involves introducing genetic elements into the cell by any suitable means.

[0124] In some embodiments, the biological and / or chemical entity is any one or more of the following entities: antibody; antigen; receptor; substrate; enzyme; ligand; nucleic acid; cell; part of a cell; extracellular vesicle; liposome; polymer; chemical substance; drug; FRET reporter gene; chemiluminescent material; tissue sample; virus or phage; cytokine; and / or protein.

[0125] Cartridge 10 further includes at least one emulsifier 14 configured to generate microdroplets. A chip 16 is disposed within cartridge 10. Chip 16 includes a first composite wall and a second composite wall having a microfluidic space therebetween for EWOD or oEWOD microdroplet manipulation. One or more emulsion processing channels 18 are provided on cartridge 10 and are configured to transport microdroplets from emulsifier 14 to chip 16 for performing EWOD or oEWOD operations. In addition, one or more aqueous processing channels 20 are provided on cartridge 10 for introducing a fluid sample such as an aqueous medium into emulsifier 14.

[0126] In use, a sample containing biological and / or chemical entities such as cells is loaded into cartridge 10 via one or more inlet ports 12. In some cases, different samples containing different cell types can be loaded into different inlet ports 12. At the start of any experiment, a sample can be loaded into cartridge 10 via inlet port 12. In some cases, additional samples can be loaded into cartridge 10 via inlet port 12 during the experiment. As Figure 1 shown, each of the inlet ports 12 is provided with a lid 22. Once a sample is loaded into inlet port 12, the user can close lid 22 to protect the sample and seal the reservoir so that it can maintain pneumatic pressure.

[0127] A pneumatic manifold 24 is disposed within cartridge 10. Pneumatic manifold 24 can be connected to an external pressure source (not shown in the figures). In use, pneumatic manifold 24 can apply pressure to cartridge 10 to move the fluid sample along channels 18, 20 within the cartridge. The pneumatic source 24 can be a pump configured to apply positive pressure to cartridge 10. Alternatively, the pump can be configured to provide a vacuum within cartridge 10. As Figure 1 shown, pneumatic manifold 24 includes a set of O-rings 25 that serve as seals to ensure that no air, gas, or liquid can escape from cartridge 10. The pneumatic manifold can provide a connection to an external instrument having a series of air outlets.

[0128] The aqueous treatment channel 20 can be used to introduce a mixture of particles, fluid components, solutions, media, and / or suspensions into one or more emulsifiers 14 under pressure. The aqueous treatment channel 20 is substantially free of corners and has smooth sidewalls to maintain the integrity of the cells. The smoothness of the sidewalls can result from the choice of material forming the sidewalls, or it can result from a post-treatment that minimizes the risk of cell adhesion to the sidewalls of the aqueous treatment channel 20. The fluid flowing through the aqueous treatment channel 20 is in laminar flow.

[0129] As Figure 1 shown, the cartridge 10 further includes an emulsion treatment channel 18. The emulsion treatment channel 18 is disposed between the emulsifier 14 and the chip 16. In use, the emulsion treatment channel 14 is configured to convey the micro-droplets generated by the emulsifier 14 to the chip 16. The emulsion treatment channel 18 has a linear geometry. As Figure 1 shown, the emulsion treatment channel 18 is a substantially straight channel in which the emulsion can flow without disruptive corners in order to maintain and preserve the integrity of the micro-droplets.

[0130] As Figure 1 shown, one or more emulsifiers 14 are provided. Up to eight emulsifiers 14 are disposed on the cartridge 10, but those skilled in the art will appreciate that the number of emulsifiers can be increased or decreased depending on the size and configuration of the cartridge 10. One or more emulsifiers 14 are configured to generate a stream of micro-droplets which can then enter the EWOD or oEWOD chip 16 via the emulsion treatment channel 18. Advantageously, if a stepped emulsifier is used, a monodisperse stream of micro-droplets with varying flow rates can be created.

[0131] Reference Figure 1 , a plurality of oil reservoirs 26, 27, 28 are shown, with one of the oil reservoirs 26 configured to supply oil to one of the emulsifiers 14. In addition, another inlet port 23 is provided to supply oil directly to the emulsifier 14. The aqueous micro-droplets generated by the emulsifier 14 are dispersed in the oil to form an emulsion. In some cases, a large single input oil reservoir 28 is provided to supply oil to all of the emulsifiers 14 disposed on the cartridge 10 simultaneously. The cartridge 10 further includes a first rotary valve 30 configured to connect the input oil reservoir 28 to the emulsifiers 14. The first rotary valve 30 can be configured to control the amount of oil flowing from the oil reservoir 28 to the emulsifiers 14. As Figure 1 shown, a second rotary valve 31 is configured to control the dispensing of micro-droplets to one or more waste reservoirs 44 on the cartridge 10. As Figure 1As shown, another oil reservoir 27 is disposed adjacent to the waste reservoir 44. In addition, the cartridge 10 houses one or more waste treatment channels 21 that connect the outlet of the EWOD or oEWOD chip 16 to the waste reservoir 44. The waste treatment channels 21 are configured to convey waste fluid from the chip 16 to the waste reservoir 44.

[0132] The cartridge 10 also includes at least one outlet port 32 for dispensing samples out of the cartridge 10. The dispensed samples may include one or more microdroplets. The outlet port 32 includes a dispensing nozzle 34. The dispensing nozzle 34 may be configured to control the flow rate of the sample fluid out of the cartridge 10.

[0133] An optical inspection area 36 is provided between the outlet port 32 and the chip 16. The inspection area 36 is suitable for imaging with a camera. The camera may be used to image the contents of the sample flowing out of the dispensing nozzle 34.

[0134] Reference Figure 1 , a dispensing path including a buffer loop 38 with ports 37 connected to at least one rotary valve 30 is shown. This allows fluid (which may contain microdroplets) to be injected into the buffer loop 38 and then, by repositioning the rotary valve 30, the fluid containing the microdroplets is ejected from the buffer loop 38 and into the dispensing nozzle 34.

[0135] A unique identifier 40 is provided within the cartridge 10 for storing information unique to each individual cartridge 10. This enables a user to identify a specific cartridge 10 for use. The unique identifier 40 on the cartridge 10 may be in the form of a series of numerical markings, barcodes, IDs, or serial codes.

[0136] As Figure 1 shown, tracks 42 located at the distal edge of the cartridge 10 mate with grooves in an instrument (not shown in the drawings) to ensure proper alignment of the cartridge 10 within the instrument. In addition, one or more alignment features 46 are provided on the surface of the cartridge 10 to ensure proper alignment of the chip 16 within the cartridge 10.

[0137] Reference Figure 2 , a shield 48 is shown that is arranged to cover the cartridge 10. The shield 48 may protect the cartridge 10 and the contents of the cartridge from exposure to environmental conditions. Thus, the shield may also effectively assist in controlling and / or maintaining the conditions inside the cartridge 10 covered by the shield 48. In one example, the shield 48 may provide a thermal seal and / or a light seal for the cartridge. As Figure 2As shown, some of the inlet ports in the inlet port 12 of the cassette 10 are partially or fully exposed so that the user can easily access or reach them to introduce a sample into the cassette. Each inlet port 12 includes a lid 22. In addition, another oil inlet port 23, oil reservoirs 26, 27, 28, the waste container 44, and the O-ring 25 of the pneumatic manifold are partially exposed to provide the user with access or reach.

[0138] Reference Figure 3 provides an illustration of a plurality of stepped emulsifiers 14 provided on the cassette 10. As Figure 3 shown, each stepped emulsifier 14 includes a branch channel 50. The curved geometry of the branch channel 50 can ensure that the distance between the inlet port 52 and the outlet port 54 of the stepped emulsifier is uniform for all parallel flow paths, and thus the flow resistance for each path through the emulsifier is substantially the same. This reduces the risk that the branch channel 50 operates at different flow rates, and thus reduces the risk that the biological material spends completely different amounts of time within the branch channel 50. The emulsifier 14 can be coupled to the surface of the cassette 10 by welding and a gasket 56.

[0139] Reference Figure 4A the stepped emulsifier 14 houses an inlet port 52 configured to receive a sample fluid from an aqueous processing channel. As Figure 4A indicated by the dashed arrow in Figure 4A and Figure 4B shown, the sample fluid flows under pressure and towards the end of the branch channel 50 where a plurality of nozzles 60 are provided, as

[0140] As Figure 4A shown, a separate port 58 is provided on the stepped emulsifier 14, and the separate port 58 is configured to introduce oil from an oil reservoir into the stepped emulsifier 14. The oil bypass represented by the solid arrow in Figure 4A bypasses the branch channel 50 and enters the micro-droplet buffer zone 59 via a bypass channel 57 through the emulsifier 14, where the formed aqueous micro-droplets are dispersed into the oil carrier phase. The droplets in the buffer zone remain under continuous low-speed flow as they pass through the buffer zone. The micro-droplets then leave the stepped emulsifier 14 via the outlet 54 of the stepped emulsifier 14 and enter the emulsion processing channel for conveyance to the chip.

[0141] Reference Figure 5 provides a nozzle 60 connected to one end of the branch channel 50 of the stepped emulsifier 14. The choice of the nozzle depth within the channel 70 of the stepped emulsifier 14 determines the micro-droplet size. AsFigure 5 and Figure 6 As shown in Figure 6 , the nozzle has a rectangular cross-section 61 with two drafted side walls 62, 64. Alternatively, the nozzle may have a different cross-sectional geometry, for example, it may be trapezoidal in profile. The nozzle 60 is configured to drip an aqueous fluid into an oil carrier phase. As the aqueous fluid flows through the nozzle 60, the height h 66 of the nozzle determines the size of the micro-droplets exiting the nozzle 60. The width w 68 of the nozzle can also affect the size of the micro-droplets formed.

[0142] In some examples, the nozzle height h 66 can be in the range between 6 μm and 30 μm, or it can be greater than 6 μm, 8 μm, 10 μm, 12 μm, 14 μm, 16 μm, 18 μm, 20 μm, 22 μm, 24 μm, 26 μm or 28 μm. In some examples, the nozzle height h can be less than 30 μm, 28 μm, 26 μm, 24 μm, 22 μm, 20 μm, 18 μm, 16 μm, 14 μm, 12 μm, 10 μm or 8 μm. The width is always greater than the height.

[0143] In addition to the height and width of the nozzle 60, the stepped change in the channel 70 of the emulsifier can also create different micro-droplet sizes as the aqueous fluid passes through the step. The stepped change in the channel 70 of the emulsifier can be between 100 μm and 250 μm, or it can be greater than 100 μm, 120 μm, 140 μm, 160 μm, 180 μm, 200 μm, 220 μm or 240 μm. In some embodiments, the stepped change in the channel 70 of the emulsifier is less than 250 μm, 240 μm, 220 μm, 200 μm, 180 μm, 160 μm, 140 μm or 120 μm.

[0144] Referring Figure 7 , a stepped emulsifier 14 is shown that includes an inlet port 52 and an outlet port 54 for introducing an aqueous fluid into the stepped emulsifier 14. A separate port 58 is provided on the stepped emulsifier 14, and the separate port 58 is configured to introduce oil into the stepped emulsifier 14. The oil bypasses the branch channels and directly enters the micro-droplet buffer zone 59 via the bypass channel 57 of the emulsifier 14, where the formed aqueous micro-droplets are dispersed into the oil carrier phase. The stepped emulsifier 14 further includes a branch channel 50 connected to a plurality of nozzles 60 and support columns 72 near the outlet port 54 of the stepped emulsifier 14. The support columns 72 provided at the outlet port 54 of the stepped emulsifier 14 can prevent the micro-droplets from overcrowding at the outlet port 54 of the emulsifier 14 and thereby minimize the risk of damaging the integrity of the micro-droplets. The support columns can be formed of the same base material as the material for the body of the stepped emulsifier.

[0145] Reference Figure 8 and Figure 9 , cassette 10 further includes one or more alignment features 46 disposed on a surface of the cassette 10 to ensure proper alignment of the chip 16 within the cassette 10. The alignment features 46 may be in the form of holes or pins 76. The holes 76 are disposed within the body of the cassette 10 and / or on the edges of the cassette 10.

[0146] In addition, the alignment features 46 disposed on the cassette 10 can be used to enable a user to properly position the cassette within an instrument (not shown in the figures) for reading out data by optical interrogation of the contents of the cassette 10. As an example, the alignment feature is an aperture 76 that is located near one of the rotary valves 31 of the cassette 10, as Figure 9 shown.

[0147] Reference Figure 10 , a cross-section of a through-hole 80 disposed between two emulsion processing channels 18 formed within the structure of the cassette 10 is shown. The through-hole 80 can be used to direct fluid between layers within the cassette structure. In addition, the through-hole 80 is sized such that the size of the through-hole 80 matches the hydraulic diameter of the emulsion processing channels 18 to which the through-hole 80 is interconnected. In addition, the through-hole 80 can be designed such that the through-hole 80 has a tapered diameter at the proximal end 83 and / or the distal end 85 to provide a smooth transition for fluid flow between the emulsion processing channels 18.

[0148] Figure 11 A shield 48 covering the body of the cassette 10 is shown. A duct 86 is disposed within the shield 48 that allows warm air to enter the body of the cassette 10 and directs the warm air to those areas where cells may be incubating.

[0149] Reference Figure 12A and Figure 12B , a plurality of gaskets 90 are shown that provide a pneumatic seal between two or more mating surfaces 92, 94, such as between the surfaces of two cassettes and / or between the surface of a cassette and the surface of an instrument. Depending on the requirements of the cassette configuration in which the gasket is deployed, the gasket 90 can be compressed over a wide range of heights.

[0150] As Figure 12BAs shown, washer 90 can be of a conical or cup-shaped configuration. The conical or cup-shaped washer 90 has a wide orifice at one end of the washer 96 that tapers gradually towards the opposite end 98 to provide a varying diameter. Various other configurations of the washer will be apparent to those skilled in the art. The conical or cup-shaped washer 90 can be collapsible and provides sealing pressure over a large compression range. Thus, the washer has the advantage that it can prevent leakage from or into mating surfaces under compression.

[0151] In some embodiments, the washer can be configured to seal rough or deformed surface(s) without leakage. In other embodiments, the washer can be configured to seal any non-linear surface(s), such as surface(s) that are substantially non-parallel.

[0152] The collapsible cup or conical structure of the washer can provide near-constant sealing pressure over a wide range of compression heights. The dimensions of the washer, such as the bead plating of the cup and / or the thickness of the wall, can be scaled to adjust the compression and sealing behavior. In some embodiments, the washer can be re-shaped to match different surfaces. This can provide multiple independent sealing sites on the first mating surface during compression without sealing the second mating surface.

[0153] The washer can be made of a wide range of materials with different hardnesses. For example, the washer can be made of a relatively soft Shore 50 silicone rubber polymer, or the washer can be made of a harder Shore 85 FKM rubber. A wide range of suitable compliant materials can be provided, including silicone elastomers, FKM, FFKM, polyurethanes, nitrile rubbers, natural rubbers, polyisoprene, neoprene, and polyamides, and more generally fluoroelastomers. Any of these materials can have a range of hardnesses.

[0154] Washers made of materials with a wide range of hardnesses can have the advantage that even if made of a high-hardness material, the washer can have soft (low Shore hardness) portions. As used herein and unless otherwise specified, the term "Shore hardness" of a material refers to an indicative measure of the compliant response of the material in a Shore hardness test. This test measures the local deformation of the material by indentation and is generally known to those skilled in the art as a suitable material selection criterion for sealing elastomeric structures. Additionally, washers made of high-hardness materials can provide a robust interface that can prevent leakage.

[0155] Another advantage of the gasket disclosed herein is that the gasket can provide a wide range of compression without collapsing or clogging the orifice of the gasket. The gasket can include a sealing range that is largely independent of the size and shape of the orifice. The gasket can be made of (one or more) materials that do not clog the orifice of the gasket when compressed. The gasket can also be suitable for molding with tapered sidewalls and a structure suitable for release from a mold by minimizing the use of overhangs and having no internal cavities.

[0156] Additionally or alternatively, the cartridge can include strengthening features (not shown in the drawings) that can be provided on the base (substrate) of the cartridge, i.e., on the bottom surface of the cartridge. The strengthening features can be made of aluminum, steel, FR4 (a composite material consisting of woven fiberglass cloth and a flame-retardant epoxy adhesive), but are not limited thereto. FR4 is a grade name for glass-reinforced epoxy laminates. The strengthening features can prevent the cartridge from bending and reduce or eliminate deformation of the cartridge when compressed or held in an instrument. Additionally or alternatively, the reinforcing material can be made of GFRP (glass fiber-reinforced polymer), carbon fiber, or other composite materials of appropriate strength.

[0157] Reference Figure 13A 、 Figure 13B and Figure 13C , a mechanical chip assembly 100 is disposed within the cartridge 10, and the mechanical chip assembly 100 allows removal and reinstallation of an EWOD or oEWOD chip 16. The mechanical chip assembly or fixture 100 can be disposed at the bottom surface or the top surface of the EWOD or oEWOD chip 16, as shown in Figure 13B and Figure 13C . The mechanical chip assembly / fixture 100 includes an upper support plate, a lower support plate, a threaded insert 102, an optional plurality of self-tapping screws, a deformable gasket that provides a seal between the chip and the substrate of the cartridge, and an optional one or more holes 104 for alignment pins to precisely position the chip within the substrate. Optionally, the fixture can be transparent. Advantageously, the fixture can allow the chip to be added to the cartridge at any point during the manufacturing process. Additionally, the fixture allows the chip to be removed for rework of the cartridge, and the fixture allows the chip to be removed and replaced by the user.

[0158] Additionally or alternatively, the mechanical assembly or fixture disposed within the cartridge can also allow removal and reinstallation of a stepped emulsifier. The features of the mechanical assembly can include a plurality of self-tapping screws, threaded inserts, an upper support plate, a lower support plate, a deformable gasket between the emulsifier and the channel plate, and optional alignment features for registering the emulsifier with the substrate of the cartridge.

[0159] Reference Figure 14A , an embodiment of a low-force spool valve 106 is shown. The spool valve 106 can be made of a thermoplastic elastomer material (such as TPE). AsFigure 14A As shown, the spool valve 106 can be disposed on the support member 108. Referring to Figure 14B , an alternative embodiment of the spool valve 106 is provided. The spool valve 106 can be disposed on the cartridge to control the opening of the aqueous reservoir in a sequential manner. By opening one reservoir at a time, the user can accurately control the amount of aqueous fluid flowing within the cartridge.

[0160] In addition, as Figure 14B shown, the spool valve 106 disposed on the substrate 110 of the cartridge can be relatively short and less flexible, which reduces the actuation force required to overcome friction. In Figure 14B , a plurality of gaskets 90 are shown disposed on both sides of the valve 106 to provide a seal in the intermediate closed position. The gaskets 90 can be independent of each other and can be lubricated and / or coated with a non-sticky coating. The gaskets 90 can be coated with a low-friction coating that prevents adhesion between the gaskets and / or prevents adhesion between the moving and stationary parts of the valve body.

[0161] In view of the present disclosure, various other aspects and embodiments of the present invention will be apparent to those skilled in the art.

[0162] As used herein, "and / or" shall be regarded as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" shall be regarded as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were listed separately herein.

[0163] Unless the context otherwise indicates, the descriptions and definitions of the above features are not limited to any specific aspect or embodiment of the present invention and apply equally to all aspects and embodiments described.

[0164] Those skilled in the art should also understand that although the present invention has been described by way of example with reference to several embodiments, the present invention is not limited to the disclosed embodiments and alternative embodiments can be constructed without departing from the scope of the present invention as defined by the appended claims.

Claims

1. A cartridge, comprising: At least one inlet port for introducing a sample into the cartridge; At least one emulsifier configured to generate micro-droplets; A chip including a first composite wall and a second composite wall having a microfluidic space therebetween for micro-droplet manipulation; An emulsion treatment channel configured to provide fluid communication between the emulsifier and the chip; And An aqueous treatment channel for introducing an aqueous medium into the emulsifier.

2. The cartridge according to claim 1, further comprising one or more through-holes connecting the emulsion treatment channels.

3. The cassette according to claim 2, wherein, The through-holes have a hydraulic diameter substantially the same as that of the emulsion treatment channels.

4. The cassette according to claim 2 or 3, wherein, The through-holes are tapered to provide a gradually changing hydraulic diameter.

5. The cassette according to any one of claims 2 to 4, wherein, The channels leading to the through-holes are tapered to provide a gradually changing hydraulic diameter.

6. The cassette according to claim 5, wherein, The aqueous treatment channel is substantially free of corners and has smooth sidewalls sized between 100 μm and 400 μm.

7. The cassette according to claim 5 or 6, wherein, The aqueous treatment channel is configured to maintain the integrity of the contents of the aqueous treatment channel during use.

8. The cassette according to any one of the preceding claims, wherein, Each emulsifier is a stepped emulsifier having a plurality of nozzles, and wherein each of the nozzles is connected by a branched channel having a curved geometry.

9. The cassette according to claim 8, wherein, The height of the nozzles is in the range of 6 μm to 30 μm, and the stepped change of the channels of the emulsifier is between 20 μm and 200 μm.

10. The cassette according to claim 8 or 9, wherein, Each stepped emulsifier in the stepped emulsifier further includes support columns located near the outlet of the emulsifier.

11. The cartridge according to any one of the preceding claims, further comprising a shield.

12. The cartridge according to any one of the preceding claims, further comprising one or more lids configured to cover each inlet port.

13. The cartridge according to any one of the preceding claims, further comprising at least one reservoir.

14. The cartridge according to any one of the preceding claims, further comprising at least one outlet port for dispensing the sample out of the cartridge.

15. The cassette according to claim 14, wherein, The outlet port includes a dispensing nozzle.

16. The cartridge according to claim 14, further comprising an optical inspection area provided between the outlet port and the chip.

17. The cassette according to claim 16, wherein, The inspection area is suitable for imaging with a camera.

18. The cassette according to claim 14, wherein, The dispensing path includes a buffer loop connected to a port on a rotary valve, the port on the rotary valve allowing droplets to be injected into the buffer loop and then, by repositioning the rotary valve, the droplets are ejected from the buffer loop into the dispensing nozzle.

19. The cartridge according to any one of the preceding claims, further comprising alignment features provided on the surface of the cartridge for ensuring correct alignment of the chip in the cartridge.

20. The cassette according to claim 13, wherein, At least one of the reservoirs is an input oil reservoir, and wherein the cartridge further comprises a rotary valve configured to connect the input oil reservoir to the stepped emulsifier.

21. The cartridge according to claim 20, further comprising a second rotary valve configured to control the output of the chip to the outlet port of the cartridge.

22. The cartridge according to any one of the preceding claims further comprises a pneumatic manifold configured to introduce pressure into the cartridge.

23. The cartridge according to any one of the preceding claims further comprises a unique identifier.

24. The cassette according to any one of the preceding claims, wherein, The plurality of stepped emulsifiers are oriented such that the inlets and outlets of the plurality of stepped emulsifiers are parallel to each other.

25. The cartridge according to any one of the preceding claims further comprises at least one waste container.

26. The cassette according to any one of the preceding claims, wherein, The microdroplet manipulation is configured to be performed by EWOD or oEWOD.

27. Use of the cartridge according to any one of the preceding claims.

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