Improvements in or relating to the composite walls of a device

Abstract

A composite wall for an electro-mechanical wire or EWOD device is provided, the composite wall comprising a substrate, a conductor layer disposed on the substrate, a dielectric layer disposed on the conductor layer, a port extending through the composite wall, and an insulator configured to separate a microdroplet from the conductor layer as the microdroplet passes through the port. A method of manufacturing the composite wall is also provided.

Description

[Technical Field]

[0001] The present invention relates to composite walls, and in particular to electrowetting-on-dielectric (EWOD) or optically-mediated electrowetting-on-dielectric (oEWOD) devices. The present invention also relates to a method for manufacturing a composite wall. [Background technology]

[0002] Electrowetting on dielectrics (EWOD) is a well-known effect in which an electric field applied between a liquid and a substrate causes the liquid to become more wettable on the substrate than it would naturally be. The electrowetting effect can be used to manipulate microdroplets (e.g., control their movement, merge them, split them, or change their shape) by applying a series of spatially varying electric fields to a substrate to increase the wettability of the surface, followed by a series of spatial variations.

[0003] A variation of this method is to use optically mediated electrowetting forces to provide a driving force and manipulate microdroplets within the device. In this optically mediated electrowetting (oEWOD) device, the microdroplets change position through a microfluidic space defined by walls, such as a pair of plates, with the microfluidic space between the walls. At least one of the walls contains what will be referred to below as "virtual" electrowetting electrode locations, which are generated by selectively illuminating regions of a semiconductor layer embedded within the virtual electrowetting electrodes. Selective illumination of this layer with light from a separate light source controlled by an optical assembly can transiently generate a virtual path of virtual electrowetting electrode locations along which the microdroplets can be moved.

[0004] To form a "virtual" electrowetting electrode within the walls of an EWOD or oEWOD, at least one of the walls involved comprises a composite layer structure, with a conductive layer embedded within this wall.

[0005] In order to use an EWOD or oEWOD device in an environment having a non-atmospheric pressure, such a device must be sealed. This is done by ensuring that the composite layer, and optionally any spacer structures within the device, enclose the entire device and define an enclosed area. This reduces the exchange of gas with the atmosphere.

[0006] Once the device is sealed, one or more ports are provided to connect the internal space of the device to the outside so that microdroplets can be introduced into the gap between two walls of the device, where they can then be manipulated by EWOD or EWOD methods. These ports can be gaps in the layers or spacer structures described above, allowing the microdroplets to enter through the side without passing through the composite wall; alternatively, they can be ports that penetrate the composite wall.

[0007] During the fabrication of the composite wall of an EWOD or oEWOD device, ports in the composite wall can be formed by laser drilling through the layers of the composite wall. This process exposes thin areas of ITO, which provide a bias voltage to the oEWOD device. As microdroplets pass through the composite wall through the ports, they must pass through the conductive layer. It is important to be able to load microdroplets into the device while a voltage is maintained across the device, as this voltage is used to hold other droplets already in the microfluidic device in place. However, when a voltage is applied to the conductive layer, one or more droplets passing through the port can come into contact with the conductive layer on the composite wall. High electric fields are known to cause droplet merging and other adverse effects, such as electrolysis of the droplet medium and droplet breakup. As a result of contact between the microdroplets and the conductive layer, the microdroplets may become trapped at the port, merge with each other, or disintegrate. This therefore means that droplets may not be loaded into the microfluidic device in an efficient manner. Microdroplets entering the microfluidic device may have large variations in their size; input droplets that are nearly monodisperse in volume may become polydisperse after passing through the port, which is highly undesirable for subsequent multiple droplet analysis (assays) and manipulation.

[0008] Furthermore, a voltage applied directly across aqueous microdroplets can drive electrolysis of the microdroplets, which can damage or even destroy biomolecules and cells contained within the microdroplets. As mentioned above, it has been hypothesized that emulsion collapse, i.e., electrolysis and merging of the microdroplets, can occur due to the voltage on the exposed conductor layer. The merging microdroplets can form large aqueous plugs, which can then cause significant damage to cells within the aqueous plugs. This effect is particularly pronounced when loading dense emulsions, particularly when the injected material is a large volume of continuous aqueous fluid (aqueous plug) rather than monodisperse microdroplets. Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, there is a need to provide a device and method for efficiently loading droplets into a microfluidic device without the droplets coalescing together or becoming trapped at a port. Additionally, there is a need to minimize or eliminate droplet collapse (electrolysis and coalescence) caused by voltages on exposed conductor layers.

[0010] It is against this background that the present invention was born. [Means for solving the problem]

[0011] According to one aspect of the present invention, there is provided a composite wall for an oEWOD or EWOD device, the wall comprising: a substrate; a conductor layer disposed on the substrate; a dielectric layer disposed on the conductor layer; a port extending through the composite wall; and an insulator configured to separate a microdroplet from the conductor layer as the microdroplet passes through the port.

[0012] The invention disclosed herein comprises a composite wall of a microfluidic device, particularly a composite wall of an EWOD or oEWOD device. According to the invention, an insulating layer of the composite wall is configured to separate and prevent contact between the microdroplets and the conductor layer as they pass through the port in the composite wall. Preventing contact between the microdroplets and the conductor layer can be advantageous because it can minimize or eliminate droplet disruption (electrolysis and coalescence) caused by voltages on exposed conductor layers.

[0013] Additionally, the composite walls offer an added advantage in that they can be used to prevent microdroplets from joining together, getting trapped around the port, or wetting on the surface of the port, allowing users to efficiently load droplets into microfluidic devices and perform experiments.

[0014] In some embodiments, the insulating layer is at least a portion of a dielectric layer disposed on the conductor layer. The conductor layer disposed around the loading port can be completely sealed by the dielectric layer. This configuration prevents droplets from contacting the conductor layer as they enter or are loaded into the device. This configuration therefore helps prevent droplet breakup by minimizing the effects of binding and / or electrolysis as microdroplets pass through the port. In some embodiments, the dielectric layer lines the inside of the port, thereby providing the insulator. In these embodiments, the dielectric layer can seal the conductor layer at least around the port, preventing the conductor layer from contacting microdroplets passing through the port. This is advantageous because the insulator therefore has the same breakdown voltage as the portion of the dielectric layer that forms the composite wall.

[0015] As used herein, "proximate a port" refers to the area of ​​the port and / or the area near the port, unless otherwise specified, and includes the walls of the port, the periphery of the port, and the area of ​​the substrate adjacent to the periphery of the port. In particular, it refers to the area of ​​the port and / or the area near the port where the conductive layer cannot contact the microdroplets as they pass through the port. For example, an insulating layer can be provided to seal off the conductive layer at least near the port. This prevents the conductive layer from contacting the microdroplets as they enter the port. In another example, the conductive layer can be removed from the vicinity of the port to form a recess, thereby preventing the conductive layer from contacting the microfluidic fluid as it passes through the port.

[0016] Furthermore, the device can provide continuous loading of emulsion into the device while continuously incorporating injected microdroplets into the device under the control of oEWOD. In some embodiments, the microdroplets can contain one or more cells. The microdroplets can also contain media such as cell culture media and / or buffer solutions. In addition, the device configuration can help preserve the integrity of the cells, biomolecules, and chemical reagents contained within the microdroplets. The device configuration can also improve the monodispersity of the microdroplets loaded into the device.

[0017] The device is particularly suitable for use with droplets delivered from an emulsifier connected upstream of the device and connected to a bore within the device. The upstream emulsifier can be a T-junction emulsifier, a cross-flow junction emulsifier, a step emulsifier, a membrane emulsifier, or any other device that applies shear forces to a fluid. Such emulsifiers can generate droplets of varying size, composition, and density. These emulsifiers can generate emulsions with varying ratios of continuous to dispersed phase. Any of these parameters can affect the susceptibility of droplets to coalescence, splitting, electrolysis, or other deleterious effects as they pass through the loading port and enter the device. Advantageously, the present invention allows a wide range of emulsions to be loaded into the device without disruption. Additionally, the present invention advantageously allows a wide range of emulsions and different types of emulsifiers to be used to deliver the device.

[0018] The device configuration can allow for the processing of weaker emulsions, such as emulsions with lower stabilizing surfactant concentrations, or emulsions containing substances that destabilize the emulsion, such as buffers, salts, proteins, or alcohols. The device configuration can allow for the processing of emulsions with very high proportions of dispersed phase within the continuous phase, or emulsions with variable ratios of dispersed to continuous phase.

[0019] In some embodiments, the insulation may be provided by a gap or recess in the conductive layer. In some embodiments, the insulation may be provided by a recess formed in at least a portion of the conductive layer. Alternative configurations of such devices may prevent or eliminate binding and / or electrolysis of microdroplets as they pass through the port.

[0020] In some preferred embodiments, the conductive layer is an indium tin oxide (ITO) layer.

[0021] Removing the ITO layer in the circular region around the port improves device performance during loading by allowing increased voltage to be applied before droplet coupling occurs. This removal can be achieved by etching, laser ablation (instantaneous removal), mechanical ablation, or any other suitable method. The shape of the ITO-free region can also be square, rectangular, oval, or any other suitable shape. In particular, this shape can follow the shape of the flow channel structure around the port.

[0022] In some embodiments, the conductive layer can be recessed near the port to provide an air gap, which is the insulator. Alternatively, the air gap can be filled with a fluid, such as oil, which provides the insulator. Creating a recess in the conductive layer, with or without the addition of oil, ensures that the conductive layer does not come into contact with the microdroplets as they pass through the port.

[0023] In some embodiments, the remaining layers in the composite wall can collapse the air gap completely, providing the insulation by collapsing into the air gap and preventing contact between the microdroplets and the conductive layer.

[0024] The dielectric layer may be made of Al2O3 or SiO2 or SiN4 (silicon nitride). In some embodiments, a dielectric layer may line the port and provide the insulation. In some embodiments, the first and / or second dielectric layers are comprised of a single dielectric material. In some embodiments, the dielectric layer may comprise a composite laminate, which contains two or more materials in a layered structure.

[0025] To form the insulator, the dielectric layer applied to the area around the port can be of a different thickness and / or made of a different material than the dielectric layer applied to the rest of the device. The dielectric material in the area around the port can be, but is not limited to, plastic, an inert coating such as Teflon AF (amorphous fluoropolymer), glass, a ceramic such as hafnia, silicon nitride, or any other suitable dielectric material.

[0026] Alternatively, the ITO layer can be coated with a dielectric layer to prevent it from contacting the microdroplets as they pass through the ports.

[0027] In some embodiments, the port can have a constant diameter. Alternatively, the diameter of the port can include at least one step change in diameter. For example, the dielectric layer can not cover the entire inner surface area of ​​the port, but can leave a portion of the substrate uncovered. This can provide a larger cross-sectional area in part of the port. In some embodiments, the port can have a diameter that increases in the direction of droplet travel. In some embodiments, the port can have a diameter that decreases in the direction of droplet travel.

[0028] In some embodiments, the substrate may define a plane and the ports extend through the composite wall in a direction generally perpendicular to the plane, which may simplify manufacturing procedures, making the manufacturing process cost-effective and efficient.

[0029] Alternatively, the port can extend at an acute angle relative to the plane of the substrate. The angled port can alter the stress on the microdroplet, causing a lower shear stress as the microdroplet transitions from the port into the microfluidic space between the first and second composite walls. By reducing the shear stress, cells retained within the microdroplet are more likely to have improved outcomes compared to higher shear stress transitions.

[0030] In some embodiments, the ports may be positioned at an acute angle relative to the composite wall. By positioning the ports at an acute angle relative to the composite wall, the microdroplets can be guided into the ports in a specific orientation, which may be useful in reducing electrolysis or electrical coupling of the microdroplets.

[0031] In some embodiments, the composite wall may further comprise a photoactive layer disposed between the conductive layer and the dielectric layer, hi some embodiments, the photoactive layer may be made of amorphous silicon.

[0032] The photoactive layer can be formed of a semiconductor material that generates localized regions of charge in response to stimulation by an electromagnetic radiation source. Examples include a hydrogenated amorphous silicon layer having a thickness in the range of 100 to 1500 nm. The amorphous silicon layer preferably has a thickness in the range of 400 to 800 nm. In some preferred embodiments, the amorphous silicon layer has a thickness of less than 100 nm. In some preferred embodiments, the amorphous silicon layer has a thickness greater than 1500 nm. In some preferred embodiments, the photoactive layer is activated by the use of visible light.

[0033] The dielectric properties of this layer are >10 7 V / m(10 7 It is preferred that the dielectric constant be greater than 3 and have a high dielectric strength of greater than 3 (V / m).

[0034] In some preferred embodiments, the dielectric layer is selected from alumina, silica, hafnia, or a thin non-conductive polymer film.

[0035] This novel device design allows for the use of holes in the device that are fabricated by a wide range of methods, including laser drilling, diamond drilling, sandblasting, and wet etching. Other methods for fabricating one or more holes include the use of laser machining followed by wet etching to modify the glass substrate of the device, and the use of isotropic etching processes such as reactive ion etching or inductively coupled plasma etching.

[0036] Some of these hole manufacturing processes leave rough and irregular surfaces that tend to expose portions of the conductive layers within the layer structure of the oEWOD device.

[0037] The device configurations disclosed herein allow for holes to be drilled in either the photoactive layer or the passive side of an oEWOD device without the drawbacks associated with bonding and electrolysis.

[0038] In some embodiments, the interior walls of the port can be annealed, polished, etched, or ablated, which is advantageous because it prevents roughness on the surface of the wall that could disrupt the integrity of the emulsion as it passes through the port.

[0039] In some embodiments, a photoactive layer may line the port to provide the insulation.

[0040] In some embodiments, a photoactive layer and a dielectric layer may line the port to provide the insulation.

[0041] In some embodiments, the composite walls disclosed herein provide a suitable surface onto which additional coating layers, such as hydrophobic and / or anti-fouling coating layers, can be deposited.

[0042] Lining the port with an insulator advantageously allows the interior surface of the port to be coated with a hydrophobic layer. The presence of a hydrophobic layer near the port can reduce the size of the contact surface between the emulsion droplets and the inner layer of the hole. The presence of a hydrophobic layer can also prevent the droplets from wetting on the wall. Thus, the addition of a hydrophobic layer can further reduce the amount of binding and droplet breakup caused by the passage of the emulsion through the port in the composite wall. The hydrophobic layer can also perform the function of being an anti-fouling layer.

[0043] The hydrophobic layer can be a monolayer of (1H,1H,2H,2H-perfluorooctyl)silane, deposited by incubation with 1H,1H,2H,2H-perfluorooctyltrimethoxysilane or 1H,1H,2H,2H-perfluorooctyltrichlorosilane, followed by low-pressure oxygen plasma treatment of the surface of the composite wall. In some cases, the coating material is introduced in several separate steps, each step removing the coating material from the atmosphere around the substrate before introducing a new pulse of material. In some cases, the substrate can be heated or incubated in water vapor, followed by each deposition stage.

[0044] In some embodiments, the recess can be removed by at least 0.001 mm from the vicinity of the port. By removing at least 0.001 mm from the vicinity of the port to create a recess in the conductor layer, the recess provides the minimum distance necessary for a droplet to pass through the port without contacting the conductor layer.

[0045] In some embodiments, the conductive layer may be recessed by removing at least 0.001 mm, at least 0.01, at least 0.1 mm, at least 1 mm, or at least 2 mm from the vicinity of the port.

[0046] The conductor layer may be recessed by a distance of 0.001 to 0.01 mm, 0.01 to 2 mm from the vicinity of the port, or by more than 0.001 mm, 0.005 mm, 0.01 mm, 0.05 mm, 0.1 mm, 0.5 mm, 1 mm, 1.5 mm, or 2 mm. In some embodiments, the conductor may be recessed by less than 2 mm, 1.5 mm, 1 mm, 0.5 mm, 0.1 mm, 0.05 mm, 0.01 mm, or 0.005 mm from the vicinity of the port.

[0047] In some embodiments, the ports can be approximately circular. Providing circular ports can be energetically more favorable because they direct droplets passing through them in a more favorable direction. Circular ports lack corners, which create low-flow regions under laminar flow conditions typically encountered in droplet devices, and these low-flow regions can trap droplets. If the ports are tilted at an angle relative to the surface of the composite wall, the port inlets and outlets can be oval, which also lack the low-flow corner features.

[0048] In some embodiments, the ports can be approximately square. Providing square ports can be advantageous because they can be more easily manufactured by processes such as laser cutting or punching. Other shapes and sizes of ports will occur to those skilled in the art. The ports can have smooth or rough surfaces. In some embodiments, the port shape can be tapered or flared.

[0049] According to another aspect of the present invention, there is provided a composite wall for an oEWOD or EWOD device, the wall comprising: a substrate; a conductor layer disposed on the substrate; a dielectric layer disposed on the conductor layer; and a port extending through the composite wall to allow micro-droplets to pass therethrough, the port being configured to insulate the micro-droplets from the conductor layer.

[0050] According to an additional aspect of the present invention, there is provided an oEWOD or EWOD device comprising a first composite wall and a second composite wall, one of which is a composite wall as described above.

[0051] According to an additional aspect of the present invention, there is provided an oEWOD or EWOD device comprising two composite walls, one of which is a composite wall as described above.

[0052] In some embodiments, the first and / or second conductor layers can be made of a transparent conductive material such as indium tin oxide (ITO), a very thin film of a conductive metal such as silver, or a conductive polymer such as PEDOT (Poly(3,4-EthyleneDioxyThiophene)). These layers can be formed as a continuous sheet or a series of discrete structures such as wires. Alternatively, the conductor layer can be a mesh of conductive material, with electromagnetic radiation directed between the lattices of the mesh.

[0053] The first substrate and first conductor layer, and / or the second substrate and second conductor layer may be transparent. The substrates may be made of glass or plastic.

[0054] The first and / or second dielectric layers may be composed of a single dielectric material or may be a composite of one or more dielectric materials, including, but not limited to, Al2O3 and SiO2.

[0055] In some embodiments, the second composite wall comprises: a substrate; a conductive layer disposed on the substrate; and a dielectric layer disposed on the conductive layer.

[0056] In some embodiments, the two composite walls can be separated by a gap of 5 microns to 2000 microns. In some embodiments, the gap can be 5 to 1750 microns, 5 to 1500 microns, 5 to 1250 microns, 5 to 1000 microns, 5 to 750 microns, 5 to 500 microns, 5 to 250 microns, 5 to 100 microns, 5 to 75 microns, 5 to 50 microns, or 5 to 25 microns. In some embodiments, the two composite walls can be separated by a gap of 20 to 120 microns. In some embodiments, the two composite walls are separated by a gap of 20 to 120 microns, but the gap can be greater than 20 microns, greater than 30 microns, greater than 40 microns, greater than 50 microns, greater than 60 microns, greater than 70 microns, greater than 80 microns, greater than 90 microns, greater than 100 microns, or greater than 110 microns. In some preferred embodiments, the two composite walls are separated by a gap of less than 120 microns, 110 microns, 100 microns, 90 microns, 80 microns, 70 microns, 60 microns, 50 microns, 40 microns, or even 30 microns. In some preferred embodiments, the two composite walls are separated by a gap of 20-40 microns. In some preferred embodiments, the gap can be 30 microns or 40 microns.

[0057] In some embodiments, an oEWOD device may comprise: a first composite wall comprising a first substrate; a first conductor layer on the substrate; a photoactive layer on the conductor layer; and a first dielectric layer on the photoactive layer, the first dielectric layer having a thickness of less than 20 nm; and a second composite wall comprising a second substrate; a second conductor layer on the substrate; and a second dielectric layer on the second conductor layer, the second dielectric layer having a thickness of less than 20 nm.

[0058] In some embodiments, the first and second composite walls are held in a spaced apart relationship to form a microfluidic space therebetween, and the walls may be separated by a spacer structure, which may be formed by an intervening structure between the first and second substrates, or may be formed by the substrates of the first or second composite walls.

[0059] The spacers may be formed by a layer of photoresist, by a layer of pressure sensitive adhesive, and / or by a layer of dry film resist. Additionally or alternatively, the spacers may be formed by etching structures and / or cavities in the glass, fused silica, or transparent plastic substrates that form the first or second composite walls.

[0060] In some preferred embodiments, the oEWOD device can be formed from two glass substrates separated by a laser-cut pressure-sensitive adhesive spacer. The holes and channels can be formed by laser drilling and ablation of the glass substrates. The remaining layers of the composite wall are then deposited, with the conductive ITO layer, the photoactive layer, and the overlying dielectric and hydrophobic layers covering the interior walls of the ports.

[0061] In some preferred embodiments, the first and / or second dielectric layers can be continuous. Furthermore, the first and / or second dielectric layers can have a thickness of 1 nm to 20 nm, or alternatively, 2 nm to 20 nm, 3 nm to 20 nm, 4 nm to 20 nm, 5 nm to 20 nm, 6 nm to 20 nm, 7 nm to 20 nm, 8 nm to 20 nm, 9 nm to 20 nm, 10 nm to 20 nm, 12 nm to 20 nm, 14 nm to 20 nm, 15 nm to 20 nm, or 18 nm to 20 nm. It can also be 1 to 15 nm, 1 to 10 nm, 1 to 5 nm, 5 to 10 nm, 5 to 15 nm, or 10 to 15 nm.

[0062] The oEWOD device further comprises a voltage source such as an A / C (Alternating / Current) voltage source; at least one electromagnetic radiation source; and a microprocessor, wherein the voltage source is connected to the first and second conductor layers to supply a voltage between the first composite wall and the second composite wall, the electromagnetic radiation source having an energy higher than the band gap of the photoexcitable layer and configured to act on the photoactive layer to cause electromagnetic induction at corresponding temporary electrowetting locations on the surface of the first dielectric layer, and the microprocessor manipulates the points at which the electromagnetic radiation acts on the photoactive layer to change the arrangement of the temporary electrowetting locations, thereby generating at least one electrowetting path and moving microdroplets along the path.

[0063] The A / C power supply may be connected to the first and second conductor layers to provide a voltage between the first and second composite walls between 1 V and 100 V. In some embodiments, the A / C power supply may be configured to provide a voltage between 0.01 V and 1 V, 0.05 V and 1 V, 1 V and 5 V, 1 V and 7 V, 1 V and 10 V, 1 V and 15 V, 1 V and 20 V, 1 V and 25 V, 1 V and 30 V, 1 V and 40 V, 1 V and 50 V, 1 V and 60 V, 1 V and 70 V, 1 V and 80 V, or 1 V and 90 V.

[0064] In some embodiments, the electromagnetic radiation source may be an LED (Light Emitting Diode) light source. In some embodiments, the electromagnetic radiation source may comprise a pixelated array of light reflected from or transmitted through an array of active pixel elements. Where the electromagnetic radiation source is pixelated, the electromagnetic radiation may be suitably provided either directly or indirectly using a reflective screen illuminated by light from the LEDs.

[0065] The first and second composite walls may further comprise first and second anti-fouling layers on the first and second dielectric layers, respectively. The anti-fouling layer on the second dielectric layer may be hydrophobic.

[0066] The first and / or second conductor layers on the substrate can have a thickness in the range of 70 to 250 nm, and in some embodiments, the first and / or second conductor layers can have a thickness greater than 70 nm, greater than 80 nm, greater than 90 nm, greater than 100 nm, greater than 110 nm, greater than 120 nm, greater than 130 nm, greater than 140 nm, greater than 150 nm, greater than 160 nm, greater than 170 nm, greater than 180 nm, greater than 190 nm, greater than 200 nm, greater than 210 nm, greater than 220 nm, greater than 230 nm, or greater than 240 nm. In some embodiments, the thickness of the first and / or second conductor layer can be less than 250 nm, less than 240 nm, less than 230 nm, less than 220 nm, less than 210 nm, less than 200 nm, less than 190 nm, less than 180 nm, less than 170 nm, less than 160 nm, less than 150 nm, less than 140 nm, less than 130 nm, less than 120 nm, less than 110 nm, less than 100 nm, less than 90 nm, or less than 80 nm.

[0067] The photoactive layer can be activated by electromagnetic radiation in the wavelength range of 400-1000 nm on the conductor layer, hi some preferred embodiments, the photoactive layer can be activated by electromagnetic radiation at wavelengths of 400-500 nm, 400-600 nm, 400-700 nm, 400-800 nm, or 400-900 nm.

[0068] The device may further include a photodetector to detect an optical signal in a microdroplet located within the device downstream, which may be a fluorescent signal.

[0069] According to an additional aspect of the present invention, there is provided a cartridge comprising: at least one inlet port; at least one emulsification apparatus; a device (or chip); and an emulsion processing flow path, wherein the inlet port is configured to introduce a sample, such as a liquid sample, into the cartridge and the emulsification apparatus is configured to generate microdroplets, the device (or chip) comprising first and second composite walls according to any of the aspects of the present invention, the walls having a microfluidic space therebetween for EWOD or oEWOD microdroplet manipulation, and the emulsion processing flow path is configured to provide fluid communication between the emulsification apparatus and the device.

[0070] A cartridge can be provided, the cartridge comprising: a reservoir; an emulsification device in a fluid circuit with the reservoir; an inlet channel downstream of the emulsification device; a composite wall or device according to any of the aspects of the invention; and a pump system, wherein the reservoir is configured to contain a sample, such as a liquid sample, the emulsification device is configured to generate a medium comprising an emulsion of aqueous microdroplets in an immiscible carrier fluid, the inlet channel is configured to receive the medium comprising the emulsion of aqueous microdroplets in the immiscible carrier fluid from the emulsification device, the device comprising at least one inlet port in fluid communication with the inlet channel, and the pump system is configured to introduce a flow of the sample, e.g., a liquid sample, into the at least one emulsification device and / or introduce a flow of the medium comprising the emulsion of aqueous microdroplets in the immiscible carrier fluid through the device.

[0071] The aqueous fluid within the cartridge may suitably be a biological fluid, such as a cell culture medium, and may contain cells, beads, particles, drugs, biomolecules, or other biological entities. These entities may be viruses, DNA (Deoxyribonucleic Acid) or RNA (Ribonucleic Acid) molecules, stimulants, cytokines, nutrients, and dissolved gases. Therefore, the design of the cartridge's flow channels and structure may be optimized to maintain the distribution and integrity of the biological fluid, particularly by selecting flow channels well-suited for uniform hydraulic diameters and minimal fluid shear.

[0072] In some embodiments, the cartridge may further comprise one or more valves located at the inlet port of the device, which control the flow of a medium comprising an emulsion of aqueous microdroplets in an immiscible carrier fluid.

[0073] In some embodiments, the emulsifier is a step emulsifier. Advantageously, a step emulsifier generates emulsions with a droplet size distribution that has minimal dependence on the flow rate at the emulsification junction. In some embodiments, a cartridge can include multiple emulsifiers. At least one of the emulsifiers is a step emulsifier. In some embodiments, several emulsifiers can be provided, each with an inlet channel. In some embodiments, a cartridge assembly can include up to eight emulsifiers. In some embodiments, a cartridge assembly can include at least one, two, three, four, five, six, or seven emulsifiers. In some embodiments, a cartridge assembly can include eight to twelve emulsifiers. In some embodiments, a cartridge assembly can include 12 to 20, 20 to 30, 30 to 50, or 50 to 100 emulsifiers. The emulsifiers are user-interchangeable, allowing a user to select the appropriate type of emulsifier for their intended purpose. For example, a user can configure a cartridge with an emulsifier that provides microdroplets in a particular size range. A user can select a set of emulsifiers, each providing microdroplets with a different size range or sub-selection of size ranges. In some embodiments, an emulsifier can be configured to generate microdroplets with a volume in the range of 14 pL (picoliters) to 180 pL, or in the range of 180 pL to 500 pL, or in the range of 500 pL to 1.2 nL (nanoliters).

[0074] Furthermore, the operation of a set of step emulsifier nozzles within a single emulsifier can be parallelized, allowing multiple emulsifier nozzles to be connected to a single aqueous input. The connected nozzles can operate independently, with variations in speed determined by the complex interactions between the interconnected junctions. All of these emulsifiers can generate microdroplets of approximately uniform size, determined by the physical size of the nozzles. This allows a large number of generators to operate in parallel at low flow rates, eliminating the adverse effects of shear forces that can damage cells and other biological materials. This also allows the emulsifier to continue generating emulsions despite the clogging or blocking of some nozzles, which is sometimes the result of particle-laden biological material flowing through a narrow, noisy opening.

[0075] In some embodiments, the pumping system may include, but is not limited to, a pump, a head reservoir, a pressure accumulator, and / or a pressure source. It is further understood that one of ordinary skill in the art will know of other pumping systems that can be used to induce the flow of the liquid sample to the emulsification device and / or induce the flow of the medium through the device.

[0076] The device may further comprise a plurality of electrowetting paths running concomitantly with one another, and a plurality of second electrowetting paths configured to intersect the first electrowetting paths to generate at least one microdroplet coalescence location.

[0077] 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 fluid, an aqueous medium, a buffer solution, a suspension, or particulate matter.

[0078] The biological and / or chemical entity can be a cell. The biological and / or chemical entity can be a nucleic acid such as RNA, DNA, or hybrids thereof, a protein, a polysaccharide, a polypeptide such as a peptide or hormone, an enzyme, a cell signaling molecule, a signal transduction molecule, an immunoglobulin, or the like, a biomolecule such as an antibody and / or an antibody fragment thereof. The biological and / or chemical entity can be a chemical substance such as a pesticide, a toxin, an antibiotic, a food, a pharmaceutical drug, a vaccine, an antiviral agent, or the like. 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.

[0079] Other biological and / or chemical entities can also be viruses, stimuli, cytokines, nutrients, and / or dissolved gases. The cells can be of the same type, for example, the cells are B cells or T cells (lymphocytes). It may be desirable to analyze co-cultures in which the cells in the droplet are heterogeneous, such as a combination of reporter cells and primary cells, or cultures combining epithelial cells of different phenotypes to form tissue-like structures. The cells can be natural. The cells can be artificial cells. The cells can be microcells. The cells can be biological cells. The biological entity can be one or more parts of a cell, for example, a nucleus and / or mitochondria.

[0080] The cells can be obtained from any suitable cell source, such as a cell sample from a human or animal, plant, or microorganism. The cells can be plant cells, insect cells, fungal cells, bacteria, or amoeba cells. The cells can be cell fusions such as hybridomas.

[0081] The cells can be obtained from cell culture, eg, stem cells, pluripotent cells, genetically modified cells, etc.

[0082] Where the cells are from a sample / biological sample, the sample may be any human, animal, environmental (native, non-native, or modified), or food sample containing at least one cell type. The sample / biological sample may be selected from: stool, peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper's fluid (pre-ejaculatory) or pre-ejaculatory fluid, female vaginal fluid, sweat, fecal material, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme (chyme), chyle, bile (bile), interstitial fluid, menstrual period, pus, sebum, vomit, vaginal secretions, mammary gland secretions, mucosal secretions, watery stool, pancreatic juice, sinus lavage fluid, bronchopulmonary aspirate, blastocyst cavity fluid, and umbilical cord blood. Alternatively, the sample may be derived from a tissue sample.

[0083] Cells can be isolated from a patient or individual. Using the cartridges of the invention described herein, these cells can be screened (selected) and returned to the patient (autologous cell transplant). These cells can be isolated from an individual and selected for administration to the patient (allogeneic cell transplant).

[0084] The cells can be human or mammalian cells. The cells can be of any suitable type, from any tissue type, such as from an organ or tissue of the body.

[0085] The cells can be immune cells, including monocytes, macrophages, osteoclasts, neutrophils (polymorphonuclear leukocytes), dendritic cells, small (micro)glial cells, mast (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 cells can be CHO cells or Jurkat (human leukemia T cell-derived) cells.

[0086] According to another aspect of the present invention, there is provided a method of manufacturing a composite wall according to any aspect and embodiment of the present invention, the method comprising the steps of: providing a substrate; depositing a conductive layer on the substrate; creating a hole in the conductive layer; depositing a dielectric layer on the conductive layer; and creating a port through the composite wall.

[0087] In some embodiments, a method of fabricating a composite wall includes the steps of: providing a conductive layer, such as an ITO layer, on the substrate; providing a dielectric layer on the conductive layer; and drilling holes in some of the layers to create ports through the composite wall.

[0088] In this case, it may be advantageous to remove 0.001 mm to 2 mm from the vicinity of the port to provide a recess in the conductor layer to increase manufacturing tolerances and thus provide a lower margin of error during drilling operations, which can create an efficient and cost-effective manufacturing process for composite walls according to any of the aspects and embodiments of the present invention.

[0089] In some embodiments, the holes in the conductor layer can have a larger diameter than the ports, which can effectively create embodiments with a recess, where the dielectric layer intrudes into the recess in the conductor layer, so there may not be an air gap recess, but rather the edges of the conductor layer are insulated by the dielectric.

[0090] In some embodiments, ports can be created through the composite wall prior to deposition of the dielectric layer, which deposition results in the dielectric layer lining the port.

[0091] In some embodiments, a method of manufacturing a composite wall is provided, where no drilling steps are required, and the method steps may include depositing the conductor layer on the substrate and depositing the dielectric layer on the conductor layer.

[0092] Gaps can be provided at the edges of the composite wall to provide ports. Recesses can be provided in the conductor layer to prevent microdroplets from contacting the conductor layer as they pass through the ports. Alternatively, the conductor layer can be coated with a dielectric layer to prevent the conductor layer from contacting the droplets as they pass through the ports.

[0093] According to one aspect of the present invention, there is provided a species screened by the device, apparatus or method disclosed herein.

[0094] According to one aspect of the present invention, a species selected by a device, apparatus, or method disclosed herein is provided.

[0095] According to one aspect of the present invention, there is provided a species separated by the device, apparatus, or method disclosed herein.

[0096] According to one aspect of the present invention, there is provided a species produced by the device, apparatus, or method disclosed herein.

[0097] These species can be chemical, biochemical, or biological in nature. For example, the present invention can provide agonists / antagonists to entities identified by the screening, selection, and / or isolation methods disclosed herein.

[0098] The present invention can provide therapeutic agonists / antagonists for entities identified by the screening, selection and / or isolation methods disclosed herein.

[0099] According to one aspect of the present invention, there is provided a method of using the device, apparatus, method, or species disclosed herein.

[0100] According to one aspect of the present invention, there is provided the use of the devices, apparatus, methods or species disclosed herein in the treatment of a species.

[0101] The present invention may provide for the use of the devices, apparatus, methods, or species disclosed herein in the production of an article of manufacture, which may be chemical, biochemical, or biological in nature.

[0102] The use can be peptide synthesis. The use can be synthetic biology. The use can be cell line generation or development. The use can be cell therapy. The use can be drug discovery. The use can be antibody development.

[0103] According to one aspect of the present invention, there is provided the use of the devices, apparatus, methods or species disclosed herein in the analysis of species.

[0104] The analysis can be a physical analysis, a chemical analysis, or a biological analysis.

[0105] The use may be sub-cellular imaging.The use may be high contrast imaging.

[0106] The use may be diagnostic.

[0107] The method of use can be a bioassay. The bioassay can be a high-throughput screening method. The bioassay can be an ELISA (Enzyme-Linked Immunosorbent Assay).

[0108] The use may be cell secretion.

[0109] The above usage can be a QC (Quality Control) safety profile.

[0110] The invention will now be further and more particularly described, by way of example only, with reference to the accompanying drawings in which: [Brief explanation of the drawings]

[0111] [Figure 1] Figures 1A and 1B show two oEWOD devices according to the present invention, and Figure 1C provides an alternative embodiment of an oEWOD device according to the present invention. [Figure 2] FIG. 1 illustrates a composite wall of a microfluidic device. [Figure 3] FIG. 1 illustrates a composite wall of a microfluidic device. [Figure 4] FIG. 1 illustrates a composite wall according to one aspect of the present invention. [Figure 5] FIG. 1 illustrates an embodiment of a composite wall. [Figure 6] Figure 6A shows another embodiment of a composite wall, Figure 6B shows an alternative embodiment of a composite wall, and Figure 6C shows an alternative embodiment of a composite wall. [Figure 7] 10A-10C illustrate additional embodiments of composite walls. [Figure 8] 8A-8C show the composite wall before, during, and after the etching process. [Figure 9] 9A and 9B provide illustrations of alternative etching processes for at least the conductor layer of a composite wall. [Figure 10] 10A-10E provide illustrations of alternative etching processes for a composite wall before, during, and after etching at least the conductor layer. [Figure 11] Figure 11A is a top view of a microfluidic device, such as an EWOD or 0EWOD device, with ports located through the side of the device wall, and Figure 11B is a cross-sectional view of the microfluidic device after etching, illustrating the etched regions created near the ports through the side of the device. [Figure 12]Figure 12A is a top view of a microfluidic device, such as an EWOD or 0EWOD device, with ports located through the side of the device wall. Figure 12B is a cross-sectional view of an alternative microfluidic device after etching, illustrating the etched regions created near the ports through the side of the device. [Figure 13] Figure 13A is a top view of a microfluidic device, such as an EWOD or 0EWOD device, with ports located through the side of the device wall. Figure 13B is a cross-sectional view of an alternative microfluidic device after etching, illustrating the etched regions created near the ports through the side of the device. DETAILED DESCRIPTION OF THE INVENTION

[0112] Referring to FIG. 1A, a microfluidic device, specifically an oEWOD device 100, is provided. The oEWOD device shown in FIG. 1A includes a first composite wall 102, the first composite wall 102 being composed of a first substrate 104, a first conductor layer 106, the first conductor layer 106 on the substrate 104, a photoactive layer 108 on the conductor layer 106, and a first dielectric layer 110 on the photoactive layer 108, where the first substrate 104 can be made of glass, the first conductor layer 106 having a thickness of 70-250 nm, and the photoactive layer 108 is activated by electromagnetic radiation in a wavelength range of 400-850 nm on the conductor layer 106 and has a thickness in the range of 300-1500 nm. The first dielectric layer 110 is formed as a continuous layer having a thickness of less than 20 nm. The lower limit of this layer thickness is determined, at least in part, by the method for providing such a thin layer, which must be continuous. However, theoretically, this layer can have a thickness of 0.1 nm to 20 nm. The first conductor can be transparent.

[0113] The device 100 also includes a second composite wall 112, which includes a second substrate 114 and a second conductor layer 116 on the substrate 114, where the second substrate 114 can be made of glass. The second conductor can be transparent. The second conductor layer 116 can have a thickness in the range of 70 to 250 nm. A second dielectric layer 118 can be present on the second conductor layer 116, where the second dielectric layer 118 has a thickness of less than 20 nm. Like the first dielectric layer, the second dielectric layer must be continuous, and the practical lower limit for thickness is determined by manufacturing constraints, but can be between 1 nm and 20 nm. The exposed surfaces of the first and second continuous dielectric layers 110 and 118 are spaced 20 to 180 μm apart to define a microfluidic space 121 configured to contain a microdroplet 122.

[0114] The photoactive layer 108 is made of amorphous silicon. The first and second conductor layers are made of ITO. An intervening bonding layer 124 is provided on the first dielectric layer 110 and may also be provided on the second dielectric layer 118. The thickness of the intervening layer may be between 0.1 nm and 5 nm. In some embodiments, not shown in the accompanying drawings, the intervening bonding layer may be omitted. In such embodiments, the hydrophobic layer is applied directly to the first dielectric layer.

[0115] A hydrophobic layer 126 is disposed on the intermediate bonding layer 124. An example of a hydrophobic layer can be fluorosilane or fluorosiloxane. The intermediate bonding layer 124 is optional; the channel walls 120 can be made of SU-8 or can be part of a glass structure. The intermediate layer 124 is disposed between the dielectric layers 110, 118 and the hydrophobic layer 126.

[0116] 1A, incident light 130 can be used to provide a light sprite pattern 131 in which the incident light 130 shines on a portion of the photoactive layer 108 to hold the microdroplets 122 in a stationary position within the microfluidic space 121. An oil carrier phase 134 can be provided to the microdroplets 122 through holes 136 in the device to replenish key nutrients and components to keep the contents within the microdroplets 122, such as one or more cells, viable and healthy. In some cases, the oil phase 134 can provide key nutrients, media, and components for cell growth, viability, and / or productivity for cell proliferation, cell survival, and / or cell growth rates.

[0117] The first and second substrates 104, 114 are made of a material with high mechanical strength. For example, the first and second substrates can be made of glass, metal, or engineered plastic. In some embodiments, the substrates have some flexibility. In some embodiments, the first and second substrates have a thickness of at least 100 μm. In some embodiments, the first and second substrates can be thicker than 2500 μm. In some embodiments, the first substrate is silicon, fused silica, or glass. In some embodiments, the second substrate is fused silica and / or glass. The glass can be, but is not limited to, soda-lime glass or float glass.

[0118] The first and second conductor layers 106, 116 are disposed on the surfaces of the first and second substrates 104, 114 and typically have thicknesses in the range of 70-250 nm, preferably 70-150 nm. At least one of these layers is made of a transparent conductive material such as indium tin oxide (ITO), a very thin film of a conductive metal such as silver, or a conductive polymer such as PEDOT. These layers can be formed as a continuous sheet or a series of discrete structures such as wires. Alternatively, the conductor layer can be a mesh of conductive material, with electromagnetic radiation being directed between the lattices of the mesh.

[0119] The photoactive layer 108 is formed of a semiconductor material capable of generating localized regions of charge in response to stimulation by an electromagnetic radiation source. Examples include a hydrogenated amorphous silicon layer having a thickness in the range of 300-1500 nm. In some embodiments, the photoactive layer is activated by the use of visible light. The dielectric properties of this layer are >10 7 It preferably includes a high dielectric strength of V / m and a dielectric constant > 3. In some embodiments, the dielectric layer is selected from alumina, silica, hafnia, or a thin non-conductive polymer film.

[0120] Instead, at least the first dielectric layer, and preferably both dielectric layers, are coated with an antifouling layer to help establish the desired microdroplet / carrier fluid / surface contact angle at various hypothetical electrowetting electrode locations. The antifouling layer is additionally intended to prevent the microdroplet contents from adhering to the surface and being reduced as the microdroplet moves through the chip. For optimal performance, the antifouling layer should help establish the microdroplet / carrier fluid / surface contact angle, which should be in the range of 50° to 180° when measured at the air-liquid-surface three-point interface at 25°C. In some embodiments, these layers have a thickness of less than 10 nm and are typically formed as monolayers.

[0121] In some embodiments, the microfluidic space includes one or more spacers to maintain a predetermined distance between the first and second walls. Spacer options include beads or pillars, or ridges made from an intermediate resist layer fabricated by photopatterning. Alternatively, deposited materials such as silicon oxide or silicon nitride can be used to fabricate the spacers. Alternatively, thin-film layers, including flexible plastic films with or without adhesive coatings, can be used to form the spacer layer. Various spacer geometries can be used to form narrow, tapered, or partially closed channels defined by pillars. Through careful design, these spacers can be used to facilitate microdroplet deformation, followed by microdroplet splitting and manipulation of the deformed microdroplets. Similarly, these spacers can be used to physically separate zones of a chip to prevent cross-contamination between droplet populations and promote the correct flow of droplets when loading the chip under hydraulic pressure.

[0122] 1B, an alternative oEWOD device 100 is provided. As shown in FIG. 1B, the oEWOD device includes a first composite wall 102, the first composite wall 102 being composed of a first substrate 104, a first conductor layer 106 on the substrate 104, a photoactive layer 108 on the conductor layer 106, and a first dielectric layer 110 on the photoactive layer 108, where the first substrate 104 may be made of glass, the first conductor layer 106 having a thickness of 70-250 nm, the photoactive layer 108 being activated by electromagnetic radiation in a wavelength range of 400-850 nm on the conductor layer 106, and the photoactive layer 108 having a thickness in the range of 300-1500 nm. The first dielectric layer 110 is formed as a continuous layer having a thickness of less than 20 nm.

[0123] The device 100 shown in FIG. 1B also includes a second composite wall 112, which includes a second substrate 114 and a second conductor layer 116 on the substrate 114, where the second substrate 114 can be made of glass. The second conductor can be transparent. The second conductor layer 116 can have a thickness in the range of 70 to 250 nm. A second dielectric layer 118 can be present on the second conductor layer 116, where the second dielectric layer 118 has a thickness of less than 20 nm. Like the first dielectric layer, the second dielectric layer must be continuous, and the practical lower limit for thickness is determined by manufacturing constraints, but can be between 1 nm and 20 nm. The exposed surfaces of the first and second continuous dielectric layers 110 and 118 are spaced 20 to 180 μm apart to define a microfluidic space 121 configured to contain a microdroplet 122.

[0124] FIG. 1B shows an alternative embodiment of the oEWOD device 100 in which the spacer layer is not formed of a separate material, but is formed as part of a structure within the first (active) substrate 104. Sub-layers of the oEWOD device, formed by the first conductor layer 106, the photoactive layer 108, the first dielectric layer 110, the intervening bonding layer 124, and the hydrophobic layer 126, can partially or completely cover the walls of the spacer structure. An additional embodiment is an alternative configuration of the device 100 in which the spacer layer is formed by structuring the second (passive) substrate 114. In some cases, the spacer can be formed by structuring both the first and / or second substrates 104, 114, or by using a combination of structures within the first and / or second substrates 104, 114 and an intervening material, such as the channel walls 120, as shown in FIG. 1A.

[0125] 1B, incident light 130 can be used to provide a light sprite pattern 131, where the incident light 130 illuminates a portion of the photoactive layer 108 to hold the microdroplets 122 in a stationary position within the microfluidic space 121. An oil carrier phase 134 can be provided to the microdroplets 122 through holes 136 in the device to replenish key nutrients and components to keep the contents within the microdroplets 122, such as one or more cells, viable and healthy. In some cases, the oil phase 134 can provide key nutrients, media, and components for cell growth, viability, and / or productivity for cell proliferation, cell survival, and / or cell growth rates.

[0126] 1C, a microfluidic device 100, such as an oEWOD or EWOD device 100, is provided. The device 100 comprises a first composite wall 102 and a second composite wall 112. The first composite wall 102 and the second composite wall 112 each comprise a substrate 104, 114, a dielectric layer 110, 118, and an ITO conductor layer 106, 116. The first composite wall 102 also comprises a photoactive layer 108 and a port 136 extending through the first composite wall 102. One or more aqueous microdroplets 122 can enter the microfluidic device 100 through the port 136. A voltage can be applied directly across the one or more aqueous microdroplets 122 as they travel through the port 136.

[0127] As shown in FIG. 1C, device 100 further comprises a hydrophobic layer 126 disposed on intervening bonding layer 124 in first composite wall 102. An additional intervening layer 107 is disposed between conductor layer 106 and photoactive layer 108 of first composite wall 102. Intervening bonding layers 107, 124 are optional; channel wall 120 may be made of SU-8 or may be part of a glass structure. An additional intervening layer 124 is disposed on second composite wall 112 between dielectric layer 118 and hydrophobic layer 126. Intervening layers 107, 124 may be zinc oxide or silicon oxide layers.

[0128] 1C, incident light 130 can be used to provide a light sprite pattern 131, where the incident light 130 illuminates a portion of the photoactive layer 108 to hold the microdroplets 122 in a stationary position within the microfluidic space. An oil carrier phase 134 can be provided to the microdroplets 122 through holes 136 in the device 100.

[0129] Referring to FIGS. 2 and 3, a microfluidic device 100, such as an oEWOD or EWOD device 100, is provided. The device 100 comprises a composite wall 102 and a second composite wall 112, as known in the art. Each of the first (102) and second (112) composite walls comprises a substrate 104, 114, a dielectric layer 110, 118, and an ITO conductor layer 106, 116. The first composite wall 102 also comprises a photoactive layer 108 and a port 136 extending therethrough. One or more microdroplets 122 can enter the microfluidic device through the port 136. As shown in FIGS. 2 and 3, the ITO layer 106 of the first composite wall 102 is exposed to the port 136. A voltage is applied across one or more aqueous microdroplets 122 as they travel through the port 136. This can promote electrolysis within the aqueous microdroplets and the formation of harmful gas bubbles 133, which can damage or even destroy biomolecules or cells contained within the aqueous microdroplets. Furthermore, emulsion disruption, such as electrolysis and bonding, can occur due to the voltage present on the exposed conductive layer.

[0130] 4, a composite wall 102 for a microfluidic device 100, such as an oEWOD or EWOD device 100, is provided in accordance with the present invention. The composite wall 102 comprises a substrate 104, a dielectric layer 110, and a conductor layer 106. The substrate 104 can be fused silica, silicon, silicon dioxide, or glass. The conductor layer 106 is disposed on the substrate 104, and the dielectric layer 110 is disposed on the conductor layer 106. Additionally, the composite wall 102 comprises a port 136 extending through the composite wall 102. The composite wall 102 also comprises an insulator configured to separate the microdroplets from the conductor layer 106 as they pass through the port 136.

[0131] The conductor layer 106 may be made of an ITO layer. The conductor layer may be connected to a voltage source, such as an A / C voltage source (not shown in the accompanying drawings). This connection ensures that a voltage, typically in the range of 1V to 100V, can be applied across the composite wall. The applied voltage is preferably in the range of 1 to 7V.

[0132] It is necessary to be able to load microdroplets through ports in the composite wall under voltage, because this voltage is used to hold other droplets already in place within the microfluidic space. However, if there is a voltage applied to the conductive ITO layer, the droplets tend to become trapped or bond together as they enter the port and pass over the exposed edge of the conductive ITO layer. Therefore, an insulator is provided to prevent the microdroplets from contacting the conductor layer as they pass through the port.

[0133] As shown in FIG. 4 , a gap or recess 138 is provided in the conductor layer 106, and the conductor layer 106 is etched away a predetermined distance from the port 136 using various etching methods, including, but not limited to, laser drilling, diamond drilling, sandblasting, and / or wet etching. Etching the conductor layer 106 away from the port 136 leaves a gap 138 between the port 136 and the conductor layer 106. This ensures that there is no contact between the droplet 122 and the conductor layer 106 when the droplet 122 passes through the port 136 under voltage. This can reduce or eliminate electrolysis and / or electrical coupling of the microdroplets, thereby ensuring that cells and / or biomolecules contained within the microdroplets remain intact. Furthermore, the gap 138 between the conductor layer 106 and the port 136 can further minimize or eliminate capture of the droplet 122 in the port and binding of the droplets to each other. The gap 138 can be filled with a fluid, such as oil.

[0134] The dielectric layer 110 can be composed of a single dielectric material or can be a composite of two or more dielectric materials. For example, the dielectric layer can be made of a single material such as, but not limited to, Al2O3 or SiO2. In other examples, the dielectric layer can be made of more than one material such as, but not limited to, Al2O3 or SiO2.

[0135] The composite layer 102 further comprises a photoactive layer 108. The photoactive layer 108 may be made of amorphous silicon. The photoactive layer 108 may be activated by electromagnetic radiation in the wavelength range of 400-1000 nm on the conductor layer. A dielectric layer may be deposited on the photoactive layer by atomic layer deposition. Additionally or alternatively, a second dielectric layer may be deposited on the photoactive layer.

[0136] Alternatively, or in addition, the insulator can be photoactive layer 108. Photoactive layer 108 can seal conductor layer 106 at port 136 to ensure that conductor layer 106 does not contact microdroplets 122 as they pass through port 136.

[0137] Under control of an applied voltage, the microdroplets 122 pass through the port 136 into a microfluidic workspace (manipulation space), where they can then be manipulated by EWOD or oEWOD forces. The port 136 can have a diameter sufficient to allow one or more microdroplets 122 to enter the microfluidic device 100. The microdroplets 122 can advance through the composite wall 102 in a sequential manner. Alternatively, two or more microdroplets can simultaneously pass through the port 136 into the device 100. The port 136 can have a uniform diameter throughout its entire length and / or width. Alternatively, the port 136 can have a tapered region.

[0138] The ports through the composite wall 102 can be formed by laser drilling through the layers of the composite wall 102. Other techniques that can be used to create ports through the composite wall include, but are not limited to, diamond drilling, sandblasting, and wet etching. Those skilled in the art will recognize that alternative techniques can be used to form ports through the composite wall.

[0139] 4 also shows a second composite wall 122, which includes a substrate 114, a conductor layer 116 disposed on the substrate, and a dielectric layer 118 disposed on the conductor layer. The two composite walls 102, 112 are separated by a microfluidic gap or space 121 between 20 and 120 microns. In some examples, the gap 121 between the two composite walls 102, 112 can be greater than 20 microns, greater than 30 microns, greater than 40 microns, greater than 50 microns, greater than 60 microns, greater than 70 microns, greater than 80 microns, greater than 90 microns, greater than 100 microns, or greater than 110 microns. In some examples, the gap between the two composite walls can be less than 120 microns, less than 110 microns, less than 100 microns, less than 90 microns, less than 80 microns, less than 70 microns, less than 60 microns, less than 50 microns, less than 40 microns, or less than 30 microns.

[0140] The two composite walls 102, 112 can be part of an EOWD or oEOWD device 100. The oEWOD device can further include an A / C power supply; at least one electromagnetic radiation source; and a microprocessor, where the A / C power supply is connected to the first and second conductor layers to provide a voltage between the first composite wall and the second composite wall, the at least one electromagnetic radiation source having an energy higher than the band gap of the photoexcitable layer and configured to act on the photoactive layer to induce electromagnetic induction at corresponding temporary electrowetting locations on the surface of the first dielectric layer, and the microprocessor is operable to manipulate the points at which the electromagnetic radiation acts on the photoactive layer to change the location of the temporary electrowetting locations, thereby generating at least one electrowetting path and moving microdroplets along the path.

[0141] An A / C power supply (not shown in the accompanying drawings) is connected to the first and second conductor layers and is configured to supply a voltage of 1V to 100V between the first composite wall and the second composite wall.

[0142] The conductor layer on the substrate can be in the range of 70 to 250 nm thick. The photoactive layer can be activated by electromagnetic radiation in the wavelength range of 400 to 1000 nm on the conductor layer.

[0143] The electromagnetic radiation source can be an LED light source or other lamps, which have an output of 0.005-0.1 Wcm -2 In some cases, the electromagnetic radiation source may provide a level of 0.005 to 0.1 Wcm -2 or the electromagnetic radiation source is at a level of 0.005 Wcm -2 Super, 0.0075Wcm -2 Super, 0.01Wcm -2 Super, 0.025Wcm -2 Super, 0.05Wcm -2 Over 0.075Wcm -2 In some embodiments, the electromagnetic radiation source can be greater than 0.1 Wcm -2 Less than 0.075Wcm -2 Less than 0.05Wcm -2 Less than 0.025Wcm -2 Less than 0.01Wcm -2 Less than 0.0075Wcm -2 Less than 0.005Wcm -2 Less than or equal to 0.0025Wcm -2 can be at a level below.

[0144] This allows highly complex patterns of virtual electrowetting electrode positions to be rapidly created and destroyed on the first dielectric layer, thereby enabling precisely controlled electrowetting forces to be used to precisely propel microdroplets along essentially any virtual path. Such electrowetting patterns can be viewed as consisting of a continuum of virtual electrowetting electrode positions on the first dielectric layer.

[0145] In some embodiments, the second composite wall may further comprise a second photoexcitable layer, and the electromagnetic radiation source may act on the second photoexcitable layer to create a second pattern of temporary electrowetting locations, and may also vary the second pattern. The electromagnetic radiation source may be an LED light source, and the LED light source may have a power of 0.005 to 0.1 Wcm. -2 It is possible to provide a level of electromagnetic radiation.

[0146] A structure can be provided between the first and second dielectric layers. The structure between the first and second dielectric layers can be made of, but is not limited to, epoxy, polymer, silicone, or glass, or a mixture or composite of these, and can have straight, sloped, curved, or microstructured walls / surfaces. The structure between the first and second dielectric layers can be connected to first and second composite walls to create a sealed microfluidic device and define channels and regions within the device. The structure can occupy the gap between the two composite walls. Alternatively, or in addition, the conductor and dielectric layers can be deposited on a molded substrate already having walls.

[0147] The devices according to Figures 4-13 may optionally be provided with a spacer structure, not shown in Figures 4-13, which, if provided, is disposed between the first composite wall 102 and the second composite wall 112 of the device 100. Additionally or alternatively, the devices according to Figures 4-13 may also comprise anti-fouling layers and / or intervening layers, also not shown in Figures 4-13.

[0148] The microdroplets can contain biological and / or chemical entities, such as biomolecules, which can be, but are not limited to, nucleic acids such as DNA, RNA, or messenger RNA (mRNA), proteins, peptides or polypeptides, enzymes, polysaccharides, peptides, proteins, antibodies, and / or antibody fragments thereof. The microdroplets can also contain other components, such as buffer solutions, vitamins, minerals, nutrients, gases such as oxygen, and / or cofactors. The microdroplets can contain one or more cells.

[0149] 5, an alternative structure for a composite wall 102 according to the present invention is provided. As shown in Figures 5 and 6A, a first composite wall 102 is provided comprising a substrate 104, a dielectric layer 110, and an ITO conductor layer 106. The first composite wall 102 also comprises a photoactive layer 108.

[0150] 5 and 6A also show a second composite wall 112, which comprises a substrate 114, a conductor layer 116 disposed on the substrate 112, and a dielectric layer 118 disposed on the conductor layer 116. The two composite walls 102 and 112 are separated by a microfluidic space or gap 121 of 20-120 microns. FIG. 5 shows that a port 136 is provided on the second composite wall 114. The port 136 extends through the second composite wall 112 to allow microdroplets 122 to be loaded into the microfluidic device 100 under voltage.

[0151] 5, etching away second conductor layer 116 away from port 136 leaves gap 138 between port 136 on second composite wall 112 and conductor layer 116. This ensures that droplet 122 does not come into contact with conductor layer 116 when droplet 122 passes through port 136 under voltage.

[0152] Additionally or alternatively, the insulator is a continuous dielectric layer 110 that seals at least the conductor layer 106 around the periphery near the port 136, as shown in FIG. 6A. Sealing the conductor layer 106 around the periphery near the port 136 ensures that the conductor layer 106 does not contact the microdroplets 122 as they pass through the port 136. In some examples, the dielectric layer 110 that seals the conductor layer 106 at the port 136 has at least the same breakdown voltage as the dielectric layer of the composite wall 102.

[0153] In situations where a pinhole defect occurs in the dielectric layer 110 forming the insulator or in a dielectric layer that is part of the composite wall 102, it may be advantageous for the dielectric layer 110 of the composite wall 102 to provide a comparable dielectric strength to the dielectric layer forming the insulator, and vice versa. This dielectric strength must be high enough to prevent breakdown at least around the port 136 under the same conditions under which the oEWOD device 100 holds the microdroplet 122. As shown in FIG. 6A , the dielectric layer 110 forming the insulator can also seal the photoactive layer 108 and a portion of the substrate 104 around the port 136. This alternative configuration ensures that the conductor layer 106 does not come into contact with the microdroplet 122 as it passes through the port 136.

[0154] As shown in FIG. 6B, an alternative device 100 to that shown in FIG. 6A is illustrated. In FIG. 6B, a hydrophobic layer 16 is provided on the first composite wall 102. A dielectric layer 110 lines the inside of the port 136 to provide insulation. Additionally, as shown in FIG. 6B, a hydrophobic layer 126 can be provided on the surface of the insulator around the periphery near the port 136. The presence of the hydrophobic layer 126 around the port 136 can reduce the size of the contact surface between the emulsion droplet 122 and the inner layer of the port 136, where the inner layer of the port 136 includes at least the conductor layer 106. Additionally or alternatively, the hydrophobic layer 126 around the periphery near the port 136 can prevent the droplet 122 from wetting the wall 102.

[0155] Referring to Figure 6C, an alternative device 100 to that shown in Figures 6A and 6B is provided. In Figure 6C, at least a portion of conductor layer 106, photoactive layer 108, dielectric layer 110, and hydrophobic layer 126 line the inside of port 136 to provide the insulation. As shown in Figure 6C, conductor layer 106 is adjacent to substrate 104 of first composite wall 102. Photoactive layer 108, dielectric layer 110, and hydrophobic layer 120 are then provided to seal conductor layer 106 lined to port 136. This alternative configuration ensures that conductor layer 106 does not contact microdroplets 122 as they pass through port 136.

[0156] 7, a composite wall 102 is shown, comprising a substrate 104, a dielectric layer 110, and a conductor layer 106. The composite wall 102 comprises a photoactive layer 108, which may be made of amorphous silicon.

[0157] 7 also shows a second composite wall 112, which comprises a substrate 114, a conductive layer 116 disposed on the substrate 112, and a dielectric layer 118 disposed on the conductive layer 116. As shown in FIG. 6, the dielectric layer 118 forms the insulator and seals the conductive layer 116 at the port 136. The two composite walls 102 and 112 can be separated by a microfluidic space or gap of 20-120 microns.

[0158] 7 illustrates an embodiment in which a port 136 is disposed at an acute angle α relative to the second composite wall 112, through which a microdroplet 122 passes. The stress on the microdroplet is different from that experienced in a port perpendicular to the microdroplet, resulting in lower shear stress as the microdroplet transitions from the angled port 136 into the microfluidic space 121. By reducing shear stress, biological entities, such as cells, held within the microdroplet are likely to have improved viability compared to transitions at higher shear stresses.

[0159] Additionally, the provision of angled ports can guide microdroplets into the microfluidic space 121 in a specific direction, which can be useful in reducing electrolysis or electrical coupling of the microdroplets, and therefore require less power to move the microdroplets through the ports in the composite wall.

[0160] The angle of the port relative to the substrate can be any angle less than 90 degrees. In some cases, the angle of the port relative to the substrate can be less than 85 degrees, less than 80 degrees, less than 75 degrees, less than 70 degrees, less than 65 degrees, less than 60 degrees, less than 55 degrees, less than 50 degrees, less than 45 degrees, less than 40 degrees, less than 35 degrees, less than 30 degrees, less than 25 degrees, less than 20 degrees, less than 15 degrees, less than 10 degrees, or less than 5 degrees. Alternatively, the angle of the port relative to the substrate can be more than 5 degrees, more than 10 degrees, more than 15 degrees, more than 20 degrees, more than 25 degrees, more than 30 degrees, more than 35 degrees, more than 40 degrees, more than 45 degrees, more than 50 degrees, more than 55 degrees, more than 60 degrees, more than 65 degrees, more than 70 degrees, more than 75 degrees, more than 80 degrees, or more than 85 degrees.

[0161] 8A-8C, a composite wall 102 is provided, comprising a substrate 104, a dielectric layer 110, an amorphous silicon photoactive layer 108, and a conductor layer 106. The substrate 104 can be fused silica, silicon, silicon dioxide, or glass. The composite wall 102 also comprises a first dielectric layer 142 made of silica and a second dielectric layer 144 made of aluminum oxide. Ports 136 allow microdroplets to enter the microfluidic workspace while a voltage is applied, allowing the microdroplets to be manipulated by oEWOD forces. It is necessary to be able to load microdroplets through the composite wall ports while a voltage is applied, because this voltage is used to hold other droplets in place within the microfluidic space. However, in the presence of a voltage applied to the conductive ITO layer, the microdroplets may become trapped or bind to each other as they pass over the exposed edge of the ITO layer.

[0162] 8B, a protective layer 146 is provided on top of layer 147 of composite wall 102. In particular, protective layer 146 is provided on dielectric layer 110. Protective layer 146, such as a photomask, can be a photoresist layer or any other suitable patternable layer, such as aluminum. The photoresist layer can be spin-coated or spray-coated onto the surface of the dielectric layer and patterned by photolithography to leave a predetermined region 148 of composite layer 147 uncovered by protective layer 146. This predetermined region 148, i.e., the distance away from the port, is approximately 0.001 to 2 mm.

[0163] Alternatively, after spin-coating or spray-coating onto a layer of the composite wall, patterning can be performed by lifting off an underlying masking material, such as polyimide tape, to expose areas where the coating layer is to be removed. This creates two areas on the surface of the composite wall: a first area on the surface of the composite wall that is covered by the protective layer; and a second area on the surface of the composite wall that is not covered by the protective layer.

[0164] Referring to FIG. 8C , the uncovered surfaces of composite layer 147 are removed down to substrate 104 by wet or dry etching with a reactive fluid, which can be a liquid, gas, or plasma. For example, a first etching step involves a 5-minute dry etch with a reactive plasma such as fluoroform, followed by a 30-minute dry etch with sulfur hexafluoride plasma. Then, in a subsequent step, dielectric layers 142, 144 are wet etched with 37% hydrogen chloride for 7 minutes. The end result of these etching processes is shown in FIG. 8C , where portions of dielectric layers 142, 144, photoactive layer 108, and conductive ITO layer 106 have been removed around port 136. This creates a recess or gap in uncovered surface 148, which is comprised of dielectric layers 142, 144, photoactive layer 108, and conductor layer 106. Thus, in use, microdroplets can be loaded into port 136 while a voltage is applied across the device without interacting with the conductive ITO.

[0165] Referring to Figure 9A, a composite wall 102 is shown, comprising a substrate 104, a photoactive layer 108, a conductor layer 106, and a dielectric layer 110. Figures 9A-9B illustrate an alternative etching process for the layers of the composite wall. As shown in Figures 9A and 9B, the conductor layer 106 is etched away from the vicinity of the port 136 to create a recess 150 in the conductor layer 106. The recess 150 can be filled with a fluid, such as oil.

[0166] During this etching process, a portion of the composite wall 102 is immersed in a protective liquid. A protective layer (not shown in FIG. 9) is formed on the immersed layer of the composite wall 102. The protective liquid can be any suitable liquid in which the etchant is insoluble. HFE-7500 can be used with a hydrogen chloride gas etchant because HFE-7500 is insoluble in hydrogen chloride and therefore forms a protective layer on the immersed layer of the composite wall.

[0167] The partially immersed composite wall 102 can be etched in a hydrogen chloride gas atmosphere. As shown in FIG. 9B, the layers not protected by the HFE-7500 protective layer, i.e., the unimmersed layers of conductive ITO layer 106 and aluminum oxide layer 144, are exposed. The unimmersed layers are etched to create gaps 150 in ITO layer 106 and gaps 151 in aluminum oxide layer 144 near port 136. By way of example, exposing the partially immersed composite wall to the hydrogen chloride gas etching atmosphere for 5 hours results in an 8-12 μm in-layer etching of the exposed ITO and aluminum oxide directly beneath the silica and / or amorphous silicon layers.

[0168] The composite wall is then ultrasonically cleaned in water to remove residual hydrogen chloride from the composite wall, which prevents further undesired etching of the conductive ITO and aluminum oxide layers when they are no longer immersed in the HFE-7500 protective solution.

[0169] FIG. 9B shows the structure of the composite wall 102 after etching, showing the conductive ITO layer 106 and aluminum oxide layer 144 underneath the silica layer 142 and amorphous silicon layer 108 etched around the periphery near the port 136.

[0170] 10A-10E, a composite wall 102 is shown, comprising a substrate 104, a photoactive layer 108, a conductor layer 106, and a dielectric layer 110. FIGS. 10A-10E provide illustrations of an alternative etching process for the composite wall 102. As shown in FIGS. 10A-10E, at least a portion of the conductive ITO layer 106 is removed from the surface of the composite wall 102. Removal of the ITO layer 106 is performed prior to deposition of an additional layer 152 on the conductive ITO layer 106 and prior to drilling ports 136 through the composite wall 102.

[0171] 10A, the entire surface of the substrate 104 may be coated with a conductive layer of ITO 106. A protective layer 146 may be deposited on top of the conductive layer to protect regions 154 of the conductive layer. The protective layer 146 may be a photoresist layer or any other suitable patternable layer, such as aluminum.

[0172] The protective layer 146 can be deposited on top of the conductive layer 106 by spin coating or spray coating and can be patterned using photolithography. This creates a first region 154 of the surface of the composite wall that is covered by the protective layer 146, while a second region 156 of the surface of the composite wall 102 is not covered by the protective layer 146. The second region 156 of the composite wall 102 comprises the exposed ITO layer 106 that is to be removed. As shown in FIG. 10B, the exposed ITO layer 106 is not covered by the protective layer 146.

[0173] A fluid capable of reacting with the conductive layer, which can be a hydrogen chloride liquid, solution, or gas, or an oxalic acid solution, can be applied to the entire surface of the composite wall. This etches the exposed conductive layer 106 down to the substrate 104, as shown in FIG. 10C. For example, the entire surface can be immersed in 37% hydrogen chloride for 7 minutes. Alternatively, a plasma capable of reacting with the conductive layer can be applied to the entire surface of the wall. This plasma can be, but is not limited to, a methane and hydrogen plasma, a chlorine and argon plasma, or a carbon tetrafluoride plasma.

[0174] Alternatively, or in addition, the exposed conductive layer can be removed from the surface using laser ablation.

[0175] After treatment with the reactive fluid or laser, the surface of the composite wall can be ultrasonically cleaned in water to remove any residual hydrogen chloride from the surface of the device and prevent any further undesired etching of the conductor layer once the protective layer has been removed. The protective layer can be removed from the surface of the device by solvent cleaning and oxygen plasma cleaning.

[0176] 10D, additional layers 152 are deposited over the entire surface of composite wall 102 by physical vapor deposition, atomic layer deposition, chemical vapor deposition, and / or evaporation. Additional layers 152 include a photoactive layer of amorphous silicon, a first dielectric layer 142 of silicon oxide, and a second dielectric layer 144 of aluminum oxide.

[0177] 10E, ports 136 are created in the composite wall 102 where the conductive ITO layer 106 was removed by drilling holes through the composite wall 102. One or more microdroplets can then be loaded into the device through the ports under voltage. It is important that the microdroplets do not interact with the conductive ITO layer.

[0178] In an alternative embodiment, the conductive ITO layer can be removed from the substrate by laser ablation. In this embodiment, the entire surface of the substrate is coated with a conductive ITO layer, as shown in Figure 10A. Optionally, a protective layer can be deposited on top of the conductive layer to protect areas of the conductive layer that should not be etched, as shown in Figure 10B.

[0179] Laser ablation is then used to remove a portion of the conductive ITO layer on the surface of the substrate, resulting in a first region 154 on the surface of the substrate 104 being covered by the conductive ITO layer 106 and a second region 156 not being covered by the conductive ITO layer, as shown in Figure 10C.

[0180] Various deposition techniques, such as physical vapor deposition, atomic layer deposition, chemical vapor deposition, and / or evaporation, can be used to deposit additional layer 152 over the entire surface of the substrate. As shown in Figure 10D, additional layer 152 comprises amorphous silicon photoactive layer 108, a first dielectric layer 142 made of silicon oxide, and a second dielectric layer 144 made of aluminum oxide.

[0181] In a subsequent step shown in FIG. 10E, ports 136 are created in the composite wall 102 by drilling holes through the composite wall 102 where the conductive layer 106 was removed.

[0182] Instead, a step of drilling holes through the composite wall can be performed before additional layers are deposited on the substrate.Through the ports, microdroplets can be loaded into the device without contacting the ITO layer.

[0183] 11A-11B, a port 136 is shown disposed on the side of device 100. Device 100 comprises a first composite wall 102 and a second composite wall 112. First composite wall 102 comprises at least a substrate 104, a dielectric layer 110, and a conductor layer 106. A photoactive layer 108 is also disposed within first composite wall 102. Second composite wall 112 comprises a substrate 114, a dielectric layer 118, and a conductor layer 116. Microdroplets are loaded into microfluidic space 121 through port 136 disposed on the side of device 100, as shown in FIG. 11B. As shown in FIG. 11A, when microdroplets are loaded into device 100 through port 136, the conductor layer 106 around at least port 136 of composite wall 102 is etched (155) by any of the etching methods described herein. This helps prevent the microdroplets from coming into contact with the conductor layer 106 on the sides of the device 100. Microdroplets passing through the port enter region 157 of the device, as shown in Figure 11A.

[0184] As part of the etching process, a protective layer is deposited over most of the layers 104, 110, 106 of the composite wall 102 and / or side port 136. The protective layer may be a photomask such as photoresist, or any other suitable patternable layer such as aluminum.

[0185] In some cases, the protective layer can be a single layer, or the protective layer can be multiple layers. A photoresist layer can be spin-coated or spray-coated onto the surface of the composite wall and patterned by photolithography, so that areas of the composite wall, generally near the ports, remain uncovered by the protective layer.

[0186] Alternatively, photoresist can be spin-coated or spray-coated, followed by lift-off patterning of an underlying masking material, such as polyimide tape, to expose the areas of the composite wall where layers are to be removed. This creates two regions: one on the surface of the composite wall covered by the protective layer, and the second uncovered. The uncovered layer is then removed down to the substrate by wet or dry etching using a reactive fluid, which can be a liquid, gas, or plasma. This is illustrated in FIG. 11B, which shows that dielectric layers 142 and 144, photoactive layer 108, and conductor layer 106 are etched away from port 136 by a distance of 0.001 to 2 mm. In addition, as shown in FIG. 11B, the silicon oxide 142 and aluminum oxide 144, which form the second dielectric layer, and conductor layer 116, respectively, of composite wall 112 are also etched away from port 136.

[0187] 12A-12B, a device is shown having a composite wall 102. The composite wall 102 comprises at least a substrate 104, a dielectric layer 110, and a conductor layer 106. The conductor layer 106 may be made of ITO. A photoactive layer 108 is also provided within the first composite wall 102. A second composite wall 112 is provided as shown in FIG. 12B. The second composite wall 112 comprises a substrate 114, a dielectric layer 118, and a conductor layer 116. As shown in FIGS. 12A and 12B, a port 136 may be located on the side of the device 100. As shown in FIG. 12A, a portion of the composite wall 102, i.e., at least the conductor layer, is etched away from the port 136. Next, a microdroplet is loaded into a microfluidic region 157 through a cavity in the side of the device 100, as shown in FIG. 12A.

[0188] As shown in Figure 12B, the conductor layers 106, 116 of the composite walls 102, 112 are etched or removed to create recesses 150 in the conductor layers 106, 116. This helps prevent microdroplets from coming into contact with the conductor layers 106 on the sides of the device 100. This therefore prevents the microdroplets from becoming trapped and binding during device installation. As shown in Figure 12B, a portion of the aluminum oxide layer 142 is also etched away to create recesses 151 in the aluminum oxide layer 142.

[0189] The composite wall 102 is immersed in a protective liquid, such as HFE-7500. This forms a protective layer on most of the surface of the composite wall. However, there are areas near the ports that do not have the protective layer, leaving this surface exposed. The entire surface of the device is then exposed to a hydrogen chloride gas atmosphere. Exposing the composite wall to the hydrogen chloride gas atmosphere causes etching of the conductive ITO 106 and aluminum oxide layer 144 exposed around the ports 136, as shown in FIG. 12B. Once the device is removed from the protective liquid, subsequent rinsing of the device removes any remaining hydrogen chloride and prevents further undesired etching.

[0190] 13A-13B, a device is shown having a composite wall 102. The composite wall 102 comprises at least a substrate 104, a dielectric layer 110, and a conductor layer 106. The conductor layer 106 may be made of ITO. A photoactive layer 108 is also provided within the first composite wall 102. A second composite wall 112 is provided, as shown in FIG. 13B. The second composite wall 112 comprises a substrate 114, a dielectric layer 118, and a conductor layer 116. As shown in FIGS. 13A and 13B, a port 136 is provided on the side of the device 100. A region of the composite wall may be etched or removed by laser ablation to prevent microdroplets from coming into contact with the ITO layer 106 as they pass through the port 136. This allows the droplets to be loaded laterally under voltage into the device region 157 while preventing the droplets from combining with each other or becoming trapped at the port.

[0191] As shown in FIG. 13B, a conductor layer 106 is deposited on the substrate 104 of the composite wall 102. A protective layer is then deposited on the conductor layer 106 to cover a portion of the conductor layer. The protective layer can be deposited by spin coating or spray coating and can be patterned using photolithography. As a result, a first region on the substrate is covered by the protective layer, and a second region on the substrate is not protected by the protective layer. The unprotected region of the substrate 104 includes the exposed ITO layer 106.

[0192] The exposed ITO layer can be removed by etching with a reactive fluid or by laser ablation. In subsequent steps, the surface of the composite wall can be cleaned by ultrasonication in water, by solvent cleaning, and by oxygen plasma cleaning. Alternatively, the exposed ITO layer can be removed from the substrate by laser ablation.

[0193] The additional layer 152 can be, but is not limited to, a dielectric layer 110 made of silicon oxide 142, a dielectric layer 110 made of aluminum oxide, and the photoactive layer 108 is deposited over the entire surface of the composite wall 102. The additional layer can be deposited on the substrate using a variety of deposition techniques, such as physical vapor deposition, atomic layer deposition, chemical vapor deposition, and / or evaporation. Other deposition techniques can also be used. In a subsequent step, as shown in FIG. 13B, ports 136 can then be created by drilling holes through the side of the device 102 where the ITO layer 106 was etched or removed.

[0194] Instead, a step of drilling holes through the composite wall can be performed before additional layers are deposited on the substrate. Microdroplets can be loaded into the device through the ports without contacting the ITO layer.

[0195] Various additional aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0196] As used herein, "and / or" should be construed as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" should be construed as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth herein.

[0197] Unless the context requires otherwise, the feature descriptions and definitions set forth above are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described.

[0198] While the present invention has been described by way of example with reference to certain embodiments, those skilled in the art will further appreciate that the invention is not limited to the disclosed embodiments and that alternative embodiments may be constructed without departing from the scope of the invention as defined in the appended claims.

Claims

1. o A composite wall for an EWOD or EWOD device, comprising: A substrate; a conductor layer provided on the substrate; a dielectric layer provided on the conductor layer; a port extending through the composite wall; an insulator; The insulator is configured to separate the droplet from the conductor layer as the droplet passes through the port.

2. The composite wall of claim 1 , wherein the insulation is provided by a recess formed in at least a portion of the conductive layer.

3. The composite wall of claim 1 , wherein the dielectric layer lines the port to provide the insulation.

4. A composite wall according to any preceding claim, wherein the ports have a constant diameter.

5. A composite wall according to any preceding claim, wherein the substrate defines a plane and the port extends through the composite wall in a direction generally perpendicular to the plane.

6. A composite wall according to any preceding claim, wherein the port is disposed at an acute angle relative to the composite wall.

7. The composite wall of any one of claims 1 to 6, further comprising a photoactive layer disposed between the conductor layer and the dielectric layer.

8. 8. The composite wall of claim 7, wherein the photoactive layer lines the port to provide the insulation.

9. 9. The composite wall of claim 7 or 8, wherein the photoactive layer and the dielectric layer line the port to provide the insulation.

10. 10. The composite wall of any of claims 3 to 9, wherein the dielectric layer lining the port and providing the insulation seals at least the conductor layer around the periphery near the port, and the dielectric layer sealing at least the conductor layer has the same breakdown voltage as the dielectric layer of the composite wall.

11. 3. The composite wall of claim 2, wherein the conductive layer has a recess that removes the conductive layer from the vicinity of the port by at least 0.001 mm.

12. 3. The composite wall of claim 2, wherein the conductive layer has a recess that removes the conductive layer from the vicinity of the port by at least 0.01 mm.

13. 3. The composite wall of claim 2, wherein the conductive layer has a recess that removes the conductive layer from the vicinity of the port by at least 0.1 mm.

14. 3. The composite wall of claim 2, wherein the conductive layer has a recess that removes the conductive layer by at least 1 mm from the vicinity of the port.

15. A composite wall according to any preceding claim, wherein the port is generally circular.

16. A composite wall according to any preceding claim, wherein the port is substantially square.

17. A composite wall according to any preceding claim, wherein the composite wall further comprises a hydrophobic layer.

18. o A composite wall for an EWOD or EWOD device, comprising: A substrate; a conductor layer provided on the substrate; a dielectric layer provided on the conductor layer; a port extending through the composite wall to allow microdroplets to pass therethrough; A composite wall wherein the port is configured to insulate the microdroplet from the conductive layer.

19. An oEWOD or EWOD device comprising a first composite wall and a second composite wall, wherein one of the first composite wall and the second composite wall is a composite wall according to any one of claims 1 to 18.

20. the second composite wall: A substrate; a conductor layer provided on the substrate; a dielectric layer provided on the conductor layer; 20. An oEWOD or EWOD device according to claim 19, comprising:

21. 20. An oEWOD or EWOD device according to claim 19, wherein the first and second composite walls are separated by a gap of 20 to 120 microns.

22. A method for manufacturing a composite wall according to any one of claims 1 to 18, providing the substrate; depositing the conductor layer on the substrate; creating a hole in the conductor layer; depositing the dielectric layer on the conductor layer; creating said port through said composite wall; A method comprising:

23. 23. The method of claim 22, wherein the hole in the conductor layer has a larger diameter than the port.

24. 23. The method of claim 22, wherein the port is created through the composite wall prior to deposition of the dielectric layer, and deposition of the dielectric layer results in the dielectric layer lining the port.

25. A cartridge, at least one inlet port for introducing a sample into the cartridge; at least one emulsifier configured to generate microdroplets; A device comprising the first and second composite walls according to any one of claims 19 to 21, wherein the first and second composite walls have a microfluidic space between the first and second composite walls for EWOD or oEWOD microfluidic manipulation; an emulsion processing flow path configured to provide fluid communication between the emulsification apparatus and the device; A cartridge comprising:

26. 26. The cartridge of claim 25, wherein the cartridge comprises a plurality of the emulsifying devices.

27. 27. A cartridge according to claim 25 or 26, wherein at least one of the emulsifiers is a step emulsifier.

28. A cartridge according to any one of claims 25 to 27, further comprising a reservoir configured to contain the sample.

29. A cartridge according to any one of claims 25 to 28, further comprising a pump system configured to introduce the sample stream into at least one of the emulsification devices.

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