Improvements in or relating to microfluidic devices

Abstract

A device for manipulating microdroplets using optically mediated electrowetting is provided, the device comprising a microfluidic space bounded by a first composite wall and a second composite wall, the first composite wall comprising a first substrate, a conductor layer on the substrate, a photoactive layer on the first conductor layer, and a first continuous dielectric layer on the photoactive layer, the first continuous dielectric layer having a thickness of less than 20 nm, and the second composite wall comprising a second substrate and a second conductor layer on the second substrate.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to microfluidic devices, and in particular to devices that use optically mediated electrowetting-on-dielectric (oEWOD) to manipulate microdroplets. [Background technology]

[0002] 2. Background of the Invention The design of devices for manipulating microdroplets using optically mediated electrowetting is driven by a number of competing effects and observed phenomena.

[0003] Focusing on the efficiency of microdroplet manipulation, in many designs it would be preferable to maximize the speed at which the microdroplets can be manipulated. Increasing the speed of droplet manipulation allows for higher throughput of biological experiments. Another aspect of the efficiency of oEWOD devices is the reliability with which the droplets can be held stationary within the device, with a minimum number of droplets being lost or moving around from their holding position. The speed at which the microdroplets can be manipulated correlates superlinearly with the voltage applied. The maximum voltage that can be applied determines the thickness of the dielectric required to ensure that the device operates below the breakdown voltage of the dielectric layer. Thus, the literature teaches that thick dielectric layers are necessary to safely operate at the high voltages required to maximize speed. The reliability of droplet retention depends on the complex interplay between the droplet retention force and the strength of any external forces that may remove the droplet from its holding position, particularly dielectrophoretic effects, and the movement of the surrounding carrier phase, and the components of this carrier phase. Summary of the Invention [Problem to be solved by the invention]

[0004] It is against this background that the present invention was born. [Means for solving the problem]

[0005] According to one aspect of the invention there is provided a device for manipulating microdroplets using optically mediated electrowetting, the device comprising a microfluidic space, the microfluidic space comprising: - The first compound wall; The boundary is with the second compound wall, The first compound wall is A first substrate; a first conductor layer on the substrate; a photoactive layer on the first conductor layer; a first continuous dielectric layer on the photoactive layer, the first continuous dielectric layer having a thickness of less than 20 nm; The second compound wall is A second substrate; and a second conductor layer on the substrate.

[0006] In some embodiments, the second composite wall further comprises a second continuous dielectric layer on the second conductor layer, the second continuous dielectric layer having a thickness of less than 20 nm.

[0007] In some embodiments, the first and second composite walls are held in spaced apart relation to form a microfluidic space therebetween, and the walls may be separated by a spacer structure, which may be formed by an interposed structure between the first and second substrates, or may be formed by the substrates of the first or second composite walls.

[0008] The spacers may be formed with 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.

[0009] According to another aspect of the present invention there is provided a device for manipulating microdroplets using optically mediated electrowetting, the device comprising: - The first compound wall; A second composite wall; The first compound wall is A first substrate; a first conductor layer on the substrate; a photoactive layer on the first conductor layer; a first continuous dielectric layer on the photoactive layer, the first continuous dielectric layer having a thickness of less than 20 nm; The second compound wall is A second substrate; a second conductor layer on the substrate; a second continuous dielectric layer on the second conductor layer, the second continuous dielectric layer having a thickness of less than 20 nm.

[0010] The design of devices to manipulate microdroplets using optically mediated electrowetting is driven by a number of competing effects and observed phenomena. A well-recognized superlinear relationship exists between the velocity of the microdroplets and the applied voltage. Thus, the maximum voltage applied determines the required dielectric thickness. Thus, to optimize the velocity of the microdroplets, it is expected that the applied voltage will be maximum and the corresponding dielectric thickness will be increased. However, the inventors have found that high voltages have their own associated practical delivery problems.

[0011] The inventors have experimentally found that as the applied voltage is increased, the maximum achievable oEWOD velocity increases rapidly, as expected. However, the inventors have also observed that the ability to hold the droplet stationary decreases with increasing voltage due to previously unobserved driving forces. Initially, this manifests as a characteristic random movement of the droplet around the target location, and as the voltage is further increased, the velocity of this random motion increases until the random motion overcomes the holding power of the oEWOD and control of the droplet is lost. This effectively imposes a maximum voltage and therefore reduces the maximum velocity well below that initially expected and predicted in the literature.

[0012] There are two states that determine the voltage-driven response of an oEWOD system: the "on" and "off" states, which correspond to the illuminated and non-illuminated areas of the device. In an ideal oEWOD device, the voltage applied within the on-areas of the device would be exactly zero, and only the "on"-state areas would apply voltage. In an oEWOD device, a spatially varying, optically controlled voltage on the surface changes the contact angle between the droplet and the surface, thus providing a propulsive or holding force to the droplet. When holding the droplet, it partially exists in an "on" state and partially exists in an "off" state, with the spatial extent of each state being determined by the size of the illuminated area. The contrast between the voltages in the "on" and "off" states creates the holding force. As the applied voltage increases, the electric field strength of both states increases. An increase in the electric field strength of the "on" state leads to an increase in device performance, since it increases the electrowetting force, while an increase in the electric field strength of the "off" state partially counters this increase in force. However, since the ratio between these two states remains constant and this force depends on the square of the electric field, there is an overall increase in the oEWOD force. Thus, the literature shows that with increasing voltage, one skilled in the art would expect an improvement in both retention and droplet movement.

[0013] This is clearly contrary to the inventors' observations, where they observe an increase in the rate of movement, but a decrease in the ability to hold the droplet stationary with increasing voltage. Thus, the inventors theorize that this phenomenon can only be explained by a supralinear (faster than squared) dependence of the unwanted propulsion force on the field strength in the "off" state. Thus, the performance of the devices disclosed herein can be improved by designing the device structure to reduce the strength of the "off" state, rather than by maximizing the strength of the "on" state, as has been the focus of literature in the field.

[0014] A theoretical shortcut to achieving this would be to increase the thickness of the photoactive layer. However, this is inappropriate for applications that require simultaneous manipulation of a very large number of droplets, as it would greatly increase the optical power requirements. Furthermore, increasing the thickness of the photoactive layer is inappropriate for facilitating simultaneous parallel manipulation of thousands of droplets. Therefore, rather than minimizing the "off" state by modifying the photoactive layer, the inventors investigated the effect of the capacitance of the dielectric layer. In this counterintuitive focus on the "off" state, the inventors found that by reducing the thickness of the dielectric layer, a higher percentage of the voltage would be dropped across the photoactive layer, and thus the electric field strength at the surface of the dielectric would be reduced. Thus, the inventors reduced the thickness of the dielectric layer of the device by a factor of approximately five, below that recommended in the literature. This results in an enormous increase in the performance of the device due to the mitigation of the droplet retention failure mode in the "off" state, allowing it to reach higher operating voltages, and therefore higher oEWOD power, while maintaining the same level of required illumination.

[0015] The first dielectric layer can be deposited on the photoactive layer by atomic layer deposition. Additionally or alternatively, a second dielectric layer can be deposited on the photoactive layer.

[0016] It has been surprisingly discovered that providing the first and / or second dielectric layers with a continuous layer thickness of less than 20 nm causes the droplets to become more stable, and therefore the droplets come to rest on the substrate. In contrast, the inventors have discovered that increasing the thickness of the first and / or second dielectric layers to more than 20 nm can cause less controlled movement of the droplets on the substrate, and thus the droplets are more likely to exhibit uncontrolled movement away from the illumination area. As a result, uncontrolled droplets can make merging and splitting of the droplets more difficult, for example, for accurate and efficient oEWOD operation. In some preferred embodiments, the first and / or second dielectric layer can be 1 nm to 20 nm thick, 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, or 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.

[0017] The first substrate and / or the second substrate may be transparent.The first conductor layer and / or the second conductor layer may be transparent.

[0018] The device may further comprise an alternating current (A / C) power source; at least one electromagnetic radiation source; and a microprocessor, wherein the A / C power source is connected to the first conductor layer and the second conductor layer to supply a voltage between the first composite wall and the second composite wall, and wherein the electromagnetic radiation of the at least one electromagnetic radiation source has an energy higher than the band gap of the first photoexcitation layer and is configured to strike the photoactive layer and induce corresponding temporary electrowetting locations on the surface of the first dielectric layer, and the microprocessor controls the electromagnetic radiation source to manipulate the point at which the electromagnetic radiation strikes the photoactive layer to change the location of the temporary electrowetting locations, thereby creating at least one electrowetting path along which microdroplets can be moved.

[0019] The device may further comprise an intervening layer of silicon oxide. The intervening layer of silicon oxide is provided on the first and / or second dielectric layer. An advantage of the intervening layer is that it can be used as a tie layer for an anti-fouling or non-fouling layer. The intervening layer is provided between the dielectric layer and the hydrophobic layer. The thickness of the intervening layer may be between 0.1 nm and 5 nm. The thickness of the intervening layer can be greater than 0.1 nm, greater than 0.25 nm, greater than 0.5 nm, greater than 0.75 nm, greater than 1 nm, greater than 1.5 nm, greater than 2 nm, greater than 2.5 nm, greater than 3 nm, greater than 3.5 nm, greater than 4 nm, or greater than 4.5 nm; or less than 5 nm, less than 4.5 nm, less than 4 nm, less than 3.5 nm, less than 3 nm, less than 2.5 nm, less than 2 nm, less than 1.5 nm, less than 1 nm, less than 0.75 nm, less than 0.5 nm, or less than 0.25 nm.

[0020] The exposed surfaces of the first and second composite walls can be spaced apart by less than 200 μm to define a microfluidic space configured to contain a microdroplet. The microfluidic space can be between 2 and 50 μm wide. In some embodiments, the microfluidic space is greater than 2 μm, greater than 4 μm, greater than 6 μm, greater than 8 μm, greater than 10 μm, greater than 12 μm, greater than 14 μm, greater than 16 μm, greater than 18 μm, greater than 20 μm, greater than 22 μm, greater than 24 μm, greater than 26 μm, greater than 28 μm, greater than 30 μm, greater than 32 μm, greater than 34 μm, greater than 36 μm, greater than 38 μm, greater than 40 μm, greater than 42 μm, greater than 44 μm, greater than 46 μm, or greater than 48 μm. In some preferred embodiments, the microfluidic space can be less than 50 μm, less than 48 μm, less than 46 μm, less than 44 μm, less than 42 μm, less than 40 μm, less than 38 μm, less than 36 μm, less than 34 μm, less than 32 μm, less than 30 μm, less than 28 μm, less than 26 μm, less than 24 μm, less than 22 μm, less than 20 μm, less than 18 μm, less than 16 μm, less than 14 μm, less than 12 μm, less than 10 μm, less than 8 μm, less than 6 μm, or less than 4 μm.

[0021] The exposed surfaces of the first and second composite walls can include one or more spacers to hold the first and second walls apart a predetermined amount to define a microfluidic space configured to contain the microdroplets. The physical shape of these spacers can be used to assist in the splitting, merging, and elongation of the microdroplets within the device. The spacers can be, but are not limited to, blade-shaped structures, wedge structures, pillars, hydrophilic patches, narrow channels, or can be dimples on a surface.

[0022] In some embodiments, the microdroplets may contain one or more cells. The microdroplets may contain a medium, such as a cell culture medium and / or a buffer solution.

[0023] The A / C power supply may be connected to the first and second conductor layers and configured to provide a voltage between the first and second composite walls between 0V and 100V. In some embodiments, the voltage provided may be between 0V and 50V, 0.1V, 0.1V to 2V, 3 to 4V, or 0V to 10V. In some embodiments, the A / C power source can be configured to provide a voltage greater than 0V, greater than 5V, greater than 10V, greater than 15V, greater than 20V, greater than 25V, greater than 30V, greater than 35V, greater than 40V, greater than 50V, greater than 60V, greater than 70V, greater than 80V, or greater than 90V, or a voltage less than 90V, less than 80V, less than 70V, less than 60V, less than 50V, less than 45V, less than 40V, less than 35V, less than 30V, less than 25V, less than 20V, less than 15V, less than 10V, or less than 5V.

[0024] The first and second composite walls may further comprise first and second antifouling layers on the first and second dielectric layers, respectively. The antifouling layer on the second dielectric layer may be hydrophobic.

[0025] The electromagnetic radiation source may comprise a pixelated array of lights, with light being reflected from or transmitted through such an array.

[0026] The electrowetting locations may be crescent shaped in the direction of microdroplet travel.

[0027] The device may further comprise a photodetector for detecting an optical signal in a microdroplet located within or downstream of the device, the optical signal being a fluorescent signal.

[0028] The device may further comprise an upstream inlet for producing a medium consisting of an emulsion of small aqueous droplets in an immiscible carrier medium, which may optionally be inert.

[0029] The device may further comprise an upstream inlet for directing a flow of a medium comprising an emulsion of microdroplets through the microfluidic space via an inlet port into the microfluidic space and into an immiscible dispersion medium.

[0030] The first and second composite walls defining the microfluidic space between them may form the periphery of a cartridge or chip.

[0031] The device may further comprise a plurality of first electrowetting paths extending parallel to each other.

[0032] The device may further comprise a plurality of second electrowetting paths which interact with the first electrowetting path to generate at least one microdroplet coalescence location.

[0033] The device may further comprise an upstream inlet for introducing microdroplets into the microfluidic space, the diameter of the microdroplets being more than 20% greater than the width of the microfluidic space.

[0034] The second composite wall may further comprise a second optical excitation layer, and the electromagnetic radiation from the electromagnetic radiation source may also impinge on the second optical excitation layer to generate a second pattern of temporary electrowetting locations, which second pattern may also be varied.

[0035] The electromagnetic wave source can be an LED (light emitting diode) light source, and the electromagnetic radiation can be 0.005~0.1Wcm- 2 In some preferred embodiments, the electromagnetic radiation source can be provided at a power of 0.005 to 0.1 Wcm- 2 or 0.005Wcm- 2 Super, 0.0075Wcm- 2 Super, 0.01Wcm- 2 Super, 0.025Wcm- 2 Super, 0.05Wcm- 2 Over 0.075Wcm- 2 In some preferred 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 It can be less than.

[0036] The first transparent conductor layer on the substrate may have a thickness in the range of 70-250 nm. The photoactive layer may be activated by electromagnetic radiation in the wavelength range of 400-1000 nm on the conductor layer, and the conductor layer may have a thickness in the range of 300-1000 nm.

[0037] In some embodiments, the photoactive layer may be made of amorphous silicon (a-Si).

[0038] In some preferred embodiments, a microdroplet can be passed through a microfluidic space defined by two opposing walls, each wall including a dielectric layer, and a sufficiently low voltage is applied across the dielectric layers, below the dielectric breakdown voltage of the dielectric layers. The use of two dielectric layers, with a sufficiently low voltage across the two dielectric layers, not only prevents destructive ionization of the conductive droplet, but also substantially eliminates the adverse effect of dielectric pinhole defects on the droplet, unexpectedly improving performance despite the reduced electrowetting force caused by the use of two dielectric layers. As a result, for example, 0.01 Wcm- 2 Optically mediated electrowetting can be achieved by simultaneously manipulating thousands of droplets using low power illumination sources such as LEDs generating low power. In preferred embodiments with large area microfluidic devices having areas larger than 1 cm x 1 cm, the devices are suitable for parallel manipulation of more than 10,000 droplets, 50,000 droplets, 100,000 droplets, or even 1,000,000 droplets for very large area devices.

[0039] In some embodiments, large area devices can be used to process thousands of droplets. The inventors have previously attempted to build larger devices that use a single dielectric layer to process droplets in parallel. However, the inventors encountered defective areas where the droplets could not move. Through experimentation and testing, the inventors found that pinhole defects are a significant limitation of device performance, especially as devices become larger.

[0040] Dielectric layers always have sparse pinhole defects, which make them conductive in small, isolated areas. Known optimized processes can achieve a thickness of 1 cm 2 This can result in approximately 38 pinhole defects per micrometer. Pinhole defects can trap droplets and make them immobile. This effect is even more severe when using conductive media such as buffer solutions.

[0041] In some embodiments, a two-dielectric layer structure is provided that can be used below the dielectric breakdown voltage. When operating below the breakdown voltage, such a two-sided dielectric layer structure can provide a novel effect of largely negating the effects of pinhole defects. With the dielectric disposed on both the top and bottom of the droplet, a conductive path can be formed only if the pinhole defect in the first dielectric layer exactly aligns with the pinhole defect in the second dielectric layer. The probability of this occurring is very, very small. This pinhole mitigation feature provided by the presence of the second dielectric layer is key to enabling the simultaneous manipulation of thousands of droplets within a relatively large area.

[0042] For large area devices or very large area devices suitable for parallel manipulation of more than 100,000 droplets or even more than 1,000,000 droplets, the number of pinhole defects becomes a significant limitation in device performance because the probability of a single droplet touching a pinhole defect becomes much higher. A single droplet trapped on a pinhole defect can block the movement of other droplets in the device and thus inhibit or interrupt the operation of the system. Thus, the advantages of the present invention in countering the effects of pinhole defects are of paramount importance in the operation of very large area devices containing a very large number of microdroplets.

[0043] According to another aspect of the invention, there is provided a cartridge comprising: a reservoir containing a liquid sample; an emulsification apparatus in a fluid circuit with the reservoir; an inlet flow path downstream of the emulsification apparatus; a device according to any aspect of the invention; and a pump system, wherein the emulsification apparatus is configured to generate a medium comprising an emulsion of microdroplets in an immiscible dispersion medium, and the inlet flow path is configured to receive the medium comprising the emulsion of microdroplets in the immiscible dispersion medium from the emulsification apparatus, wherein the device comprises at least an inlet port, the device is in fluid communication with the inlet flow path, and the pump system is configured to direct a flow of the liquid sample into the emulsification apparatus and / or direct a flow of the medium comprising the emulsion of microdroplets through the device into the immiscible dispersion medium.

[0044] The aqueous fluids in the cartridge may be biological fluids such as cell culture media, and these fluids may suitably contain cells, globules, particles, drugs, biomolecules, or other biological entities. These entities may be viruses, DNA or RNA molecules, stimulants, cytokines, nutrients, and dissolved gases. Thus, the design of the cartridge flow channels and structures may be optimized to preserve the dispersion and integrity of the biological fluids, particularly by selection of well-matched flow channels of uniform hydraulic diameter and minimal fluid shear forces.

[0045] In some embodiments, the cartridge may further comprise one or more valves at the inlet port of the device which control the flow of a medium comprising an emulsion of microdroplets in an immiscible dispersion medium through the device.

[0046] In some embodiments, the emulsifier may be a step emulsifier. In some embodiments, several emulsifiers may be provided, each of which is provided with an inlet channel.

[0047] In some embodiments, the pumping system may include, but is not limited to, a pump, a head reservoir, an accumulator, and / or a pressure source, It is further understood that other pumping systems are known to those of skill in the art that may be used to induce the flow of liquid sample to an emulsifier and / or induce the flow of media through the device.

[0048] Numerous techniques are currently known in the art for the formation of aqueous emulsions of microdroplets surrounded by an immiscible carrier medium. These techniques include cross-flow emulsion generators, T-junction generators, and step emulsifiers. Cross-flow emulsion generators, T-junction emulsion generators, and other related devices are typically used to create variable size microdroplets. The size distribution of the microdroplets depends on the flow conditions created at the junction where the oil and water feedstock meet. In addition, the size of the microdroplets depends on the fluid properties such as the interfacial tension and viscosity of the flowing fluid. Therefore, precise control and regulation of the flow rate of the fluids entering these types of emulsion generators is required to deliver droplets of uniform and repeatable size distribution into the oEWOD device.

[0049] Advantageously, the step emulsifier produces an emulsion with a microdroplet size distribution that is minimally dependent on the flow rate at the emulsification junction. The size of the microdroplets is determined primarily by the physical dimensions of the emulsification nozzle as well as the material properties of the flowing fluid. While both step emulsifiers and other emulsification devices are sensitive to the properties of the flowing fluid, the degree of dependence on interfacial tension and viscosity is greatly reduced in the step emulsifier. Thus, precise control and adjustment of flow parameters is not required to modify the size distribution of the microdroplets exiting the emulsifier. The step emulsifier can be operated with a simple fixed flow rate or fixed pressure system. The step emulsifier is particularly suited for operation with oEWOD devices because it avoids the need for inspection and optical access of the emulsifier at a location that would otherwise overlap with the optical assembly used for operation of the oEWOD device. The step emulsifier avoids the complexity and cost of introducing multiple inspection and microdroplet size monitoring devices to monitor and control multiple emulsification devices operating within one cartridge assembly. Thus, multiple independent step emulsifiers can be connected to different inlets on the oEWOD device to provide a fluidically isolated emulsion production input path between the water content input and the oEWOD device. The use of fluidically isolated input paths allows the oEWOD device to receive a set of independent emulsion inputs made with different input water contents without the possibility of cross-contamination between them.

[0050] In some embodiments, the cartridge assembly may include up to 8 emulsifiers. In some embodiments, the cartridge assembly may include at least 1, 2, 3, 4, 5, 6, or 7 emulsifiers. In some embodiments, the cartridge assembly may include 8 to 12 emulsifiers. In some embodiments, the cartridge assembly may include 12 to 20, 20 to 30, 30 to 50, or 50 to 100 emulsifiers.

[0051] These emulsifiers can be user-interchangeable, allowing the user to select the type of emulsifier that is appropriate for the purpose he or she intends. For example, the user can configure a cartridge with emulsifiers that provide a particular microdroplet size range. The user can select a set of emulsifiers, each providing microdroplets with a different size range, or sub-selection of size ranges. In some embodiments, the emulsifiers can be configured to generate microdroplets with a volume in the range of 14 pL to 180 pL, or in the range of 180 pL to 500 pL, or in the range of 500 pL to 1.2 nL. These emulsifiers can also be configured to provide microdroplets with a volume less than 14 pL, particularly in the size range of 10 fL to 50 fL or 50 fL to 14 pL. In some embodiments, the emulsifiers can be configured to generate microdroplets with a volume greater than 1.2 nl, including at least the range of 1.2 nL to 4 nL. If the emulsifier is a step emulsifier, the volume of the microdroplets can be varied by changing the geometry of the emulsifier nozzle, in particular by changing the height of the minor axis of a rectangular nozzle.

[0052] Furthermore, the operation of a set of step emulsifier nozzles in a single emulsifier can be parallelized, connecting multiple emulsifier nozzles to a single water-containing input. The connected nozzles can operate independently with varying speeds determined by the complex interactions between the interconnected junctions. The emulsifiers can all 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, reducing the adverse effects of shear that can damage cells and other biological material. This also allows the emulsifier to continue to generate emulsion despite partial blockage or blocking of some nozzles, which is sometimes a result of biological material containing particulates passing through narrow nozzle openings.

[0053] According to one aspect of the invention there is provided a species sorted by the device, apparatus, cartridge or method disclosed herein.

[0054] According to one aspect of the present invention, there is provided a species selected by the device, apparatus, cartridge, or method disclosed herein.

[0055] According to one aspect of the invention, there is provided a species sequestered by the device, apparatus, cartridge, or method disclosed herein.

[0056] According to one aspect of the invention there is provided a species produced by the device, apparatus, cartridge or method disclosed herein.

[0057] The species may be chemical, biochemical, or biological in nature.

[0058] For example, the invention can provide agonists / antagonists to an entity identified by the screening, selection, and / or isolation methods disclosed herein. The invention can provide agonists / antagonists to an entity identified by the screening, selection, and / or isolation methods disclosed herein for use in therapy. The entity can be chemical, biochemical, or biological in nature.

[0059] According to one aspect of the present invention, there is provided a method of using the device, apparatus, cartridge, method or species disclosed herein.

[0060] According to one aspect of the present invention there is provided a method of use of the device, apparatus, cartridge, method or species disclosed herein in therapy.

[0061] The invention provides for the use of the devices, apparatus, cartridges, methods, or species disclosed herein in the production of an article of manufacture.

[0062] Such a use may be peptide synthesis. Such a use may be synthetic biology. Such a use may be cell line engineering or development. Such a use may be cell therapy. Such a use may be drug discovery. Such a use may be antibody discovery.

[0063] According to one aspect of the invention there is provided a method of use of a device, apparatus, cartridge, method or species disclosed herein in an assay.

[0064] The analysis may be a physical, chemical, or biological analysis.

[0065] The method of use may be intracellular imaging.The method of use may be high content imaging.

[0066] The above method of use may be diagnostic.

[0067] The method of use may be a bioassay. The bioassay may be a high-throughput screen. The bioassay may be an enzyme-linked immunosorbent assay (ELISA).

[0068] The method of use may be cell secretion.

[0069] The method of use may be QC (quality control) safety profiling.

[0070] The invention will now be described in more detail, by way of example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0071] [Figure 1]1A and 1B are diagrams illustrating two oEWOD device configurations according to the present invention. [Diagram 2] FIG. 2 is an equivalent circuit diagram of the OEWOD configuration shown generally in FIGS. 1A and 1B. [Diagram 3] FIG. 1 is a voltage plot of the critical voltage in OEWOD devices having dielectric layers of different thicknesses. [Figure 4] 1 is a flow diagram of the surfactant equilibrium between various states within an OEWOD device. [Diagram 5] 5A and 5B are diagrams illustrating the undesired movement of a droplet in an oEWOD device when the droplet is held close to stationary. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] With reference to FIG. 1A, a microfluidic device, in particular an oEWOD device 100, is provided. The oEWOD device 100 shown in FIG. 1A comprises: a first composite wall 102 made of a first substrate 104; a first conductor layer 106 on the substrate 104; a photoactive layer 108 activated by electromagnetic radiation in the wavelength range 400-850 nm on the conductor layer 106; and a first dielectric layer on the photoactive layer 108, where the first substrate 104 can be made of glass, the first conductor layer 106 has a thickness in the range of 70-250 nm, and the photoactive layer 108 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 the thickness of this layer is determined, at least in part, by the method of preparing such a thin layer, which must be continuous. However, theoretically it can have a thickness of 0.1 nm to 20 nm. The first conductor layer can be transparent.

[0073] The device 100 also comprises a second composite wall 112, which comprises: a second substrate 114 and a second conductor layer 116 on the second substrate 114, which may be made of glass. The second conductor layer may be transparent. The second conductor layer 116 may have a thickness in the range of 70-250 nm. A second dielectric layer 118 may be present on the second conductor layer 116, the second dielectric layer 118 having a thickness of less than 20 nm. Like the first dielectric layer, the second dielectric layer must be continuous and its lower limit for thickness is determined by manufacturing constraints, but may be between 1 nm and 20 nm. The exposed surfaces of the first continuous dielectric layer 110 and the second continuous dielectric layer 118 are spaced 20-180 μm apart to define a microfluidic space 121 configured to contain a microdroplet 122.

[0074] The photoactive layer 108 is made of amorphous silicon. The first and second conductor layers are made of ITO (indium tin oxide).

[0075] An intermediate 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 this intermediate layer may be between 0.1 nm and 5 nm. The thickness of this intermediate layer may be greater than 0.1 nm, greater than 0.25 nm, greater than 0.5 nm, greater than 0.75 nm, greater than 1 nm, greater than 1.5 nm, greater than 2 nm, greater than 2.5 nm, greater than 3 nm, greater than 3.5 nm, 4, or greater than 4.5 nm, or less than 5 nm, less than 4.5 nm, less than 4 nm, less than 3.5 nm, less than 3 nm, less than 2.5 nm, less than 2 nm, less than 1.5 nm, less than 1 nm, less than 0.75 nm, less than 0.5 nm, or less than 0.25 nm. An advantage of the intermediate layer is that it may be used as a bonding layer for an anti-fouling layer or a non-fouling layer. In some embodiments, although not shown in the accompanying drawings, the intermediate bonding layer may be omitted. In such an embodiment, the hydrophobic layer may be applied directly to the first dielectric layer.

[0076] The 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 and 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.

[0077] 1A, an incident light 130 can be used to provide a light sprite pattern 131, where the incident light 130 provides light on 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 the macronutrients and ingredients to keep the contents within the microdroplets 122, such as one or more cells, alive and healthy. In some cases, the oil phase 134 can provide macronutrients, one or more media, and ingredients for cell growth, survival, and / or proliferation.

[0078] 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 can have a certain degree of 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 have a thickness of more than 2500 μm, such as 3000 μm, 3500 μm, or 4000 μm. In some embodiments, the first and second substrates can have a thickness in the range of 100 to 2500 μm. In some embodiments, the first and second substrates can have a thickness of greater than 100 μm, greater than 200 μm, greater than 300 μm, greater than 400 μm, greater than 500 μm, greater than 600 μm, greater than 700 μm, greater than 800 μm, greater than 900 μm, greater than 1000 μm, greater than 1100 μm, greater than 1200 μm, greater than 1300 μm, greater than 1400 μm, greater than 1500 μm, greater than 1600 μm, greater than 1700 μm, greater than 1800 μm, greater than 1900 μm, greater than 2000 μm, greater than 2100 μm, greater than 2200 μm, greater than 2300 μm, or greater than 2400 μm. In some embodiments, the first and second substrates can have a thickness of less than 2500 μm, less than 2400 μm, less than 2300 μm, less than 2200 μm, less than 2100 μm, less than 2000 μm, less than 1900 μm, less than 1800 μm, less than 1700 μm, less than 1600 μm, less than 1500 μm, less than 1400 μm, less than 1300 μm, less than 1200 μm, less than 1100 μm, less than 1000 μm, less than 900 μm, less than 800 μm, less than 700 μm, less than 600 μm, less than 500 μm, less than 400 μm, less than 300 μm, or less than 200 μm. In some embodiments, the first substrate has a thickness of approximately 1100 μm and the second substrate has a thickness of approximately 700 μm. In other embodiments, the first and second substrates can have a thickness of 800 microns. 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 may be, but is not limited to, soda-lime glass or float glass.

[0079] The first and second conductor layers 106, 116 are disposed on one surface of the first and second substrates 104, 114 and typically have a thickness in the range of 70-250 nm, with 70-150 nm being preferred. At least one of these layers is made of a very thin film of a transparent conductive material such as indium tin oxide (ITO), 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 as 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 openings of the mesh.

[0080] The photoactive layer 108 is formed of a semiconductor material capable of generating localized regions of charge in response to stimulation by a source of electromagnetic radiation. 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 has a high dielectric strength (V / m) and a dielectric constant (dielectric constant) of > 3. In some embodiments, the dielectric layer is selected from alumina, silica, hafnia, or a non-conductive polymer thin film.

[0081] Alternatively, at least the first dielectric layer, and preferably both dielectric layers, can be coated with an anti-fouling layer to help establish desired microdroplet / dispersion medium fluid / surface contact angles at various hypothetical electrowetting electrode locations, the anti-fouling layer intended to additionally prevent the contents of the microdroplets from adhering to the surface and being reduced as the microdroplets move through the chip.

[0082] For optimal performance, the antifouling layers should help establish a microdroplet / dispersion medium fluid / surface contact angle that should be in the range of 50°-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. Alternatively, these layers can be composed of polymers of acrylate esters such as methyl methacrylate or its derivatives substituted with hydrophobic groups, e.g., alkoxysilyl groups. Either or both of the antifouling layers are hydrophobic to ensure optimal performance. In some embodiments, an intervening layer of silica less than 20 nm thick can be inserted between the antifouling coating and the dielectric layer to provide a chemically compatible bridge.

[0083] The first and second dielectric layers, and thus the first and second walls, define a microfluidic space that is at least 10 μm wide, preferably in the range of 20-180 μm wide, within which the microdroplets are contained. Before being contained, the microdroplets themselves preferably have a characteristic diameter, which is 10% larger, or alternatively 20% larger than the width of the microfluidic space. Thus, when the microdroplets enter the chip, they are subjected to compression, leading to deformation of the spherical microdroplets, resulting in enhanced electrowetting performance, e.g., due to better microdroplet splitting ability. In some examples, the first and second dielectric layers can be coated with a hydrophobic coating, such as fluorosilane.

[0084] In some embodiments, the microfluidic space includes one or more spacers to hold the first and second walls apart by a predetermined amount. Spacer options include beads or pillars, ridges generated from an intermediate resist layer, which are generated by photopatterning. Alternatively, deposition materials such as silicon oxide or silicon nitride can be used to create the spacers. Alternatively, layers of film can be used to form the spacer layer, including flexible plastic films with or without adhesive coatings. Various spacer geometries can be used to form narrow channels defined by straight lines of pillars, tapered channels, or partially closed channels. With careful design, these spacers can be used to assist in the deformation of microdroplets, after which microdroplet splitting can be performed and operations can be performed on the deformed microdroplets. Similarly, these spacers can be used to physically separate zones of the chip to prevent cross-contamination between droplet constituents and direct the flow of droplets when loading the chip under hydraulic pressure.

[0085] An A / C power supply attached to the conductor layer can be used to bias the first and second walls to provide a potential difference between them; suitably this potential difference is in the range of 0 to 50 volts. These oEWOD structures are typically used with electromagnetic radiation sources having wavelengths in the range of 400 to 850 nm, e.g. 550, 620 and 660 nm, and energies above the band gap of the photoactive layer. The photoactive layer is suitably activated at a virtual electrowetting electrode location, where the incident intensity of the radiation used is between 0.005 and 0.1 Wcm. -2 The electromagnetic radiation source is in the range of 0.005 to 1 Wcm -2 or 0.005 Wcm -2 Super, 0.0075Wcm -2 Super, 0.01Wcm -2 Super, 0.025Wcm -2 Super, 0.05Wcm -2 or 0.075Wcm -2In 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 made to a level less than

[0086] When the electromagnetic radiation sources are pixelated, they are powered either directly or indirectly using a reflective screen such as a digital micromirror device (DMD) illuminated by LEDs or other lamps. This allows highly complex patterns of virtual electrowetting electrode positions to be rapidly created and destroyed on the first dielectric layer, thereby enabling the microfluid to be precisely directed along essentially any virtual path using tightly controlled electrowetting forces. Such an electrowetting path can be viewed as consisting of a continuum of virtual electrowetting electrode positions on the first dielectric layer.

[0087] The first and second dielectric layers may be composed of a single dielectric material or may be a composite of two or more dielectric materials, including, but not limited to, Al2O3 and SiO2.

[0088] 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, silicon or glass, or a mixture thereof, and has planar, angled, curved or microstructured walls / surfaces. The structure between the first and second dielectric layers can be connected to the top and bottom 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 conductors and dielectrics can be deposited on a molded substrate that already has walls.

[0089] The oEWOD device 100 shown in Figure 1B provides an alternative oEWOD configuration. As shown in Figure 1B, the oEWOD device comprises: a first composite wall 102 made of a first substrate 104, a conductor layer 106 on the substrate 104, a photoactive layer 108, 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 has a thickness in the range of 70-250 nm, the photoactive layer 108 is activated by electromagnetic radiation in the wavelength range of 400-850 nm on the conductor layer 106, and the photoactive layer 108 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.

[0090] The device 100 shown in FIG. 1B also comprises a second composite wall 112, which comprises: a second substrate 114 and a second conductor layer 116 on the substrate 114, which may be made of glass. The second conductor layer may be transparent. The second conductor layer 116 may have a thickness in the range of 70-250 nm. A second dielectric layer 118 may be present on the second conductor layer 116, the second dielectric layer 118 having 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 its thickness is determined by manufacturing constraints, but may be between 1 nm and 20 nm. The exposed surfaces of the first continuous dielectric layer 110 and the second continuous dielectric layer 118 are spaced 20-180 μm apart to define a microfluidic space 121 configured to contain a microdroplet 122.

[0091] 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 the structure within the first (active) substrate 104. The secondary layers of the oEWOD device, formed of the first conductor layer 106, the photoactive layer 108, the first dielectric layer 110, the intermediate 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.

[0092] In some cases, spacers can be formed by structuring the first and / or second substrates 110, 114 together, or by using a combination of structures in the first and / or second substrates 104, 114 and an insert material such as the channel walls 120, as shown in FIG. 1A.

[0093] 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 the macronutrients and macroingredients to keep the contents within the microdroplets 122, such as one or more cells, alive and healthy. In some cases, the oil phase 134 can provide macronutrients, medium, and contents for cell growth, survival, and / or proliferation.

[0094] Referring to FIG. 2, an equivalent circuit diagram of the device of FIGS. 1A and 1B is shown. FIG. 2 shows that the photoactive layer employs a light-dependent resistor 128 and a capacitor 129. Illumination lowers the resistance of resistor 128, which causes the resistor to form a conductive path. In the "off" state area where the photoactive layer is not illuminated, the resistor forms a substantially non-conductive path. Ideally, a zero voltage is applied to dielectric layer 118 during the "off" state, or during the period when the applied voltage is as close to zero as possible. As a result, in the equivalent circuit diagram of FIG. 2, the photoactive layer 108 contributes a significant amount of resistance and capacitance to the circuit. For an ideal photoactive layer, in the "off" state, the resistance would be infinitely high, leaving a purely capacitive element as the representation of the photoactive layer. In reality, as shown in FIG. 2, all photoactive materials have some resistance in the absence of illumination. Conversely, in the "on" state shown in FIG. 2, illumination of the photoactive layer ideally provides a conductive path across the photoactive layer 108. This would effectively eliminate the photoactive layer as a resistive and capacitive element, and therefore would present the full applied voltage to the underlying dielectric layer 110. With realistic, non-ideal photoactive layers, there would be residual resistance in the illuminated portion of the photoactive layer because the resistance of the photoactive layer 108 does not drop to zero.

[0095] When a non-zero voltage is applied during the "on" state, this voltage is used to hold the microdroplet 122 at a spot of illumination or to propel the movement of the microdroplet along a predetermined electrowetting path. The difference between the "on" and "off" state voltages affects the maximum speed at which the microdroplet can be manipulated.

[0096] During the "on" state, the photoactive layer 108 can provide an applied voltage to the dielectric layer 110 that is attenuated only by the residual resistance of the illuminated photoactive layer 108, whereas the "off" state provides a voltage that is significantly attenuated by the resistance of the non-illuminated photoactive layer. The electrowetting force on each droplet is governed by the difference between the contact angle of the illuminated portion of the microdroplet and the contact angle of the non-illuminated portion. The contact angle of each of these regions is determined by the applied voltage that reaches the dielectric layer 110. Thus, the residual resistance in the photoactive layer 108 in the "off" state changes the contact angle at that portion of the microdroplet 122, thus modifying the electrowetting force. In an equivalent circuit model, the resulting voltage drop across the dielectric layer is the result of the interaction of the complex impedance of the photoactive layer 108 with the impedance of the dielectric layer 110. In the "on" state, light is provided to the microdroplets for the purpose of manipulating the microdroplets. This manipulation includes, but is not limited to, retaining, moving, splitting, and merging the microdroplets. A voltage source 140 can provide a voltage to the microdroplet 122 to cause the microdroplet 122 to move.

[0097] FIG. 2 illustrates the control of the "off" state and its role in optimizing the design of the device shown in FIGS. 1A and 1B. Optimizing the "off" state voltage is a consideration that is relevant only for optically mediated electrowetting systems. The velocity of the microdroplets 122 is determined, at least in part, by the difference between the "on" and "off" state voltages. Ideally, for optically mediated systems, the "off" state should tend toward 0V. The efficiency of the movement also depends on the absolute voltage during each of the "on" and "off" states. Movement is more efficient if the voltage difference between the "on" and "off" states spans a significant change in the degree of wetting. For example, an "on" state at 11V results in the array being largely wetted, as opposed to an "off" state at 1V where the array is totally unwetted. This can be contrasted with a scenario where the "off" state is 100V and the array is completely wetted, so there is no change in the degree of wetting at the 110V "on" state. Both of these scenarios have a 10 V voltage difference between the "on" and "off" state voltages, but the degree of wetting varies much more over a range of 1 to 11 V. Thus, in optically mediated systems, there is a desire to minimize the "off" voltage so imaging can occur during the "off" phase. Within this voltage regime, the optimum dielectric thickness is much thinner.

[0098] An additional experimental phenomenon observed while optimizing the device design is the random motion of the microdroplets near the point of illumination. Without wishing to be bound by theory, it appears that the microdroplets move randomly when the contrast between the "on" and "off" state voltages is reduced. This random motion appears to be minimized in a system where the "off" state voltage tends to zero (0V). This can be achieved by reducing the capacitance of the system and thus providing a thin dielectric layer rather than the much thicker dielectric layers taught in the current technology.

[0099] Referring to FIG. 3, a plot of the electric field gradient is shown, illustrating the size of the electric field and the field gradient across various locations in an oEWOD device 100 with a photoactive layer 108 as shown in FIG. 3. In particular, the voltage magnitude is shown between illuminated regions 132, 134 and non-illuminated regions 136, 138 for a device with an aluminum oxide dielectric layer 111 having a thickness of 120 nm, and for a device with an aluminum oxide dielectric layer having a thickness of less than 20 nm. The voltage plot is the output of a one-dimensional (1D) model, which is constructed by calculating the applied voltage at each material boundary in the system and calculating the potential, and therefore the electric field drop across each material block. The model is calculated over a small region of the device between the transparent conductor layer 116 shown in FIG. 1A and the base of the microdroplet 122 shown in FIG. 1A, where the device has a dielectric layer 110 having a thickness of 20 nm (thin dielectric device), and where the device has a dielectric layer having a thickness of 120 nm (thick dielectric device).

[0100] When it is desired to use the EWOD device at its full capacity, i.e., at the maximum possible motion speed and the highest level of force applied to the droplet, it is necessary to increase the driving voltage, which is governed by the following equation:

number

[0101] Here, the electrowetting force F is the capacitance of the device C d , and on-state voltage V on,d is proportional to.

[0102] Actual maximum operating voltage V max is limited for any given device by the dielectric breakdown (voltage) of the insulating layer; above the breakdown threshold there is undesirable electrolysis of the water containing droplets.

number

[0103] The above equation 2 is the maximum voltage V max is the thickness d of the dielectric and the dielectric breakdown strength E BD This shows that it is a product of

[0104] For this reason, oEWOD devices can optionally provide a maximum force F at a voltage just below the breakdown threshold. max Can work with:

number

[0105] Therefore, the maximum level of electrowetting force that can be applied to a droplet at any one time follows the proportionality relationship of Equation 3.

[0106] However, for the specific case of driving droplet motion with oEWOD, another unexpected factor exists, which is that the velocity of droplet motion is determined not by the total electrowetting force, but by the localized electric field gradient across the dielectric below the droplet, especially in the vicinity of the three-way contact line between the droplet, the carrier phase, and the active oEWOD surface. The droplet motion in an oEWOD device is driven by the asymmetry of surface energy between the illuminated and non-illuminated areas of the droplet; the motion is the result of the droplet relaxing its surface energy to the lowest possible energy state. Thus, the maximum possible surface energy difference between the illuminated and non-illuminated areas, which is increased by maximizing the electric field gradient in the dielectric layer below the droplet, determines the velocity of the droplet motion.

[0107] The electric field gradient in this local contact line region can be calculated for both the thick dielectric 111 device known in the art and the thin dielectric 110 device disclosed herein, as shown previously in Figure 2. When both devices are operated at the same voltage, e.g., well below the breakdown threshold of both devices, the electric field gradient across the microdroplet is higher in the device with the thin dielectric layer 110, even though the same absolute electric field exists in the thick dielectric 111 device. This increased electric field gradient across the droplet results in faster and more controlled droplet movement at a fixed operating voltage, which means that devices with the thin dielectric layer 110 disclosed herein can operate effectively at lower operating voltages.

[0108] Furthermore, there are likely to be other confounding effects driven by the electric field gradient within the device, and therefore it would be advantageous to have a device that operates at lower voltages to reduce these confounding effects, such devices being disclosed in more detail below.

[0109] Alongside the electric field gradient across the microdroplet, there may be an electric field gradient generated within the surrounding carrier phase. The carrier phase is a mixture of a fluorocarbon oil, such as HFE7500, and a PEG-PFPE (polyethylene glycol-perfluoropolyether) based triblock surfactant. It is well known that this class of surfactant forms complex molecular structures on the surface of the chip and within the carrier phase. These structures include Langmuir-Blodgett films on the chip interface, dimers, micelles, vesicles, and other supermolecular structures of surfactant (SUMO) within the carrier layer. There are multi-directional equilibrium states formed between surfactant molecules within the carrier phase, as free surfactant, as oligomers, in the surface layer of the microdroplet, and in the depletion layer at the chip surface. Transitions between any of these states and any of the others are possible, since all of these states are in direct fluid communication. The equilibrium between the states and the associated interactions are illustrated by the block diagram in FIG.

[0110] As shown in Figure 4, a diagram is shown illustrating the connections between the chip surface 140 with the surface layer 142, the surfactant micelles 144, the free surfactant 140, and the surface layer 148 of the microdroplet. The surfactant molecules 141 can transition between the states on the chip interface 142, on the droplet interface 148, and in two states in solution: the free surfactant form as isolated molecules 146, and the free surfactant form as supramolecular structures such as micelles and dimers 144, as indicated by the arrows in Figure 4.

[0111] The presence of an electric field gradient in the carrier phase gives rise to a second unexpected effect, which is dielectrophoresis of non-dropletized material, especially supramolecular structures formed with surfactants such as micelles 144, vesicles, and oligomers in the carrier phase. Near the contact line between the droplet 148 and the droplet surface 150, the aqueous droplet 148 distorts the local electric field to provide a gradient, which causes supramolecular structures within this gradient to rapidly attach to the droplet surface 150. There may also be a slower drift of SUMO towards the chip surface 140. If the droplet is already deformed by electrowetting forces that force the surfactant surface to deform and possibly wrinkle, inducing the coalescence of an excess surfactant layer into micelles, which is expelled by capillary snapping that provides a thrust force. Once the droplets start moving, they can encounter micelles by advection, and the same DEP forces rapidly stratify these droplets onto the preceding surface. At the rear of the microdroplets, surfactant accumulates due to the droplet surface flow, resulting in a surface thrust force. This is a feedback cycle, with the force increasing with speeds of several cm s -1 The rear surface of the microdroplet remains anisotropically layered in the surfactant for a significant amount of time after the enforcing field is removed.

[0112] The result of such electric field gradient driven acceleration is that the microdroplets move with forces that are not determined by the optical electrowetting control; this can occur in non-illuminated areas as well as in partially illuminated microdroplets. This can manifest as microdroplets moving in an uncontrolled manner. In extreme cases, such uncontrolled motion can pull the microdroplets away from the sprites that hold them and move them a significant distance within the chip. As a result, the moving microdroplets can disrupt the hold of other microdroplets within the device, an undesirable effect.

[0113] These effects, and the optimal behavior of the present invention to mitigate them, are further illustrated in Figures 5A and 5B, which are a series of time-lapse micrographs showing the movement of droplets on two different devices: a thin dielectric device (Figure 5A) and a thick dielectric device (Figure 5B), both of which are filled with an aqueous droplet 152 of approximately 70 μm diameter, which is then captured on an illumination spot or sprite 154 of the oEWOD and held within the device by a combination of light and an external voltage (not shown in the accompanying drawings) applied to the conductor layers of the device.

[0114] The droplet 152 in FIG. 5A is held stationary under a voltage of 5V in a thin dielectric device with a dielectric layer approximately 20 nm thick. Under these conditions, the device is capable of moving the droplet 152 over the entire surface under the control of oEWOD at over 4 mm / s. The three images in the time series are taken at an interval of 1 s (one second), during which the droplet moves a distance of less than one tenth of its diameter away from the sprite 154. When the droplet 152 is held, there is very little movement of the droplet 152 and of the central light-holding spot or sprite 154.

[0115] FIG. 5B shows the results of performing a similar test on a thick dielectric device known in the current art, with a 120 nm thick dielectric layer. The device is operated with an AC bias of 10 V, and the droplet motion speed can reach 3 mm / s. However, under these conditions, there is a significant degree of droplet motion in the vicinity of the holding sprite 154 when the droplet 152 is held in a stationary position using the oEWOD force. The time-lapse images in FIG. 5B are again taken at 1 s intervals, but during this time frame, the droplet 152 is significantly displaced from its holding spot 154 due to the effects of dielectrophoresis of the supramolecular surfactant structures contained within the surrounding carrier phase. At the extreme end of the motion, the droplet 152 is displaced away from the sprite 154 by as much as half its diameter. These adverse effects on the device with the thick dielectric layer shown in FIG. 5B are not observed in the device with the thin dielectric layer, i.e., less than 20 nm thick, shown in FIG. 5A.

[0116] Various additional aspects and embodiments of the present invention will be apparent to those skilled in the art in view of this disclosure.

[0117] As used herein, "and / or" should be interpreted as a specific disclosure of each of the two specified features or components with the other or without the other. For example, "A and / or B" should be interpreted as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, just as if each were individually set forth herein.

[0118] Unless otherwise indicated by context, 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.

[0119] Furthermore, while the present invention has been described by way of example with reference to certain embodiments, it will be understood by those skilled in the art that the invention is not limited to the disclosed embodiments and that alternative embodiments may be constructed without departing from the scope of the present invention as defined in the appended claims.

Claims

1. A device for manipulating micro-droplets using optically mediated electro-wetting, wherein the device comprises a micro-fluidic space, and the micro-fluidic space is bounded by a first composite wall and a second composite wall, wherein the first composite wall comprises a first substrate, a first conductor layer on the first substrate, a photoactive layer on the first conductor layer, and a first continuous dielectric layer on the photoactive layer, the first continuous dielectric layer having a thickness of less than 20 nm, wherein the second composite wall comprises a second substrate, and a second conductor layer on the second substrate. Device.

2. The device according to claim 1, wherein the second composite wall further comprises a second continuous dielectric layer on the second conductor layer, the second continuous dielectric layer having a thickness of less than 20 nm.

3. further comprising an alternating current (A / C) power supply, at least one electromagnetic radiation source, and a microprocessor, wherein the A / C power supply is connected to the first conductor layer and the second conductor layer to supply a voltage between the first composite wall and the second composite wall, and the electromagnetic radiation of the at least one electromagnetic radiation source has an energy higher than the bandgap of the first photoexcitation layer and is configured to impinge on the photoactive layer to induce corresponding temporary electro-wetting positions on the surface of the first dielectric layer. The microprocessor controls the electromagnetic radiation source to manipulate the point at which the electromagnetic radiation strikes the photoactive layer, thereby changing the arrangement of the temporary electro-wetting positions, creating at least one electro-wetting path, and enabling the micro-droplets to move along the at least one electro-wetting path. The device according to claim 1.

4. The device according to claim 1, further comprising an intervening layer of silicon oxide provided on the first continuous dielectric layer and / or on the second continuous dielectric layer, the thickness of the intervening layer being from 0.1 nm to 5 nm.

5. The exposed surface of the first composite wall and the exposed surface of the second composite wall are arranged at a distance of less than 200 μm from each other, defining a microfluidic space configured to contain the micro-droplets, the width of the microfluidic space being from 2 to 50 μm. The device according to claim 1.

6. The exposed surface of the first composite wall and the exposed surface of the second composite wall include one or more spacers, the spacers holding the first composite wall and the second composite wall apart by a predetermined amount and defining a microfluidic space configured to contain the micro-droplets. The device according to claim 1.

7. The A / C power supply is connected to the first conductor layer and the second conductor layer, supplying a voltage of 0 V to 50 V between the first composite wall and the second composite wall. The device according to claim 1.

8. The first composite wall and the second composite wall each further comprise a first anti-fouling layer and a second anti-fouling layer on the first continuous dielectric layer and the second dielectric layer respectively, the anti-fouling layers on the first continuous dielectric layer and the second continuous dielectric layer being hydrophobic. The device according to claim 1.

9. The electromagnetic radiation source comprises an array of pixelated light, the light being reflected from or transmitted through the array. The device according to claim 3.

10. The device according to claim 1, further comprising a photodetector for detecting an optical signal within a microdroplet located within or downstream of the device.

11. The device according to claim 1, further comprising a plurality of first electro-wetting paths extending parallel to each other.

12. The device according to claim 11, further comprising a plurality of second electro-wetting paths, the second electro-wetting paths interacting with the first electro-wetting paths to generate at least one microdroplet coalescence position.

13. The device according to claim 1, wherein the second composite wall further comprises a second photoexcitation layer, and the electromagnetic radiation from the electromagnetic radiation source also impinges on the second photoexcitation layer to generate a second pattern of temporary electro-wetting positions, and the second pattern can also be changed.

14. The device according to claim 1, wherein the electromagnetic wave source is an LED light source.

15. The device according to claim 1, wherein the electromagnetic radiation source is at a level of 0.005 to 0.1 W cm- 2 .

16. The device according to claim 1, wherein the first conductor layer on the first substrate is transparent and has a thickness in the range of 70 to 250 nm.

17. The device according to claim 1, wherein the photoactive layer is activated by the electromagnetic radiation within the wavelength range of 400 to 1000 nm.

18. A cartridge comprising a storage container containing a liquid sample, an emulsifying device within a fluid circuit having the storage container, an inlet flow path provided downstream of the emulsifying device, a device having a microfluidic space, and a pump system. The emulsifying device is configured to produce a medium consisting of an emulsion of minute water droplets in a non-miscible dispersion medium. The inlet flow path is configured to receive the medium consisting of the emulsion of the minute water droplets in the non-miscible dispersion medium from the emulsifying device. The microfluidic space is bounded by a first composite wall and a second composite wall, The first composite wall comprises a first substrate, a first conductor layer on the first substrate, a photoactive layer on the first conductor layer, and a first continuous dielectric layer on the photoactive layer, the first continuous dielectric layer having a thickness of less than 20 nm. The second composite wall comprises a second substrate, and a second conductor layer on the second substrate. The device comprises at least an inlet port, and the device is in fluid communication with the inlet flow path. The pump system is provided for guiding the flow of the liquid sample to the emulsifying device and / or for guiding the flow of the medium consisting of the emulsion of the minute water droplets into the non-miscible dispersion medium through the device. Cartridge.

19. The cartridge according to claim 18, further comprising one or more valves provided at the inlet port of the device, the valves controlling the flow of the medium consisting of the emulsion of the minute water droplets in the non-miscible dispersion medium through the device.

20. The cartridge according to claim 19, wherein the emulsifying device is a step emulsifying device.

Images