Multi-cartridge digital microfluidic device and method of use

Abstract

The invention relates to a multi-cartridge digital microfluidic device and a method of use. High throughput digital microfluidic (DMF) systems and methods (including apparatuses, systems, cartridges, DMF devices, etc.) are described herein. The system, device and method integrate liquid treatment with a DMF device, providing flexible and efficient sample reaction and sample preparation. These systems, devices, and methods may be used for a wide variety of cartridge configurations and sizes.

Description

[0001] This application is a divisional application of an application with an application date of April 8, 2020, application number 202080042230.8, and invention name “Multi-box digital microfluidic device and method of use”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 831,171, filed April 8, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0004] Incorporated by Reference

[0005] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0006] field

[0007] The present application generally relates to digital microfluidic (DMF) devices and methods. In particular, the systems, devices and methods described herein relate to an air-gap DMF apparatus, which includes a cartridge containing an air matrix and a ground electrode and a durable component containing a drive electrode. background

[0009] Digital microfluidics (DMF) has become a powerful preparation technology for a wide range of biological and chemical applications.DMF can be used to control a variety of samples and reagents including solids, liquids and irritating chemicals in real time, accurately and flexibly without the need for pumps, valves or complex pipeline arrays.DMF can be referred to as (or can include) so-called electrowetting on demand (EWOD).In DMF, discrete droplets of nanoliter volume to microliter volume are distributed from a reservoir onto a flat surface coated with a hydrophobic insulator, on which a series of potentials are applied to an electrode array to manipulate (transport, separate (split), merge, mix) the discrete droplets.Complex reaction series can be carried out using DMF alone or using a hybrid system, in which DMF is integrated with channel-based microfluidics.

[0010] It will be very advantageous to have a high-throughput air matrix DMF device (e.g., device and / or system), which can carry out multiple determinations and / or sample preparation on multiple boxes of current identical or different configurations. Device includes a DMF device integrated with a mechanical arm and a liquid handling subsystem, which can run a workflow on one or more boxes arranged on a DMF electrode array, and the device can provide reliable and cost-effective sample processing. High-throughput air-matrix DMF device adopts disposable boxes, which are cheap to manufacture, and provides flexible configuration as required. This paper describes methods and devices that can solve these problems, including systems and equipment.

[0011] Overview of the Disclosure

[0012] High-throughput digital microfluidics (DMF) methods and devices (including devices and systems, such as boxes, DMF readers, etc.) are described herein. Although the methods and devices described herein may be particularly suitable for air matrix DMF devices (also referred to herein as air gap DMF devices), these methods and devices may be configured for other DMF devices (e.g., oil gaps, etc.). The methods and devices described herein can be used to process relatively larger volumes than those that may be processed by conventional DMF devices, in part because the air gaps of DMF devices may be larger (e.g., greater than 280 microns, 300 microns or greater, 350 microns or greater, 400 microns or greater, 500 microns or greater, 700 microns or greater, 1 mm or greater, etc.). In addition, any of the devices and methods described herein may be configured to include or work with one or more disposable boxes having a dielectric layer forming the bottom of the box; the drive electrode need not be part of the box. These devices may be adapted to allow the dielectric to be firmly held to the electrode during operation, which has proven to be very challenging, especially when the dielectric layer is slightly flexible. The term "box" can refer to a container that forms an area in which droplets move by electrowetting forces, which can include an air gap and can be inserted into a DMF reading / driving device. The box can be disposable (e.g., one-time use or limited use). The box can be a double-plate box (e.g., having a top plate and a bottom plate forming a surface) or a single-plate box (having a bottom plate / surface that is open at the top). The box can be configured to allow visualization of the fluid (droplets) in the air gap.

[0013] The larger air gap of the device described here can be provided by using a double plate box, wherein the interval between the plates forms an air gap. Alternatively, a larger air gap can be provided by using a single plate box, which only has a bottom dielectric layer covering the electrode array on the DMF device. Finally, the device and method described herein can provide multiplexing capabilities, which can be used more easily and faster. The device can include a more effective and intuitive user interface, as well as the ability to create, modify, store and / or transmit various microfluidic control schemes.

[0014] For example, a dual-plate box for a digital microfluidics (DMF) device may have a bottom and a top, and may include: a sheet of dielectric material having a first side and a second side, the first side forming an exposed bottom surface on the bottom of the box, wherein at least the second side of the sheet of dielectric material includes a first hydrophobic surface; a top plate having a first side and a second side; a ground electrode on the first side of the top plate. The ground electrode may include a grid pattern forming a plurality of open cells. The box may also include: a second hydrophobic surface covering the ground electrode on the first side of the top plate; and an air gap separating the first hydrophobic layer and the second hydrophobic layer, wherein the air gap includes a spacing greater than 280 microns (e.g., greater than 300 microns, greater than 400 microns, etc.). The top plate may be formed of any suitable material, particularly including clear or transparent materials (e.g., acrylic resins, etc.).

[0015] The top plate of the dual-plate cartridge may include one or more ports or openings to the air gap that may provide a fluid input or fluid output for entry by the liquid handling tips of the robot / liquid handling subsystem.

[0016] In any of the dual-panel boxes described herein, the top plate may include a plurality of cavities within the thickness of the top plate; these cavities may be closed (e.g., sealed) and / or filled with a thermally insulating material having a low thermal mass and a low thermal conductivity. In some variations, the insulating material comprises air. The cavities may be located above an air gap region, which would correspond to a heating and / or cooling region (e.g., a thermal control region); the lower thermal mass in these regions may allow significantly faster heating / cooling of droplets in the air gap below one or more cavities. Thus, the thickness of the top plate in these regions may include cavities; the bottom of the cavities (corresponding to the bottom surface of the top plate) may be less than 1 mm thick (e.g., less than 0.9 mm, 0.8 mm, 0.7 mm, 0.6 mm, 0.5 mm, 0.4 mm, 0.3 mm, 0.2 mm, 0.1 mm, 90 microns, 80 microns, 70 microns, 60 microns, 50 microns, 40 microns, 30 microns, etc.). The cavity bottom may preferably be as thin as possible while providing structural support for the electrodes and any dielectric coating on the bottom surface of the top plate. The cavity top surface may be substantially thicker (e.g., 1.5 times, 2 times, 3 times, 4 times, 5 times, etc.) than the cavity bottom surface.

[0017] Alternatively, a single plate box can be used for high throughput DMF devices and methods as described herein. A single plate box can be formed similar to a double plate box, but without an upper plate. The single plate box can also be without an integrated electrode grid and can be operated in a single-sided electrowetting mode on a DMF device.

[0018] In a single-plate box or a double-plate box, the dielectric material forming the bottom surface can be made hydrophobic (e.g., by coating, including dip coating, etc., impregnation with a hydrophobic material, etc.) and / or it itself can be hydrophobic. For example, the bottom surface (e.g., the bottom surface of the box) can be formed by a film that is both a dielectric material and a hydrophobic material. For example, the bottom surface can be a polytetrafluoroethylene film that is both hydrophobic and acts as a dielectric (which may include an adhesive or an adhesive portion, such as a polytetrafluoroethylene tape). Other films may include plastic paraffin films (e.g., "Parafilm", such as PARAFILM M). However, in particular, films that can withstand high temperatures (e.g., 100° C. and above) (e.g., polytetrafluoroethylene films) are preferred.

[0019] In any of the cartridges described herein, the sheet of dielectric material can be flexible. Such flexibility can help secure the dielectric to the drive electrode to ensure full contact between the dielectric and the drive electrode. Typically, the sheet of dielectric material can be sufficiently compliant that it can be bent or flexed under relatively low forces (e.g., a pressure of 50 kPa or greater). The dielectric sheet can be any suitable thickness; for example, the sheet can be less than 30 microns thick (e.g., less than 20 microns thick, etc.).

[0020] As mentioned, the dielectric material sheet can be flexible and can form the bottommost surface of the box. The sheet can be generally flat (flat), although it can be flexible. The outer surface can be protected by a removable (e.g., peeling) cover. The dielectric properties can be those generally consistent with DMF (particularly air-based DMF) devices. The dielectric can be coated with a first hydrophobic layer on the inside (second side). The hydrophobic layer can be a relatively inert (e.g., not reacting with aqueous droplets moving in the air gap) hydrophobic material coating.

[0021] Any dual-plate box can also include a window from the top of the box to the air gap, through which the air gap can be seen. This can allow imaging into the air gap. Single-plate boxes can be imaged anywhere within the single-plate box. This imaging can be used to detect outputs (e.g., reaction outputs, such as binding, colorimetric assays, RT-PCR, etc.). In addition, the window of the dual-plate box or the open air gap of the single-plate box can provide an inlet for photoactivation of reagents present in the droplets in the box. The window can be any suitable size; for example, the window can be formed as between 2% and 50% of the top of the box. The window can be on one side of the box and / or one end of the box. Multiple imaging windows can be used. Optionally, the top plate of the dual-plate box can be transparent or substantially translucent, allowing imaging through the top plate.

[0022] The box can include one or more reagent reservoirs. For example, in the reservoir or in the air gap, the box can include one or more reagents, particularly freeze-dried (e.g., "freeze-dried") reagents. For example, a double-plate box can include one or more freeze-dried reagent reservoirs on the second side of the top plate. Alternatively, a double-plate box or a single-plate box can include one or more freeze-dried reagent reservoirs on the bottom plate of the box.

[0023] Also described herein is a DMF device for use with any of the boxes described herein to measure them, including detecting the results of the measurements. These devices may be referred to as DMF driver devices here. For example, the DMF driver device may be configured to apply a vacuum on the dielectric bottom surface of the box (particularly a subset of the ports below a particular box or multiple boxes) so that the electrode is in uniform and close contact with the dielectric forming each unit cell pattern, thereby moving the droplets of the fluid in the air gap. The applicant was surprised to find that simply bonding the dielectric material to the electrode is not enough because it results in unequal contact and power variations required to move the droplets, as well as inefficiencies in droplet movement, control, and consistency. In addition, even when used in combination with an adhesive, the use of vacuum has similar problems, especially when the dielectric is flexible. This article describes devices and methods for using them, in which a vacuum is used to fix the dielectric bottom of the box by driving multiple openings in the electrode itself or around / close to the driving electrode. In a variation where a vacuum is applied through all or some of the drive electrodes (e.g., spaced apart in a pattern on the mounting surface (e.g., at a corner), the dielectric is consistently held to the drive electrodes in a uniform manner, even when a relatively low vacuum negative pressure is used. This configuration may also allow for the formation of partitions or barriers within the cartridge by including protrusions on the cartridge holding surface on which the cartridge is held.

[0024] Specifically, described herein is a digital microfluidic (DMF) device that simultaneously controls and drives the movement of one or more droplets through one or more, especially several, DMF boxes. As used herein, driving electrowetting and other reactions simultaneously in multiple different boxes (or in a single box in some variations) can include operating the electrowetting operation (DMF control) simultaneously in multiple different boxes and / or in a single box, or operating the electrowetting operation continuously in multiple different boxes and / or in a single box, for example by rapidly cycling multiple different operations.

[0025] For example, described herein is a digital microfluidic device comprising: a placement surface configured to simultaneously place one or more boxes; a plurality of drive electrodes disposed on the placement surface, wherein the drive electrodes are configured to apply a voltage to move droplets within the box when the box is placed on the placement surface; a plurality of vacuum ports distributed across the placement surface; a vacuum control configured to selectively apply a vacuum through a subset of the vacuum ports below one or more boxes placed on the placement surface when the one or more boxes are placed on the placement surface; an electrical control for applying energy to the drive electrodes to move droplets within an air gap of the one or more boxes when the one or more boxes are placed on the placement surface; and a liquid handling subsystem for dispensing liquid to or removing liquid from the one or more boxes when the one or more boxes are placed on the placement surface.

[0026] Typically, any of these devices (e.g., systems, equipment, etc.) can include a robotic arm. For example, the liquid handling subsystem can include a robotic arm. The liquid handling subsystem can include a liquid handling head and at least one liquid handling tip. In some variations, the liquid handling head is configured to dispense or extract a volume of about 1 microliter to about 100 milliliters.

[0027] In any of these devices, the vacuum control may further include a vacuum pump for applying vacuum to the plurality of vacuum ports. In any of these variations, each of the plurality of vacuum ports may be coupled to an opening via a drive electrode in a plurality of drive electrodes. The vacuum control may be configured to determine which vacuum ports are located below one or more boxes. The vacuum control may further include a manifold and a plurality of valves.

[0028] The plurality of drive electrodes may include between about 408 and about 20,000 drive electrodes.

[0029] Typically, any of these devices can include a master control subsystem for coordinating activation of multiple drive electrodes, multiple vacuum ports, and a liquid handling subsystem. In some variations, vacuum controls and / or electrical controls can be part of the master control subsystem, or can be coupled to the master control subsystem and / or to each other. The master control subsystem can include non-transient instructions for controlling multiple vacuum ports, multiple drive electrodes, and a liquid handling subsystem. The master control subsystem can be configured to simultaneously move droplets in the air gaps of two or more boxes when the box is loaded onto a placement surface.

[0030] The devices described herein may also include one or more temperature control zones within the drive electrode array.Alternatively or additionally, any of these devices may include one or more magnetic control zones within the drive electrode array, each magnetic control zone including a magnet configured to deliver a magnetic field.

[0031] Generally speaking, these digital microfluidic devices can include one or more cartridge alignment features located on a mounting surface.

[0032] Methods are also described herein, including methods of operating any of these devices. For example, a method of selectively securing one or more cartridges in a digital microfluidic device having a plurality of drive electrodes is described herein, the method comprising: placing one or more cartridges on a mounting surface of the digital microfluidic device to generate one or more corresponding mounting locations of the one or more cartridges, wherein each of the one or more cartridges is located on a sub-area of ​​the mounting surface; sensing each corresponding mounting location of the one or more cartridges; determining a subset of the plurality of drive electrodes located below the corresponding mounting location of the one or more cartridges; selectively applying a vacuum to the corresponding mounting location of the one or more cartridges to secure the one or more cartridges to the mounting surface of the digital microfluidic device.

[0033] Selectively applying a vacuum may include applying a vacuum through a subset of vacuum ports located below the respective placement locations. In some variations, applying a vacuum may include applying a vacuum through an opening that passes through all or some of the drive electrodes located below the respective placement locations. Sensing each respective placement location may include sensing an electrical difference in a plurality of drive electrodes below the respective placement location. Alternatively or additionally, sensing each respective placement location may include sensing a resistance to airflow through a subset of vacuum ports on a placement surface in the respective placement location.

[0034] Each of the one or more cassettes may include a base plate configured to deform upon application of a vacuum so as to adhere to the seating surface.

[0035] In some variations, each respective seating position of the one or more boxes may have a respective area corresponding to the size of each box.At least one of the one or more boxes may have a different size than the other boxes of the one or more boxes.

[0036] For example, described herein is a digital microfluidic (DMF) actuator device configured to operate with one or more cartridges having a bottom dielectric surface, an optional top plate having a ground electrode, and an air gap between the bottom dielectric and the top plate (in the case of a dual-plate cartridge), the device comprising: a mounting surface for mounting the one or more cartridges; a plurality of drive electrodes located on the mounting surface, wherein each drive electrode comprises an opening therethrough; a vacuum pump for applying a vacuum to a vacuum port; and a controller for applying energy to sequentially activate and deactivate one or more selected drive electrodes to move a droplet within the air gap of the cartridge along a desired path within the air gap, wherein the DMF device is configured to apply a vacuum to a vacuum manifold to secure each drive electrode to the bottom dielectric of the disposable cartridge when the disposable cartridge is placed on the mounting surface; and a robotic arm and liquid handling subsystem.

[0037] In some variations, the apparatus includes a vacuum manifold coupling a vacuum pump to a plurality of vacuum ports to apply the vacuum.

[0038] The DMF driver device described herein can be configured to operate with any box described herein, and can be suitable for use with such box. However, it should be understood that box is not an essential part of the DMF driver. In general, these devices can operate with one or more boxes (e.g., reusable or disposable boxes), and these boxes have bottom dielectric surfaces, optional top plates with ground electrodes and gaps (e.g., typically air gaps but not necessarily air gaps, and in the case of double-plate boxes, air gaps are arranged between bottom dielectrics and top plates).

[0039] The DMF driver device can also include a placement surface for placing one or more boxes usually. The placement surface can have a width of about 50, 60, 70, 90, 100, 150, 200, 250, 400, 500, 600, 700, 780 or about 800 millimeters, or have a width of any value therebetween. The placement surface can have a length of about 80, 100, 110, 150, 200, 400, 600, 800, 1000, 1300, 1500, 1750, 1800, 2000, 2050 or about 2100 millimeters. The placement surface can include a drive electrode and / or any projection, and the drive electrode can be flush or substantially flush with the placement surface, and the projection can be used to form a cutoff in the gap region (e.g., air gap) of the box by predictably deforming the dielectric into the gap region. A plurality of drive electrodes on the placement surface can be formed on the placement surface or milled into the placement surface. For example, the mounting surface can be a substrate such as a printed circuit board (e.g., an electrically insulating surface) to which the drive electrodes are attached or formed. There can be about 300, 400, 500, 600, 800, 900, 1000, 1500, 2000, 5000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000 or about 70,000 drive electrodes.

[0040] Typically, as mentioned above, all or most (e.g., >50%, >60%, >70%, >80%, >90%, >95%, etc.) of the drive electrodes in the electrode array may include openings that pass through the drive electrodes and are connected to a vacuum source. The vacuum source may be a vacuum manifold that connects these openings through the drive electrodes to a source of vacuum, such as a vacuum pump that is part of the device, or a separate vacuum pump that is connected (e.g., wall vacuum) to the device. The openings through the electrodes may be the same size, and they may be located anywhere on / through the drive electrodes. For example, they may pass through the center of the drive electrode, and / or through the edge region of the drive electrode, etc. The openings may be of any shape (e.g., circular, oval, square, etc.). In some variations, the size of the opening may be about 1 mm in diameter (e.g., 1.2 mm in diameter, 1.1 mm in diameter, 1.0 mm in diameter, 0.9 mm in diameter, 0.8 mm in diameter, etc.).

[0041] Typically, a vacuum manifold can be coupled to and / or can include a plurality of vacuum ports, each vacuum port coupled to one (or in some variations, more than one) of the openings in the drive electrode. The vacuum manifold can be located below the mounting surface. For example, the vacuum manifold can be a pipe or other passage below the mounting surface that connects to the openings in the drive electrode.

[0042] The amount of negative pressure (vacuum) applied by the vacuum manifold to hold the cassettes can be adjusted, selected and / or regulated to prevent deformation of the membrane of each cassette (and therefore the bottom surface of the air gap). The device can apply vacuum to hold the cassettes only where there are cassettes, and can avoid applying vacuum to areas where cassettes can be placed but are not currently placed. This also helps maintain the pressure used to hold the cassettes placed in the DMF reading device. For example, the pressure can be maintained between -0.5 inches of mercury (in Hg) and -25 in Hg (e.g., between a lower limit of about -0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9, 10, 11, 12, 13, 14, 15 in Hg, etc. and an upper limit of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 in Hg, etc., including, for example, less than about 4 in Hg, less than about 5 in Hg, less than about 6 in Hg, less than about 7 in Hg, less than about 8 in Hg, less than about 9 in Hg, less than about 10 in Hg, less than about 12 in Hg, less than about 15 in Hg, less than about 17 in Hg, less than about 19 in Hg, Hg, less than about 20 in Hg, less than about 22 in Hg, etc.).

[0043] Therefore, the high-throughput DMF device can include a mechanical arm and a liquid handling subsystem (or a liquid handling subsystem including a mechanical arm). The mechanical arm can be a three-axis mechanical arm, allowing access to any position on the placement surface. The mechanical arm can also include a metal probe, which is configured to electroporate biological cells in a droplet in at least one of the one or more boxes placed on the placement surface. The mechanical arm can be fully integrated into the process of the operation in the control DMF driver device, including distributing, mixing, adding, removing, reacting the fluid in one or more boxes and otherwise engaging with the fluid in one or more boxes. In some variations, the mechanical arm is a part of the liquid handling subsystem, or can be separated therefrom. The term "mechanical arm" is intended to be widely used as any mechanical positioning subsystem that can operate in 3D space here.

[0044] The liquid handling subsystem may further include a liquid handling head. The liquid handling head may be configured to distribute and / or remove any suitable volume (e.g., a volume from about 1 microliter to about 100 microliters) through one or more liquid handling tips. Therefore, the liquid handling subsystem may further include at least one liquid handling tip. The liquid handling subsystem may also include multiple liquid handling tips, such as 4, 8, 12 or 96 liquid handling tips. The liquid handling subsystem may be transported or extracted from one or more boxes placed on the placement surface. The liquid handling subsystem may be transported or extracted from a liquid droplet by a fluid input or output (e.g., port) on the top plate of a double-plate box. The liquid handling subsystem may be transported or extracted from any position in a single-plate box by an air gap. The liquid handling subsystem may further include a reagent / sample storage subsystem, which may be temperature controlled. The reagent / sample storage subsystem may further include an orifice plate accommodating area, which may include any one of 24 holes, 48 ​​holes, 96 holes and 384 hole orifice plates. The liquid handling subsystem can retrieve reagents or samples from the reagent / sample storage subsystem and transport the reagents or samples to one or more boxes. The samples may contain biological cells. The liquid handling subsystem can further extract product droplets from one or more boxes and place the product droplets in the holes of the orifice plate in the orifice plate receiving area.

[0045] A high-throughput DMF apparatus may include a housing surrounding a DMF electrode array and mounting surface, a robotic arm, and a liquid handling subsystem, wherein the housing controls one or more of humidity, temperature, and gas environment.

[0046] The DMF drive described herein may include a main controller for coordinating an electrode array and a mechanical arm / liquid handling subsystem. The main controller may adjust the housing to adjust one of humidity, temperature, or gaseous environments. The controller may include one or more processors, memories, and any other circuits necessary or useful for operating the device, the operation including coordinating the application of energy to activate / deactivate the drive electrode, guiding the mechanical arm / liquid handling subsystem to distribute or extract droplets; operating a pump for vacuum control and / or microfluidic control, one or more valves (e.g., for microfluidic control, vacuum control), a temperature controller (e.g., a resistive heater, a Peltier cooling, etc.), a motor (e.g., for a mechanical arm, a liquid handling subsystem, an optical device, etc.), one or more displays, etc.

[0047] As mentioned, any of these means may include one or more protrusions extending from the seating surface, wherein the one or more protrusions are configured to form a barrier in the air of the cartridge when a vacuum is applied through an opening in the drive electrode.

[0048] Any of these devices can include an optical subsystem. The optical subsystem can include one or more illumination sources, such as visible illumination sources, infrared illumination sources and / or ultraviolet illumination sources and excitation light sources for visualization. The optical subsystem can also include an imaging device, which is configured to detect optical signals from a box placed on a placement surface. The optical subsystem can be movable or fixed. The optical subsystem can be used to detect (e.g., sense) feeds or changes caused by one or more interactions (e.g., binding, enzymatic reactions, etc.) in the droplets. The optical subsystem can be configured to detect optical signals from a box placed on a placement surface. Therefore, an optical sensor can provide detection of readout from a device. Any of these devices can include one or more motors, for example, the motor is configured to move an optical device. In some variations, the optical subsystem can be connected to a robotic arm or included as part of a robotic arm.

[0049] The apparatus described herein may also include one or more temperature sensors (e.g., thermistors, etc.). For example, the apparatus may include one or more temperature sensors coupled to the mounting surface. In certain variations, the thermistors may protrude from the mounting surface and form a barrier or chamber in the air gap of the box. Alternatively or in addition, one or more temperature sensors may be within the substrate of the mounting surface and in thermal contact with the mounting surface, for example, via a thermally conductive material (e.g., copper).

[0050] As mentioned, the apparatus described herein may include one or more heaters, particularly resistive heaters. For example, the apparatus may include resistive heaters below (or above) at least some of the drive electrodes; this may allow for temperature-regulated sub-regions of the device. The entire drive electrode surface may also be cooled (e.g., by circulation of a cooling fluid) to slightly below room temperature (e.g., between 15 degrees Celsius and 25 degrees Celsius, between 15 degrees Celsius and 22 degrees Celsius, between 15 degrees Celsius and 20 degrees Celsius, between 15 degrees Celsius and 18 degrees Celsius, etc.).

[0051] The device can also include one or more magnets on or below one or more of the driving electrodes, and the magnet is configured to be activated to apply a magnetic field. Therefore, magnetic beads can be used for other reactions in binding materials or DMF devices, and magnetic beads can be selectively maintained in one or more regions of the equipment. For example, one or more neodymium magnets can be used, for example, by moving the magnet closer or further away from the box to keep the magnetic particles in place (for example, the magnet is moved upwards to the electrode 3mm, 4mm, 5mm, 6mm, 7mm, 8mm, etc.). Electromagnets can be selectively activated or deactivated to keep / release magnetic particles. The mechanical arm can include one or more magnets for selectively applying a magnetic field.

[0052] Any of the devices described herein may also include one or more Peltier coolers beneath at least some of the drive electrodes, wherein the Peltier coolers are configured to cool to 10 degrees Celsius or less (e.g., 5 degrees Celsius or less, 7 degrees Celsius or less, 11 degrees Celsius or less, 12 degrees Celsius or less, 15 degrees Celsius or less, 20 degrees Celsius or less, etc.).

[0053] Typically, any of these devices can include an external housing, a front panel display and one or more inputs (e.g., a touch screen display, a dial, a button, a slider, etc.) and / or a power switch. The device can be configured to operate in conjunction with one or more other DMF devices. In some variations, a single housing can enclose a plurality of box placement surfaces, each box placement surface having (by a single or multiple controllers) individually addressable / controllable drive electrode arrays, which allows the parallel processing of a plurality of boxes; in these variations, all or some of the components (pumps, motors, optical subsystems, controllers) can be shared between different box placement surfaces.

[0054] Any of these devices may include an output configured to output signals detected by the device. The outputs may be on one or more displays / screens, and / or they may be electronic outputs that are transmitted to a memory or remote processor for storage / processing and / or display. For example, any of these devices may include a wireless output.

[0055] Also described herein is a method for preventing or reducing evaporation in any of these devices. For example, described herein is a method for preventing droplet evaporation in an air matrix digital microfluidics (DMF) device, the method comprising: introducing a water-containing reaction droplet into an air gap of an air matrix DMF device, the air gap being formed between a first plate and a second plate of the air matrix DMF device; sequentially energizing a driving electrode on or in the first plate to move the water-containing reaction droplet in the air gap of the air matrix DMF device, so that the water-containing reaction droplet is combined with a droplet of a non-polar fluid in the air gap of the air matrix DMF device, which forms a coated reaction droplet, in which the non-polar fluid coats the water-containing reaction droplet and protects the reaction droplet from evaporation; and sequentially energizing a driving electrode to move the coated reaction droplet in the air gap of the air matrix DMF device.

[0056] The volume of non-polar fluid can be less than the volume of moisture reaction droplet.Any of these methods can be included in the air gap of air matrix DMF device and one or more other moisture droplet combinations of the droplet of coating.Any of these methods can also include by the droplet of coating being at least partially extracted out in the air gap of air matrix DMF device into microfluidic channel to remove the coating of non-polar fluid.The method can also include by the opening in the first plate or the second plate the droplet of non-polar fluid is added in the air gap of air matrix DMF device.Usually, the droplet of non-polar fluid can be liquid between 10 degrees Celsius and 100 degrees Celsius.

[0057] For example, a method of preventing evaporation of droplets within an air-matrix digital microfluidics (DMF) device may include introducing additional aqueous material into an aqueous reaction droplet into an air gap of the air-matrix DMF device, including from a robotic arm and / or a fluid dispensing subsystem.

[0058] Method and apparatus described herein can be particularly suitable for use and processing with large volume droplets.Usually, most of the unit droplets of DMF devices and particularly air matrix DMF devices are limited to about 4 microliters or less aqueous fluids, and air gap is limited to an interval less than about 250 microns or 300 microns between drive electrode and ground electrode (top plate and base plate in air gap region).The method for larger volume operation is described herein, wherein the interval between drive electrode (for example, base plate) and ground electrode (for example, top plate) can be much larger (for example, between about 280 microns and 3mm, between about 300 microns and 3mm, between about 400 microns and 1.5mm, for example, between 400 microns and 1.2mm, etc., or 400 microns or larger, 500 microns or larger, 1mm or larger, etc.). Thus, the unit droplet size (the droplet on a single unit cell driven by a single drive electrode can be much larger, for example, 5 microliters or more, 6 microliters or more, 7 microliters or more, 8 microliters or more, 9 microliters or more, 10 microliters or more, 11 microliters or more, 12 microliters or more, 13 microliters or more, 14 microliters or more, 15 microliters or more, etc., for example, between 5 microliters-20 microliters, between 5 microliters-15 microliters, between 7 microliters and 20 microliters, between 7 microliters and 15 microliters, etc.).

[0059] Dispensing large droplets using electrowetting is conventionally performed with smaller volumes (e.g., less than 5 microliters), however, dispensing larger volumes into single units has proven difficult, particularly with high accuracy and precision. Methods of dispensing a predetermined volume of liquid using electrowetting are described herein. For example, a method of dispensing a predetermined volume of fluid into an air gap of an air matrix microfluidic (DMF) device is described herein, wherein the air gap is greater than 280 microns (e.g., 300 microns or greater, 400 microns or greater, etc.) wide, and wherein the DMF device includes a plurality of drive electrodes adjacent to the air gap, the method comprising: flooding a portion of the air gap with fluid from a port connected to the air gap; applying energy to activate a first drive electrode adjacent to a portion of the air gap filled; and when the first electrode is activated, applying suction to draw the fluid back into the port, which leaves the droplet of the fluid in the air gap adjacent to the activated first electrode.

[0060] Applying energy to activate the first drive electrode may include applying energy to activate one or more drive electrodes connected to the first drive electrode, and further wherein applying suction to draw fluid back into the port when the first drive electrode is activated includes: extracting fluid when the first drive electrode and one or more drive electrodes connected to the first drive electrode are active, which leaves droplets of fluid in an air gap adjacent to the activated first drive electrode and the one or more drive electrodes connected to the first drive electrode.

[0061] The first drive electrode may be spaced from the port by at least one drive electrode. Any of these methods may also include deactivating one or more drive electrodes adjacent to a second portion of the air gap, the second portion being in a filled portion of the air gap and between the port and the first drive electrode. The air gap may be greater than 500 microns.

[0062] Filling the portion of the air gap may include applying a positive pressure to expel fluid from the port. The method may also include sequentially energizing drive electrodes adjacent to the air gap to move the droplet in the air gap of the air matrix DMF device.

[0063] Applying suction to draw the fluid back into the port when the first electrode is activated may include leaving a droplet of fluid having a volume of 10 microliters or more in the air gap adjacent to the activated first electrode.

[0064] For example, a method for dispensing a predetermined volume of fluid into an air gap of an air matrix digital microfluidics (DMF) device may include (wherein the air gap is greater than 280 microns wide (e.g., 300 microns or greater, 400 microns or greater, etc.), and further wherein the DMF device includes a plurality of drive electrodes adjacent to the air gap): filling a portion of the air gap with fluid from a port connected to the air gap; applying energy to activate a first drive electrode or a first group of connected drive electrodes adjacent to the portion of the filled air gap, wherein the first drive electrode or the first group of connected drive electrodes are separated from the port by one or more drive electrodes that are not activated; and when the first electrode or the first group of connected electrodes is activated, applying suction to draw the fluid back into the port, which leaves a droplet of the fluid in the air gap adjacent to the first electrode or the first group of connected electrodes.

[0065] Also described herein is a control system for a DMF device, such as those described herein. In particular, described herein is a control system including a graphical user interface for operating any of these devices. These control systems (subsystems) may include software, hardware and / or firmware. Therefore, any of these devices may be configured to be stored in a non-transitory medium (e.g., memory) for performing any of the methods and programs described herein.

[0066] For example, the present invention describes a method for controlling a digital microfluidic (DMF) device, the method comprising: providing a graphical user interface, the graphical user interface including a menu of fluid handling control commands, the fluid handling control commands including one or more of the following: move, heat, remove, circulate, wait, interrupt, mix and dispense; receiving a fluid handling protocol, the fluid handling protocol including fluid handling control commands selected by a user; based on the fluid handling protocol, calculating a path for moving a fluid in an air gap of the DMF device, wherein the path minimizes the amount of overlap in the path to avoid contamination; and based on the calculated path, executing the fluid handling protocol using the DMF device as described in more detail below, any of these methods may also or alternatively include controlling the operation of a robotic arm in conjunction with the DMF and / or suction (as part of a main controller).

[0067] Fluid handling control commands may include at least one of the following: move, heat, remove, wait and mix. For example, fluid handling commands may include all of the following: move, heat, remove, wait and mix. The user may select an icon corresponding to each of these commands, and may enter them in sequence and / or may indicate incubation time and temperature conditions. The device may automatically determine the optimal path in the air gap region of the box to perform each of these steps (e.g., by moving the droplet to a suitable region of the box, including a heater, a magnet, a microfluidic port, etc.) so that the droplet may be manipulated as needed. For example, receiving a fluid treatment scheme may include receiving a string of fluid handling control commands. Calculating a path may include calculating a path based on the arrangement of a heating zone and a cooling zone in a DMF device. Calculating a path may include determining the shortest path that does not intersect itself. Typically, executing a fluid treatment scheme on a DMF device may include executing a fluid treatment scheme in a disposable box connected to the DMF device.

[0068] Also described herein is a digital microfluidic (DMF) device configured to operate with one or more removable and / or disposable cartridges having a bottom dielectric surface, an (optional) top plate having a ground electrode, and an air gap between the bottom dielectric and the top plate, the device comprising: a mounting surface for mounting the disposable cartridge on the top surface; a first plurality of drive electrodes on the mounting surface, wherein all or some of the drive electrodes include openings therethrough; a thermal control for applying thermal energy to a first area of ​​the mounting surface; a plurality of thermal vias; The invention relates to a cartridge comprising a thermal via, wherein the thermal via comprises a thermally conductive material and is thermally connected to a first area of ​​the mounting surface but is electrically isolated from a subset of the electrodes, and further wherein the thermal via is thermally connected to a thermal control; a plurality of vacuum ports, wherein each vacuum port is coupled to one or more of the openings through the drive electrodes; a vacuum pump for applying a vacuum to the vacuum ports; and a control for applying energy to sequentially activate and deactivate one or more selected drive electrodes to cause a droplet to move within an air gap of the cartridge along a desired path within the air gap.

[0069] As described in more detail below, the mounting surface can be configured to individually mount a plurality of different cartridges, including cartridges of different types and configurations.

[0070] The thermal vias can have any suitable size. For example, each thermal via can have a diameter between about 0.5 mm and about 2 mm (e.g., between about 0.5 mm and about 1.8 mm, between about 0.5 mm and about 1.5 mm, between about 0.5 mm and about 1.2 mm, between about 0.8 mm and about 1.2 mm, etc.). Any number of thermal vias can be used per unit (e.g., there can be between about 5-15 thermal vias associated with a region corresponding to a single electrode in the first region).

[0071] The thermal vias may each be filled with a thermally conductive material; the material may be electrically conductive or electrically insulating. In certain variations, the thermally conductive material is a metal. The DMF drive may also include one or more resistive heaters beneath at least some of the drive electrodes.

[0072] The mounting surface may be formed or at least partially formed on a printed circuit board (PCB) (including on an electrode array formed on the PCB). As mentioned above, any device described herein may include one or more magnets; in certain variations, the magnet may be below one or more of the drive electrodes, the magnet being configured to be activated to apply a magnetic field. For example, the magnetic field may pass through an opening in the drive electrode. The device may include one or more Peltier coolers below at least some of the drive electrodes, which are configured to cool to less than 10 degrees Celsius.

[0073] Also described herein are methods for detecting the location and / or identity of a material in an air gap of a digital microfluidics (DMF) cartridge. The material may include droplets (e.g., aqueous droplets), wax, droplets coated / wrapped in wax (e.g., liquid wax), oil droplets, droplets with magnetic particles, etc. The identity may be determined for a material at a specific location in the air gap, such as between an upper surface and a lower surface forming an air gap in the cartridge. The cartridge may be divided into cells (e.g., areas above individual drive electrodes).

[0074] In the devices and methods described herein, adjacent drive electrodes may be configured as reference electrodes, even in variations where the cartridge may include a reference electrode, for example, on the top plate. Thus, the devices described herein may not use a built-in reference electrode in the cartridge.

[0075] For example, a method for detecting position and / or identity may include: designating an electrode adjacent to a drive electrode as a reference electrode and disconnecting the reference electrode from a drive circuit; setting a voltage of one or more drive electrodes of a drive electrode array to a high voltage while setting all other drive electrodes of the drive electrode array to ground; sensing a voltage at the reference electrode; determining a capacitance based on the voltage sensed at the reference electrode; and identifying a material in an air gap adjacent to one or more drive electrodes based on the determined capacitance.

[0076] The method may also include reconnecting the reference electrode to the drive circuit and driving the droplet in the air gap by applying a voltage between the reference electrode and one of the drive electrodes. These steps may be repeated iteratively to track the movement of the material in the air gap, and the various drive and reference electrodes (as well as the grounded reference electrode) may be changed to scan through the cartridge.

[0077] Disconnecting the reference electrode may include allowing the reference electrode to float (e.g., not grounded). The reference electrode may be the entire upper electrode (on a first side of the air gap, opposite the array of drive electrodes). Disconnecting the reference electrode from the drive circuit (e.g., from a controller that drives movement of droplets in the air gap by digital microfluidics) may include connecting the reference electrode to a sensing circuit for detecting a voltage at the reference electrode and thus detecting the capacitance of the air gap. The reference circuit may include one or more reference capacitors arranged to allow measurement of the air gap capacitance.

[0078] Setting the voltage of one or more of the drive electrodes to a high voltage may include setting one or more of the drive electrodes to between 10 V and 400 V (eg, between 100 V and 500 V, such as approximately 300 V, etc.).

[0079] Any of these methods may include determining a total capacitance for the air gap. The method may also include determining the total capacitance using one or more reference capacitors connected to the reference electrode when the reference electrode is disconnected from the drive circuit. For example, determining the capacitance between the first side of the air gap and the second side of the air gap based on the voltage sensed at the reference electrode may also include using the total capacitance.

[0080] Identifying the material in the air gap may include identifying the material in the air gap based on the determined capacitance using a reference database, the reference database including a plurality of capacitance ranges.

[0081] Also described herein are cartridges (e.g., disposable cartridges and / or removable cartridges) for digital microfluidics (DMF) devices that include a tensioning frame to keep a bottom dielectric material in tension and thus flat. For example, any cartridge described herein may include: a sheet of dielectric material having a first side and a second side, the first side forming an exposed bottom surface on the bottom of the cartridge, wherein at least the second side of the sheet of dielectric material includes a first hydrophobic surface; a tensioning frame that holds the sheet of dielectric material in tension so that it is substantially flat; in the case of a dual-plate cartridge, the cartridge further includes: a top plate having a first side and a second side and a thickness between the first side and the second side; a ground electrode on the first side of the top plate; a second hydrophobic surface covering the ground electrode on the first side of the top plate; and an air gap separating the first hydrophobic layer and the second hydrophobic layer, wherein the air gap includes a spacing greater than 280 microns. Any other cartridge features described herein may be included with these cartridges.

[0082] Any of these boxes may also include a lip extending at least partially (including completely) around the sheet of dielectric material and protruding from the sheet of dielectric material. The lip may engage with a channel or groove on the seating surface. Alternatively or in addition, the box may include a peripheral channel or groove into which a protrusion on the seating surface of the device engages.

[0083] The tensioning frame may include an outer frame and an inner frame. The sheet may be held between the outer frame and the inner frame. The boxes may include any other box features mentioned herein.

[0084] In a first aspect, a digital microfluidic device is provided, the device comprising: a mounting surface configured to simultaneously mount one or more boxes; a plurality of drive electrodes disposed on the mounting surface, wherein the drive electrodes are configured to apply a high voltage to move droplets within the box when the box is mounted on the mounting surface, and wherein all or some of the drive electrodes include openings through the drive electrodes; a plurality of vacuum ports, wherein each vacuum port is coupled to one or more openings through the drive electrodes; a vacuum control configured to selectively apply a vacuum through a subset of the vacuum ports below one or more boxes mounted on the mounting surface when the one or more boxes are mounted on the mounting surface; an electrical control for applying energy to sequentially activate and deactivate one or more selected drive electrodes to move droplets within an air gap of one or more boxes, the one or more boxes being mounted on the mounting surface; a vacuum pump for applying a vacuum to the plurality of vacuum ports; and a robotic arm and liquid handling subsystem for dispensing liquid to or removing liquid from one or more boxes when the one or more boxes are mounted on the mounting surface.

[0085] The digital microfluidic device can be configured to house one or more single-plate cartridges and / or dual-plate cartridges. On a digital microfluidic device, a mixture of single-plate cartridges and dual-plate cartridges can be used simultaneously.

[0086] In some embodiments, the apparatus can include about 408 to about 20,000 drive electrodes in the plurality of drive electrodes on the mounting surface. In some embodiments, the robotic arm of the apparatus can be a three-axis robotic arm.

[0087] In some embodiments, the liquid handling subsystem may include a liquid handling head and at least one liquid handling tip. In some embodiments, the liquid handling subsystem may include a plurality of liquid handling tips. The liquid handling head may be configured to distribute or extract a volume of about 1 microliter to about 100 milliliters. In some embodiments, the liquid handling head may also be configured to distribute biological cells suspended in the liquid. In some embodiments, at least one liquid handling tip may also be configured to mix the fluid in the droplet in one or more boxes when one or more boxes are placed on a placement surface.

[0088] In various embodiments of the apparatus, the robotic arm may further include a metal probe configured to electroporate biological cells within droplets within the one or more cartridges when the one or more cartridges are seated on the seating surface.

[0089] In some embodiments, the apparatus may further include a master control subsystem for coordinating activation of the plurality of drive electrodes, the plurality of vacuum ports, the robotic arm, and the liquid handling subsystem. In some embodiments, the master control subsystem may include non-transitory instructions for executing a method of controlling the plurality of vacuum ports and the plurality of drive electrodes, a method of controlling liquid handling, or a combination thereof.

[0090] In various embodiments of the device, the device may further include one or more temperature control areas in the drive electrode array. The temperature control area may be configured to heat and / or cool. In certain embodiments, the device may further include one or more areas in the drive electrode array, each area including a magnetic field. In certain embodiments, the device may further include one or more box alignment features on the placement surface.

[0091] On the other hand, a method for selectively fixing one or more boxes in a digital microfluidic device having multiple drive electrodes is provided, the method comprising: placing one or more boxes on a mounting surface of the digital microfluidic device to produce one or more corresponding mounting positions of the one or more boxes, wherein each of the one or more boxes is located on a sub-area of ​​the mounting surface; sensing each corresponding mounting position of the one or more boxes; determining a subset of the multiple drive electrodes located below the corresponding mounting position of the one or more boxes; and selectively applying a vacuum to the corresponding mounting position of the one or more boxes to fix the one or more boxes to the mounting surface of the digital microfluidic device.

[0092] In some embodiments, each of the one or more boxes can include a base plate that is configured to deform when a vacuum is applied so as to adhere to the placement surface. Each corresponding placement position of the one or more boxes can have a corresponding area corresponding to the size of each box. In some variations, at least one of the one or more boxes can have a different size than other boxes in the one or more boxes.

[0093] On the other hand, a method for coordinating droplet movement and liquid handling in a box within a digital microfluidic device is provided, the method comprising: placing one or more boxes on a placement surface of the digital microfluidic device to produce one or more corresponding placement positions of the one or more boxes, wherein each of the one or more boxes is located on a sub-area of ​​the placement surface; sensing each corresponding placement position of the one or more boxes; determining a subset of multiple drive electrodes located below the corresponding placement position of the one or more boxes; selectively applying a vacuum to the corresponding placement position of the one or more boxes to fix the one or more boxes to the placement surface of the digital microfluidic device; identifying the position of the fluid input portion of each of the one or more boxes; placing droplets from a liquid handling subsystem in each of the one or more boxes via the corresponding fluid input portion; and activating a first selected drive electrode located below each of the one or more boxes to drive each corresponding droplet to a corresponding second position within each of the one or more boxes by electrowetting.

[0094] In some variations, each second location within one or more of the cartridges may include a fluid output.

[0095] In some variations, the method can further include extracting a corresponding fluid droplet at each corresponding second location within the one or more cartridges, wherein the extracting includes applying suction via at least one liquid handling tip. In some embodiments, when the liquid handling subsystem includes multiple liquid handling tips, extracting the corresponding droplets can be performed in parallel.

[0096] In some variations, activating at least a first selected drive electrode of each respective region of the plurality of drive electrodes may further include grounding the first drive electrode to an adjacent second drive electrode of each respective region.

[0097] In another aspect, a method for simultaneously operating a single-plate cartridge and a dual-plate cartridge within a digital microfluidic device having a plurality of drive electrodes is provided, the method comprising: placing the single-plate cartridge at a first placement position on a placement surface and placing the dual-plate cartridge at a second placement position on the placement surface; selectively fixing the single-plate cartridge and the dual-plate cartridge to the placement surface of the digital microfluidic device; identifying a first subset of a plurality of drive electrodes located below the first placement position and a second area including a second subset of a plurality of drive electrodes located below the second placement position; placing a first droplet from a liquid handling subsystem within the single-plate cartridge and a second droplet within the dual-plate cartridge via a robotic arm; and activating at least a first selected drive electrode of each respective subset to drive each respective droplet to a respective second position within each of one or more cartridges by electrowetting.

[0098] In some variations, disposing the first droplet may include disposing through an air gap of a single plate cartridge to any location within an inner surface of the cartridge. In some embodiments, disposing the second droplet within a dual plate cartridge may include disposing the second droplet through a port at one or more predetermined locations of an upper plate of the dual plate cartridge. In some variations, disposing the first droplet within the single plate cartridge and disposing the second droplet within the dual plate cartridge may include disposing the first droplet and the second droplet substantially simultaneously.

[0099] In some variations, activating at least a first selected drive electrode of each respective subset to drive each respective droplet to the respective second position may further include grounding the first selected drive electrode to an adjacent second drive electrode of each respective subset of the plurality of drive electrodes. In some embodiments, activating at least a first selected drive electrode of each respective subset to drive each respective droplet to the respective second position may be performed substantially simultaneously.

[0100] In some variations, each respective second position may further include a fluid output. In some variations, the fluid output at the second position of a single plate cartridge may include extracting droplets from any location within an interior surface of the cartridge through an air gap of the single plate cartridge; and further wherein the fluid output at the second position within a dual plate cartridge may include ports at one or more predetermined locations on an upper plate of the dual plate cartridge.

[0101] In some variations, the method may further include extracting a corresponding fluid droplet at each corresponding second position within the single-plate cartridge and the dual-plate cartridge, wherein the extracting includes applying suction via at least one liquid handling tip of the liquid handling subsystem. In some embodiments, when the liquid handling subsystem includes a plurality of liquid handling tips, extracting the corresponding droplets may be performed in parallel.

[0102] In another aspect, a digital microfluidic device is provided, comprising: a mounting surface configured to simultaneously mount one or more cartridges; a plurality of drive electrodes disposed on the mounting surface, wherein the drive electrodes are configured to apply a high voltage to move a droplet within the cartridge when the cartridge is mounted on the mounting surface, and wherein all or some of the drive electrodes include openings through the drive electrodes; a plurality of vacuum ports, wherein each vacuum port is coupled to one or more openings through the drive electrodes; and a vacuum control configured to selectively apply a vacuum through a subset of the vacuum ports below the one or more cartridges mounted on the mounting surface when the one or more cartridges are mounted on the mounting surface. an electrical control for applying energy to sequentially activate and deactivate one or more selected drive electrodes to move droplets within an air gap of one or more boxes, the one or more boxes being placed on a placement surface; a vacuum pump for applying a vacuum to a plurality of vacuum ports; a robotic arm and liquid handling subsystem for dispensing liquid to or removing liquid from one or more boxes when the one or more boxes are placed on the placement surface; and a main controller for coordinating activation and deactivation of one or more selected drive electrodes with dispensing liquid to or removing liquid from one or more boxes when the one or more boxes are placed on the placement surface.

[0103] In some embodiments, the liquid handling subsystem may further include a liquid handling head. In some embodiments, the liquid handling head may be configured to distribute a volume from about 1 microliter to about 100 microliters. In some embodiments, the liquid handling subsystem may further include at least one liquid handling tip. In some embodiments, the liquid handling subsystem may further include a plurality of liquid handling tips. In some embodiments, the robotic arm may be a three-axis robotic arm.

[0104] In some embodiments, the master controller may be configured to direct the robotic arm and the liquid handling subsystem to dispense droplets at a selected position within at least one of the one or more boxes disposed on the mounting surface based at least in part on the position of each of the one or more boxes disposed on the mounting surface. In some embodiments, the master controller may be configured to direct the robotic arm and the liquid handling subsystem to dispense droplets at a selected position within at least one of the one or more boxes disposed on the mounting surface based at least in part on the position of a pre-spotted reagent within at least one of the one or more boxes disposed on the mounting surface. In some embodiments, the master controller may be configured to direct the robotic arm and the liquid handling subsystem to dispense droplets at a selected position within at least one of the one or more boxes disposed on the mounting surface based at least in part on whether at least one of the one or more boxes is a single-plate box or a dual-plate box. When the at least one box is a single-plate box, the selected position may be any position within the single-plate box, and when the at least one box is a dual-plate box, the selected position may be the position of a fluid input / output port on the upper plate of the dual-plate box.

[0105] In some embodiments, the master controller may also be configured to sequentially mix the droplets by the liquid handling tip at a selected position within at least one of the one or more cartridges disposed on the mounting surface. In some embodiments, the master controller may be configured to select one or more selected drive electrodes based in part on the position of each of the one or more cartridges disposed on the mounting surface. In some embodiments, the master controller may be configured to select one or more selected drive electrodes based in part on the position of the droplets within each of the one or more cartridges disposed on the mounting surface.

[0106] In some embodiments, the master controller may be configured to sequentially activate and deactivate one or more selected drive electrodes based in part on the position of each of the one or more cartridges disposed on the mounting surface. In some embodiments, the master controller may be configured to sequentially activate and deactivate one or more selected drive electrodes based in part on the position of a droplet within each of the one or more cartridges disposed on the mounting surface. In some embodiments, the master controller may be configured to sequentially activate and deactivate one or more selected drive electrodes based in part on detecting completion of dispensing of a droplet within each of the one or more cartridges disposed on the mounting surface.

[0107] In some embodiments, the master controller can be configured to sequentially dispense droplets to each of the one or more cartridges or extract droplets from each of the one or more cartridges based at least in part on determining that one or more selected drive electrodes in each of the one or more cartridges positioned on the positioning surface are not activated to drive droplets during the dispensing or extraction of droplets.

[0108] In some embodiments, the digital microfluidics device may further include a temperature controlled reagent / sample storage subsystem.

[0109] In some embodiments, the master controller can also be configured to identify a selected reagent or sample in a droplet and transfer the selected reagent or sample in the droplet to at least one of the one or more boxes when the one or more boxes are placed on the placement surface. In some embodiments, the selected reagent or sample can be different for each of the one or more boxes.

[0110] In certain embodiments, the reagent / sample storage subsystem includes an orifice plate accommodating area. In certain embodiments, the orifice plate accommodating area can be configured to accommodate any one of 24 holes, 48 ​​holes, 96 holes and 384 hole orifice plates. In certain embodiments, the master controller can also be configured to extract a sample from the orifice plate in the orifice plate accommodating area when one or more boxes are placed on the placement surface, when the sample of the droplet form is distributed to at least one of the one or more boxes.

[0111] In some embodiments, the master controller may be further configured to deliver droplets to the well plate in the well plate receiving area when droplets are extracted from at least one of the one or more cartridges when the one or more cartridges are seated on the seating surface.

[0112] In some embodiments, the robotic arm may further include a metal tip configured to electroporate a biological cell within a droplet at a location within one of the one or more cartridges when the one or more cartridges are positioned on the positioning surface. In some embodiments, the master controller may be configured to direct the robotic arm to deliver the metal tip to the droplet location within the cartridge. In some embodiments, the master controller may be configured to schedule electroporation of the biological cell based at least in part on determining that one or more selected drive electrodes in the cartridge are not activated to drive the droplet during electroporation.

[0113] In another aspect, a system is provided, comprising: a digital microfluidic device, the device comprising: a mounting surface configured to simultaneously mount one or more boxes; a plurality of drive electrodes disposed on the mounting surface, wherein the drive electrodes are configured to apply a high voltage to move droplets within the box when the box is mounted on the mounting surface, and wherein all or some of the drive electrodes include openings through the drive electrodes; a plurality of vacuum ports, wherein each vacuum port is coupled to one or more openings through the drive electrodes; a vacuum control configured to selectively apply a vacuum through a subset of the vacuum ports below one or more boxes mounted on the mounting surface when one or more boxes are on the mounting surface; an electrical control for applying energy to sequentially activate and deactivate one or more selected drive electrodes to move droplets within an air gap of one or more boxes, the one or more boxes being mounted on the mounting surface; a vacuum pump for applying a vacuum to the plurality of vacuum ports; and a robotic arm and liquid handling subsystem for dispensing liquid to or removing liquid from one or more boxes when the one or more boxes are mounted on the mounting surface. In some embodiments, the system further comprises a housing, wherein the housing controls one or more of humidity, temperature, and gas environment.

[0114] In some embodiments, the placement position of one or more boxes on the placement surface may not be predefined.In some embodiments, the size of the placement position of at least one of the one or more boxes is different from the size of the placement position of another one of the one or more boxes.

[0115] In some embodiments, the digital microfluidics device may be configured to house one or more single-plate cartridges and / or dual-plate cartridges.

[0116] In some embodiments, the liquid handling subsystem may include at least one liquid handling tip. In some embodiments, the liquid handling subsystem may include multiple liquid handling tips. In some embodiments, at least one liquid handling tip or more liquid handling tips may be further configured to mix droplets within at least one of the one or more boxes disposed on the mounting surface.

[0117] In some embodiments, the liquid handling subsystem may include a liquid handling head. In some embodiments, the liquid handling head may be configured to dispense a volume of about 1 microliter to about 100 milliliters. In some embodiments, the liquid handling head may also be configured to dispense biological cells suspended in a liquid.

[0118] In some embodiments, the robotic arm may be a three-axis robotic arm.In some embodiments, the robotic arm may further include a metal probe configured to electroporate biological cells within a droplet within at least one of the one or more cartridges disposed on the mounting surface.

[0119] In certain embodiments, the system may further include a temperature controlled reagent / sample storage subsystem. In certain embodiments, the reagent / sample storage subsystem may include an orifice plate accommodating area. In certain embodiments, the orifice plate accommodating area may be configured to accommodate any one of 24, 48, 96 and 384 orifice orifice plates.

[0120] In some embodiments, the system may further include a master control subsystem configured to coordinate activation of the plurality of drive electrodes, the plurality of vacuum ports, the robotic arm, and the liquid handling tip. In some embodiments, the master control subsystem may include non-transitory instructions for executing a method of controlling the plurality of vacuum ports and the plurality of drive electrodes, a method of controlling liquid handling, or a combination thereof.

[0121] In some embodiments, the master control subsystem may be further configured to control at least one of the humidity, temperature, and gas environment of the enclosure. In some embodiments, the master control subsystem may include non-transitory instructions for executing a method of controlling at least one of the humidity, temperature, and gas environment of the enclosure.

[0122] In some embodiments, the system may further include an optical subsystem. In some embodiments, the optical subsystem may include an illumination source. In some embodiments, the illumination source may include one or more of a visible light source or an ultraviolet light source. In some embodiments, the optical subsystem may further include an imaging device configured to detect a bright field image, a fluorescent image, or a luminescent image. In some embodiments, the optical subsystem may be configured to control one or more illumination sources and imaging devices when one or more illumination sources and imaging devices are present.

[0123] In some embodiments, the optical subsystem may be further configured to communicate with the main control subsystem. In some embodiments, the non-transitory instructions of the main control subsystem may further include instructions for sequentially activating the drive electrodes and activating liquid handling based on the imaging communications received from the optical subsystem. In some embodiments, the main control subsystem may also include non-transitory instructions for executing the method of controlling the optical subsystem. In some embodiments, the non-transitory instructions may include instructions for activating and deactivating bright field and / or fluorescent illumination and / or capturing images of selected areas of the placement surface of the digital microfluidic device.

[0124] In some embodiments, the digital microfluidic device may further include one or more regions within the drive electrode array, wherein one or more regions further include a magnet. In some embodiments, the digital microfluidic device may further include one or more regions within the drive electrode array, including heating and / or cooling controllability. In some embodiments, the system may further include more than one digital microfluidic device. On the other hand, a method of operating a system including a digital microfluidic device is provided, the method comprising: for each of one or more boxes, identifying a corresponding subset of drive electrodes under each of the one or more boxes; for each of the one or more boxes, determining a plurality of associated ground electrodes adjacent to the corresponding subset of drive electrodes; selectively fixing each of the one or more boxes on a mounting surface of the digital microfluidic device; and coordinating for each of the one or more boxes: determining a type of reaction to be performed in each of the one or more boxes; distributing a sample into each of the one or more boxes, thereby forming a corresponding sample droplet in each of the one or more boxes; distributing one or more reagents / buffers into each of the one or more boxes; to each of one or more boxes, thereby forming corresponding reagent / buffer droplets in each of the one or more boxes; optionally, driving corresponding reagent droplets / sample droplets to reaction positions in each of the one or more boxes; merging corresponding sample droplets with their corresponding reagent droplets, thereby forming corresponding reaction droplets in each of the one or more boxes; performing corresponding reactions in corresponding reaction droplets, thereby forming corresponding product droplets in each of the one or more boxes; when corresponding reactions in corresponding reaction droplets of one or more boxes are assays, determining assay results of corresponding reaction droplets of one or more boxes; and when corresponding reactions in one or more boxes are sample preparation reactions, outputting corresponding product droplets.

[0125] In some variations, forming the corresponding reagent / buffer droplets may further include mixing the dispensed reagent / buffer with a pre-installed reagent disposed at a selected position within one or more boxes. In some variations, forming the corresponding reaction droplets may further include mixing the sample droplets with a pre-installed reagent disposed at a selected position within one or more boxes.

[0126] In some variations, when the system further includes one or more illumination sources, forming the corresponding reagent droplets or forming the corresponding reaction droplets may further include photoactivating the corresponding reagent droplets or reagents within the corresponding reaction droplets.

[0127] In some embodiments, when the drive electrode is not activated, the corresponding sample droplet is distributed, the corresponding reagent / buffer is distributed, or the corresponding product droplet is extracted. In some embodiments, when the drive electrode is not activated, the corresponding sample droplet, the corresponding reagent / buffer droplet, the corresponding reaction droplet, or the corresponding product droplet is formed. In some embodiments, forming the corresponding sample droplet can further include electroporating the biological cells in the sample droplet.

[0128] In some variations, conducting the reactions may further comprise driving the respective reaction droplets to respective reaction locations within one or more cartridges, wherein the respective reaction locations comprise magnets and / or temperature controllers.

[0129] In some variations, the method may further include regulating at least one of temperature, humidity, or gas environment.

[0130] In some variations, when the system includes a main control subsystem, the corresponding process of coordination can be automatically performed, including: determining, distributing, mixing, merging, driving, performing reactions or outputs. In certain embodiments, the corresponding process of coordinating one or more boxes can be performed independently. In some variations, the corresponding process of coordinating one or more boxes can further include scheduling mechanical arms and / or liquid handling subsystems and scheduling corresponding multiple electrodes, thereby reducing conflicting instructions for mechanical arms, liquid handling subsystems and / or corresponding multiple electrodes in one or more boxes.

[0131] In some variations, when the system includes an imaging device, scheduling the robotic arm and / or the liquid handling subsystem and scheduling the corresponding plurality of electrodes of one or more cartridges can further include receiving imaging information from the imaging device, confirming the completion of a previous corresponding process within the one or more cartridges. In some variations, when the system includes an imaging device, determining the assay results of the corresponding reaction droplets of the one or more cartridges can further include detecting a visible signal, a fluorescent signal, or a luminescent signal from the corresponding reaction droplets.

[0132] In some variations, when the system includes a master control subsystem, the master control system may regulate at least one of the temperature, humidity, or gas environment of the enclosure.

[0133] In some variations, when the system further comprises a reagent / sample storage subsystem (including a well plate receiving area), outputting the corresponding product droplets may further comprise depositing the output product droplets within the wells of the well plate. BRIEF DESCRIPTION OF THE DRAWINGS

[0134] The novel features of the invention are set forth with particularity in the claims that follow. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description and the accompanying drawings, which set forth illustrative embodiments in which the principles of the invention are utilized, wherein:

[0135] Figure 1 A is a schematic diagram from a top-down perspective of one example of an air-matrix digital microfluidics (DMF) device.

[0136] Figure 1 B shows the passage through Figure 1 An enlarged view of a cross section of a portion of an air matrix DMF device shown in A, the cross section being taken through a thermal conditioning region (hot zone).

[0137] Figure 1 C shows the passage through Figure 1 An enlarged view of a second cross-section of a region of the air-matrix DMF device of A; the region includes holes through the base plate and the actuating electrodes and is configured such that replenishing droplets can be delivered from the holes (which are connected to a reservoir of solvent, shown in this example as an attached syringe) into the air gap of the air-matrix DMF device.

[0138] Figure 2 is similar to Figure 1 A- Figure 1 C shows an example of a DMF surface using a rigid box including electrodes and air gap regions.

[0139] Figure 3A An example of a typical DMF arrangement using, for example, a rigid box is shown; Figure 3B An example of a DMF configuration is shown in which cartridge 315 is a disposable portion that does not include the electrodes but is held to the reusable electrodes by a plurality of partial vacuum ports (adjacent to or through the electrodes).

[0140] Figure 4A A top view of an electrode (e.g., an electrode array) formed as part of a device is shown. As shown, the electrode can include multiple vacuum openings therethrough. The electrode can define different regions, including thermal control regions (e.g., regions with thermistors and / or cooling and / or heating). Figure 4A , 18 rows and 10 columns are shown; larger or smaller arrays may be used.

[0141] Figure 4B An enlarged region of the electrodes forming the upper electrode layer is shown showing vacuum openings through a majority (e.g., >50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) or all of the electrodes. Although square electrodes (with central vacuum openings) are shown, other electrode shapes (e.g., interlocking, rectangular, circular, etc.) or vacuum opening locations (off-center, etc.) through the electrodes may be used. Figure 4B , a temperature sensor (eg, a thermistor) is shown.

[0142] Figure 4C The diagram shows that it can exist in (for example Figure 4B A resistive heating layer beneath the electrode layer (shown in FIG. 1 ). Under the array, a continuous or multiple separate traces of resistive material may be used. Black dots indicate vacuum manifolds (forming multiple vacuum openings through the electrodes). The resistive heating layer may be electrically isolated from the electrodes above it; the current applied through the resistive heating layer may be controlled regionally by a controller. The controller may include PID control.

[0143] Figure 5A A partially disassembled view of the device is shown, illustrating the connections that can be made between the PCB containing the electrodes, the liquid coolant, and the vacuum used to secure the box dielectric to the electrodes.

[0144] Figure 5B Examples of fans and radiators, reservoirs, and pumps are shown that may be used for liquid coolant that includes the box contacting surfaces of the electrodes. The pumps, pipes, fans, radiators, and reservoirs may be used to move water or liquid coolant under the electrodes so that the coolant may absorb heat as it passes under the electrodes, where the coolant may then be recirculated after being cooled again as it passes through the fans and radiators.

[0145] Figure 5C shows a similar Figure 4A-4C Another view of the electrode's PCB shown in , which is connected to a vacuum pump and liquid coolant (input and output).

[0146] Figure 5D and Figure 5E The figure shows the application of vacuum to secure the box (shown here only through the dielectric material as a proof of concept). Figure 5D In , the vacuum is off and the dielectric is not secured against the electrodes. The dielectric may wrinkle and may include areas of poor contact, which includes poor electrical contact. By comparison, Figure 5E The dielectric is shown held against the electrode by multiple openings through the electrode, which hold the dielectric uniformly against the electrode and result in surprisingly uniform electrical properties between the removable cartridge and the electrode.

[0147] Fig. 5F An example of a top view of a PCB is shown showing an array of small electrodes with holes formed through the central region of each electrode.

[0148] Figure 5G Shown below the electrode Fig. 5F A portion of a PCB (other layers may be formed above the electrodes) showing a hole formed through the PCB that may be connected to a vacuum pump.

[0149] Figure 6 The figure shows different functional areas that can be formed by electrode arrays and / or removable boxes. Figure 6 In , the removable box has been made transparent (the microfluidic area above the top plate, air gap and dielectric forming the DMF part of the box has been made transparent). Different areas are indicated by different boxes and can be distributed over the array in a specific arrangement. For example, in Figure 6 In FIG. 6 , seven of the electrodes are configured as magnetic regions 605 that can exert a local magnetic force (to the electrodes) to retain the magnetic beads or particles in a droplet on the electrodes. Eight of the peripheral regions (each spanning six electrodes) are configured as cooling zones that can be in thermal contact with a Peltier device or other thermal cooling zone. In addition, in FIG. Figure 6 In the figure, the six 16-electrode regions on the left are configured as cooling zones, which can also be in thermal contact with the same or different Peltier devices (e.g., to keep them below 10 degrees Celsius). Two central heating zones (one across five electrodes and the other across 32 electrodes) are also included, and the two central heating zones can be thermally cycled over all zones or regions of zones. Four optical reading zones (each across four electrodes) are spaced apart from each other on the right perimeter of the device. Typically, the heating and / or thermal cycling zones are located in the center, separated from the cooling / storage zones at the periphery. There can be overlap between these zones (e.g., between the magnetic zone and the heating / cooling zone).

[0150] Figure 6 Also shown in transparent view is a microfluidic portion that can be formed above the air gap ((and in the top plate, as described). For example, in Figure 6 In the embodiment, the microfluidic portion 611 includes a pair of serpentine microfluidic channels 615, 616, each connected to an opening into the air gap (the opening can be adjusted by a valve). The microfluidic portion may also include a valve. Figure 6 In the embodiment of the present invention, the microfluidic channel also includes a pair of ports 617, 618, through which positive and / or negative pressures can be applied to (along with any valves) regulate the movement of fluids in the microfluidic region and (in some variations) regulate the movement of fluids into or out of the air gap. The microfluidic portion may also include one or more waste chambers 621.

[0151] Fig. 7Ais a top view of an exemplary box as described herein. In this example, the box includes: a DMF portion including a top plate and a dielectric separated by an air gap; and a microfluidic portion that is connected to the air gap and can be externally connected to a channel input and / or output. Fluid can be applied to the box through one or more openings (shown as small openings) into the air gap and / or through the channel input / output. The right side of the box includes a window area, which allows optical observation through the box.

[0152] Figure 7B Show Fig. 7A A top perspective view of a box.

[0153] Figure 7C is from Fig. 7A and Figure 7B An end or side view of the left side view of the box showing the upper microfluidic channel and the lower DMF portion (showing the spacing between the top, ground, plate and dielectric forming the air gap).

[0154] Fig.7D yes Figure 7A-7C A top view of a box with the cover for the microfluidic channels removed. Fig.7D The channels are shown.

[0155] Figure 8 is an example of a disposable box, including a plastic top plate and a dielectric.

[0156] Fig. 9 Paper-based digital microfluidics that can be used as part of a cartridge are shown.

[0157] Fig.10 An example of an open array of electrodes beneath a disposable plastic top plate and dielectric is shown.

[0158] Fig.11 Shown is a double plate box over an open array, held in place by a vacuum to keep it rigidly attached over the electrodes.

[0159] Fig.12 The figure shows the use of openings through the electrode array; these openings can be used to apply sufficient suction (e.g., vacuum) to keep the box (e.g., bottom, dielectric layer) aligned and fixed to the device. Positive pressure can be applied to release the box.

[0160] Figure 13-Figure 15 An example of a microfluidic channel interfacing with a DMF air gap region as described herein is shown. Fig.13In the , the microfluidic portion of the cartridge is shown as a pair of channels, each connected to an inlet / outlet, and each terminating in a bridge region that forms an opening in the air gap to the DMF portion of the cartridge (in this example, below the microfluidic portion). Fluids can be removed, added, washed, etc. in and out of the air gap of the DMF portion. Fig.14 and Fig.15 In the example, by alternating between inlet / outlet and applying suction, the fluid is washed through the bridging droplet and into the air gap, as shown. In this example, the external fluid components (e.g., tubing and reservoir) are integrated into the top plate of the DMF part, which allows for a compact form factor. The microfluidic channels can be used to add / remove reagents (e.g., remove waste, wash, etc.). The bridging droplet can be an electrode or group of electrodes, and the size of the droplet can be adjusted by the DMF.

[0161] Fig.16A and Fig. 16B The figure shows the extraction and mixing of fluids in a DMF device (e.g., a cartridge) as described herein using a fluid application and extraction technique comprising a bifurcated channel, which allows large volumes of fluid to be exchanged between two reservoirs. Fig.16A In the , fluid application and extraction equipment is connected through the top plate. Fig. 16B In the embodiment, fluid application and extraction equipment is connected from the side panels.

[0162] Fig.17A is another example of a DMF cartridge configured to mix a fluid with one or more droplets in the air gap of the DMF cartridge, extract a fluid, add a fluid, etc. Fig.17A In the embodiment, an interface 1127 for a fluid line is connected through the top plate, and the fluid line can be a microfluidic channel, including a microfluidic channel formed partially through the top plate 1117, and (different from Fig.16A )The air gap in this interface area can be larger than the air gap in other parts of the DMF box.

[0163] exist Fig. 17B In the embodiment, the interface 1127 of the fluid pipeline is located at the edge of the air gap, similar to Fig. 16B ;exist Fig. 17B In the embodiment of the present invention, the air gap area is larger than the air gap area in other areas of the box.

[0164] exist Figure 16A-16B , Figure 17A-17B In any of the above, the fluid lines (e.g., 1143, 1145) and reservoirs (1105, 1107) can form part of the DMF apparatus and can be connected to ports and / or one or more valve interfaces on the box (e.g., the top surface of the box).

[0165] Figures 18A-18C The diagram is similar to Fig.17A Operation of the fluid application and extraction apparatus shown in .

[0166] Figure 19A-Figure 19C The graph shows the effect of evaporation on a droplet in an air-gap DMF apparatus maintained at 95 degrees Celsius for 2 minutes, showing substantial evaporation.

[0167] Figure 20A-20C It is shown that when a cover of a non-polar material (such as liquid paraffin) is used, after one hour ( Fig. 20B ) and two hours later ( Fig. 20C )'s resistance to evaporation, which shows little or no evaporation.

[0168] Figure 21A-Figure 21D The figure shows the use of non-polar jacket materials in an air matrix DMF device. Figure 21A-21B Shown is the movement of an aqueous (polar) droplet when coated with a non-polar cover material that moves along with the droplet. Figure 21C-Figure 21D The addition of additional polar material to the droplet is shown, which expands to include the additional polar material.

[0169] Figure 21E-Figure 21I The diagram shows adding a large sample to the jacket material, and mixing the sample.

[0170] Figure 22A-22D The figure shows the control of the volume of the droplet when dispensing a droplet (e.g., a reagent) into the air gap of a DMF device. In particular, the air gap described herein can be a large air gap (e.g., a spacing between the top and bottom dielectrics greater than 280 microns, greater than 300 microns, >400 microns, >500 microns, >600 microns, etc.). In such a case, the electrowetting force alone may not be sufficient to dispense a droplet of a predetermined volume. Figure 22A-22D As shown in , droplets broken off from a large volume can be used to dispense a predetermined volume. Fig.22A In the distribution electrode is activated, and is spaced from the distribution port (tube). Fig. 22B In the embodiment of the present invention, the reagent to be dispensed is applied to the air gap, which fills the area including the dispensing electrode, which is separated from the dispensing port by at least one electrode. Fig. 22C In the embodiment, the reagent is then drawn back into the dispensing port while the dispensing electrode is active, but the electrode between the dispensing port and the dispensing electrode is not active, which forms a neck (e.g., a neck) that is eventually disconnected. Fig.22D), which leaves a droplet of a predetermined volume on the dispensing electrode.

[0171] Figure 23A-23F The diagram shows the use of the above Figure 22A-22D An example of a technique described herein that dispenses droplets of a predefined volume.

[0172] Fig.24 An example of a method of controlling a DMF apparatus as described herein is shown, the method comprising programming the apparatus using a graphical user interface.

[0173] Figure 25A-25B Diagram showing a visual control or command ( Fig.25A ) and scenarios describing the use of these visual controls / commands ( Fig.25B ) example.

[0174] Figure 26A-26H An example of a user interface for controlling a DMF device as described herein is shown.

[0175] Fig.27A and Fig.27B Illustrated are top and bottom perspective views, respectively, of one example of a top portion of a digital microfluidics cartridge as described herein.

[0176] Fig.28 The figure shows an example of a portion of a box, showing the thermal control area.

[0177] Fig.29 is an example of a portion of a device (e.g., a cartridge seat portion) having a reduced thermal mass to increase the rate of temperature regulation of a cartridge held on the seat portion.

[0178] Fig.30 is another example of a portion of a device (e.g., a cartridge seat portion) having a reduced thermal mass to increase the rate of temperature regulation of a cartridge held on the seat portion.

[0179] Fig.31A and Fig.31B The figure shows an example of an apparatus including thermal vias for helping to control the temperature of a cartridge (eg, of one or more cells of an air gap of a cartridge).

[0180] Fig.32 is an example of a cartridge that includes an opening in the top plate for sampling or adding fluid to droplets in the cartridge.

[0181] Fig.33A An ITO sensing circuit with switches is shown.

[0182] Fig.33B Another example of a capacitance sensing circuit is shown, which includes a plurality of reference capacitors.

[0183] Figure 34A-Figure 34C One method of identifying and / or locating a droplet in an air gap as described herein is illustrated. Fig.34A One example of a range of capacitances corresponding to the presence or absence of various materials (eg, aqueous droplets, wax, etc.) at specific cells in the air gap is shown. Fig.34B is a graph showing exemplary voltage measurements from a sensing electrode (top electrode). Fig.34C is a graph showing an example of the change in the electrical permittivity of water as a function of temperature.

[0184] Fig.35A is a top view of an example of a vacuum chuck.

[0185] Fig.35B yes Fig.35A Cross-sectional view of a vacuum chuck.

[0186] Fig.36 Shows Figure 35A-Figure 35B An isometric view of the chuck is shown in FIG.

[0187] Fig.37 Shows something like Figure 35A-Figure 35B A top view of the chuck is shown in FIG.

[0188] Fig.38A Another example of a vacuum chuck is shown.

[0189] Fig.38B A cross-sectional and enlarged view of the chuck is shown.

[0190] Fig.39 Shows something like Figure 35A-Figure 35B A bottom view of the chuck is shown in FIG.

[0191] Fig.40 Shows something like Fig.35A An isometric view of the chuck is shown in FIG.

[0192] Fig.41A An example of a heat dissipation system is shown, which may be included in any of the devices described herein.

[0193] Fig.41B It passes through Fig.41A Cross-sectional view of the chuck.

[0194] Fig.42 A front view of the chuck and fan is shown.

[0195] Fig.43 An example of an arrangement of a chuck, a fan, and a PCB (a portion of a mounting surface) is shown.

[0196] Fig.44 is a perspective view of a chuck, which may include a thermal (eg, heat) dissipation system for regulating the temperature of the cartridge.

[0197] Fig.45A yes Fig.44 A top view of the chuck.

[0198] Fig.45B It passes through Fig.45A Cross-sectional view of the chuck.

[0199] Fig.46 A side view of the assembly of the chuck, heat sink and a pair of cooling fans is shown, with arrows indicating the flow of temperature (cooling the chuck and therefore the cartridge when loaded onto a device).

[0200] Figures 47A-47C Components of a vacuum chuck and cooling subsystem (eg, heat sink block and cooling fan) are illustrated.

[0201] Fig.48 The figure shows an example of components for an apparatus comprising: a PCB having an array of electrodes for applying DMF to a box (not shown); a vacuum block for holding the bottom of the box on the PCB; and a thermal regulator subsystem comprising a heat sink / heat block and a pair of cooling fans.

[0202] Fig.49A and Fig.49B A tensioning frame and a film frame are shown respectively for fixing and keeping a film (eg a dielectric film) smooth which may form the bottom of the box.

[0203] Fig.49C is a side view of the assembled tension frame.

[0204] Fig.49D is a perspective view of the assembled tension frame.

[0205] Fig.50A is an example of an exploded view of a two-panel box.

[0206] Fig.50B is another example of an exploded view of a two-panel box.

[0207] Fig.51 is an exploded view of an example of a cartridge and cartridge housing portion of a device.

[0208] Fig.52A is a top view of the device's PCB, on which the cartridge may be placed.

[0209] Fig.52B is Fig.52A A side view of a PCB portion is shown in FIG.

[0210] Fig.52C is an example of a side view showing a cassette on a mounting surface of the device.

[0211] Fig.52D yes Fig.52C A magnified view of .

[0212] Fig.53 is an exploded view of the box and mounting surface / area of ​​the device.

[0213] Fig.54A is a top view of the device's PCB (which may form a mounting surface).

[0214] Fig.54B It passes through Fig.54A A side cross-sectional view of a portion of the device shown in FIG.

[0215] Fig.55A An example of an electrode grid setup with independent action areas is shown.

[0216] Fig.55B Another example of an electrode grid arrangement with independent action areas is shown.

[0217] Figure 56A-Figure 56D An example of a thermal regulation subsystem of an apparatus as described herein is illustrated.

[0218] Figure 57A-Figure 57B An example of a magnetic subsystem of an apparatus as described herein is illustrated.

[0219] Figure 58A-Figure 58B An example of an electrode subsystem of a device as described herein is illustrated.

[0220] Fig.59 An example of a device including a vortex (mechanical vibration) subassembly is schematically shown.

[0221] Figures 60A-60B An example of a vacuum chuck for use with the apparatus described herein is shown.

[0222] Fig.61 Is the user interface of the device.

[0223] Fig.62 is an example of a user interface for guiding the use of the devices described herein.

[0224] Fig.63 is another example of a user interface for a device.

[0225] Fig.64 A market use of a laboratory solution for the device described herein is schematically shown.

[0226] Figures 65-68 An example of a user interface for controlling the operation of the apparatus described herein is shown.

[0227] Fig.69 is a schematic diagram of a system according to some embodiments of the present disclosure.

[0228] Figures 70A-70C is a graphical representation of a system according to some embodiments of the present disclosure.

[0229] Fig.71 is a schematic representation of a software architecture used in a system according to some embodiments of the present disclosure. Detailed Description

[0231] Digital microfluidics (DMF) has become a popular technology with a wide range of applications in chemistry, biology, and medicine. Like microchannel-based fluidics, DMF is being used to miniaturize a variety of applications with advantages such as reduced consumption of reagents and solvents, faster reaction rates, and integration capabilities (i.e., lab-on-a-chip concept). In DMF, nanoliter to microliter droplets containing samples and reagents are controlled (i.e., moved from reservoirs, merged, mixed, and dispensed) by applying a series of potentials to an array of electrodes coated with a hydrophobic insulator. DMF differs from microchannel-based fluidics in that it allows precise control of multiple reagent phases (liquids and solids) in heterogeneous systems without the need for complex microvalve networks.

[0232] For many applications, it may be most convenient to perform DMF on an open surface so that the matrix surrounding the droplets is ambient air. Figure 1 A illustrates an example of an air-matrix DMF apparatus. Typically, an air-matrix DMF apparatus (such as Figure 1 The air-matrix DMF device shown in FIG. 1 includes a plurality of unit cells 191 adjacent to each other and defined by a single actuation electrode 106 opposite to a ground electrode 102; each unit cell may be of any suitable shape, but may generally have the same approximate surface area. Figure 1In A, the unit cell is rectangular. One or more droplets to be manipulated by the actuation electrode can be isolated from the electrode by a hydrophobic insulator. When an electric potential is applied, charge accumulates on both sides of the insulator, and this phenomenon can be used to move, merge, mix, separate, and disperse the droplets. Examples of other DMF devices and / or cartridges that may be suitable for the uses described herein are described, for example, in International Application No. PCTUS2018049415, filed on September 4, 2018, entitled “DIGITAL MICROFLUIDICS DEVICES AND METHODS OF USING THEM,” which claims priority to U.S. Provisional Patent Application No. 62 / 553,743, filed on September 1, 2017, and U.S. Provisional Patent Application No. 62 / 557,714, filed on September 12, 2017, and International Application No. PCT / US2020 / 020255, filed on February 28, 2020, entitled “DIGITAL MICROFLUIDICS DEVICES AND METHODS OF USING THEM,” which claims priority to U.S. Provisional Patent Application No. 62 / 811,540, filed on February 28, 2019, each of which is incorporated herein by reference in its entirety.

[0233] Despite the enthusiasm for the simplicity that digital microfluidics can offer, Figure 1 The low throughput capability of the system / device shown in A limits its application to technical problems that require high-throughput automation of complex processes, such as DNA library preparation for next-generation sequencing (NGS), DNA assembly for synthetic biology, all types of detection (including but not limited to cell culture analysis, qPCR, etc.) and drug discovery. The applicant describes the first system architecture that combines the reaction capabilities and processing of digital microfluidics with the capabilities of liquid handling robots to provide DMF systems and devices that can meet these requirements for growing analysis and sample preparation processes. The devices described herein can distribute selected amounts of reagents, samples, or other liquids to designated areas on the surface of a box supporting DMF, thereby providing multiplexing capabilities. These devices can provide fast and inexpensive high-throughput processing and analysis in a digital microfluidic environment.

[0234] This paper describes a new architecture that pairs digital microfluidics with liquid handling robots. The new multilayer architecture system is formed by integrating the DMF platform into commercially available or custom liquid handling robots. Fig.70A As shown, the two fluid manipulation paradigms, the electromechanical droplet manipulation platform and the liquid handling subsystem (including liquid handling head, liquid handling tip) are orthogonal and independent. Fig.70ANot shown in the figure, the robot guides the distribution of liquid to multiple positions on the DMF electrode array surface (e.g., the placement surface of one or more boxes) / extracts fluid from these positions, providing maximum flexibility for multiplexing experiments in terms of quantity and type. The DMF device can also include additional features / components to achieve a series of determinations / sample preparations described below. In addition, both fluid manipulation paradigms can be enclosed using a chamber (e.g., housing) that provides precise humidity, temperature, and gas control.

[0235] like Fig.70B As shown, a novel feature of these open array DMF systems / devices is that single plate box format and double plate box format of different sizes can be accommodated simultaneously.In double plate format, droplet is sandwiched between two substrates, and in single plate format, droplet is positioned on the top of a single substrate.In both configurations, droplet to be manipulated is isolated from electrode by hydrophobic insulator.The key feature of this integrated system includes using a liquid pipetting head with three-axis delivery capability (for example, using a mechanical arm to approach the delivery point) to transport reagent droplet high throughput and automatically transport it onto the DMF platform.A further description of system, device and its method of use is provided.

[0236] Device. A digital microfluidic device or a system comprising a digital microfluidic device may include a placement surface configured to place one or more boxes simultaneously. The placement surface may have a width of about 50 mm, 60 mm, 70 mm, 90 mm, 100 mm, 150 mm, 200 mm, 250 mm, 400 mm, 500 mm, 600 mm, 700 mm, 780 mm or about 800 mm, or a width of any value therebetween. The placement surface may have a length of about 80 mm, 100 mm, 110 mm, 150 mm, 200 mm, 400 mm, 600 mm, 800 mm, 1000 mm, 1300 mm, 1500 mm, 1750 mm, 1800 mm, 2000 mm, 2050 mm or about 2100 mm. A plurality of drive electrodes may be arranged on the placement surface, wherein the drive electrode is configured to apply a high voltage to move the droplet in the box when the box is placed on the placement surface. There can be about 300, 400, 500, 600, 800, 900, 1000, 1500, 2000, 5000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000 or about 70,000 drive electrodes, or there can be a plurality of drive electrodes of any value between these values. In some variations, there can be about 300 to about 65000, about 350 to about 50,000, about 400 to about 25,000 or about 400 to about 20,000 electrodes in the electrode array of the device. The electrode can have any shape and can be made of any material described herein. All or some drive electrodes include an opening passing therethrough, and as described below, the opening can be configured in the drive electrode with any suitable size and position. The device can also include a plurality of vacuum ports, each of which is coupled to one or more openings passing through the drive electrode. A vacuum control included in the device is configured to selectively apply vacuum through a subset of vacuum ports below a box disposed on a placement surface to selectively secure one or more boxes to the placement surface. A vacuum pump for applying vacuum to a plurality of vacuum ports may be incorporated into the device or a system comprising the device. The device may also include an electrical control for applying energy to sequentially activate and deactivate one or more selected drive electrodes to move droplets within an air gap of a box disposed on the placement surface. The device includes a robotic arm and a liquid handling subsystem for dispensing liquid to or removing liquid from a box on the placement surface.

[0237] In some variations of the device, the robot arm of the device can be a three-axis robot arm. The three-axis function will allow dispensing to or extraction from any suitable position on the placement surface, for example, the robot arm can approach any box placed on the placement surface, and can further approach any suitable position within the upper surface of a double-plate box or any suitable position within the air gap of a single-plate box. This ability to dispense fluid directly to or extract from any desired position on the placement surface can further allow the size of the box to be reduced compared to the size of the box used in a DMF device without mechanical delivery / extraction, because the droplet may not need to be driven to a second or additional position to add reagents or buffers to the droplet.

[0238] In some variations, the robotic arm may further include a metal probe configured to electroporate biological cells within droplets within one or more cartridges when the one or more cartridges are positioned on the positioning surface. The metal probe may be energized to provide electroporation energy to the selected droplets, thereby electroporate biological cells present in the droplets.

[0239] The liquid handling subsystem of the device may include a liquid handling head and at least one liquid handling tip. In certain embodiments, the liquid handling subsystem may include a plurality of liquid handling tips, which may be approximately 4, 8, 12 or 96 liquid handling tips. The liquid handling head may be configured to distribute or extract the volume of any value in any range from about 0.1 microliter to about 500 milliliters, from about 0.5 microliter to about 250 milliliters, from about 1 microliter to about 100 milliliters or any range therebetween. The fluid to be distributed may be a reagent, a reagent mixture, a sample or any combination thereof. In some cases, the liquid handling head may be further configured to distribute biological cells suspended in the liquid, such as samples of biological cells to be processed or measured. Processing used herein refers to the manipulation or processing of biological cells, such as but not limited to destroying cell walls or cell membranes to obtain cell contents. The contents of biological cells to be approached for processing may include but are not limited to nucleic acids or proteins. In some variations, the at least one liquid handling tip may be further configured to mix the fluid in the droplets in one or more boxes disposed on the surface of the placement. The mechanical arm or liquid handling head may be provided with an airflow, which may be guided to mix the fluid in the droplets through the liquid handling tip.

[0240] The digital microfluidic device can be configured to place one or more single plate boxes and / or double plate boxes. On the digital microfluidic device, a mixture of single plate boxes and double plate boxes can be used simultaneously. These boxes can all be the same size (width × length), or there can be one or more different sizes. As mentioned above, for a specific reaction or sample preparation workflow, relative to the box used on the DMF device without mechanical distribution, the size of the box can be reduced, because the extra movement of the droplet is not required, for example, to merge with reagent or buffer droplets. The placement position of one or more boxes on the placement surface can be uncertain. That is, according to the requirements of the reaction to be carried out or the preparation, the box can be placed at the placement position of any suitable position so that the box covers any required heating zone or magnetic control area. The area on the placement surface can be used for single plate boxes or double plate boxes at will, and allows the single plate box to be placed near the double plate box in any arrangement.

[0241] The placement surface of the device may also include alignment features as described herein to help place the cartridge on the placement surface. The alignment features may be any alignment features described herein, and may include one or more of alignment pins, camera-based visual algorithms, and / or radio frequency identification.

[0242] In some variations, the apparatus may further include a master control subsystem for coordinating activation of the plurality of drive electrodes, the plurality of vacuum ports, the robotic arm, and the liquid handling subsystem. The master control subsystem may also include non-transitory instructions for executing a method of controlling the plurality of vacuum ports and the plurality of drive electrodes, a method of controlling liquid handling, or a combination thereof, as described more fully below.

[0243] In some variations, as described herein, the device may further include one or more temperature controlled areas within the drive electrode array. The temperature controlled area may be configured to heat and / or cool. The selected area of ​​the placement surface may be operated at a temperature between about 4°C and about 100°C, between about 10°C and about 100°C, between about 15°C and about 100°C, between about 20°C and about 100°C, between about 25°C and about 100°C, between about 30°C and about 100°C, between about 37°C and about 100°C, or any temperature therebetween. There may be one or more such temperature controlled areas in the placement surface. In some variations, there may be 1, 4, 8, 10, 16, 20, 32 or more temperature controlled areas in the placement surface. The device may also include one or more areas within the drive electrode array, each area including a magnetic field.

[0244] The digital microfluidic device may also include a temperature controlled reagent / sample storage subsystem. In some variations, the reagent / sample storage subsystem may include an orifice plate accommodating area. In some embodiments, the orifice plate accommodating area may be configured to accommodate any one of 24-well, 48-well, 96-well, or 384-well orifice plates.

[0245] In some variations, the main controller of the device can be configured to direct the robot and liquid handling subsystem to distribute droplets at a selected position in at least one of the one or more boxes placed on the placement surface based at least in part on the position of each of the one or more boxes placed on the placement surface. The main controller can be further or optionally configured to direct the robot and liquid handling subsystem to distribute droplets at a selected position in at least one of the one or more boxes placed on the placement surface based at least in part on the position of the pre-installed reagent in at least one of the one or more boxes placed on the placement surface. In some variations, the main controller can be configured to direct the robot and liquid handling subsystem to distribute droplets at a selected position in at least one of the one or more boxes placed on the placement surface based at least in part on whether at least one of the one or more boxes is a single-plate box or a double-plate box. When the at least one box is a single-plate box, the selected position can be any position in the single-plate box, and when the at least one box is a double-plate box, the selected position can be the position of the fluid input / output port on the upper plate of the double-plate box.

[0246] In some variations, the main controller of the device can also be configured to sequentially mix the droplets at selected locations within at least one of the one or more boxes placed on the placement surface through the liquid handling tip. The mixing capability can be provided by a gas flow included in the robotic arm, as described above, and a gas (e.g., nitrogen or atmospheric air) can be ejected through the liquid handling tip to cause agitation and mixing of the droplets. The main controller can sequence the mixing at one or more boxes to proceed in a serial manner, such as one mixing event at a time, or can sequence the mixing at one or more boxes to proceed in a parallel manner, such as all mixing events are performed at the same time.

[0247] In some variations, the master controller may be configured to select one or more selected drive electrodes to drive droplets within the one or more cartridges disposed on the seating surface based in part on the position of each of the one or more cartridges disposed on the seating surface. Alternatively or additionally, the master controller may be configured to select one or more selected drive electrodes based in part on the position of droplets within each of the one or more cartridges disposed on the seating surface.

[0248] In some variations, the master controller may be configured to sequentially activate and deactivate one or more selected drive electrodes. The order of activation and deactivation may be based at least in part on the position of each of the one or more boxes disposed on the placement surface. Additionally or alternatively, the master controller may be configured to sequentially activate and deactivate one or more selected drive electrodes based in part on the position of a droplet within each of the one or more boxes disposed on the placement surface. In some variations, the master controller may be further configured to sequentially activate and deactivate one or more selected drive electrodes based in part on detecting completion of dispensing or extraction of a droplet within each of the one or more boxes disposed on the placement surface.

[0249] In some embodiments, the master controller can be configured to sequentially dispense droplets to each of the one or more cartridges or extract droplets from each of the one or more cartridges based at least in part on determining that one or more selected drive electrodes in each of the one or more cartridges disposed on the seating surface are not activated to drive droplets during dispensing or extraction of droplets.

[0250] In some variations, the master controller may also be configured to identify a selected reagent or sample in a droplet and distribute the selected reagent or sample in the droplet to at least one of the one or more boxes when the one or more boxes are placed on the placement surface. The selected reagent or sample may be different for each of the one or more boxes, or the same reaction or sample preparation may be performed simultaneously in multiple boxes, and the master controller may control multiple additions or extractions.

[0251] When the reagent / sample storage subsystem includes a well plate receiving area, the main controller can also be configured to extract the sample from the well plate in the well plate receiving area when the sample in the droplet is distributed to at least one of the one or more boxes when one or more boxes are placed on the receiving surface. In some variations, the main controller can also be configured to transport the droplet to the well plate in the well plate receiving area when the droplet is extracted from at least one box of the one or more boxes placed on the receiving surface.

[0252] When the robotic arm further comprises a metal tip configured to electroporate a biological cell within a droplet at a location within one of the one or more cartridges disposed on the mounting surface, the master controller may be configured to direct the robotic arm to deliver the metal tip to the location of the droplet within the cartridge. Additionally or alternatively, the master controller may be configured to schedule electroporation of the biological cell based at least in part on determining that one or more selected drive electrodes in the cartridge are not activated to drive the droplet during electroporation.

[0253] Based on the foregoing, another exemplary digital microfluidic device may include: a mounting surface configured to simultaneously mount one or more cartridges; a plurality of drive electrodes disposed on the mounting surface, wherein the drive electrodes are configured to apply a high voltage to move a droplet within the cartridge when the cartridge is mounted on the mounting surface, and wherein all or some of the drive electrodes include openings through the drive electrodes; a plurality of vacuum ports, wherein each vacuum port is coupled to one or more openings through the drive electrodes; and a vacuum control configured to selectively apply a vacuum through a subset of the vacuum ports below the one or more cartridges mounted on the mounting surface when the one or more cartridges are on the mounting surface. a vacuum; an electrical control for applying energy to sequentially activate and deactivate one or more selected drive electrodes to move droplets within an air gap of one or more boxes, the one or more boxes being placed on a placement surface; a vacuum pump for applying a vacuum to a plurality of vacuum ports; a robotic arm and liquid handling subsystem for dispensing liquid to or removing liquid from one or more boxes when the one or more boxes are placed on the placement surface; and a main controller for coordinating the activation and deactivation of one or more selected drive electrodes with the dispensing of liquid to or removing liquid from the one or more boxes when the one or more boxes are placed on the placement surface.

[0254] System. Figures 70A-70CAs shown, a system for high-throughput digital microfluidics may include a digital microfluidics device, the device including: a placement surface configured to simultaneously place one or more boxes; a plurality of drive electrodes disposed on the placement surface, wherein the drive electrodes are configured to apply a high voltage to move droplets within the box when the box is placed on the placement surface, and wherein all or some of the drive electrodes include openings through the drive electrodes; a plurality of vacuum ports, wherein each vacuum port is coupled to one or more openings through the drive electrodes; a vacuum control configured to selectively apply a vacuum through a subset of the vacuum ports below one or more boxes placed on the placement surface when the one or more boxes are placed on the placement surface; an electrical control for applying energy to sequentially activate and deactivate one or more selected drive electrodes to move droplets within an air gap of one or more boxes, the one or more boxes being placed on the placement surface; a vacuum pump for applying a vacuum to the plurality of vacuum ports; a robotic arm and a liquid handling subsystem for dispensing liquid to or removing liquid from one or more boxes when the one or more boxes are placed on the placement surface. In some variations, the system may further include a housing, wherein the housing controls one or more of humidity, temperature, and gas environment. The controlled humidity, temperature, and gas environment may be used to limit drying of droplets within a cartridge placed on a placement surface. The gas environment may also include atmospheric air and added 5% carbon dioxide, which may be useful for maintaining viability of cells within a closed system. The humidity may be a relative humidity that reduces evaporation from the liquid droplets while not condensing moisture in the gas environment onto housing surfaces or other surfaces of system components.

[0255] The system may include any features of the digital microfluidics device for high-throughput digital microfluidics as described herein, and in any combination, as may be envisioned by the skilled person. Some exemplary but non-limiting features are described in the following paragraphs.

[0256] Thus, the system may include a placement surface, wherein the placement position of one or more boxes on the placement surface may not be predefined. In some variations, the size of the placement position of at least one of the one or more boxes is different from the size of the placement position of another of the one or more boxes. In some variations, the system may be configured to place one or more single-plate boxes and / or dual-plate boxes.

[0257] In some variations, the liquid handling subsystem of the system may include at least one liquid handling tip. The liquid handling subsystem may include a plurality of liquid handling tips, which may be 4, 8, 12, or 96 liquid handling tips. In some variations, at least one liquid handling tip or more liquid handling tips may be further configured to mix droplets within at least one of the one or more cartridges disposed on the mounting surface.

[0258] The liquid handling subsystem of the system may include a liquid handling head. In some variations, the liquid handling head may be configured to dispense a volume of about 1 microliter to about 100 milliliters, but as described above, other volumes are also possible. The liquid handling head may also be configured to dispense biological cells suspended in a liquid.

[0259] In some variations, the robotic arm may be a three-axis robotic arm. In some variations, the robotic arm may further include a metal probe configured to electroporate biological cells within a droplet within at least one of the one or more cartridges disposed on the mounting surface.

[0260] The system may further include a temperature controlled reagent / sample storage subsystem. In some variations, the reagent / sample storage subsystem may include an orifice plate accommodating area. The orifice plate accommodating area may be configured to accommodate any one of 24, 48, 96 and 384 orifice orifice plates.

[0261] The system for high-throughput digital microfluidics may further include a master control subsystem configured to coordinate activation of the plurality of drive electrodes, the plurality of vacuum ports, the robotic arm, and the liquid handling tip. In some embodiments, the master control subsystem may include non-transitory instructions for executing a method of controlling the plurality of vacuum ports and the plurality of drive electrodes, a method of controlling liquid handling, or a combination thereof.

[0262] In some variations, the master control subsystem may be further configured to control at least one of humidity, temperature, and gas environment of the enclosure. The master control subsystem may include non-transitory instructions for executing a method of controlling at least one of humidity, temperature, and gas environment of the enclosure.

[0263] Optical subsystem. In some variations, the system for high-throughput digital microfluidics may further include an optical subsystem. In some embodiments, the optical subsystem may include an illumination source. In some embodiments, the illumination source may include one or more of a visible light source or an ultraviolet (UV) light source. The light source may be an LED or any other light source capable of providing light in the desired wavelength range. The illumination source may be used to image the placement surface and / or one or more boxes placed thereon. An illumination source with an appropriate wavelength (such as, but not limited to, UV) may also be used to photoactivate reagents in droplets on the surface of one or more boxes placed on the placement surface. In another example, caged fluorescent dyes may be uncaged using photoactivation in the UV range. The optical subsystem may further include an imaging device configured to detect a bright field image, a fluorescent image, or a luminescent image. In some variations, the optical subsystem may be configured to control one or more illumination sources and imaging devices when one or more illumination sources and imaging devices are present.

[0264] The optical subsystem may be further configured to communicate with a master control subsystem. In some variations, the non-transitory instructions of the master control subsystem may further include instructions for sequentially activating drive electrodes and activating liquid handling based on imaging communications received from the optical subsystem. The master control subsystem may also include non-transitory instructions for executing a method of controlling the optical subsystem. In some variations, the non-transitory instructions may include instructions for activating and deactivating brightfield and / or fluorescent illumination and / or capturing images of selected areas of a placement surface of a digital microfluidic device.

[0265] In some variations of the high-throughput digital microfluidic system, the digital microfluidic device may further include one or more regions within the drive electrode array, wherein one or more regions further include a magnet. In some variations, the digital microfluidic device of the system may further include one or more regions within the drive electrode array including heating and / or cooling controllability. In some variations of the system, the system may further include more than one digital microfluidic device.

[0266] A method for using the device. A method for selectively fixing one or more boxes in a digital microfluidic device for high-throughput digital microfluidics is provided for a digital microfluidic device, the digital microfluidic device comprising: a placement surface configured to place one or more boxes at the same time; a plurality of drive electrodes disposed on the placement surface, wherein the drive electrodes are configured to apply a high voltage to move a droplet within the box when the box is placed on the placement surface, and wherein all or some of the drive electrodes include openings passing through the drive electrodes; a plurality of vacuum ports, wherein each vacuum port is connected to one or more openings passing through the drive electrodes; a vacuum control configured A method for selectively applying vacuum through a subset of vacuum ports below one or more boxes placed on the placement surface when one or more boxes are placed on the placement surface; an electrical control for applying energy to sequentially activate and deactivate one or more selected drive electrodes to move droplets within the air gap of one or more boxes, one or more boxes are placed on the placement surface; a vacuum pump for applying vacuum to multiple vacuum ports; a robotic arm and a liquid handling subsystem for dispensing liquid to one or more boxes or removing liquid from one or more boxes when one or more boxes are placed on the placement surface. The method includes placing one or more boxes on the placement surface, thereby generating corresponding placement positions of one or more boxes; sensing each of the one or more placement positions; and determining an area including a subset of multiple drive electrodes located below each placement position, thereby identifying one or more areas of drive electrodes located below the multiple placement positions. The vacuum control is activated to apply vacuum to one or more areas of the drive electrodes; and one or more boxes are selectively fixed to the placement surface of the digital microfluidic device.

[0267] In some variations, each of the one or more boxes can include a base plate that is configured to deform when a vacuum is applied so as to adhere to the placement surface. Each respective placement location can have a respective area corresponding to the size of each box. In some embodiments, at least one of the one or more boxes has a different size than the other boxes in the one or more boxes.

[0268] The digital microfluidic device for high-throughput digital microfluidics may include any other features of the digital microfluidic device described in this method. In addition, it is contemplated by those skilled in the art that this method of selectively fixing one or more boxes may be combined with any other method described herein. In some variations, the device may include a main control subsystem, and the method may be performed by the main control subsystem. In some variations, the method performed by the main control subsystem may be performed automatically.

[0269] A method for coordinating droplet movement and liquid handling in a cartridge within a digital microfluidic device for high-throughput digital microfluidics is provided for a digital microfluidic device, the digital microfluidic device comprising: a placement surface configured to simultaneously place one or more cartridges; a plurality of drive electrodes disposed on the placement surface, wherein the drive electrodes are configured to apply a high voltage to move droplets within the cartridge when the cartridge is placed on the placement surface, and wherein all or some of the drive electrodes include openings through the drive electrodes; a plurality of vacuum ports, wherein each vacuum port is coupled to one or more openings through the drive electrodes; a vacuum control, which is A vacuum pump configured to selectively apply vacuum through a subset of vacuum ports beneath one or more cartridges disposed on the mounting surface when one or more cartridges are disposed on the mounting surface; an electrical control for applying energy to sequentially activate and deactivate one or more selected drive electrodes to move a droplet within an air gap of one or more cartridges, the one or more cartridges being disposed on the mounting surface; a vacuum pump for applying vacuum to a plurality of vacuum ports; a robotic arm and liquid handling subsystem for dispensing liquid to or removing liquid from one or more cartridges when the one or more cartridges are disposed on the mounting surface. . The method includes placing each of one or more cartridges on a placement surface; determining a placement position of each of the one or more cartridges on the placement surface; determining an area including a subset of a plurality of drive electrodes located below each placement position, thereby identifying one or more areas of drive electrodes located below the plurality of placement positions; selectively fixing each of the one or more cartridges on the placement surface of the digital microfluidic device; identifying a position of a fluid input of each of the one or more cartridges; and placing a droplet from a liquid handling subsystem in each of the one or more cartridges via a corresponding fluid input; identifying the fluid input of each of the one or more cartridges may include, for each of the one or more cartridges, determining whether each cartridge is a single-plate cartridge or a dual-plate cartridge. Since the entire upper surface of the bottom plate of the single-plate cartridge is open to the robot arm and the liquid handling subsystem, the fluid input of the single-plate cartridge may be any position on the surface facing the air gap of the single-plate cartridge. The fluid input of the dual-plate cartridge may be a port or other opening in the upper plate of the dual-plate cartridge, and the dual-plate cartridge may have one or more fluid inputs, and in some variations, may have multiple fluid inputs. Identifying the fluid input of the dual-plate cartridge may include identifying the location of one or more ports or openings on the upper plate of the dual-plate cartridge. Distributing the droplets into each of the one or more cartridges may include distributing the droplets sequentially to each of the one or more cartridges serially, e.g., one droplet at a time. Alternatively, distributing may include distributing the droplets into each of the one or more cartridges on the placement surface in parallel, e.g., distributing all the droplets substantially simultaneously.The method may include associating the position of each respective droplet within each of the one or more cartridges with a respective first selected drive electrode of a respective region of a plurality of drive electrodes; and activating the first selected drive electrode of each respective region to drive each respective droplet to a respective second position within each of the one or more cartridges by electrowetting. In some variations, activating at least the first selected drive electrode of each respective region of the plurality of drive electrodes may further include grounding the first drive electrode to an adjacent second drive electrode of each respective region.

[0270] In some variations, each second position in one or more boxes may include a fluid output. The method may further include extracting a corresponding fluid droplet at each corresponding second position in the one or more boxes, wherein the extraction includes applying suction via at least one liquid handling tip. When the liquid handling subsystem includes a plurality of liquid handling tips, the corresponding fluid droplets may be extracted in parallel (e.g., simultaneously for each of the one or more boxes). In some variations, the extraction of the corresponding fluid droplets may be performed serially.

[0271] The digital microfluidic device for high-throughput digital microfluidics may include any other features of the digital microfluidic device for the method described herein. In addition, it can be envisioned by those skilled in the art that this method for coordinating the movement of droplets and liquid handling in a box within a digital microfluidic device can be combined with any other method described herein. In some variations, the device may include a main control subsystem, and the method may be performed by the main control subsystem. In some variations, the method performed by the main control subsystem can be performed automatically.

[0272] A method for simultaneously operating a single-plate cartridge and a dual-plate cartridge in a digital microfluidic device is provided, the digital microfluidic device comprising: a mounting surface configured to simultaneously mount one or more cartridges; a plurality of drive electrodes disposed on the mounting surface, wherein the drive electrodes are configured to apply a high voltage to move a droplet within the cartridge when the cartridge is mounted on the mounting surface, and wherein all or some of the drive electrodes include openings through the drive electrodes; a plurality of vacuum ports, wherein each vacuum port is coupled to one or more openings through the drive electrodes; a vacuum control configured to selectively apply a vacuum through a subset of the vacuum ports below one or more cartridges mounted on the mounting surface when the one or more cartridges are mounted on the mounting surface; an electrical control for applying energy to sequentially activate and deactivate one or more selected drive electrodes to move a droplet within an air gap of one or more cartridges, the one or more cartridges being mounted on the mounting surface; a vacuum pump for applying a vacuum to the plurality of vacuum ports; and a robotic arm and liquid handling subsystem for dispensing liquid to or removing liquid from the one or more cartridges when the one or more cartridges are mounted on the mounting surface. The method includes placing a single-plate box at a first placement position on a placement surface and placing a double-plate box at a second placement position on the placement surface; identifying a first area and a second area, the first area including a first subset of multiple drive electrodes located below the first placement position, and the second area including a second subset of multiple drive electrodes located below the first placement position; selectively fixing the single-plate box and the double-plate box to the placement surface of the digital microfluidic device; identifying the position of a first fluid input portion in the single-plate box at the first placement position and the position of a second fluid input portion in the double-plate box at the second placement position; placing a first droplet from a liquid handling subsystem in the single-plate box and placing a second droplet in the double-plate box; associating the position of the first droplet with a first selected drive electrode of a first subset of multiple drive electrodes; associating the position of the second droplet with a first selected drive electrode of a second subset of multiple drive electrodes; activating at least the first selected drive electrode of each corresponding subset to drive each corresponding droplet to a corresponding second position in each of one or more boxes by electrowetting.

[0273] Selectively securing the single plate cassette and the dual plate cassette to the mounting surface may also include activating a vacuum control in an electrode region below the cassette. In some variations, when the droplet is dispensed through the air gap of the single plate cassette, the first fluid input in the single plate cassette may include any location within the surface of the cassette. In some variations, the first fluid input in the dual plate cassette may be a port at one or more predetermined locations on the upper plate of the dual plate cassette. Disposing the first droplet in the single plate cassette and disposing the second droplet in the dual plate cassette may include substantially arranging the first droplet and the second droplet simultaneously, such as in parallel, or may include arranging the first droplet and the second droplet in succession.

[0274] In some variations, activating at least a first selected drive electrode of each respective subset to drive each respective droplet to the respective second position may further include grounding the first selected drive electrode to an adjacent second drive electrode of each respective subset of the plurality of drive electrodes. Activating at least a first selected drive electrode of each respective subset to drive each respective droplet to the respective second position may be performed substantially simultaneously. Alternatively, activating at least a first selected drive electrode of each respective subset to drive each respective droplet to the respective second position may be performed sequentially.

[0275] In some variations, each corresponding second position may further include a fluid output. The fluid output at the second position of the single-plate cartridge may be anywhere within the inner surface of the cartridge, and extraction is performed through an air gap of the single plate. The fluid output at the second position within the dual-plate cartridge may be a port at one or more predetermined positions on the upper plate of the dual-plate cartridge. The method may further include extracting a corresponding fluid droplet at each corresponding second position within the single-plate cartridge and the dual-plate cartridge, wherein the extraction may include applying suction via at least one liquid handling tip of the liquid handling subsystem. In some variations, when the liquid handling subsystem includes a plurality of liquid handling tips, the extraction of the corresponding droplets may be performed in parallel.

[0276] The digital microfluidic device for high-throughput digital microfluidics may include any other features of the digital microfluidic device for the method described herein. In addition, it can be envisioned by those skilled in the art that this method for operating a single-plate box and a double-plate box simultaneously in a digital microfluidic device can be combined with any other method described herein. In some variations, the device may include a main control subsystem, and the method may be performed by the main control subsystem. In some variations, the method performed by the main control subsystem may be performed automatically.

[0277] A method for operating a system including a digital microfluidic device is provided, wherein the digital microfluidic device includes: a mounting surface configured to simultaneously mount one or more cartridges; a plurality of drive electrodes disposed on the mounting surface, wherein the drive electrodes are configured to apply a high voltage to move a droplet within the cartridge when the cartridge is mounted on the mounting surface, and wherein all or some of the drive electrodes include openings through the drive electrodes; a plurality of vacuum ports, wherein each vacuum port is coupled to one or more openings through the drive electrodes; a vacuum control configured to selectively apply a vacuum through a subset of the vacuum ports below one or more cartridges mounted on the mounting surface when the one or more cartridges are mounted on the mounting surface; an electrical control for applying energy to sequentially activate and deactivate one or more selected drive electrodes to move a droplet within an air gap of one or more cartridges, the one or more cartridges being mounted on the mounting surface; a vacuum pump for applying a vacuum to the plurality of vacuum ports; and a robotic arm and liquid handling subsystem for dispensing liquid to or removing liquid from the one or more cartridges when the one or more cartridges are mounted on the mounting surface. In some embodiments, the system further comprises a housing configured to control one or more of humidity, temperature, and gas environments. The method comprises, for each of the one or more boxes, identifying a corresponding subset of drive electrodes under each of the one or more boxes; for each of the one or more boxes, determining a plurality of associated ground electrodes adjacent to the corresponding subset of drive electrodes; and selectively fixing each of the one or more boxes on the placement surface of the digital microfluidic device. The method comprises, for each of the one or more boxes, coordinating the activities to be performed in each box, and comprising sequencing the activities. Sorting the activities may include configuring resources, such as a robotic arm and a liquid handling subsystem, for efficient and non-overlapping actions. The efficient action of the robotic arm may include designing a series of paths for the robotic arm / liquid handling subsystem to travel to minimize the total length of travel. Sorting the activities may include determining the appropriate reaction cycle for a specific workflow in each box, and planning to start each workflow to allow for timely distribution, mixing, heating, and cooling events on the multiplexed placement surface of the digital microfluidic surface. The method coordinates activities such as determining a reaction type to be performed in each of the one or more boxes; dispensing a sample into each of the one or more boxes to form a corresponding sample droplet in each of the one or more boxes; and dispensing one or more reagents / buffers into each of the one or more boxes to form a corresponding reagent / buffer droplet in each of the one or more boxes. The method may also include: for each of the one or more boxes on the placement surface; coordinating driving a corresponding reagent droplet and / or sample droplet to a reaction position in each of the one or more boxes.Coordinating the driving of corresponding reagent droplets and / or sample droplets can include, for each of one or more boxes, identifying the position of the corresponding reagent droplet and / or sample droplet; identifying one or more driving electrodes under the position of the corresponding reagent droplet and / or sample droplet; activating the driving electrodes under the corresponding reagent droplet and / or sample droplet, and grounding one or more driving electrodes to corresponding adjacent electrodes, thereby driving the corresponding reagent droplet and / or sample droplet to the next position by electrowetting, and repeating these processes to reach the reaction position of the corresponding reagent droplet and / or sample droplet.

[0278] The method may further include, for each of the one or more cartridges, coordinating merging a corresponding sample droplet with its corresponding reagent droplet to form a corresponding reaction droplet within each of the one or more cartridges; and performing a corresponding reaction in the corresponding reaction droplet to form a corresponding product droplet within each of the one or more cartridges. In some variations, the coordination may further include, for each of the one or more cartridges, coordinating driving the reaction droplets to a reaction location within one or more cartridges, wherein the corresponding reaction location includes a magnet and / or a temperature control.

[0279] The method may also include coordinating each of the one or more cartridges to determine an assay result of the corresponding reaction droplets of the one or more cartridges when the corresponding reaction in the corresponding reaction droplets of the one or more cartridges is an assay. The method may also include, for each of the one or more cartridges, outputting a corresponding product droplet when the corresponding reaction in the one or more cartridges is a sample preparation reaction.

[0280] In some variations, coordination may further include, for each of the one or more cartridges, coordinating the mixing of dispensed reagents / buffers with pre-installed reagents disposed at selected locations within each of the one or more cartridges to form corresponding reagent / buffer droplets. In some variations, coordination may further include, for each of the one or more cartridges, coordinating the mixing of sample droplets with pre-installed reagents disposed at selected locations within the one or more cartridges to form corresponding reaction droplets.

[0281] In some variations, when the system further includes one or more illumination sources, coordinating the formation of corresponding reagent droplets or forming corresponding reaction droplets may further include coordinating photoactivation of reagents in corresponding reagent droplets or corresponding reaction droplets. When imaging one or more boxes placed on the placement surface, the dual-plate box may have a transparent or translucent upper plate, thereby allowing imaging. When photoactivating one or more boxes placed on the placement surface, the dual-plate box may have a transparent or translucent upper plate that does not significantly absorb the photoactivation wavelength, allowing the photoactivated illumination to fully pass through the upper plate to reach the photoactivated reagents. In other variations, the dual-plate box may include a window cut into the upper plate that allows the photoactivated illumination to reach the photoactivated reagents in the box.

[0282] In some variations, dispensing corresponding sample droplets, dispensing corresponding reagents / buffers, or extracting corresponding product droplets can be coordinated to be performed when the drive electrode is not activated. In some embodiments, forming corresponding sample droplets, corresponding reagent / buffer droplets, corresponding reaction droplets, or corresponding product droplets can be coordinated to be performed when the drive electrode is not activated.

[0283] In some embodiments, forming the corresponding sample droplets may further include coordinating electroporation of biological cells within the sample droplets.

[0284] In some variations, the method may further include regulating at least one of temperature, humidity, or gas environment in the housing of the system.

[0285] In some variations, when the system includes a master control subsystem, coordinating corresponding processes, such as determining, allocating, mixing, merging, driving, performing reactions or outputs, can be performed automatically. In some embodiments, coordinating corresponding processes of each of the one or more boxes can be performed independently, for example, running the workflow of each of the one or more boxes without synchronizing the workflow activities with the workflow activities of other boxes on the placement surface.

[0286] In some embodiments, coordinating the corresponding processes of one or more boxes may further include scheduling the robotic arms and / or liquid handling subsystems and scheduling the corresponding multiple electrodes, thereby reducing conflicting instructions for the robotic arms, liquid handling subsystems and / or corresponding multiple electrodes in one or more boxes.

[0287] In some variations, when the system includes an imaging device, scheduling the robotic arm and / or the liquid handling subsystem and scheduling the corresponding plurality of electrodes of one or more cartridges can further include receiving imaging information from the imaging device, confirming the completion of a previous corresponding process within the one or more cartridges. In some variations, when the system includes an imaging device, determining the assay results of the corresponding reaction droplets of the one or more cartridges can further include detecting a visible signal, a fluorescent signal, or a luminescent signal from the corresponding reaction droplets.

[0288] In some variations, when the system includes a master control subsystem, the master control system may regulate at least one of the temperature, humidity, or gas environment of the enclosure.

[0289] In some variations, when the system further comprises a reagent / sample storage subsystem (including a well plate receiving area), outputting the corresponding product droplets may further comprise depositing the output product droplets within the wells of the well plate.

[0290] The digital microfluidic device for high-throughput digital microfluidic technology can further include any other features of the digital microfluidic device described herein, for use in the present method of operating a system including a digital microfluidic device. In addition, as can be imagined by those skilled in the art, the method can be combined with any other method described herein. In some variations, the device can include a main control subsystem, and the method can be performed by the main control subsystem. In some variations, the method performed by the main control subsystem can be automatically performed.

[0291] DMF device. By returning to the figure, the present invention can be better understood. Figure 1 A- Figure 1 In C, the droplets (e.g., reaction droplets) fit on the first plate 153 and the second plate 151 (in Figure 1 A- Figure 1 The entire air matrix DMF device can have any suitable shape and thickness. Figure 1 B is through Figure 1 A is an enlarged view of a cross section of a hot zone of an air matrix DMF shown in Figure 1, which shows a layer of a DMF device (e.g., a layer forming a base plate). Typically, a DMF device (e.g., a base plate) includes several layers, which may include layers formed on a printed circuit board (PCB) material; these layers may include a protective cover layer, an insulating layer, and / or a supporting layer (e.g., a glass layer, a ground electrode layer, a hydrophobic layer; a hydrophobic layer, a dielectric layer, an actuating electrode layer, a PCB, a thermal control layer, etc.). Any of these surfaces may be rigid (e.g., glass, PCB, a polymer material, etc.). The air matrix DMF device described herein also includes both a sample reservoir and a reagent reservoir, as well as a mechanism for supplementing reagents.

[0292] exist Figure 1 A- Figure 1In the example shown in C, the top plate 101 (in this case a glass material (although plastic / polymer materials including PCBs can be used)) provides support and protects the layers below from damage by external particles, as well as providing a certain amount of insulation for the reactions occurring within the DMF device. Therefore, the top plate can confine / sandwich the droplets between the plates, which can enhance the electric field compared to an open air matrix DMF device (without a plate). The upper plate (in this example, the first plate) can include a ground electrode and can be transparent or translucent; for example, the substrate of the first plate can be formed of glass and / or transparent plastic. However, although the upper plate is transparent, it can be coated with a conductive material and / or can include a ground electrode (ground electrode layer 102) adjacent to and below the substrate for the DMF circuit. In some cases, the ground electrode is a continuous coating; alternatively, multiple ground electrodes, such as adjacent ground electrodes, can be used. Below the ground electrode layer is a hydrophobic layer 103. The hydrophobic layer 103 is used to reduce the wetting of the surface and helps to keep the reaction droplets in a cohesive unit.

[0293] As Figure 1 A- Figure 1 The second plate shown in the lower plate or bottom plate 151 in C may include an actuation electrode defining a unit cell. In this example, as in the first plate, the outermost layer facing the air gap 104 between the plates also includes a hydrophobic layer 103. The material forming the hydrophobic layer may be the same on both plates, or it may be a different hydrophobic material. The air gap 104 provides a space in which the reaction droplet is initially contained in the sample reservoir and is moved for running a reaction step or multiple reaction steps and for holding various reagents for each reaction step. Adjacent to the hydrophobic layer 103 on the second plate is a dielectric layer 105, which can increase the capacitance between the droplet and the electrode. Adjacent to the dielectric layer 105 and below the dielectric layer 105 is a PCB layer, which includes an actuation electrode (actuation electrode layer 106). The actuation electrode can form each unit cell. The actuation electrode can be energized to move the droplet to different regions in the DMF device so that each reaction step can be carried out under different conditions (e.g., temperature, combination with different reagents, magnetic region, pump inlet region, etc.). Support substrate 107 (eg, PCB) (on Figure 1 B and Figure 1C) may be adjacent to and below the actuation electrode layer 106 to provide support and electrical connections for components including the actuation electrodes, traces connecting them (which may be insulated), and / or additional control elements including a thermal regulator 155 (shown as a TEC), temperature sensors, optical sensors, magnets, pumps, etc. One or more controllers 195 for controlling the operation of the actuation electrodes and / or controlling the application of droplet replenishment to the reaction droplets may be connected but separate from the first plate 153 and the second plate 151, or they may be formed on and / or supported by the second plate. Figure 1 A- Figure 1 C, the first plate is shown as the top plate, and the second plate is the bottom plate; this orientation can be reversed. Also shown is a source or reservoir 197 of solvent (make-up fluid) connected to a hole in the second plate via a tube 198.

[0294] As mentioned, the air gap 104 provides a space in which the reaction steps can be performed, which provides an area in which reagents can be held and in which the reagents can be processed (e.g., by mixing, heating / cooling, combining with reagents (enzymes, labels, etc.)). Figure 1 In A, the air gap 104 includes a sample reservoir 110 and a series of reagent reservoirs 111. The sample reservoir may also include a sample loading feature for introducing the initial reaction droplet into the DMF device. Based on the needs of the reaction to be performed, the sample loading can be loaded from the top, from the bottom or from the side, and can be unique. Figure 1 The sample DMF equipment shown in A comprises six sample reagent reservoirs, and wherein each sample reagent reservoir comprises opening or port for introducing every kind of reagent in the corresponding reservoir.According to the reaction to be performed, the number of reagent reservoirs can be variable.Sample reservoir 110 and reagent reservoir 111 are communicated by reaction zone fluid.Reaction zone is electrically communicated with actuating electrode layer 106, and wherein actuating electrode layer 106 is located below the reaction zone.

[0295] The actuation electrode 106 is Figure 1A is depicted as a grid or unit cell. In other examples, based on the needs of the reaction, the actuating electrodes can be in completely different patterns or arrangements. The actuating electrodes are configured to move the droplet from one area of ​​the DMF device to another area or multiple areas. The motion and to some extent the shape of the droplet can be controlled by switching the voltage of the actuating electrodes. One or more droplets can be moved along the path of the actuating electrodes by sequentially energizing and deenergizing the electrodes in a controlled manner. In the example of the DMF device shown, one hundred actuating electrodes (forming about one hundred unit cells) are connected to seven reservoirs (a sample reservoir and six reagent reservoirs). The actuating electrodes can be made of any suitable conductive material, such as copper, nickel, gold or a combination thereof.

[0296] exist Figure 1 A- Figure 1 In the exemplary device shown in C, the DMF device is usually integrated so that an electrode (e.g., an actuating electrode and a ground electrode) is a part of the same structure that can be loaded with a sample and / or a fluid. The electrode can be a part of a box, and the box can be removable. Although box has been described (see, for example, US20130134040), it has been shown that such a box is difficult to use, particularly when being imaged by the device and when operating in an air matrix device.

[0297] Generally, digital microfluidic devices and methods are described herein. In particular, air matrix digital microfluidic devices including systems and equipment are described herein, and methods for operating the air matrix digital microfluidic devices to process fluid samples. For example, a DMF device can include a compact DMF driver / device, which is configured to work with a removable box / disposable box. The DMF driver / device can include an array of drive electrodes, which are suitable for applying negative pressure and / or positive pressure at multiple points on the box and particularly at the electrode contact point, aligning the box and fixing it in place. The box can include an air gap, which is open to the environment (for example, for air) via an opening (for example, a side (lateral) opening and / or a top opening). The air gap can be formed between two dielectric layers. The upper top area can include one or more ground electrodes. The ground electrode can advantageously be formed by an opaque material, which is patterned to include one or more windows allowing imaging through the top. The windows may be arranged above the electrodes such that the ground region extends opposite the drive electrodes and around and / or between the drive electrodes.

[0298] Any device described herein may also include a fluid application and extraction component (e.g., a fluid application and / or extraction device) that is connected to the air gap through the top or through the side of the box. Any device described herein may include or use a non-polar cover material (e.g., a non-polar liquid, such as room temperature wax) that forms a protective cover around the aqueous droplets in the device and can move with the droplets. Also described herein is a user interface for interfacing with the device, including a user interface for controlling device movement, mixing, combining, washing, magnetically concentrating, heating, cooling, etc. These user interfaces can allow manual, automatic or semi-automatic input, control and / or execution of protocols.

[0299] Figure 2 The diagram shows something like Figure 1 A- Figure 1 An example of a DMF apparatus is shown in C. Figure 2 In the DMF device, a plurality of driving electrodes 211 are formed into non-square / non-rectangular shapes and are positioned adjacent to each other in rows or columns. Figure 2 In the DMF apparatus, four reservoir areas 203, 205, 207, 209 are located on the right and may be preloaded or otherwise hold droplets of material to be added during operation of the DMF apparatus. Some or all of the electrodes may be heated or cooled.

[0300] exist Figure 2 In the device, the DMF drive electrode 211 is a solid, flat electrode. The application of energy between the drive electrode and a ground or reference electrode causes the movement of aqueous (e.g., polar) droplets. Figure 2 In the embodiment of the present invention, the ground electrode or reference electrode is formed as a conductive transparent coating (such as ITO) on the upper plate, which is also clear (transparent). This allows the device to be monitored from above the air matrix / air gap, including monitoring any unit, such as a unit cell.

[0301] However, it would be beneficial to provide a DMF apparatus (e.g., device, system, etc.) that can be used with a disposable cartridge that does not include a drive electrode. Figure 3A and Figure 3B A DMF system including integrated drive electrodes is shown ( Figure 3A ) and different configurations of systems where the drive electrode is part of the device but the box only includes a ground electrode (e.g., a top plate), an air gap, and a dielectric bottom. For example, in Figure 3AIn the embodiment, an air gap is formed between the grounded top plate 303 and the drive electrodes and dielectric film 305 (e.g., Teflon film). The drive electrodes and dielectric film can be part of a box including the top plate and can be separately attached to a substrate (switch board 307) connected to the main processor 309 and the power board 311.

[0302] In contrast, Figure 3B , the cartridge does not include a drive electrode 313, but rather includes a top plate / ground electrode, a dielectric, and an air gap 315 therebetween. As will be described in greater detail herein, a vacuum device (e.g., a vacuum manifold) can be positioned beneath the electrode 313 to apply a pressure (e.g., between 50 kPa and 250 kPa, 50 kPa or greater, 60 kPa or greater, 70 kPa or greater, 80 kPa or greater, 90 kPa or greater, 100 kPa or greater, 110 kPa or greater, etc.) to completely secure the dielectric, and thus the remainder of the cartridge, to the device. The electrode can be supported on a substrate, such as a printed circuit board or switch board 317, which can also be connected to a main processor 319 and a power supply 321. As will be described in greater detail herein, a vacuum device (e.g., a vacuum manifold) can be positioned beneath the electrode 313 to apply a pressure (e.g., between 50 kPa and 250 kPa, 50 kPa or greater, 60 kPa or greater, 70 kPa or greater, 80 kPa or greater, 90 kPa or greater, 100 kPa or greater, 110 kPa or greater, etc.) to completely secure the dielectric, and therefore the remainder of the cartridge, to the device. Figure 3B As shown in , the dielectric film may also be hydrophobic (eg, a Teflon film may be used), or may be treated, coated, sprayed, dipped, etc., into a hydrophobic material so that at least the side facing the air gap is hydrophobic.

[0303] exist Figure 4A-4C and Figure 10-12 The mounting surface of an exemplary DMF device is shown in more detail in FIG. Figure 4A In FIG. 4 , the mounting surface includes an array of drive electrodes 401 (labeled in rows 0-9 and columns AR). Each of these drive electrodes includes a central hole or opening through the electrode through which a vacuum can be applied to hold the dielectric of the cartridge against the drive electrode. Figure 4A In, the mounting surface also includes a temperature sensor (thermistor 405) which is positioned between the electrodes in different orientations. Figure 4B A slightly enlarged view of the mounting surface including the drive electrodes is shown, showing the thermistor 405 between the drive electrodes. The vacuum opening 407 is located at Figure 4B . Any shape and size of drive electrodes may be used, including interlocking drive electrodes. In addition, patterns of drive electrodes that are not monolithic may be formed; for example, an electrode pattern may include open areas that do not include drive electrodes (e.g., areas surrounding drive electrodes, etc.), such as Figure 1 A and Figure 2 As shown in .

[0304] Figure 4C An example of a heater is shown, which may be positioned at certain portions of the drive electrodes (eg Figure 4B In this example, the resistive heating circuit 409 can be located below the driving electrodes (e.g., embedded at any layer of the PCB forming the mounting surface). In general, the resistive heating and thermistor can be embedded at any layer of the electrode PCB board. The heater can be part of the PCB with the electrodes and thermistor, such as Figure 4A-4C The current and therefore the temperature of the drive electrode and / or adjacent dielectric (and therefore any droplets on the cell below the dielectric / drive electrode) can be regulated, for example, by a PID control loop in combination with a thermistor. To cool the dielectric (and the entire mounting surface), a liquid cooler can be circulated through the substrate, for example on the bottom of the mounting surface. Figure 4C In the example of FIG. 5 , the resistive heater is shown as a continuous trace of low-resistance material (eg, having a resistance between approximately 10-15 ohms).

[0305] Any suitable temperature regulation technique can be used. For example, stirring (e.g., magnetic stirring) can be used. Even small volume droplets can contain a local temperature range, so the temperature distribution can have a standard deviation. This can be reduced by stirring (e.g., via magnetic beads). With sufficient stirring, the droplets can be close to isothermal. In any of these variations, the top plate can be used to help regulate the temperature. For example, the top plate can be used to dissipate heat. A heat conductor (e.g., a steel block) on top of the top plate can greatly speed up the time it takes for the top plate to cool. If the top plate has a large thermal mass, or mass is added to the top plate, this can reduce the time required for a set number of thermal cycles.

[0306] The temperature difference between the top plate and the bottom heater (e.g., buried heater) can help determine the temperature standard deviation. Heating the top plate in conjunction with the electrodes can reduce the time required to raise the temperature. For example, the top plate can include a Figure 4C The localized resistive heater shown in FIG. A heated / cooled top plate can be implemented separately from the cassette by including a top thermal mass that engages the top of the cassette when the cassette is on the seating surface. For example, the heated and / or cooled top thermal mass can be a manifold that presses down onto the cassette.

[0307] As mentioned, liquid coolant can be applied to the bottom and / or top of the box. In particular, a circulating liquid coolant can be used. In some variations, the entire bottom of the box can be cooled (e.g., to within 3 degrees -5 degrees of room temperature, e.g., between 15 degrees Celsius -35 degrees Celsius). Figure 5A, an example of a mounting surface 501 is shown removed from the device to illustrate that a liquid coolant is coupled to the base of the mounting surface so that the coolant can be pumped into 503 and out of 505 through the mounting surface 501 .

[0308] Figure 5B Pump 511, piping 517, fan 515, radiator 516 and reservoir 513 are shown being used to move water or liquid coolant under the electrodes. The coolant absorbs heat as it passes under the electrodes and is cooled again as it passes through the fan and radiator.

[0309] As mentioned above, applying a vacuum by the device through the openings in the electrodes allows the dielectric of the cartridge to be securely and releasably held. Openings that do not pass through the electrodes will not hold the dielectric smoothly on the mounting surface. However, when a vacuum is applied through all of the drive electrodes that can be activated, the dielectric is held flat against the drive electrodes and a continuously lower energy can be applied. For example, Figure 5D and Figure 5E The figure shows a dielectric (shown unattached to the box for illustrative purposes) being secured to a mounting surface having an electrode with an opening through which a vacuum is applied. Figure 5D In the embodiment of FIG. 5 , the vacuum is turned off and the dielectric 555 rests loosely on the mounting surface in a number of folds. Figure 5E In the process, a vacuum is applied via electrodes.

[0310] Using a vacuum in this manner allows for reduced dielectric thickness and, thus, lower power (eg, voltage) requirements. Compared to using an adhesive or using a vacuum applied externally to the electrode, Figure 5A-5E The configuration shown in leads to a reduction in half of the power requirement for the DMF. In the example shown, the thickness of the dielectric can be between 7 microns and 13 microns. When an adhesive is used, the dielectric is almost twice as thick (e.g., 25 microns).

[0311] exist Figure 5C , pump 560 is shown connected via a tube to a vacuum manifold configured to pull air through the holes in the electrodes. The dielectric membrane sits on top and remains rigid as long as the pump is pulling air. In addition, any protrusions in the surface of the dielectric (particularly protrusions around the air gap of the box or slightly smaller than the width of the air gap of the box) will not interfere with the seal, but will form a shell, channel, barrier or other structure within the air gap, which can help to separate the air gap.

[0312] Fig. 5F and Figure 5G The diagram shows the upper and middle layers of the mounting surface, which shows the connection between the vacuum source (via connector 565) through mechanical and / or pipe manifolds ( Figure 5G), and out of the opening through the electrode ( Fig. 5F ).

[0313] Figures 10 to 12 The figure shows an example of a mounting surface 900 to which a cartridge may be held by a vacuum port through an electrode. Fig.10 In , a mounting surface is formed on a substrate (e.g., a PCB or other electrically insulating surface) and includes an array of electrodes 901, which in this example are shown as quadrilateral (e.g., square) shapes. Any other suitable shape may be used. The drive electrodes 901 are thin conductive surfaces that may be flush or substantially flush with the mounting surface, or may protrude slightly above the mounting surface. In Fig.11 In the embodiment, the box 905 is shown as being placed on top of the array of driving electrodes 901 on the mounting surface 900. The box can be placed on the mounting surface (as described above) by a drawer. Figure 5E and Fig. 5F ). When on a placement surface, a vacuum may be applied by all or a subset of the drive electrodes (e.g., the drive electrodes over which fluid may be transported in the air gap) to hold the dielectric (and thus the cartridge) in place. As mentioned above, without applying a vacuum by the electrodes themselves, more energy may be required to reliably drive fluid in the air gap, and the dielectric must be thicker. Fig.12 An enlarged view of a portion of mounting surface 900 is shown showing electrode 901 having a central opening 909 into a vacuum manifold.

[0314] The mounting surface of the device can be divided into functional areas that control the position and operation of different parts, including heating, magnetic bead control, washing, adding solutions, cooling, imaging / detection, etc. These areas can be defined in the DMF device. For example, now returning to Figure 6 , Figure 6 The diagrams illustrate different functional areas defined based on connections within and / or below (or in some variations, above) the mounting surface. Figure 6 In the case of a PDMS device, a solution can be dispensed through the top of the box (e.g., top plate) via one or more holes. Thus, the drive electrodes below the fixed dielectric can form multiple unit cells (one drive electrode per unit cell), and each cell or region of cells (multiple cells) can be controlled to perform a specific function. For example, in Figure 6In the DMF device, the DMF device includes an arrangement of zones or unit cells arranged around the periphery of the box, such as a cooling zone (e.g., cooled via a Peltier zone below) 605. These areas can also be used to store solutions and can be kept between 3 degrees Celsius and 20 degrees Celsius (e.g., less than 10 degrees Celsius, between about 2 degrees Celsius and 25 degrees Celsius). A central heating zone 609 can be used to heat droplets. One or more magnetic zones 603 can be used to turn on / off a magnetic field that can be used to fix magnetic particles (e.g., for removing material, etc.). Any zones can overlap. For example, at least one unit cell in a heating zone can also be a magnetic zone. Other functional zones include imaging / optical zones. In this case, dual functionality may be possible because a magnet can be positioned directly below the heating zone when resistive heating is used.

[0315] In addition to the zones formed by the configuration of the mounting surface of the DMF apparatus, functional zones for providing aliquots of solution, mixing solutions and / or removing solutions may also be formed in the cartridge, for example, by cutting into the top plate to provide intimate access to the air gap. Figure 6 In the embodiment of the present invention, the upper (top) plate microfluidic area has been made transparent. Usually, the microchannel can be used for mixing, distributing waste and taking out the waste on the top plate from the air gap area. In addition, any of these boxes can also include a reagent reservoir in the top plate. Microfluidics can be controlled by one or more valves (e.g., valve controls) for distributing and mixing and taking out waste.

[0316] box

[0317] Typically, a dual-plate cartridge as described herein may include a dielectric, a first hydrophobic coating on the dielectric, a second hydrophobic coating on a ground electrode (and / or a top plate), and a top plate to which the ground electrode is coupled. For example, the hydrophobic coating may be a Teflon coating. The cartridge may also include one or more microfluidic channels, particularly microfluidic channels having controlled access to an air gap formed directly in the top plate.

[0318] For example, Figure 7A-7D An example of a dual plate box 700 is shown, which includes a microfluidic area 703 on the upper surface, which is covered by a cover 703 having one or more access ports 705, 707 for entering the microfluidic portion of the device. The cover 703 may also include one or more valves and / or one or more openings that can be used to deliver and remove fluids and / or gases (e.g., air). The box may also include an opening through the top plate 713, which includes an opening that connects the microfluidic channel to the air gap area within the channel.

[0319] Any of the boxes described herein may also include one or more transparent window areas 711 for optically imaging one or more areas (readout areas) within the air gap. Optionally, the dual-panel box may have a transparent, translucent, substantially transparent, or substantially translucent top panel. In embodiments using a single-panel box, the lack of a top panel may provide full access to any point within the interior surface of the single-panel box.

[0320] Figure 7B yes Fig. 7A A top perspective view of a box. Figure 7B A side view of the box is shown, showing the bottom dielectric film material at the bottom. Figure 7C It is not visible in the figure, but may refer to the spacing 753 between the dielectric and the ground electrode. Fig.7D The top plate is shown with the covering removed. FIG. 7A to FIG. 7D , with the top removed, showing both the first microfluidic channel and the second microfluidic channel, each microfluidic channel having an opening from the microfluidic channel into the air gap. Fig.7D In the , both channels can be used simultaneously by pushing / pulling fluid through one channel into the unit below them for flushing, mixing, waste removal, etc. Figure 7A-7D In the embodiment, there are through holes through the top plate for air to enter. Although the top plate can be thicker, in some variations, it may be beneficial to include more reagents, including freeze-dried reagents that can be rehydrated.

[0321] Figure 8-Figure 9 Different examples of boxes that can be used are shown. Figure 8 In the exemplary dual-plate box 800 (similar to Figure 7A-7D ) is shown on a mounting surface 803 including electrodes. The box 800 includes an air gap ( Figure 8 The other end of the cartridge includes a window region 807 through which a portion of the air gap can be imaged. Both the front (window) region and the back (microfluidic) region of the cartridge may include an access region for accessing the air gap and / or the microfluidic portion. Fig. 9 In Figure 2, three different DMF design configurations on paper are shown. The paper DMF devices are formed by inkjet printing an array of silver drive electrodes with reservoirs connected to contact pads onto a paper substrate.

[0322] In the box, the top plate can be any suitable material, including transparent materials, such as acrylic resin. The top plate can be formed of (or can include) one or more conductive polymers. A ground electrode can be formed on the top plate.

[0323] As already discussed above, any of these devices and methods may include one or more microfluidic channels integrated into the cartridge. In particular, the device may include a microfluidic mixing and extraction region. Figure 13-15 1504. The microfluidic channels 1501 and 1503 are shown in the figure. For example, two microfluidic channels 1501, 1503 can be formed into the top plate of the air gap, and the openings leading to the air gap can be positioned to be no more than a fixed distance from each other. The fluid can flow from one microfluidic channel to another microfluidic channel through the air gap. The area of ​​the air gap between these openings can bridge these two areas 1505. Compared to being easily carried out in the air gap, this configuration can be used to mix larger droplets (e.g., greater than 5 microliters, greater than 7 microliters, greater than 10 microliters, greater than 15 microliters, greater than 20 microliters, greater than 25 microliters, greater than 30 microliters, greater than 1 ml, etc.).

[0324] For example, in Fig.13 , a first pressure source 1507 (negative pressure and / or positive pressure) is shown attached to one end of a microfluidic channel, and a second pressure source 1509 (positive pressure and / or negative pressure) is shown attached to another microfluidic channel. Fluid can be drawn from the air gap into the first channel 1501 through opening 1505; alternatively or in addition, by applying positive pressure 1507, fluid can move from the first channel 1501 into the air gap through opening 1505; at the same time, by applying negative pressure 1509 in the second channel, fluid can be drawn from the air gap into the second channel at or near the same opening 1505. Alternating positive and negative pressures can allow relatively large volumes of solution to enter and exit the air gap between two microfluidic channels, as shown in FIG. Fig.14 and Fig.15 As shown in .

[0325] exist Figure 13-Figure 15 In the example shown in , the top plate integrates the microfluidic channels and the reservoirs and tubes; alternatively or in addition, one or more ports (e.g., for connection to a pressure source, valves, and the like) may be included. For example, a cover over the microfluidic channels may be included with ports and / or valves and the like. Positive and negative pressures may be applied in the microfluidic channels, for example, by reversing the polarity of a peristaltic pump.

[0326] Fig.16A and Fig. 16B Schematic diagrams illustrate examples of methods for applying fluid to the air gap of DMF apparatus 1120 and removing fluid from the air gap of DMF apparatus 1120 (including washing). Fig.16A1104, an air gap 1121 of the cartridge is formed between the top plate 1117 and the bottom dielectric 1126. A connector interface 1127 connects the combined inlet / outlet ports of the first fluid channel 1143 and the second fluid channel 1145. These fluid channels may be connected to one or more reservoirs 1105, 1107. As already described above, in some variations, two separate connector interfaces (ports) may be used, with each fluid line (e.g., a fluid line may be a microfluidic channel, as described above) being connected to one connector interface (port). The bridging droplets in the air gap region 1121 can be connected to both inlet and outlet lines, and fluid can be drawn into and out of fluid lines 1143, 1145 to mix the droplets, add fluid to the droplets, remove fluid from the droplets, expose solid phase capture elements (e.g., magnetic beads, non-magnetic beads, etc.) to the same fluid to repeatedly deplete the fluid from the analyte of interest, for example, to concentrate the analyte on a solid phase or other surface), etc.

[0327] Alternatively, if Fig.17A and Fig. 17B As shown in FIG. 1 , the box may include air gaps of different heights. For example, in Fig.17A , the air gap around the connector interface 1127 can be larger (e.g., between 0.5 mm and 2 mm) than the air gap between other areas of the top plate and the dielectric 1126 because a portion of the top plate 1115 (or a separate top plate 1115 connected to another top plate 1117) can be spaced farther from the dielectric 1126. Similarly, in Fig. 17B For example, by spacing a portion of the top plate 1117 further away from the dielectric 1126 bottom layer, the air gap 1119 near the connector interface at the edge of the device can be larger than the air gap 1121 in other areas.

[0328] The diagram shows Fig.17A The schematic diagram of the prototype DMF device and the cartridge shown in Figures 18A-18C is illustrated in , and was used to demonstrate proof of principle for mixing larger volumes of solutions in the air gap of a DMF box. Fig.18A In the embodiment, the upper plate of the DMF box includes an opening through the top plate 1801, which is connected to the first fluid line 1843 and the second fluid line 1845. By alternating negative pressure (suction) between the first fluid line and the second fluid line, the fluid moves back and forth between the first reservoir 1805 and the second reservoir 1807, as shown in FIG. Fig.18A , Fig.18B and Fig. 18CIn this example, when fluids are exchanged between reservoirs, magnetic particles holding the analyte of interest are magnetically held in the air gap (e.g., against a bottom, e.g., hydrophobic coated dielectric) by DMF device 1809, which enhances binding and / or washing.

[0329] In any of the air gap devices described herein, evaporation can be controlled or reduced, particularly when droplets in the air gap are heated. Figure 19A-Figure 19C The figure shows the effect of evaporation on the droplet 1903 after only a few minutes. The intact droplet Fig.19A After one minute at 95 degrees Celsius, the droplet volume has been significantly reduced (e.g., a loss of between 5%-15% of the droplet volume, as shown in FIG. Fig.19B Two minutes later ( Fig.19C ), the droplets are between 20% and 34% smaller. To prevent this loss due to evaporation, the droplets in the air gap can be sheathed or covered in a non-polar sheath, such as Figure 20A-20C For example, a liquid paraffin material (e.g., a non-polar material that is liquid within the working range described herein, e.g., between 10 degrees Celsius and 99 degrees Celsius) may be used. Fig. 20A In the embodiment, the shielded droplets 2003 in the liquid paraffin 2005 are heated (e.g., to 65 degrees Celsius or higher). Fig. 20B ), the droplets had not evaporated significantly. Similarly, after 2 hours ( Fig. 20C ), the droplets maintain approximately the same volume.

[0330] In use, the non-polar cover material can be added and removed at any point during the DMF process, such as Figure 21A-Figure 21I . Surprisingly, removal can be accomplished, for example, by aspirating the shrouded droplet upward from the air gap, for example, from a port into the microfluidic channel, as described above. For example, liquid paraffin can be removed to a waste reservoir by applying negative pressure to the droplet from a port across the top or side of the air gap. The lower density liquid paraffin can be the first layer to be aspirated upward, which leaves the aqueous droplet behind. Previously, it was believed that removing the shroud of a non-polar liquid was difficult or impossible.

[0331] For example, Fig.21A A shrouded droplet is shown in which an aqueous droplet 2101 is surrounded by a non-polar liquid 2103 (e.g., liquid paraffin). In this example, small bubbles have also formed in the liquid paraffin. The droplet can be easily moved, such as Fig.21B , which shows the movement of droplets by coordinated application of energy to drive electrodes to alter the electrowetting of aqueous droplets. Fig.21BIn the embodiment of the present invention, the hooded droplet has been moved to the right side. Initially, by directly applying the non-polar liquid that enters the air gap on the droplet, or applying in the region of the air gap that the droplet can move to, the aqueous droplet can be combined with the non-polar liquid. The hooded droplet can also be combined with one or more other droplets, and this other droplet can include the non-polar liquid droplet itself, or can not be hooded. In some modifications, the hooded droplet (comprising small aqueous droplets and relatively large-volume non-polar solution) can be combined with the target droplet, so that the target droplet is hooded. A small amount of aqueous liquid in the hooded droplet can be a buffer, a diluent or other solutions that allow the hooded droplet to move in the air gap. When used together with the DMF box with a larger (e.g., 0.5mm or larger) gap width, this technology is particularly useful. Larger gap width may originally make larger droplets difficult to keep the hood of the non-polar hood material that is usually less dense. Fig. 21C and Fig.21D The figure shows a droplet 2101 that has been combined with another droplet to form a larger jacketed droplet 2101'. The larger droplet can also be moved by controlled actuation of the drive electrode, such as Fig. 21C and Fig.21D As shown in .

[0332] FIG. 21E to FIG. 21I The figure shows the use of a non-polar liquid mantle in a sample including magnetic bead material. Fig.21E In the embodiment of the present invention, the jacketed droplet includes a small amount of aqueous liquid 2121 and a relatively large volume of non-polar jacket material 2123, which can be combined, for example, by moving the jacketed droplet 2123 into the sample droplet 2121, such as Fig.21F This allows them to be combined so that the cover material now covers the sample droplet. In this case, the sample droplet is quite large and includes a certain concentration of sample absorbing magnetic beads.

[0333] Once assembled, the jacketed droplet 2121' can be moved (via DMF) to a port leading into an air gap from which the solution can be extracted, such as Fig.21H In this example, the solutions can be mixed by applying positive and negative pressures to move the solutions into and out of the fluid channel 2131. The non-polar solution that jackets the microdroplet can be removed by applying negative pressure to draw the solution out of the air gap through the top port; the first solution removed is the jacket material. Thereafter, as Fig.21I As shown in , the magnetic particles to which the desired analyte has been bound can be held on the bottom side of the air gap, for example by applying a magnetic field, and the droplet solution can be removed and / or washed in the absence of a non-polar sheathing solution, which might otherwise interfere with the binding of the analyte to the magnetic particles or the release of the analyte from the magnetic particles. Fig.21IIn the embodiment of the present invention, the magnetic particles 2133 are retained in the air gap and a separate wash buffer can be applied by moving a washing and / or elution droplet 2135 over the magnetic particles.

[0334] In addition to the techniques discussed above for controlling evaporation (e.g., using a sleeve of a nonpolar liquid), any of the methods and devices described herein may also include controlling the partial pressure of water vapor inside the box to produce a "zero evaporation" condition, for example by balancing the rates at which water molecules leave and enter the water surface. The balance need not be perfect, but can be adjusted by adjusting the temperature and pressure so as to get as close to a zero evaporation condition as possible. This can vary with temperature; for example, when controlling relative humidity, it may be best to adjust the humidity up and down with temperature, such as during hybridization or PCR cycles using the device. Alternatively or in addition, any of these devices may use local replenishment to regulate evaporation by slightly moving droplets to recapture nearby condensation (see, e.g., Figure 19B-Figure 19C , which shows an evaporative droplet surrounding a main droplet). Any of these methods and devices may also or alternatively use a walled-inheating zone to reduce the surface area from which evaporation can occur. For example, as mentioned above, in certain variations, the mounting surface of the DMF device may include protrusions that form localized areas in the box, because the vacuum can be precisely applied to control the contact between the flexible dielectric and the electrode, the protrusions on the mounting surface can create chambers or channels in the air gap, which includes partially forming a walled-inheating zone that can reduce the evaporation surface area. In certain variations, the top plate can be spaced differently across the box; the evaporation rate can be lower for thinner droplets than for thicker droplets. Therefore, any heating zone can have a narrower air gap width to reduce evaporation.

[0335] In any large volume droplet DMF cartridge, for example, a DMF cartridge having a gap spacing of 0.5 mm or greater (e.g., 0.6 mm or greater, 0.7 mm or greater, 0.8 mm or greater, 0.9 mm or greater, 1 mm or greater, e.g., between 0.4 mm and 2 mm, between 0.5 mm and 2 mm, between 0.5 mm and 1.8 mm, between 0.5 mm and 1.7 mm, etc.), it has proven particularly difficult to dispense droplets with predictable volumes because the surface tension of relatively large droplets may require a larger amount of energy to release smaller droplets from larger droplets. Typically, in digital DMF systems, the ratio between the spacer (air gap) thickness and the electrode size determines the volume of droplet dispensed. In conventional digital microfluidics methods, a spacer thickness of less than about 500 microns (0.5 mm) allows electrowetting forces to separate a unit droplet from a larger volume of liquid; this is not possible at higher spacer thicknesses (e.g., greater than 500 microns). Methods for separating a unit droplet from a larger volume in an air gap having a width (e.g., spacer thickness) of 500 μm or more are described herein. In certain variations, this can be performed, for example, by filling an area of ​​the air gap with a solution dispensed from a port (the port can be a side port, a top port, or a bottom port), and then selectively activating a unit (corresponding to a drive electrode) in the filled area, and then pumping the solution back into a port (or another port) that is offset from the activated electrode so that the droplet remains on the activated electrode when the solution is pumped into the port; the droplet on the activated electrode is disconnected from the larger filled volume (e.g., by necking off), which leaves the dispensed droplet behind, where it can then be driven by the drive electrode, combined with one or more other droplets, etc.

[0336] For example, an integrated companion pump can be used to drive a large volume of aqueous solution into the DMF device (e.g., into the air gap of a DMF box) and onto an activated electrode. The aqueous solution can then be pumped away from the DMF device, which dispenses the unit droplets onto the activated electrode. Figure 22A-22D An example of this method is shown in Figure 1. Fig.22A In the embodiment, the port 2201 leading to the air gap 2205 of the DMF box is connected to a fluid channel (e.g., a microfluidic channel as described above), which is connected to the fluid channel at Fig.22A2209 is shown as holding an aqueous solution (reagent 2203). In this example, a single drive electrode 2207 has been actuated; alternatively, in some variations, the electrode is not activated until after the area of ​​the DMF device is filled. Pre-activating the electrode can help distribute a predetermined amount to the unit cell defined by the drive electrode. In any of these examples, more than one adjacent drive electrode can be activated to dispense a larger volume droplet.

[0337] Next, if Fig. 22B As shown in , the air gap region including the activated drive electrode is filled with aqueous solution 2203. Fig.22A The release of a large volume (e.g., 250 μL) from a channel (tube 2209) is shown. In certain variations, when the reagent approaches the distal channel 2209, the drive electrode 2207 is activated (e.g., an AC potential of 390 Vrms, or an alternating field effect is otherwise generated by using a DC potential), which can generate electrowetting forces that further promote transfer of the reagent from the tube 2209 to the activated drive electrode 2207; further flow from the channel occurs, causing the droplet to grow to completely cover the activated drive electrode.

[0338] exist Fig. 22C , the aqueous solution (reagent 2203) is then drawn from the air gap through the same port 2201 or a separate port, where the activated drive electrode is separated from the port into which the solution is drawn by a certain distance (e.g., the distance can be approximately equal to the width of the activated electrode); the distance is sufficient so that the droplet on the activated drive electrode is disconnected from the liquid necking drawn back into the channel 2209. For example, as Fig. 22C As shown in , aspiration of the reagent back into the tube can cause the droplet to neck off from the rest of the solution; the neck region continues to shrink until a unit droplet (e.g., 10 μL) is left on the activated drive electrode, as shown in FIG. Fig.22D The same process can be repeated with activation of two, three, and five electrodes to dispense approximate multiples of unit droplets (e.g., 20 μL, 30 μL, and 50 μL), respectively, as shown in FIG. Figure 23A-23E Multiple droplets can be dispensed and combined individually, or alternatively multiple electrodes can be used to dispense larger volumes at once, as mentioned. The size of the droplet (droplet volume) can be based in part on the size of the drive electrode and the spacing of the air gap.

[0339] Figure 23A-23F The figure shows the dispensing of various predetermined volumes of solution from the reservoir on the top of the box using the method described above. Fig.23AIn the example, the area of ​​the air gap including the port connected to the channel is filled with solution 2301, which holds the solution above the larger air gap (e.g., 0.5 mm width), as shown, and a single activated electrode is used to interrupt a predetermined volume of solution (e.g., 10 microliters), as shown. Fig. 23B The droplet can be moved away from the filled area and the process repeated multiple times to produce multiple droplets of approximately uniform volume (e.g., 10 microliters + / - 5%, 10%, 15%, 20%, 25%, etc.). Fig.23D , a first unit droplet 2303 (e.g., having a volume of 10 microliters) is shown adjacent to two combined unit droplets 2305 that form a second droplet having a 2× volume (e.g., 20 microliters). Similarly, Fig.23E A large droplet 2307 (eg, 50 microliters) is shown formed by combining five unit droplets. Fig.23F The figure shows that a larger drive electrode 2315 (eg, having approximately 4× the surface area) is used that can be activated when filling the air gap area to form a larger unit droplet 2311 (eg, a 40 μL unit droplet).

[0340] Thus, by filling or flushing the dispensing area of ​​the air gap with a large volume of aqueous solution, and activating the drive electrode (or over an already activated drive electrode), and then removing the solution (e.g., pumping it out), a relatively precise volume of droplets can be left behind. As mentioned, when using a large volume DMF device (box), for example, with a gap between 0.4 mm or 0.5 mm and up to 3 mm, the technology can be used to dispense a smaller volume of droplets from a large volume reservoir with a reasonable amount of force; unlike an air gap DMF device with a smaller air gap, which can directly dispense a smaller volume of droplets from a larger volume by applying electrowetting energy, the larger force effectively prevents direct dispensing by DMF in a larger air gap device. In many of the examples provided herein, the gap spacing of the air gap is between 1 mm and 1.3 mm (e.g., about 1.14 mm), although at least up to 3 mm spacing has been successfully used.

[0341] As described herein, dispensing of solutions can be particularly important in processing samples (eg, mixing, etc.) and in replenishing solution lost due to evaporation in such systems.

[0342] User Control Interface

[0343] In any device and method described herein, the DMF device can be controlled by a user so that the DMF device can perform one or more schemes (e.g., laboratory procedures) to the sample inserted into the DMF device (e.g., box). For example, the DMF device can include a user interface that dynamically and flexibly allows the user to control the operation of the DMF device to perform a scheme selected by the user or input by the user. Typically, when the treatment scheme for operation is converted by the DMF device, there are many considerations, including preventing contamination during the program. Contamination may occur when moving sample droplets on the path taken by the earlier steps (or parallel steps) in the program, and the scheme is being executed in this movement. Typically, one or more reaction droplets being processed may need to be moved to different positions in the air gap of the DMF box, and / or temporarily move out of the air gap area. In addition, it will be difficult for the user to coordinate these movements to avoid earlier paths or future paths (e.g., contamination), and to remember which positions are suitable for heating, cooling, mixing, adding, removing, thermal cycling, etc.

[0344] This article describes a user interface for controlling the operation of a DMF device, which allows the user to more easily input scheme information / steps into the DMF. This can be achieved in part by the following operations: a set of graphical step representations (e.g., mixing, adding, heating, cooling, circulation, washing, etc.) of the steps that can be performed are provided, and the user is allowed to select / input these steps in a manner that the duration of the step or the degree (e.g., temperature, etc.) applied are equally intuitively provided. After input, the device can then determine effective access, to perform the scheme of input within the predetermined layout constraints of the DMF device and / or box, to avoid contamination. For example, any of these devices can determine the access (pathfind (pathfind)) that prevents or reduces the path intersection in the air gap, and such intersection in the air gap can cause contamination.

[0345] Fig.24 is an exemplary schematic diagram illustrating the steps involved in controlling any DMF device described herein. For example, Fig.24 In the example, a user can enter a solution using a graphical / visual user interface (referred to herein as "SAM"). This can be referred to as Figure 25A-Figure 26B ). The graphical scheme can then be converted into a series of target destinations, and the target scheme can then be used by the device to adapt the scheme to the DMF device. Fig.24 In the process, the system can determine the path and obtain control of the drive electrodes, heaters, cooling (e.g., Peltier), magnetism, microfluidics (pumps), etc.), etc., in order to complete the protocol. The path can be optimized to require the shortest path, but constrained by limiting or reducing overlap in the path to prevent contamination, loss of materials (including reagents and / or Teflon), heat dissipation, etc.

[0346] As mentioned, Fig.25A and Fig.25B An example of a visual interface (e.g., a graphical user interface) for inputting a desired solution is shown. Fig.25A In FIG. 1 , a set of control icons (“move”, “heat”, “remove”, “circulate”, “mix”, “interrupt”, “dispense” and “wait”) are shown. The user can select or arrange these icons to provide a graphical representation of the treatment scheme, such as Fig.25B Each icon may have an associated duration, and thus, the icons may be used to select processing instructions or steps for a sample. In this example, the icons are uniquely identified by one or more of: color, image, and text.

[0347] The user may enter the protocol directly into the device, or into a computer or other processor in communication with the DMF device.

[0348] After input, the scheme can be converted into a data structure format (e.g., a JSON format indicating the name of the scheme and sample, where the sample goes, how much volume to use, etc.). Then, the data structure can be used directly or converted into a format (e.g., java script) so that the device can determine the path taken in the box to achieve the desired scheme. Path discovery can be performed locally (e.g., in a DMF device) or remotely, and communicated to the DMF device. Path discovery can be configured to maximize based on the shortest path length, which also avoids crossing or certain crossings to prevent contamination. Therefore, the device can determine the shortest route to avoid contamination. Typically, the user interface can allow the user to easily select the desired action and elements (e.g., mixing, etc.); the device may already be familiar with reagents (e.g., components of the device). The user can then select an action, duration, temperature, etc.

[0349] Figure 26A-26H The figure shows an example of a device for determining a path based on an input scheme. For example, Fig.26A A graphical representation of a specific configuration of the DMF box air gap for planning the first set of steps (e.g., sample preparation) is shown. The device can know the distribution of units in the air gap, as well as the configuration of functional areas (heater, cooler, mixing / microfluidics, waste removal, distribution, etc.) in the DMF box. Fig.26B is a graphic representation of an apparatus for determining a path for labeling a sample having genomic DNA (or a fragment of DNA) with an adapter tag. Fig.26CIn, a step of moving a first buffer (e.g., SureSelect QXT buffer) to an appropriate location for future processing is performed. The path can be selected based on both past movements and future movements, and can be recursively modified as future protocol steps are defined. In Fig.26D , the path used to move the DNA sample is shown (black). Fig.26E shows the movement of the enzyme mix from a cooling area where it is stored for combination with a sample; Fig.26F The user is shown mixing a sample with a buffer and enzyme mixture. The mixed sample can then be moved along the calculated pathway ( Figure 26G ) to the heating / cooling zone for circulation ( Fig.26H ). Additional steps may then be performed as indicated.

[0350] Figures 65-68 Examples of user interfaces and methods including them for controlling the operation of the systems described herein and for selecting, editing, and storing regimens are illustrated.

[0351] exist Fig.61 In the present invention, the user interface can be displayed on a display of the device (e.g., a touch screen) and / or a remote computer device (e.g., a smart phone, a laptop computer, a desktop computer, etc.). Fig.62 An example of a user interface is shown that instructs the user what to apply to various box inputs. Fig.63 It is an example of a cloud interface for selecting, modifying (editing) and / or sharing protocols using the visual scheme design language described herein. The user interface is an open canvas interface that allows users to create, edit, delete and save any scheme in a drag-and-drop interface. Users can select the unit operations (heating, cooling, mixing, elution, washing, incubation, thermal cycling) of reagents, samples, systems and build their own schemes. As mentioned above, a pathfinder (block to the conversion of actions on the device) algorithm can adopt the constraints (pollution, volume and viscosity) of samples and reagents, electrode grids and box constraints, and find the best path between two points to avoid all the constraints mentioned above. Users can share the constructed schemes made on their open canvas in the scheme storage. The open canvas unit operation can be automatically converted into a scripting language for scheme execution by the device.

[0352] For example, users can share recipes from other users or labs. For example, a user from organization A has created recipe X for x application in the cloud interface, with their preferred conditions and volumes. The user from organization A can share recipe X with the community in the marketplace. The user from organization B can read and download recipe X, edit it or load it directly into their machine and run it. The recipe can have a fee paid by the user from organization B, and the machine provider and the user from organization A can share the revenue. This is done in Fig.64 Shown in.

[0353] Fig.65 and Fig.66 Examples of user interfaces for selecting a plan and viewing a plan are shown, respectively.

[0354] Figure 66 to Figure 68 The diagram illustrates a user interface for programming and operating the device both on the device and / or on a remote processor (e.g., a desktop computer, laptop computer, tablet computer, etc.).

[0355] Thermal Control

[0356] Any of the devices described herein can include features for thermal control (e.g., heating and / or cooling) and / or droplet detection (e.g., tracking and / or identification). For example, an apparatus including a cartridge and device can be configured to quickly and accurately cycle the temperature of a droplet. Alternatively or in addition, droplet detection can quickly and accurately scan an electrode grid for droplets (including, but not limited to, reagents, wax, water, etc.).

[0357] As described above, the device can be configured to include one or more thermal control elements, including cooling and / or heating. For example, the device can include resistive heating in some of the units to heat the droplets in the air gap. For example, in some variations, the resistive heater can be included in layer 2 of a printed circuit board (PCB), such as a portion of a first copper layer below the surface of the PCB. The device can also include a heat sink or cooling element, such as a liquid cooler (chiller) that is continuously thermally connected to the PCB. Any of these variations can also include one or more of thermal mass reduction and / or heat conduction through the PCB (e.g., through electrodes that form part of the PCB in the device), which can increase the rate of temperature change in the unit.

[0358] Thermal mass reduction can refer to reducing or removing thermal mass from a device (e.g., a system, an apparatus, etc.) to reduce the total amount of energy required to reach a temperature or temperature range. Ideally, when there is less thermal mass, less energy needs to be taken out of the system to reduce the sample temperature during thermal cycling, thereby enabling faster cycle rates without the need for very large heating and cooling systems (i.e., no more liquid cooling of the stack). The devices and methods described herein can reduce thermal mass by reducing / removing thermal mass from above the droplet or above the area in the upper (top) plate of the box that holds one or more droplets. For example, when the upper plate / top plate is formed of an acrylic material or a polycarbonate material, the thermal mass above the air gap area can be reduced by including one or more cavities (e.g., a polycarbonate structure and / or an acrylic structure) in the top plate and filling the cavities with an insulating material or a material with low thermal conductivity (e.g., air). The cavity may be positioned in the top plate of the box above the thermal controller region so that when a droplet of material is below the cavity, heating / cooling applied by the device (e.g., by the PCB) may more rapidly change the temperature of the droplet in the air gap region. Removing thermal mass above the droplet may be incorporated into the design of any of the boxes described herein. The cavity may be formed near the bottom surface of the top plate (e.g., immediately on one side of the air gap); the cavity may partially pass through the thickness between the top and bottom surfaces of the top plate. Fig.28 The figure shows an example of a portion of a box, which shows a thermal control area in the top plate 2801 of the box 2804. The box can be positioned on the device 2803. The droplet 2807 in the air gap area of ​​the box (e.g., the area defined by the bottom surface of the upper plate 2801 and the top surface of the lower dielectric material sheet 2809). Thus, in a variation in which the box body including the top plate is formed by a solid polycarbonate sheet on the top plate, one or more cavities (e.g., Fig.29 ) and can be enclosed or filled with an insulating material with low thermal mass. This can prevent heat from transferring from the sample to the storage area above it. The void replacement material can be air or a similar material with low thermal conductivity and low thermal mass.

[0359] Alternatively or additionally, thermal mass can be removed from the PCB by removing material (e.g., with precision milling) and / or using material with very low thermal mass. For example, one or more layers of the PCB can be removed in the heater zone (e.g., the heating area or the thermal control area) to reduce the thermal mass. This can be done from the bottom side of the board so as not to damage the surface finish of the electrode.

[0360] Fig.29is an example of a milled area in the PCB of the device having a lower thermal mass in order to increase the response time to temperature changes of the droplets in the air gap of the box. In this schematic example showing a cross-sectional view, the layers of the bottom (e.g., PCB) can include one or more layers of, for example, copper, and the dielectric below the droplets (in the PCB of the device) has been milled to create a cavity or void that can be filled with an insulating material (including air). Therefore, heat conduction through the PCB can be reduced. Typically, the cavity in the top plate and / or the bottom plate can help thermally isolate the droplets in the air gap between the top plate and the bottom plate.

[0361] In addition to accelerating temperature changes in the droplet by reducing thermal mass, any of the methods and apparatus described herein can also increase thermal conductivity between the heater source and the electrode to improve performance. For example, if the heater layer on the PCB is in layer 2, then using a high thermal conductivity dielectric layer will increase heat transfer from the heater layer to the electrode, such as Fig.30 As shown in . Fig.30 A highly conductive dielectric 3005 is shown between the heater 3003 and the electrode 3001 copper region.

[0362] In certain variations, the device (and in particular the PCB portion of the device) may optionally or additionally be configured to increase thermal conductivity by including one or more thermal vias near each active (e.g., drive) electrode / cell. A thermal via may be a channel or passageway in thermal contact with an area near the electrode, including an area beneath the electrode (such as PCB material) of a thermal control area, and may be filled with any thermally conductive material. For example, filling the vias with a thermally conductive material (e.g., but not limited to: copper, epoxy, resin, etc.) may further increase thermal conductivity and may significantly increase the thermal response time of droplets or other materials in the air gap. Thus, heating and / or cooling may be much more rapid than without the vias. The thermally conductive vias may be implemented with or without milled areas in the PCB ( Fig.31A and Fig.31B As shown in Fig.31A shows the milled area with thermal vias, Fig.31B shows thermal vias without milled areas). For example, Fig.31A The figure shows a plurality of thermally conductive vias 3105 in an example of a base plate (eg, a PCB) where the base plate has been milled to provide areas of thermal isolation around areas of thermal control activity.

[0363] The vias may be filled with any suitable thermally conductive material. In some variations, the vias are filled with a non-conductive thermally conductive material (eg, epoxy, resin, etc.).

[0364] One end of the through hole can be in thermal contact (e.g., can touch) with the final upper surface of the device (e.g., the box contact surface) and / or an area adjacent to the electrode. In particular, when the thermal via is filled with a conductive material (e.g., copper), the thermally conductive via can contact an area that is closely adjacent to the electrode but not in electrical contact with the electrode. Another portion of the thermal via can be in thermal contact with the heat sink below the upper surface (e.g., on the side surface and / or bottom surface). In some variations, the opposite end of the through hole can be in contact with a temperature control surface (e.g., a cooling surface, a heating surface, etc.). In some variations, the through hole can be thermally connected to a thermal controller (e.g., a heater, a cooler, a heat sink, etc.) at one end region; the through hole can pass through a vacuum chuck on which the PCB can be located.

[0365] The vias may be of any suitable size. For example, the thermally conductive vias (referred to herein as thermal vias or simply vias) may have a diameter between 0.1 mm and 3 mm, between 0.1 mm and 2 mm, between 0.5 mm and 1.5 mm, about 0.8 mm, about 1 mm, about 1.2 mm, about 1.4 mm, etc. The thermal vias may have a circular cross-section, an oval cross-section, a rectangular cross-section, a square cross-section, a triangular cross-section, or any other cross-section and may be cylindrical, extending from a thermal control (e.g., one or more of a heater, a cooler, a heat sink, etc.) through the printed circuit board to an area immediately below or closely adjacent to the electrode (in some variations, not contacting the electrode so that they remain electrically isolated from the electrode, but not thermally isolated).

[0366] As mentioned, any suitable number of through-holes may be formed per cell (e.g., associated with each electrode that drives the movement of fluid in the air gap of the cartridge). For example, each cell in a thermal control region (which may include multiple thermal control units) may be in contact with 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc., or more through-holes. For example, each thermal control unit may be in contact with more than 8 through-holes.

[0367] The use of thermal vias may provide significant improvements in the rate of change of heating and / or cooling of a thermally controlled area as compared to systems that do not include thermal vias.

[0368] Box Features

[0369] In addition to the features described above, any cartridge may alternatively or additionally include one or more openings into or through certain upper top plates of the cell (e.g., areas that would correspond to one or more drive electrodes). These openings may be open and may allow direct imaging 3221, such as Fig.32 Alternatively or in addition, the openings may be used to passively distribute fluid from the air gap. For example, Fig.32, an opening 3203 in the top plate of the box 3205 can be used to passively dispense fluid from a droplet 3211 positioned below the opening; the droplet can move via DMF below the opening, as described above. After being positioned, a predetermined amount of fluid can be passively dispensed from the droplet into the opening, for example via capillary action, and the droplet can move away from the opening. The sampled material can then be analyzed or processed using microfluidics in the top of the box, and / or can be analyzed in place. Optionally, the sampled material can be added to another droplet 3219 after the first droplet 3211 has moved away; the second droplet is positioned below the opening through the top plate including the sampled material 3203. Based on the size of the opening 3203, the sampled material (fluid) from the first droplet can be a metered amount. The top plate can include a hydrophilic surface or a hydrophilic surface coating. In certain variations, the openings in the top plate can be preloaded with a material, such as liquid wax or other coating material that can combine with the droplets as they move beneath the openings (e.g., to dispense a coating material, such as an anit-evaporation coating of liquid paraffin, oil, etc.). The openings in the top plate can also act as an insulator. The openings can extend over a portion of the cell so that the return electrode can be on the edge of the opening. The openings can be of any size and dimension (e.g., circular, square, etc.). Although Fig.32 The variation shown in shows imaging through the top plate (using optics 3221), however in some variations, imaging can be done from the bottom through the bottom of the box. For example, an area of ​​the bottom of the box (e.g., a dielectric film) can be transparent or optically permeable for imaging (e.g., fluorescence).

[0370] In any of the cassettes described herein, the top plate may include a plurality of manifolds for delivering one or more materials into the air gap. Fig.27A and Fig.27B The figures show one example of a top plate formed from a polymer material (eg, acrylic and / or polycarbonate). Fig.27A An upper region of the top plate is shown (which may be covered by one or more covers, not shown). Fig.27AIn, multiple distribution areas 2704, 2706, 2708 of different sizes are included. For example, a smaller 2706 (e.g., 2 microliters-20 microliters in size), a medium 2704 (e.g., 100 microliters to 1mL) and a large 2708 (e.g., 1mL to 5mL) are shown, and a waste and / or mixing area 2710 is also shown. These chambers can be preloaded with fluid, and each chamber can include an opening leading to an air gap area. A pressure control can be used to apply pressure to drive the fluid out of the opening in the distribution area and drive it into the air gap, which can be controlled by the device or other equipment holding the box. Therefore, the device can include one or more pressure interfaces, which can be used to control the release of the fluid from the top plate and the fluid handling in the top plate.

[0371] In some embodiments, the top of the box can be covered with a protective film, such as the 200 μm thick top cover film shown. The bottom surface of the box body (the top surface forming the air gap) can be covered with a conductive base material, which can be hydrophobic or can include a hydrophobic coating. For example, the film can be a COC film sputtered with ITO (conductive material) and CPT (all-hydrophobic base) to seal the channel on the bottom side of the main box body.

[0372] Fig.27B The diagram shows Fig.27A The bottom side of the top plate portion shown in FIG. The bottom side may be coated or covered with an electrode and / or a dielectric and / or a hydrophobic coating, as described above. Fig.27B In the embodiment of the present invention, the top plate may also or alternatively include one or more channels in the surface of the plate that can allow mixing, as described above. The bottom surface of these channels can be formed by the upper dielectric and / or return electrode (which, in certain variations, can include dielectric, hydrophobic membrane and / or electrode layers). In general, the boxes described herein may include one or more serpentine mixing channels that can provide a fluid path for the entire volume of liquid so that they can mix disorderly in the EWOD region.

[0373] In any box described herein, the bottom surface of the drive electrode in the placement surface and particularly the contact device that can be configured to contact the device is formed by a dielectric material, as described above. The bottom surface can be a sheet of dielectric material having a first side and a second side (the first side forms an exposed bottom surface on the bottom of the box). The second side of the dielectric material sheet can include a hydrophobic surface and can form one side of the air gap. The bottom surface can be, for example, a film that is itself a dielectric and / or coated with a dielectric material. For example, in some variations, the film is a dielectric and / or a hydrophobic film. It can be beneficial to make the bottom surface substantially flat. Any box described herein can be configured to apply tension to a sheet of dielectric material. For example, any of these boxes can include a frame to keep the dielectric material in a tensioned state. Therefore, the box can include a tensioning frame that holds the bottom sheet of the box.

[0374] The dielectric and / or hydrophobic membrane tensioning design can pretension the sheet (e.g., dielectric and / or hydrophobic membrane) so that the surface of the sheet is always flat and remains flat during its interface with the device mounting surface (e.g., PCB) and during use of the DMF device. The purpose of the tensioning frame that holds the (e.g., dielectric and / or hydrophobic) membrane in the box is to interface with the mounting surface (e.g., PCB interface) to ensure that the membrane remains in full contact with the electrode grid (e.g., drive electrode) throughout the use of the device.

[0375] In any of the boxes described herein, the bottom of the box may include a sheet of dielectric material having a first side and a second side, the first side forming an exposed bottom surface at the bottom of the box, as described above. Any of the boxes described herein may include a tensioning frame to keep the sheet flat by applying tension. The sheet, when exposed as the bottom of the box, may be slightly recessed compared to the outer perimeter of the bottom of the box, which may fit into a flange or recess on the device, as will be described in additional detail below. Therefore, the sheet of dielectric material at the bottom of the box does not need to be the bottom-most surface.

[0376] For example, Figure 49A-Figure 51 The figure shows an example of a box assembly that includes a frame that stretches / smooths the bottom of the box (e.g., a dielectric sheet). Figure 49A-49D An example of a tensioning frame is shown. In this example, the box body features a two-part film tensioning mechanism. Figure 49A-49BThe two parts shown in (and the assembled views in 49C-49D) can include a tensioning frame 4901 and a dielectric film frame and / or a hydrophobic film frame 4903. When assembled, the membrane forming the bottom of the box can be adhered to the dielectric film frame and / or the hydrophobic film frame 4903. The membrane and membrane frame 4903 assembly can be inserted into a groove in the tensioning frame 4911 using a connector (e.g., a snap-fit ​​mechanism). After snapping into the tensioning frame, the membrane can be pulled taught in all directions in the XY plane. The frame assembly can then be fastened to the box body. The assembled frame can include a lower contour (e.g., a cutout) area 4909, which can provide a path to electrically connect the return electrode on the upper plate bypassing the membrane on the bottom surface of the box.

[0377] An example of a box including a frame for keeping the bottom film flat is Fig.50A is shown in an exploded view. Fig.50A , the individual components of the box and film tensioning assembly are shown. The figure also outlines their arrangement during assembly. The first two components assembled may include, for example, an optically clear double-sided adhesive 5002 and a sheet of dielectric material 5003 (e.g., coated on a conductive material). A frame (e.g., a tensioning frame 5004) and a sheet including a dielectric material 5005 may also be included, and the film is fixed in place by a second portion of the film frame 5006. An air gap 5009 may be formed between the bottom surface 5003 of the film 5005 and the top sheet (which may include a return electrode).

[0378] Fig.50B Depicted are the individual components in the box and film tensioning assembly after assembling the optically clear double-sided adhesive and the dielectric material and / or hydrophobic material coated on the conductive material. The conductive material can be any conductive material, such as ITO, aluminum film, copper, and others.

[0379] The membrane / cassette and PCB interface may include a membrane tensioning frame as described above, and grooves (slots) drilled from the top surface of the PCB may form a border around the electrode grid of the device. Fig.51 An isometric exploded view of an example of the components of a box, including a membrane 5120 and a membrane tensioning frame (an outer frame 5121 and an inner frame 5123), and an upper (top) portion 5109 of the box; Fig.51Also shown is a portion of the device, including a PCB 5111 that forms a placement surface for the box. The placement surface also includes a groove 5105 to receive the flange around the bottom film of the box (in this example, formed by the tensioning frame 5103). The groove can be a groove drilled around the perimeter of the electrode grid. As shown in the component arrangement in this embodiment, the film tensioning frame 5103 can be slotted into the groove 5105 around the electrode grid. After assembly, the film tensioning frame 5103 can tension the film in X and Y, but also pull downward in the Z direction at the edge of the film. The film can be wrapped on the filleted edges of the groove, just slightly outside the boundary of the electrode grid (not shown).

[0380] Fig.52A and Fig.52B Shown are a top view and a cross-sectional view, respectively, of one example of a box including a bottom dielectric (and hydrophobic or hydrophobically coated) film, and a film tensioning frame positioned over a PCB assembly portion of a device. Fig.52B The cross section in the highlights how the dielectric film and / or hydrophobic film can be tensioned across the electrodes and sealed down using vacuum ports for at least some of the electrodes (drive electrodes) that pass through the PCB, and also illustrates the placement of the edges (extending from the film) in grooves formed in the PCB placement surface to seat the film. When fully assembled, these components can allow a secure, fully tensioned and flat dielectric (and / or hydrophobic) film to be secured to the drive electrode grid on the PCB. Fig.53 is an exploded view showing the individual components and their arrangement in an assembly which includes a box upper body frame 5306, a dielectric film 5305 held in tension by a tensioning frame 5304, a PCB 5302 forming a mounting surface on the device, grooves or channels on the mounting surface around the perimeter of an array of drive electrodes (driving electrodes) on the PCB, and a vacuum chuck 5301.

[0381] Fig.54A and Fig.54B A top view and a cross-sectional view of the assembly are shown, respectively. The cross-sectional view emphasizes the relationship of the vacuum chuck 5411 on the box 5413 and the membrane assembly and on the PCB 5415. Fig.54B The cross section in also highlights some of the different effects of the system. Arrow 5405 depicts the flow path of the vacuum from the diaphragm vacuum pump 5407 on the exterior of the chuck. This can be similar to the above Fig.35BThe same flow path described in . The arrows summarize the downward force applied to the membrane by the vacuum through the through holes in the PCB. The vacuum chuck and interface with the PCB firmly adhere the membrane to the electrode and apply force downward in Z. The membrane tensioning mechanism and PCB slot ensure that the membrane remains flat by applying force in X and Y, while maintaining contact around the edges due to the fillet along the inner edge of the slot.

[0382] Single-plate cartridge. In some embodiments, one or more single-plate cartridges may be used in a multiplex system. In this case, the single-plate cartridge includes a single plate and may be open to air above a single (e.g., first) plate; an "air gap" may correspond to an area above a plate where one or more droplets may travel when the droplets are on the single plate. A ground electrode may be positioned adjacent to (e.g., immediately adjacent to) each actuation electrode, e.g., in, on, or under the single plate. The plate may be coated with a hydrophobic layer (and an additional dielectric layer may be between the hydrophobic layer and the dielectric layer, or the same layer may be both a dielectric layer and a hydrophobic layer).

[0383] The single-panel cassette may be prepared similarly to the double-panel cassette described herein, but does not require the various layers to close and form the FIG. 49- Fig.51 The second plate of the dual-plate box of any one of the above. Similarly, the tension frame and bottom plate can be prepared using the same method to provide a sufficiently rigid single-plate box. The single-plate box has the advantage of providing complete access to both droplet distribution / extraction and optical imaging / activation from above the open air gap. The single-plate box can have any of the above-mentioned suitable features of the dual-plate box in any combination. In some embodiments, the bottom of the single-plate box can be optically permeable or transparent to allow imaging / photosensitization from below.

[0384] In any device described herein, the box material of the double plate or single plate box can take into account dimensional accuracy, hydrophobicity of the channel surface and biocompatibility. As described above, it may be useful to use one or more thermal windows above the area of ​​the thermal control zone. Generally, when the system is used to perform PCR on the device, the reduction of materials in the thermal heating zone can reduce thermal mass and increase the PCR ramp rate.

[0385] Features of the device

[0386] Typically, any device described herein may include a PCB portion that may include an electrode array, an active thermal control (e.g., a heater, cooling, etc.), a magnetic field applicator, etc., and a chuck (e.g., a vacuum chuck) that may be mounted to the PCB. This portion of the device may form a placement surface at the bottom of the box so that it may be securely and in a predetermined orientation on the device. For example, the box may be keyed to fit onto the placement surface in a predetermined manner (e.g., by including one or more directional slots, pins, etc.). The device may also include one or more control units, which include one or more processors that may control the activity of the device and may be configured to drive droplets and analyze information from the box. The controller may also include a memory, one or more data storages.

[0387] The placement surface of the device can be configured to place the box, and also prevent arcing, sparking or short circuiting between multiple electrodes on the placement surface. For example, the placement surface can be coated with another dielectric (the dielectric bottom surface of the box can be located thereon), such as polyparalyene and / or optional or other materials. The dielectric bottom surface can prevent arcing between electrodes in the array of electrodes (driving electrodes) on the placement surface. The spacing between the driving electrodes can be between about 50 microns-120 microns. This close packing (closepacking) between electrodes on other flat surfaces can be additionally easy to form arcs / short circuits between electrodes, so that the use of external dielectric coatings (except the dielectric layer of the box) can limit arcing / short circuits between electrodes.

[0388] As discussed and described above, some or all of the electrodes may include openings therethrough that may be connected to a vacuum source for positioning the electrodes on the device. For example, in some variations, each electrode in the array includes an opening therethrough; in other variations, every other electrode may include an opening (e.g., alternating). In some variations, every third electrode, every fourth electrode, etc. In some variations, only the corner electrodes may include openings.

[0389] Droplet detection

[0390] Any of the devices described herein may include droplet detection. As described above, droplet detection can be performed based on the capacitance of electrodes in an array of drive electrodes by monitoring the current passing through the electrodes. Also described herein are devices (e.g., systems or devices, including devices) in which droplet detection is based on capacitance measurement by creating a capacitive voltage divider. In this example, the top plate can form a reference frame (e.g., a reference electrode, such as an ITO electrode) and can be driven typically between 0V and 300V to generate an AC signal; during droplet detection, the reference electrode (top electrode) can be disconnected from the drive signal and its voltage is sensed by a controller (e.g., a microprocessor) and the voltage is sensed by a controller (e.g., a microprocessor). Fig.33A and Fig.33B In the ITO sensor, it is called "ITO sensing" because it can act as a sensing electrode and can be electrically coupled to one or more reference capacitors. One or a group of electrodes can be activated at a higher known voltage (e.g., 300V DC) while all other electrodes are grounded. This produces a Fig.33A The voltage divider shown in FIG. Fig.33A An ITO sensing circuit is shown with a switch to switch between sensing (eg, capacitive sensing from a reference plate / top plate) and driving, eg, to move one or more droplets.

[0391] exist Fig.33A In the , the voltage at the ITO sensing node (ITO sensing electrode) is driven by the ratio of C_A to the total capacitance (C_A + C_B). The capacitance of C_A varies based on the material dielectric constant between the plates of the capacitor (electrode and ITO). The capacitance of C_B also varies relative to what is present between the ITO and the remaining electrodes. Air, wax, water, and reagents have different dielectric constants and thus change the capacitance and voltage at the ITO sensing. This enables the droplet detection method to not only detect droplets (e.g., presence / absence of droplets), but also to distinguish between droplets and identify specific reagents in the electrode grid.

[0392] Due to the variability of base capacitance, two calibration capacitors may be included (e.g., Fig.33B C_REF and C_REF_LARGE). Fig.33B Another example of a capacitance sensing circuit is shown, which includes multiple reference capacitors. By driving all electrodes (e.g., all drive electrodes) to 300V, the total capacitance C_total can be calculated using the reference capacitors. If there is a large enough C_total to saturate the voltage at the ITO sensing, the reference capacitance can be increased. The regulation circuit for ITO sensing can isolate the voltage from the small leakage current.

[0393] Fig.34AExemplary values ​​of capacitance are shown, which may indicate the presence or absence of a droplet (and / or the identity of the material) in one or more cells within the air gap. As discussed above, when a cartridge including an air gap is placed into a DMF apparatus, a "cell" in the air gap may correspond to an area above a drive electrode, which may have an array of drive electrodes on the cartridge placement area. Fig.34A In , "ITO" corresponds to the upper (e.g., return) electrode on the upper plate of the box. In this example, C18, C21, C24, C27, C30 are reference capacitors (e.g., 11.9pF in this case), and C16, C19, C1, C25, C28 are capacitances measured as described above, corresponding to capacitances with or without droplets when measuring different drive electrodes (e.g., set to high voltage while other drive electrodes are grounded). Water, wax, and air (no droplets) have very different capacitances, which can be used to identify the presence or absence of droplets (e.g., capacitance greater than or equal to 0.09pF, greater than or equal to 0.1pF, etc.). In this example, capacitances above this threshold (e.g., greater than 0.06pF, 0.07pF, 0.08pF, 0.09pF, 0.1pF, 0.11pF, etc.) indicate the presence of material in the air gap, higher than the check (set to a high voltage, such as 300V). Additionally, the range of measured capacitance above the threshold can indicate the composition of the droplet, such as aqueous (water) and / or wax / oil. For example, a capacitance greater than about 3 pF (e.g., 3 pF, 3.1 pF, 3.2 pF, 3.3 pF, 3.4 pF, 3.5 pF, etc.) can indicate that the droplet is aqueous, while a capacitance between about 0.09 pF and about 3 pF can indicate that the droplet is wax or oil (e.g., between about 0.07 pF and about 3.3 pF, between about 0.09 pF and about 3.0 pF, etc.).

[0394] Fig.34B is a graph showing an example of voltages measured using this technique, based on which the difference between different voltages measured with various droplets (water, wax) versus without droplets (air) on a single test cell is shown. Fig.34B , when aqueous droplets are present, the detected voltage is approximately 3.3V, compared to 0.085V when no droplets are present, and 0.176V when wax is present. The measured value for wax is twice that of air (no droplets / material), and much higher for water; in this example, the circuit caps this value at 3.3V. Different materials can be detected by their different dielectric constants. The dielectric constant of water can also be a function of temperature. Therefore, in some variations, when droplets are present, the capacitance can vary depending on the temperature. This property can further be used to identify water, and can also be used to estimate temperature. Therefore, in some variations, capacitance measurements of droplets can also be used to estimate their temperature. For example, Fig.34C is a graph showing the static relative dielectric constant of water, which shows the change in relative dielectric constant with temperature (between 0 degrees Celsius - 300 degrees Celsius).

[0395] Chuck design

[0396] Any device described herein may include a chuck (e.g., a vacuum chuck) that may form part of a placement surface, as mentioned above. The vacuum chuck may be attached to an electrode array (e.g., a drive electrode that may be part of a printed circuit board) and may also be integrated with a magnet and / or heat dissipation feature. Any of these elements or portions of these elements may be included or omitted and may be used in any combination.

[0397] The vacuum chuck design can help ensure that a reliable and effective vacuum adheres the bottom of the box (e.g., in some variations, the dielectric layer and / or the hydrophobic layer forming the dielectric layer) to the electrode grid. The vacuum can be applied through one or more (e.g., a manifold) of the through holes (e.g., copper through holes).

[0398] In addition, any device described herein may include a magnet that is integrated into a base that includes the chuck and / or the placement surface. The integrated magnet may be configured to allow the actuable magnet to engage with the material in the cartridge (e.g., a magnetic bead in a droplet in an air gap) through the vacuum chuck. The magnet may rest slightly below the PCB that forms the placement surface of the device without affecting vacuum performance or functionality.

[0399] Any reader described herein may also or alternatively include one or more thermal regulators including one or more heat dissipation elements that can quickly and accurately dissipate heat from a heater in the device that controls the temperature of one or more cells in the cartridge when the cartridge is placed and held on a placement surface of the device. For example, two designs of heat dissipation elements are described herein that can be used alone or more closely. One exemplary heat dissipation design is configured to dissipate heat from a thermoelectric heater, while another design is configured to dissipate heat from an embedded heater.

[0400] Figure 35A-Figure 48The figure shows a vacuum chuck portion of the device, which can be used with any of the devices described herein. In general, the vacuum chuck can be configured so that negative pressure is applied through the chuck (e.g., by a vacuum pump) and is directed under the mounting surface (e.g., a PCB forming part of the mounting surface) in an area pneumatically isolated, for example, by an O-ring. The mounting surface can have a through hole (e.g., in the PCB) that allows the negative pressure to act directly on the bottom of the box (e.g., the dielectric film and / or the hydrophobic film), which is located on the top side of the mounting surface (e.g., the PCB forming the mounting surface), pulling the bottom of the box downward in the Z direction and adhering it to the electrode grid.

[0401] The vacuum chuck may include one or more of: a vacuum channel with ports on either end, a groove for an O-ring, threaded holes for attaching a PCB, and a recess under the electrode grid. Fig.35A is a top view of an example of a vacuum chuck 3500, and Fig.35B is a cross-sectional view of an example of a vacuum chuck 3500. Section AA emphasizes the vacuum channel and its accompanying ports. Pneumatic flow 3505 follows Fig.35B Path of the arrow shown in FIG: first pulled through at least one inlet port, then flowing through channel 3507, and finally out of side port 3509. A portion of the chuck (on which the seating surface formed by the PCB will be placed) is surrounded by an O-ring 3503.

[0402] For example, Fig.36 Shows Figure 35A-Figure 35B . An isometric view of the chuck shown in FIG. . The groove 3509 (which can be designed using, for example, Parker O-ring design standards) is configured to accommodate an O-ring. Once in place, and with the chuck secured to the PCB, the O-ring can pneumatically isolate the vacuum directly below the electrode grid. The seating surface can be formed by securing a PCB with electrodes (not shown) to the chuck. For example, Fig.37 As shown in FIG. 3 , the chuck may include a plurality of threaded holes 3701 for attachment to a mounting surface (eg, a PCB). Fig.37 Shows something like Figure 35A-Figure 35B In some embodiments, the chuck includes at least four threaded holes ( Fig.37 ), each equally spaced in at least the X or Y direction and centered about the origin of the chuck. The screw holes can serve a dual purpose: firstly to secure the PCB to the chuck so that the interface of the two components is flat, and secondly to apply a downward force in the Z direction about the perimeter of the O-ring, effectively creating a pneumatic seal.

[0403] Fig.38A Shows something like Figure 35A-Figure 35B A top view of the chuck is shown in FIG. Fig.38B An enlarged cross-sectional view of the chuck is shown. Fig.35B An enlarged image of section AA is shown, which shows the boundaries of recesses 3801, 3803 (along the X-axis), which can create space between the surface of the PCB and the chuck, but only in the isolated area where the vacuum is active. This space can optimize the pneumatic flow of the vacuum, as described herein. In Figure 38, an opening 3805 for a magnet is present on the upper area, and can include enough space for the magnet to move to / from the box (e.g., by moving up / down in the space, or laterally in some variations). The area around the magnet opening can include a gasket or sealing ring (e.g., an O-ring) 3809 for isolating the magnet area from the vacuum area, similar to an external O-ring.

[0404] As mentioned, any of the devices described herein may include integrated magnets. Figure 35A-Figure 39 In the embodiment of the present invention, the recessed area 3905 can be used to hold an integrated magnet that can be moved up / down by the system to engage / disengage the magnetic field. Alternatively, in some variations, the magnet can be stationary but can be switched (on / off and / or change strength) by the controller of the device.

[0405] Thus, the vacuum chuck may include an integrated magnet, and therefore may include one or both of: a cutout that allows the magnet to travel through the chuck; and a second o-ring groove that isolates the magnet area from the pneumatic flow of the vacuum. Fig.39 Shows something like Figure 35A-Figure 35B 39. A through-cut region 3905 is shown and can be sized to fit the desired magnet and allow uninterrupted travel of the actuable magnet. The magnet can pass through the cutout and land directly under the PCB when engaged, or can be disengaged through the cutout when not in use.

[0406] Fig.40 Shows something like Fig.35A 4001 can accommodate an O-ring. Once in place, and with the chuck secured to the PCB, the O-ring can pneumatically isolate the magnet cutout area from the rest of the vacuum chuck, in particular ensuring that the vacuum is not compromised by the magnet cutout.

[0407] Fig.41A and Fig.41B The diagrams are similar to Fig.35A and Fig.35B4105. The top view and side cross-sectional view of the chuck shown in FIG. 4106, but includes a gap 4115 for thermal access to a heating component, such as a heater (e.g., a resistive heater) 4105. The heater 4105 is shown above the cavity 4115 in the chuck so that it can be easily thermally regulated (e.g., cooled). The resistive heater can be in the PCB (in Fig.41A and Fig.41B not shown).

[0408] For example, Fig.41A An example of a heat dissipation system is shown, which can be included in any device described herein. The heat dissipation system can be constructed so that any heat load generated by a heater 4105 in a device (e.g., in a PCB) can be appropriately and effectively dissipated. The first heat dissipation configuration can be constructed to dissipate the heat generated by a heater embedded in a PCB, and is described below as the heat dissipation of an embedded heater. The second heat dissipation design can be constructed to dissipate the heat generated by a thermoelectric cooler embedded in a vacuum chuck, and is described below as the heat dissipation of a thermoelectric cooler. Both heat dissipation designs can use unique features in a vacuum chuck, as well as accompanying components to dissipate heat. The two designs can be used together, also in an assembly, or used alone.

[0409] For example, heat dissipation for embedded heaters in a vacuum chuck can be configured as a plenum. Fig.41A , a top view of the chuck illustrates the heat dissipation aspect of the chuck; Fig.41B A pair of air channels 4101 are shown that feed into a cooling chamber 4103, which may be part of or below (or otherwise connected to) the area in which the heater is located. Fig.41B , the flow paths of multiple air elements (channels 4101, 4101') that function in the system are shown. Air drawn in 4101 can be warmed by heat (including residual heat) from a heater in a PCB (e.g., a mounting surface, not shown), and can flow through a through cutout 4115 area in the vacuum chuck, which can be covered or partially covered, or open to the heater in the PCB (or one or more thermal vias in thermal communication with the heater). Section AA ( Fig.41B4101 ' and 4102 ') shows the pneumatic flow of the two air elements (warm air 4105 and ambient air) when the fan is turned on, the fan is flush against the chuck and centered on the through-cut 4115. The fan (not shown) can push the warm air generated by the heater out of the through-cut of the vacuum chuck. At the same time, the fan can pull ambient air into the chuck and through the two channels 4101, 4101' through-cut in the chuck. The system can continuously or intermittently circulate ambient air into the chuck and warm air out of the chuck, which effectively dissipates any heat generated by the PCB heater.

[0410] Also described herein is a system for dissipating heat from an embedded heater. For example, Fig.42 The assembly shown in can be configured to include both a chuck 4203 and a fan 4205. The pneumatic flow described above can be controlled by a fan 4205 fastened to the bottom of the chuck 4203. Fig.42 Shown is a front view of the chuck 4203 and fan 4205. A first arrow 4221 points to the vacuum chuck (top structure) and a second set of arrows 4201, 4201' depicts the airflow path. Fig.43 An example of an arrangement of a chuck 4303, a fan 4307, a PCB 4305 forming a seating surface (e.g., including an array of electrodes, not shown), and a box 4311 is shown. The box can be held down by a vacuum through openings (e.g., in some of the electrodes).

[0411] Fig.44 An example of a heat dissipation system for regulating the temperature of a thermoelectric cooler via a vacuum chuck is shown. Fig.44 , an isometric view of the chuck (similar to Fig.35A The chuck shown includes a recess 4509 designed so that a thermoelectric cooler (TEC) can be slotted into it.

[0412] Figure 45A-Figure 45B They show similar Fig.35A A top view and a cross-sectional view of the chuck are shown in FIG. Fig.45B The cross section (through AA) shown in FIG. 4 highlights the thermal path of the heat generated by the thermoelectric cooling element 4525. Rectangle 4525 represents the TEC, and the arrows in the chuck depict the heat spreading throughout the chuck. The device may include one or more heat sinks of desired size that may be fastened to the bottom of the chuck and below the TEC, which then absorbs the heat. Finally, fastened to the heat sink ( Fig.46 Two fans on either side of the ) can work together to push hot air away from the entire system and rush ambient air into the system.

[0413] Figure 47A-Figure 47C The figure shows the components of one or more devices configured for heat dissipation of a thermoelectric cooler. For example, Fig.46 A front view of the chuck is shown. Fig.46 The downward arrow 4613 in the figure shows the heat path of the heat in the chuck as described in Figure 45. Arrows 4611, 4611' depict the flow path of the air pushed into the heat sink by the fan and the path of the air exhausted from the heat sink by the fan. The fans act simultaneously in the same direction. Figure 47A-Figure 47C The assembly process and various components that may be included in the device and its method of use are shown. For example, Fig.47A A chuck 4701 is shown, Fig.47B A chuck 4701 plus a heat sink 4703 is shown, and Fig.47C A chuck 4701 plus a heat sink 4703 plus two fans 4709, 4709' are shown. Fig.48 An exploded view depicting a partial arrangement of the device components, including Fig.47C Components in (eg, chuck 4801, heat sink 4803, fans 4809, 4809') and PCB 4807, which includes drive electrodes and heaters (not visible); in addition, box 4811 is attached to the mounting surface of the PCB via vacuum.

[0414] Figures 60A-60B Another example of a vacuum chuck is shown that can be used with any of the devices described herein, for example, below and coupled to an electrode plate, as shown above. Fig.60A , the upper surface of the vacuum chuck is shown and includes an O-ring 6035 channel (eg, Fig.56B ). The chuck includes one or more vacuum holes 6009 and placement locations for the TEC 6054 and a magnet pass-through area 6068. Fig.60A The illustrated chuck also includes a plurality of alignment pins 6071 . Fig.60B Shows Fig.60A The bottom of the chuck is formed by a vacuum pump connection 6074, a heat sink connection location 6088 and a magnet channel 6068'.

[0415] Action Zone

[0416] Any device described herein may include one or more action zones that strategically locate different possible actions that droplets can experience for protocol execution. The purpose of a plexing strategy is to be adapted to different laboratory needs in a more flexible, modular manner. The different stages of a protocol to be executed can be strategically grouped into action zones to allow the protocol designer to define abstract goals on a plate. An action zone can be a fixed area below or above an electrode plate for reaction (i.e., mixing, merging, heating, cooling, thermal cycling, magnet capture, waste, optical detection, etc.).

[0417] Fig.55A An example of an electrode grid setup is shown with separate action zones for either magnetic capture 5501 (three magnetic control zones are shown that can be used as mixing chambers), a heater that can be either an isothermal or a thermal cycler (five heating zones 5503 are shown), a Peltier 5505 that is an active cooling zone for cooling to 4°C and can also be heated, and a waste connection through the channel to the top plate and into the waste chamber (three waste zones 5507 are shown that can be connected to separate or the same waste chamber). The box setup may also include a mixing connection through the channel to the top plate (e.g., one or more waste areas / zones 5507 can be used for mixing as described herein) and one or more optical detection areas 5511. Thus, Fig.55A An electrode grid with different action zones is shown. These zones can be determined by the box and the device. For example, as described above, the box can determine the waste zone, and the unit cells corresponding to heating and / or cooling (e.g., thermal control), optics, and magnets can correspond to areas of the device.

[0418] Fig.55B Another example of a system (box and device) having various action zones defined by either or both of the box and the device is illustrated. Fig.55B In FIG. 5 , the system includes 912 drive electrodes corresponding to 912 (0-911, e.g., 38×24 grid) unit cells. Some of these cells within the air gap of the cartridge may be action areas for loading, mixing, washing, imaging, etc. Typically, these systems may include one or more loading ports 5551 (in Fig.55A , 10 loading inlets are shown, each corresponding to a single drive electrode unit cell; more than one unit cell may be used). Three thermal cycling zones 5553 are shown in this example. One or more pinning features (e.g., protrusions, walls, barriers, etc.) may extend at least partially into the air gap to hold or retain the droplet and, in particular, the external hydrophobic (e.g., liquid wax) material to maintain position and the droplet. Fig.55B, 10 pinning features 5555 are shown. These pinning features can be barriers (e.g., fences, walls, blocks, etc.). Generally, the pinning features can be formed of hydrophobic, oleophilic, hydrophilic, etc. materials that can retain a coating material (e.g., a hydrophobic liquid wax material) that at least partially surrounds the encapsulated (e.g., coated) reaction droplets. Fig.55B As shown, the barrier can form a chamber that is open on one or more sides, using two or four pinning features at the corners of the three thermal cycling zones 5553. The barrier can extend from the top to the bottom of the air gap or partially into the air gap. For example, the barrier can be formed of a material including a wax (e.g., paraffin wax), such as a polymeric material mixed with paraffin wax. Fig.55B In the embodiment, the pinning feature is shown as a PTFE post that can be inserted into the main box body (e.g., the top plate) and is hydrophobic but oleophilic and thus attracts paraffin when the droplet is in the thermal cycling zone, which can keep the droplet in the center of the thermal cycling zone during use. In some variations, the pinning feature can be made of a material such as acrylic, polycarbonate, DuraSeal TM , high melting temperature fluoro wax / solid ski wax, etc. The pinning feature can be formed as part of the top plate or the bottom plate and / or can be connected to both. In use, the barrier can fix the wax droplets around the reaction droplets. For example, the wax droplets can surround the aqueous reaction droplets 1501 and remain in the open chamber in the air gap formed by the barrier.

[0419] The systems described herein may also include one or more waste areas 5557 (in Fig.55B In FIG. 1 , two zones are shown), which can be connected to a vacuum region for extracting all or part of the droplets from the air gap by suction. Fig.55B In the embodiment, one of the waste areas may be a lower capacity (eg, 1 mL) waste area 5559, while the other may be a higher capacity (eg, 2 mL, 3 mL, 5 mL, etc.) waste area 5557.

[0420] Any system described herein may also include one or more magnetic regions 5563. Fig.55A In the embodiment, the system includes four magnetic unit cells distributed in an air gap, which in some cases overlap with other regions (e.g., thermal control and / or isothermal regions). In general, any of the regions described herein can overlap (e.g., magnetic, thermally regulated inlets, mixing channels, waste channels, etc. can overlap with each other).

[0421] The system may also include one or more isothermal regions 5561 (in Fig.55B , a single isothermal region with 16 unit cells 4×4 is shown, where two of the unit cells are configured as a magnetic control zone 5563 and a waste zone 5557.

[0422] Any of these systems may also include one or more mixing channels 5565. Fig.55B The example of 5500 also shows a plurality of reservoir outlet holes 5569 from which fluid contained in one or more reservoirs of the cartridge can be added to the air gap. Any of these systems may also include one or more recovery holes 5571 (in Fig.55B A recovery hole is shown in FIG. 1 ). Typically, the cartridge may include an area smaller than the number of possible active electrodes. For example, in Fig.55B In , as mentioned above, the working area includes 912 active unit cells, however they are surrounded by non-working (inactive) unit cells / electrodes 6673 which may be part of the device. Fig.55B In the Figures, the approximate dimensions of the box base (e.g., 3.17×4.75 inches) (forming the air gap dimensions) are shown by way of example only. As in any of the figures shown herein, these dimensions may be approximate only and may be + / - 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, etc.

[0423] Thermal Subsystem. The thermal subsystem can be similar to any of the subsystems described herein. Figures 56A-56B Another example of a portion of a thermal subsystem is shown, similar to that shown above in Fig.42 , Fig.43 , Fig.46 and Figures 47A-47C Described in. Fig.56A In the embodiment, the thermal subsystem includes one or more TEC5605, which can be sandwiched between a pair of thermal conductors (graphite pads 5607, 5607') ​​and fixed in a TEC slot 5611 on a vacuum chuck 5609. The chuck can then be positioned under an electrode plate 5615, which is located under a box seat (including a box edge 5617, which is keyed to receive the box and can be sealed with a clamp frame as described above). As shown, the chuck can be coupled to a frame 5619 (e.g., a housing frame) within the housing and can be located under one or more fans 5621 and one or more heat sinks 5622. Fig.56B Shown by Fig.56A A cross section of a side view of the thermal subsystem is shown. The PCB of the electrode plate 5615 is placed on top of the chuck 5609 that holds the TEC. The chuck can be thermally conductive (e.g., formed of a thermally conductive metal and / or polymer), and one or more heat sinks 5622 and cooling fans 5621 can be located under each TEC.

[0424] Fig.56C A top perspective view of a portion of an apparatus including a thermal subsystem is shown. Fig.56C5604 is shown as being housed in a cassette holder on an electrode plate 5615. A pump 5631 and an additional front cooling fan 5633 (fan assembly) are mounted in the housing as part of a thermal control subsystem. The fan assembly, pump, and housing frame are all mounted on a base plate 5639, which may be part of the housing or coupled to the housing. Fig.56D An enlarged view of pump 5631 is shown.

[0425] As mentioned above, any thermal control subsystem described herein may also include one or more resistive heater traces, drive circuitry, and thermal protection (e.g., insulation); as described above with reference to Fig.55B As described, the resistive heater can provide isothermal heating up to about 75 degrees Celsius in the action zone (and may also include magnets).

[0426] The resistive heater may include active cooling or passive (eg air) cooling, and the resistive heater may be in an electrode plate that is integral to, for example, the second layer side.

[0427] The TEC heat transfer region may include a TEC, drive circuitry, and protection (e.g., insulation), and may be configured to transfer energy from the TEC to the EWOD, including thermal cycling at temperatures between approximately 4 degrees C and 98 degrees C. Any of the devices described herein may also include a customized TEC and bracket that may be used to provide a robust TEC that achieves ramp rates up to 10 degrees C / second and may have a high degree of temperature measurement accuracy.

[0428] In any of the devices described herein, the TEC can be a high power thermal cycling TEC (e.g., 30W) welded directly to the bottom of the electrode plate. In some variations, the ramp rate can be 3 degrees Celsius per second or higher and can be controlled by controlling the current applied to the TEC. For some variations of the control system, a closed feedback loop system can be used in terms of ramp rate and steady state with precision temperature control of at least 0.5 degrees Celsius accuracy. For example, the heater (and ramp rate) can be configured in a 4×4 electrode grid array (heater zone) to accommodate approximately 200 μL droplets per heater zone.

[0429] Magnetic Subsystem. A magnet control system (magnet control) may be included within the housing and may coordinate (via a controller) one or more magnets to apply a localized magnetic field to one or more regions of the cartridge. This is described above in conjunction with Fig.38A , 39 , 40 and 41A-41B, 57A-57B are briefly described. Figures 57A-57B Also shown are examples of magnetic subsystems that may be included as part of the device to apply and / or remove local magnetic fields to regions (zones) of the cartridge. Fig.57A , the box 5705 is placed in a box seat connected to the drive electrode array (on the electrode plate 5715) below the vacuum chuck 5709. In this example, the magnet is shown as a Haibach array of magnets 5722 (an arrangement of permanent magnets that increase the magnetic field on one side of the array and cancel the magnetic field to near zero or approximately zero on the other side), and a magnetic sheath 5726 surrounding the low field side of the array; the sheath can be connected to a column including a biasing member (e.g., a spring) 5724. The magnet on the column (e.g., the sheath) can also be connected to a motor (e.g., a stepper motor 5729) that can move the magnet up and down (e.g., in the z-axis, to and from the box). A sensor such as an optical sensor 5733 can determine the position of the magnet, and the position can be used for feedback to help adjust the position of the magnet relative to the box. For example, a marker 5737 or a mark can be connected to the magnet (e.g., through a column or sheath) and can be tracked by an optical sensor. The movement of the magnet may also be limited to prevent it from hitting the cartridge; for example, a hard stop 5738 (lip, flange, etc.) may be connected to the sheath or post to engage a corresponding limit (flange, edge, etc.) on the chuck. The bias may help the magnet return to a retracted position away from the cartridge. Fig.57B Picture shows Fig.57A . In this example, the spring compliance of the magnet head has a tolerance of approximately 1.5 mm, and the motor resolution is approximately 18° / step for approximately 80 steps / mm. As described above, the Halbach magnet array focuses the magnetic field and amplifies the magnetic flux (three neodymium magnets in this example) at a point with a diameter of approximately 3.0 mm (roughly the size of a unit cell, such as the size of an electrode) and can generate sufficient force to successfully capture the magnetic beads in the box. The magnet array housing ("magnet sheath") can secure the Halbach magnet array. A magnet actuator (e.g., a constrained linear actuator or a stepper motor) can vertically actuate the magnet housing and magnet array to move them to both an engaged position and a disengaged position. The magnet assembly can also include an optical home position sensor that detects the "home" position of the stepper motor (e.g., a disengaged position).

[0430] Electrode Array. The apparatus described herein generally includes an electrode subsystem including an array of drive electrodes and return electrode connections, and control circuitry for controlling actuation of the EWOD to move a droplet on the device. Fig.58A is an example of a top portion of an electrode subsystem that may be included in an apparatus as described. Fig.58A, the electrode subsystem includes an electrode array 5805 (as mentioned, all or some of the electrodes in the array, e.g., the peripheral rows of electrodes may include vacuum openings formed through the electrodes) and one or more return (e.g., ground) contacts 5807 for connecting to a return electrode in the box. The electrode array and return contacts may be mounted or formed on a circuit board (e.g., PCB) 5801, which may be referred to as an electrode board. The electrode board may include a high voltage power supply 5809 for providing a high voltage to the EWOD (e.g., a drive electrode). The electrode board may also include the above-mentioned box detection subsystem 5811 (e.g., one or more sensors for detecting the presence of a box in a desired position) and / or a clamp detection subsystem 5815 (including one or more clamp lock sensors) and / or a lid detection subsystem (including one or more lid sensors for detecting when the lid is closed).

[0431] The electrode plate may also include an identification tag device (e.g., an optical device, an RFID device) and / or a near field communication device (NFC device) 5830 for reading an identification tag from a box placed in the device. The electrode plate may also include a high voltage regulating circuit 5833 and / or a high voltage measuring resistor string 5835, and a decoupling capacitor 5841 that can prevent electric shock. Any of these plates may also include a circuit that includes one or more thermistor amplifiers, a TEC interlock, and an optional accelerometer 5844.

[0432] Fig.58B The bottom side of the electrode plate is shown, including the TECs (TEC 1, TEC2, TEC3) as described above, as well as an isothermal heater power supply, a TEC power supply, a high voltage power supply regulation circuit system and a circuit system for power supply regulation, droplet detection, a digital-to-analog isolation circuit system, a solid-state relay, a thermistor amplifier, TEC and heater protection logic, a vibration motor (vibration motor) for vortexing, and one or more pressure sensors.

[0433] Typically, as mentioned, the electrode plate forming at least a portion of the electrode subassembly may include a polyparaxylene (parylene) coating. The electrode plate may also include a controller (e.g., one or more processors), which may be a part of a separate plate. The electrode plate may also include a fan and / or a vacuum pump driver for applying a suitable voltage to the fan and vacuum pump in the device housing. As mentioned above, the electrode plate may include NFC electronic equipment and / or antennas for reading and writing to the NFC tags in the box.

[0434] Vortex on DMF. As above, and as Fig.58BAs shown, any device described herein can comprise mechanical vibration (for example vortex oscillator) for example on electrode plate, and it is configured to apply mechanical vibration to one or more regions of the DMF device comprising any sub-region or district.The dynamic characteristics of vortex liquid is the key to realize many standard molecular biology scheme steps including thorough mixing, compound dissolution in solution, emulsion formation, cell and tissue decomposition and / or depolymerization.As a rule, many use vortex oscillator equipment in these processes are performed, and the bottle of liquid is placed in the vortex oscillator on their base, is squeezed, and therefore the bottle is oscillated fast in circular motion, is produced in the vortex of liquid interior.Standard vortex oscillator can have the variable speed control of scope from 100 to 3200rpm.

[0435] The device described herein can mimic this process on DMF. Although the DMF chamber is stationary and circular motion cannot occur, the dynamic characteristics of the vortex in the droplet can be achieved by coupling a vibration motor to the bottom of the DMF PCB board. The vibration motor speed can be controlled in the range of 0 to 10,000 RPM, with a minimum force of 50 Newtons (11.24 lbf).

[0436] like Fig.59 As shown, vortexing in DMF can achieve partitioning reactions, which are useful in a wide range of protocols and applications (e.g., single cell biology, single cell RNA-seq, droplet digital PCR, droplet barcoding, and single molecule sequencing), all of which can be performed in the systems described herein. For example, a mechanical shaker motor (in Fig.58B ) can be mounted to an electrode plate below or near an electrode array (driving electrode array). Fig.59 , a schematic diagram of a vibration motor beneath a drive electrode ("actuating electrode") is shown, where a droplet is held in an air gap formed between an upper (top plate) and lower (bottom plate); the bottom plate can be a dielectric film of a box that is vacuum attached to the drive electrode in the device). Examples of different procedures for using this vibration motor are shown. The vibration motor operates at a speed of approximately 14,000 RPM at a voltage of, for example, 3V DC (and is approximately 6×14 mm). In Fig.59In the embodiment of the present invention, a vortex is applied by a vibration force generated from a digital microfluidic PCB board. On the left, an emulsion formation produced by vortexing two droplets is schematically shown, which merge to form a water / oil mixture using an electrowetting force. It is possible for hydrogel particles and sample solutions or single cells to be contained in a monodisperse oil emulsion when vortexed on DMF. Using DMF, heterogeneous mixtures such as slurries and solid tissue blocks can be loosened and manipulated in the protocol steps. Tissue decomposition on DMF can be mechanically achieved by vortexing. Combining a set of DMF features can enhance the ability to dissociate difficult tissues by vortexing (mechanical features) on a parallel chip and incubation with a dissociating enzyme (e.g., trypsin, papain, collagenase) (enzymatic incubation at a set temperature). Dissociation of tissue / organ / organism on DMF can be followed by single cell segmentation by applying vortexing forces to cells in emulsion as described above, and the use of mixing / heating / cooling / magnetically actuated DMF features can allow for continuation with downstream single cell protocol steps followed by library preparation steps to produce sequence-ready single cell libraries. Vortexing on DMF can help resuspend slurries or heterogeneous mixtures (e.g., magnetic or paramagnetic bead particles) in suspension after they have settled during extended storage / incubation steps.

[0437] Non-transitory computer-readable instructions. Any of the methods described herein (including user interfaces) may be implemented as software, hardware, or firmware, and may be described as a non-transitory computer-readable storage medium storing a set of instructions executable by a process (e.g., a computer, tablet, smartphone, etc.), which when executed by the process causes the process control to perform any steps, including but not limited to: displaying, communicating with a user, analyzing, modifying parameters (including timing, frequency, intensity, etc.), determining, alarming, etc.

[0438] To better suit different user needs and laboratory spaces, independent single modules can be multiplexed together, each with its own power supply, environment, internal computer and connection to the console unit of the user interface. In addition, the console unit of the user interface can be integrated to control the different modules and other laboratory functions required, such as scanning sample IDs and reagent kit IDs and integrating this information into the local laboratory or sample management system. The connection to the console unit can be wireless or it can be wired.

[0439] Example

[0440] Fig.69An example of a system 7300 is schematically shown, which includes a DMF device and a housing 7303, wherein the DMF device is contained in a housing 7308, wherein the device includes a cassette holder 7302 located on a placement surface, a robotic arm 7305, and a liquid handling subsystem 7307, all of which are enclosed in the housing 7303. The DMF device also includes an electrode subsystem 7311, a vacuum port 7313, a thermal subsystem 7315, a magnet subsystem 7317, and an optional software subsystem 7319 for operating any component. The system 7300 also includes a main controller subsystem 7321, which may include software, firmware, etc. (e.g., a desktop, laptop, mobile device, tablet, etc.) that can be run remotely for communicating with the system (including the device), controlling and / or creating, transmitting or modifying the scheme and other operating parameters of the system (including the device). The system also includes a display 7323, which is combined with an input subsystem 7325. In some variations, the system includes an optical subsystem 7327, which may include one or more illumination sources 7329 and an imaging device 7331. In method 7301 performed with system 7300, one or more reagent kits 7333 and one or more reagents 7335 may be used. The DMF device may have any combination of features described herein.

[0441] The device may also include a controller (including one or more processors, circuit systems, clocks, power regulators, wireless communication circuits, memory, etc.) to control the operation of the DMF and microfluidics on the control box. The controller may include a microcontroller, an input interface (e.g., a touch screen, buttons, knobs, etc.) circuit system, an output interface (e.g., Ethernet, WiFi, etc.), and the like. The controller may communicate and coordinate with any of the following: a vacuum subsystem, an electrode subsystem, a thermal control subsystem, a magnet control subsystem, and / or a software subsystem 7319; any or all of these subsystems may communicate with and / or be coordinated by the controller 7321.

[0442] For example, the vacuum subsystem may include a vacuum chuck, a vacuum pump, and one or more pressure sensors for detecting (and / or providing feedback to control vacuum) pressure. The software subsystem may include software, hardware, or firmware, such as a non-transitory computer-readable storage medium that stores a set of instructions that can be executed by one or more processors of a controller to coordinate the operation of the system (including any subsystem). The thermal subsystem may include a TEC, a heat sink / fan, and one or more thermal sensors (including a thermal sensor configured to monitor the temperature of a box (e.g., an air gap region), and / or one or more thermal sensors configured to monitor the temperature of a housing of a TEC or within a housing of a TEC, etc.). The magnetic subsystem may include, for example, one or more magnets (e.g., one or more Halbach array magnets), one or more actuators for all or some of the magnets, and one or more position sensors (e.g., home sensors) for monitoring / detecting the position of the magnets.

[0443] The housing may be connected to one or more inputs and / or outputs (e.g., display 7323 and input subsystem 7325) and / or may partially enclose one or more inputs and / or outputs. The display may be a touch screen and / or one or more buttons, dials, etc.

[0444] The electrode subsystem may include a drive electrode array (e.g., an electrode array) located below the box base, one or more high-voltage drivers, one or more TEC drivers, safety interlocks, one or more resistive heaters, etc.

[0445] As mentioned above, any of these systems can be used with and / or can include one or more reagents. Reagents can typically include a buffer (e.g., PBS, etc., including those with one or more antifouling agents), but can also include a sheath material (e.g., liquid paraffin material or other hydrophobic material).

[0446] In general, the systems described herein can be configured to perform thermal cycling in one or more regions (e.g., one or more separate or adjacent unit cells) between about 15°C-99°C (e.g., - / +0.5°C). These systems can be configured to manipulate reagent volumes between about 10-350 microliters through EWOD (e.g., through DMF). As will be described in more detail below, these devices can be customized to allow a user to create, modify, save, load, and transfer one or more protocols for operating the system (e.g., performing operations on a box).

[0447] In the example system shown, the device can include more than 900 independent electrodes (drive electrodes), and in some embodiments can have about 408, 912, 1824, 3648, 5472 or about 20,000 independent electrodes (drive electrodes), and can include one or more thermoelectric coolers (TECs) for better thermal control, uniformity, and reduced footprint. In this example, the device and box form 3, 4, 6, 8, 10 or more independent thermal cycling zones (controlled by TECs in the device) and one or more isothermal zones (e.g., controlled by one or more resistive heaters). The device also includes multiple magnetically independent control zones.

[0448] As described herein, any of the devices described herein may also include one or more vibration motors for mechanically vibrating all or some of the electrodes (e.g., in vibration zones, which may be separate or overlapping, e.g., with thermal control zones).

[0449] The system can be used to run multiple library preparation kits and workflows (e.g., Kapa HyperPrep PCRFree, SureSelect XTHS Sample Prep, SureSelect XTHS Hyb+Capture, etc., including custom workflows).

[0450] One or more boxes can be any box as described herein, and can be configured to be loaded and stored for reagents generally. One or more boxes can be selected as single plate box or double plate box independently. One or more boxes can include air gap (e.g., EWOD chamber), can be configured to tension base film (bottom forming air gap), and can include readable identification, including but not limited to near field communication (NFC identification, e.g., chip, circuit, etc.). Other readable identification can include RFID circuit, bar code, etc.

[0451] The housing may also enclose thermal control elements, such as one or more TECs, which are used to heat / cool and thermally cycle specific areas of the air gap within the box, as described above. One or more resistive heaters may also be included. Cooling vents and / or fans may be included in the housing to regulate the temperature therein. Figures 70A-70C As shown in the exemplary device of , the housing can also at least partially form the placement surface of the box. The electrode plate defining the drive electrode array can be inside the housing, below the box seat.

[0452] Go to Figures 70A-70C, a portion of an exemplary system 7400 is shown. A placement surface 7401 is disposed on a portion of the housing of the DMF device and is located within the chamber of the housing, where a partially enclosed wall 7403 is shown. The placement surface includes a plurality of temperature control regions 7405 and a plurality of magnetic control regions 7407 distributed throughout the placement surface. Using alignment features 7409, various boxes can be placed, and the temperature control region and magnetic control region of each box can be accessed. The DMF device of system 7400 also includes a three-axis liquid pipetting head, which includes a mechanical arm (not shown) and a liquid handling tip 7411. The liquid handling tip 7411 can retrieve reagent droplets from a reagent container 7413 to be transported to the box. The liquid handling tip 7411 can retrieve product droplets from a box placed on the placement surface and transport them to a well plate container 7415 for further processing. The liquid handling tip 7411 can also recycle droplets of waste fluid from a box placed on the placement surface and transport them to a waste container 7417 for processing. The area 7419 where the reagent containers 7413 and the well plate container 7415 are located can be temperature controlled.

[0453] The placement surface of one or more boxes (70mm×110mm to 780mm×2050mm, width×depth, electrode array) is located below the liquid pipetting head (4, 8, 12 or 96 tips). The digital microfluidic platform can be integrated with thermoelectric and resistive temperature detectors for precise temperature control (-20-100°C), magnets for controlling magnetic beads, alignment features for box positioning (e.g., alignment pins, camera-based vision algorithms and radio frequency identification). The liquid handling robot includes multiple different components: a liquid handling head for dispensing reagent and sample volumes (1 microliter to 100 milliliters) onto the DMF platform and performing thorough mixing at the pipette tip-DMF interface; reagent and waste containers for storing reagents / samples and handling waste (i.e., used tips and boxes), respectively; well plates (24, 48, 96 and 384); and a temperature-controlled area for storing reagents at precise temperatures (-20-100°C) before use and after collecting samples. Finally, both fluid manipulation modes can be enclosed by an enclosure that provides precise humidity, temperature, and gas control.

[0454] exist Fig.70BIn, the placement surface after a plurality of boxes have been arranged on the surface and the vacuum port of the DMF device is selectively fixed is shown. As shown, both single-plate or double-plate boxes can work on the surface. Box 7421 is an example of a single-plate box, and box 7423 and box 7425 are examples of double-plate boxes. The box can have different sizes, as shown by boxes 7423 and 7425. The box can be arranged on the placement surface without relying on a predefined position. Any position can be used, which is aligned with alignment features 7409 and includes a thermal control area and a magnetic control area as required. The illustration shows a schematic diagram of droplet positioning in a single-plate box and a double-plate box. In both configurations, the droplet to be manipulated is isolated from the electrode by a hydrophobic insulator. When an electric potential is applied, charges accumulate on both sides of the insulator, and this phenomenon can be used to move, merge, mix, separate and distribute the droplets.

[0455] exist Fig.70C , the placement surface of the system after the droplet is dispensed by the three-axis liquid pipetting head, which includes a mechanical arm (not shown) and a liquid handling tip 7411. In some boxes, pre-installed reagent points are set at locations within the box. The three-axis liquid pipetting head including the mechanical arm (not shown) and the liquid handling tip 7411 can dispense a drop of buffer to dissolve the pre-installed reagent for use.

[0456] exist Fig.71 In the figure, a schematic diagram of the control and sequencing of activities in one or more boxes on the placement surface is shown. In some embodiments, the software architecture can be implemented as follows, but the present invention is not limited to this. Those skilled in the art can envision many different architectures and schemes included in the teachings here.

[0457] Architecture: High-level overview. Each electrode plate layout can have a separate electrode plate-SW (software that controls the electrode plate). Multichannel pipettes can be connected using a multichannel pipette-SW (software that commands the multichannel pipette). The electrode plate-SW and the multichannel pipette-SW can exchange messages in the form of request-response, for example: the electrode plate-SW sends an action to the multichannel pipette indicating the layout, action (dispense, mix, pick up), and electrode layout; the multichannel pipette-SW receives the response (e.g., the multichannel pipette-SW processes the instruction information to see if it can run, the multichannel pipette-SW checks pre-run conditions, the multichannel pipette-SW performs the action, the multichannel pipette-SW checks post-run conditions, etc.). The multichannel pipette-SW may respond with an acknowledgment. The electrode plate-SW can receive actions and responses from the multichannel pipette-SW.

[0458] Algorithm: High-Level Overview. The algorithm may perform one or all of the following functions: pick reagents; select the layout to use (e.g., size of cartridge, requirements for temperature control and / or magnetic control); and select the script to run.

[0459] The script playback (on-script) may include (e.g., for each line): If indicated, electrode plate related execution instructions. If indicated, multi-channel pipette-software related: check multi-channel pipette position (should be resting position); move multi-channel pipette to desired position; check multi-channel pipette position (should be desired position); perform multi-channel pipette action; check multi-channel pipette position (should be desired position); return to rest position; and check multi-channel pipette position (should be resting position). After the script is completed: check multi-channel pipette position.

[0460] When a feature or element is referred to herein as "on another feature or element", it can be directly on another feature or element, or there can also be intermediate features and / or elements. In contrast, when a feature or element is referred to as "directly on another feature or element", there are no intermediate features or elements. It will also be understood that when a feature or element is referred to as "connected", "attached" or "coupled" to another feature or element, it can be directly connected, attached or coupled to other features or elements, or there can be intermediate features or elements. In contrast, when a feature or element is referred to as "directly connected", "directly attached" or "directly coupled" to another feature or element, there are no intermediate features or elements. Although described or shown with respect to one embodiment, the features and elements described or shown in this way can be applied to other embodiments. Those skilled in the art will also recognize that the reference to a structure or feature that is set to be "adjacent" to another feature can have a portion that covers or is located below an adjacent feature.

[0461] The terms used herein are only used for the purpose of describing specific embodiments and are not intended to be limitations of the present invention. For example, as used herein, the singular forms "a", "an" and "the" are also intended to include plural forms, unless the context clearly indicates otherwise. It will be further understood that when used in this specification, the terms "comprises" and / or "comprising" specify the presence of stated features, steps, operations, elements and / or parts, but do not exclude the presence or addition of one or more other features, steps, operations, elements, parts and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ".

[0462] Spatially relative terms such as "under", "below", "lower", "over", "upper" and similar terms may be used herein to easily describe the relationship between one element or feature and another element or feature, as illustrated in the accompanying drawings. It will be understood that spatially relative terms are intended to cover different orientations of the device in use and operation except for the orientation depicted in the accompanying drawings. For example, if the device in the accompanying drawings is inverted, the elements described as "below" or "below" other elements or features will be oriented to be "above" other elements or features. Therefore, the exemplary term "below" can cover both the orientation above and below. The device can be oriented in other ways (rotated 90 degrees or in other orientations), and the spatially relative descriptors used herein are interpreted accordingly. Similarly, the terms "upwardly", "downwardly", "vertical", "horizontal" and similar terms are used herein only for the purpose of explanation unless otherwise specifically indicated.

[0463] Although the terms "first" and "second" may be used herein to describe various features / elements (including steps), these features / elements should not be limited by these terms unless the context otherwise indicates. These terms may be used to distinguish one feature / element from another feature / element. Therefore, without departing from the teachings of the present invention, the first feature / element discussed below may be referred to as the second feature / element, and similarly, the second feature / element discussed below may be referred to as the first feature / element.

[0464] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," mean that various components (e.g., compositions and apparatus including devices and methods) ...

Claims

1. A digital microfluidic device, the device include: a mounting surface configured to mount one or more cartridges simultaneously; a plurality of drive electrodes disposed on the seating surface, wherein the drive electrodes are configured to apply a voltage to cause a droplet to move within one or more cartridges seated on the seating surface; a plurality of vacuum ports distributed across the mounting surface; a vacuum control configured to selectively apply a vacuum through a subset of the plurality of vacuum ports disposed beneath the one or more cassettes disposed on the mounting surface; an electrical control for applying energy to the drive electrode to move a droplet within an air gap of the one or more cartridges disposed on the mounting surface; and A liquid handling subsystem for dispensing liquid to or removing liquid from the one or more cartridges. 2 . The digital microfluidic device according to claim 1 , wherein the liquid handling subsystem comprises a robotic arm.

3. The digital microfluidic device according to claim 1, in, The vacuum control also includes a vacuum pump for applying a vacuum to the plurality of vacuum ports.

4. The digital microfluidic device according to claim 1, in, The subset of the vacuum ports are each coupled to an opening through a drive electrode of the plurality of drive electrodes.

5. The digital microfluidic device according to claim 1, in, The vacuum control is configured to determine which vacuum ports are located beneath the one or more cassettes.

6. The digital microfluidic device according to claim 1, in, The vacuum control also includes a manifold and a plurality of valves.

7. The digital microfluidic device according to claim 1, in, The plurality of driving electrodes includes about 408 to about 20,000 driving electrodes.

8. The digital microfluidic device according to claim 1, in, The liquid handling subsystem includes a liquid handling head and at least one liquid handling tip.

9. The digital microfluidic device according to claim 8, in, The liquid handling head is configured to dispense or withdraw volumes ranging from about 1 microliter to about 100 milliliters.

10. The digital microfluidic device of claim 1, further comprising a main control subsystem for coordinating activation of the plurality of drive electrodes, the plurality of vacuum ports, and the liquid handling subsystem.

11. The digital microfluidic device according to claim 10, in, The master control subsystem includes non-transitory instructions for controlling the plurality of vacuum ports, the plurality of drive electrodes, and the liquid handling subsystem.

12. The digital microfluidic device according to claim 10, in, The main control subsystem is configured to simultaneously move droplets in the air gaps of the one or more cartridges seated on the seating surface. 13 . The digital microfluidic device according to claim 1 , further comprising one or more temperature controlled regions within the plurality of driving electrodes. 14 . The digital microfluidic device according to claim 1 , further comprising one or more magnetic control regions within the plurality of driving electrodes, each magnetic control region comprising a magnet configured to deliver a magnetic field.

15. The digital microfluidics device of claim 1, further comprising one or more cartridge alignment features located on the seating surface.

16. A method for selectively fixing one or more cartridges in a digital microfluidic device having a plurality of drive electrodes, the method include: placing one or more cartridges on a mounting surface of a digital microfluidic device, thereby creating one or more respective mounting locations of the one or more cartridges, wherein each of the one or more cartridges is located on a sub-area of ​​the mounting surface; sensing each respective placement position of the one or more cartridges; determining a subset of the plurality of drive electrodes underlying the respective placement locations of the one or more cartridges; and A vacuum is selectively applied to the corresponding seating locations of the one or more cartridges to secure the one or more cartridges to the seating surface of the digital microfluidic device.

17. The method according to claim 16, in, Selectively applying the vacuum includes applying the vacuum through a subset of vacuum ports underlying the respective seating locations.

18. The method according to claim 16, in, Applying the vacuum includes applying the vacuum through openings that pass through all or some of the drive electrodes underlying the respective seating locations.

19. The method according to claim 16, in, Sensing each respective placement location includes sensing an electrical difference in a plurality of drive electrodes underlying the respective placement location.

20. The method according to claim 16, in, Sensing each respective seating location includes sensing air flow resistance through a subset of the vacuum ports on the seating surface in the respective seating location.

21. The method according to claim 16, in, Each of the one or more cassettes includes a base plate configured to deform when a vacuum is applied so as to adhere to the seating surface.

22. The method according to claim 16, in, Each respective placement location of the one or more boxes has a respective area corresponding to the size of each box.

23. The method according to claim 16, in, At least one of the one or more boxes has a different size than other boxes of the one or more boxes.

24. The method of claim 16, wherein the seating surface comprises one or more cartridge alignment features.

25. The method of claim 16, further comprising selectively delivering a magnetic field within the region of the plurality of drive electrodes.

26. The method of claim 16, wherein applying a vacuum comprises selectively applying a vacuum through a manifold and a plurality of valves.

27. A system, include: a mounting surface configured to mount one or more cartridges simultaneously; a plurality of drive electrodes disposed on the seating surface, wherein the drive electrodes are configured to apply a high voltage to move a droplet within the cartridge when the cartridge is seated on the seating surface, and wherein all or some of the drive electrodes include openings therethrough; a plurality of vacuum ports, wherein each vacuum port is coupled to one or more openings through the drive electrode; a vacuum control configured to selectively apply a vacuum through a subset of the vacuum ports beneath the one or more cartridges seated on the seating surface when the one or more cartridges are seated on the seating surface; an electrical control for applying energy to sequentially activate and deactivate one or more selected drive electrodes to move a droplet within an air gap of the one or more cartridges disposed on the mounting surface; a vacuum pump for applying a vacuum to the plurality of vacuum ports; a robotic arm and liquid handling subsystem for dispensing liquid to or removing liquid from the one or more cartridges when the one or more cartridges are seated on the seating surface; and The enclosure is configured to control one or more of humidity, temperature, or a gas environment.

28. The system according to claim 27, in, The placement position of the one or more boxes on the placement surface is not predefined.

29. The system of claim 27, further comprising a master control subsystem configured to coordinate the vacuum control and the electrical control.

30. The system according to claim 29, in, The main control subsystem is also configured to control at least one of humidity, temperature, or gas environment of the enclosure.

31. The system according to claim 27, in, The system also includes an optical subsystem.

32. The system according to claim 31, in, The optical subsystem includes an illumination source.

33. The system of claim 32, the illumination source comprising one or more of a visible light source or an ultraviolet light source.

34. The system according to claim 33, in, The optical subsystem also includes an imaging device configured to detect a bright field image, a fluorescent image, or a luminescent image.

35. The system of claim 27, further comprising a manifold and a plurality of valves configured to selectively apply vacuum through the subset of the vacuum ports.

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