Digital microfluidic device and method of use thereof

Abstract

The application relates to digital microfluidic devices and methods of use thereof. Digital microfluidic (DMF) methods and apparatus (including equipment, systems, boxes, DMF readers, etc.), and are particularly suitable for large-volume DMF devices and methods. For example, methods and apparatus for DMF using an air gap are described herein, wherein the air gap has a width that can be between 0.3 mm and 3 mm. DMF readers for use with DMF boxes are also described herein, including those suitable for use with large air gaps / large volumes, although smaller volumes may also be used.

Description

[0001] This application is a divisional application of an application filed on September 4, 2018, with application number 201880070164.8 and invention name “Digital microfluidic device and method of use thereof”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This patent claims priority to U.S. Provisional Patent Application No. 62 / 553,743, filed on September 1, 2017, entitled “DIGITAL MICROFLUIDICS DEVICES AND METHODS OF USING THEM,” and U.S. Provisional Patent Application No. 62 / 557,714, filed on September 12, 2017, entitled “DIGITAL MICROFLUIDICS DEVICES AND METHODS OF USING THEM,” each 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. Technical Field

[0006] The present application generally relates to digital microfluidic (DMF) apparatuses and methods. Specifically, the apparatus and methods described herein relate to an air-gap DMF apparatus comprising a cartridge including an air matrix and a ground electrode, and a durable component including a drive electrode. Background Art

[0007] In recent years, lab-on-a-chip and biochip devices have attracted great interest in scientific research applications and potentially point-of-care applications because they perform highly repetitive reaction steps with small reaction volumes, which saves materials and time. Although traditional biochip-type devices utilize micro- or nano-sized channels and corresponding micropumps, microvalves, and microchannels coupled to the biochip to manipulate the reaction steps, these additional components increase the cost and complexity of the microfluidic device.

[0008] Digital microfluidics (DMF) has emerged as a powerful preparation technology for a wide range of biological and chemical applications. DMF can control a variety of samples and reagents, including solids, liquids, and irritating chemicals, in real time, accurately, and with high flexibility, without the need for pumps, valves, or complex pipe arrays. In DMF, discrete droplets of nanoliter to microliter volume are dispensed from a reservoir onto a flat surface coated with a hydrophobic insulator, where they are manipulated (transported, separated, merged, mixed) by applying a series of potentials to an array of electrodes. Complex reaction series can be performed using DMF alone, or using a hybrid system in which DMF is integrated with a channel-based microfluidic system. Hybrid systems provide enormous versatility; conceptually, each reaction step can be performed in a microfluidics format that best accommodates it.

[0009] For many applications, it is most convenient to perform DMF on an open surface so that the matrix surrounding the droplet is the ambient air. Figure 1A-Figure 1C An example of an air matrix DMF apparatus is shown. Figure 1A An example of an air matrix DMF device 100 is shown. In general, the air matrix DMF device includes a plurality of unit cells 191 adjacent to each other and defined by having a single actuation electrode 106 opposite a ground electrode 102; each unit cell can be of any suitable shape, but can generally have the same approximate surface area. Figure 1A The unit cell is rectangular. The droplet (eg, a reaction droplet) fits between the first plate 153 and the second plate 151 (in Figure 1A-Figure 1C The entire air matrix DMF device can have any suitable shape and thickness. Figure 1B It passes through Figure 1A , which illustrates a cross-section of a hot zone of an air-based 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, a PCB, a polymer material, etc.). The air-based DMF device described herein also includes both a sample reservoir and a reagent reservoir, as well as a mechanism for replenishing reagents.

[0010] exist Figure 1A-1CIn the example shown in , the top plate 101 (in this case a glass material (although plastic / polymer materials including PCBs can be used)) provides support and protects the underlying layers from damage by external particulate matter, as well as providing a certain amount of insulation for the reactions performed in the DMF device. Therefore, the top plate can confine / sandwich droplets between the plates, which can enhance the electric field compared to an open air matrix DMF device (without plates). 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 surface wetting and helps to keep the reaction droplets in a cohesive unit.

[0011] As Figure 1A-Figure 1C The second plate, shown as the lower or bottom plate 151 in FIG, may include actuation electrodes that define the unit cells. In this example, as with 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 can be the same on both plates, or it can be different hydrophobic materials. The air gap 104 provides a space in which reaction droplets are initially contained in the sample reservoir and moved to run the reaction step or steps and to hold 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 droplets and the electrodes. Adjacent to and below the dielectric layer 105 is a PCB layer, which includes actuation electrodes (actuation electrode layer 106). Actuation electrodes can form each unit cell. The actuation electrodes can be energized to move the droplets to different areas in the DMF apparatus, allowing each reaction step to be performed under different conditions (e.g., temperature, with different reagent combinations, magnetic regions, pump inlet area, etc.). Support substrate (eg, PCB 107) (on Figure 1B and Figure 1C153 and the second plate 151 to provide support and electrical connections for the actuation electrodes, the traces connecting them (which may be insulated), and / or additional control elements including a thermal regulator 155 (shown as a TEC), a temperature sensor (shown as an RTD 157), an optical sensor, a magnet, a pump, 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 separated from the first plate 153 and the second plate 151, or they may be formed on and / or supported by the second plate. Figure 1A-Figure 1C In FIG, 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 the perforations in the second plate via tubing 198.

[0012] As mentioned, the air gap 104 provides a space in which reaction steps can be performed, which provides an area in which reagents can be held and processed by mixing, heating / cooling, combining with reagents (enzymes, labels, etc.). Figure 1A In the embodiment of the present invention, 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 from the top, from the bottom or from the side, and can be unique. Figure 1A The sample DMF equipment shown in Figure 1 comprises six sample reagent reservoirs, and wherein each sample reagent reservoir comprises opening or port for every kind of reagent to be introduced in the corresponding reservoir.According to reaction to be performed, the number of reagent reservoirs can be variable.Sample reservoir 110 and reagent reservoir 111 are communicated with by reaction zone fluid.Reaction zone is electrically communicated with actuating electrode layer 106, and wherein actuating electrode layer 106 is positioned below the reaction zone.

[0013] The actuation electrode 106 is Figure 1AIn the embodiment of the present invention, the actuating electrodes are 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 movement 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 approximately one hundred unit cells) are connected to seven reservoirs (one 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.

[0014] exist Figure 1A-Figure 1C In the exemplary device shown, the DMF device is typically integrated so that electrode (for example, actuating electrode and ground electrode) is a part for the same structure that can be loaded with sample and / or fluid.Electrode can be a part for box, and described box can be removable.Although box has been described (see, for example US20130134040), it has been proved that such box is difficult to use, particularly when by equipment imaging and when operating in air matrix device. Summary of the Invention

[0015] It would be highly advantageous to have an air matrix DMF apparatus that includes a cartridge that is easy to use and that can be made reliably and inexpensively. Methods and apparatus, including systems and devices, are described herein that can address these problems.

[0016] Digital microfluidics (DMF) methods and apparatus (including devices, systems, cartridges, DMF readers, etc.) are described herein. While the methods and apparatus described herein may be particularly suitable for air-matrix DMF devices (also referred to herein as air-gap DMF devices), these methods and apparatus can be configured for use with other DMF devices (e.g., oil-gap, etc.). The methods and apparatus described herein can be used to operate relatively large volumes, which is already possible with conventional DMF devices, in part because the spacing between the plates forming the air gap of a DMF device can be large (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.). Furthermore, any of the apparatus and methods described herein can be configured to include a disposable cartridge having a dielectric layer forming the bottom of the cartridge; the drive electrodes do not need to be part of the cartridge; and the apparatus can be adapted to allow the dielectric to be securely held to the electrodes during operation, which has proven to be very challenging, particularly when the dielectric layer is somewhat flexible.

[0017] Any of the methods and devices described herein may include a box in which a ground electrode is included as part of the box. In certain variations, the ground electrode may be formed as a grid pattern, the grid pattern forming a plurality of cells. The grid pattern may create a clear window, allowing visualization of the ground electrode even when an opaque ground electrode (e.g., an opaque or translucent material, such as a metal coating containing silver conductive ink) is used to form the ground electrode. The grid pattern may reflect the arrangement of the drive electrodes in the DMF device, and the box may be placed on the DMF device. For example, when the ground electrode is adjacent to the drive electrode across an air gap, the grid pattern covers the space between the adjacent electrodes. Alternatively, the ground electrode may be formed of a transparent or sufficiently transparent material so that it can be imaged. In certain variations, the ground electrode is a conductive coating. The ground electrode may be electrically continuous (e.g., electrically connected), but may include one or more openings, for example, through which droplets in the air gap may be visualized. Therefore, in any of these variations, the upper plate of the box may be transparent or sufficiently transparent to be visualized in at least one or more areas.

[0018] For example, a cartridge 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 cartridge, wherein at least the second side of the sheet of dielectric material comprises a first hydrophobic surface; a top plate having a first side and a second side; and 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 cartridge may also include: a second hydrophobic surface on the first side of the top plate covering the ground electrode; and an air gap separating the first hydrophobic layer and the second hydrophobic layer, wherein the air gap comprises a spacing greater than 280 microns.

[0019] In any of the cartridges described herein, the top plate can include a plurality of cavities within the thickness of the top plate; these cavities can be enclosed (e.g., sealed) and / or filled with a thermally insulating material having low thermal mass and low thermal conductivity. In some variations, the insulating material comprises air. The cavities can be positioned above air gap regions that will correspond to heating and / or cooling regions (e.g., thermal control regions); the lower thermal mass in these regions can allow significantly more rapid heating / cooling of droplets in the air gap below the cavity / cavities. Thus, the thickness of the top plate in these regions can include cavities; the bottom of the cavity (corresponding to the bottom surface of the top plate) can 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 can 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 can be substantially thicker (e.g., 1.5 times, 2 times, 3 times, 4 times, 5 times, etc.) than the cavity bottom surface.

[0020] 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 can itself be hydrophobic. For example, the bottom surface (e.g., the bottom surface of the cartridge) can be formed from a film that is both a dielectric and a hydrophobic material. For example, the bottom surface can be a Teflon film (which may include an adhesive or adhesive portion, such as Teflon tape), which is both hydrophobic and acts as a dielectric. Other films can include plastic paraffin film (e.g., "Parafilm," such as PARAFILM M). However, films that can withstand high temperatures (e.g., 100 degrees Celsius and above), such as Teflon film, are particularly preferred.

[0021] A box for a digital microfluidics (DMF) device can generally include a bottom and a top, and can 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; a first hydrophobic layer on the second side of the sheet of dielectric material; a top plate having a first side and a second side; a ground electrode on the first side of the top plate, wherein the ground electrode includes a grid pattern forming a plurality of open cells; a second hydrophobic layer 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.).

[0022] The term "box" can refer to a container that forms an air gap and can be inserted into a DMF reading / driving device. The box can be disposable (e.g., single use or limited use). The box can be configured to allow visualization of the fluid (droplet) in the air gap. The grid pattern can be particularly useful for allowing visualization while still providing a suitable ground reference to the drive electrode. The entire grid can be electrically coupled to form a single return (return) (ground) electrode, or multiple ground electrodes can be positioned on the top plate (via separate and / or adjacent grids).

[0023] As mentioned, the grid pattern of the ground electrode is formed of an opaque material.

[0024] As used herein, the term "grid" may refer to a pattern of repeating open cells ("windows") of any suitable shape and size, wherein the boundaries forming the open cells are formed by an integrated (and electrically continuous) material (e.g., conductive ink, metal coating, etc.). As used herein, a grid is not limited to a network of lines that intersect each other to form a series of squares or rectangles; a grid pattern may be formed by forming openings in an otherwise continuous plane of conductive material forming a ground electrode.

[0025] Therefore, typically, the grid pattern of the ground electrode can be formed from a conductive ink. For example, the grid pattern of the ground electrode can be formed from silver nanoparticles. The grid pattern can be printed, screened, sprayed, or otherwise layered onto the top plate.

[0026] Typically, the boundaries between the open cells forming the grid pattern can have a minimum width. For example, the minimum width of the grid pattern between the open cells can be 50 microns or greater (e.g., 0.1 mm or greater, 0.2 mm or greater, 0.3 mm or greater, 0.4 mm or greater, 0.5 mm or greater, 0.6 mm or greater, 0.7 mm or greater, 0.8 mm or greater, 0.9 mm or greater, 1 mm or greater, etc.). As mentioned, the open cells (e.g., "windows") formed by the grid pattern can be any shape, including quadrilateral shapes (e.g., square, rectangular, etc.) or elliptical shapes (e.g., oval, circular, etc.) and / or other shapes (+ shape, H shape, etc.).

[0027] Typically, the grid pattern of the ground electrode can extend over a majority of the top plate (and / or a majority of the box). For example, the grid pattern of the ground electrode can extend over 50% or more (e.g., 55% or more, 60% or more, 65% or more, 70% or more, 80% or more, 90% or more, etc.) of the first side of the top plate.

[0028] In any of the cartridges described herein, the sheet of dielectric material can be flexible. This flexibility can help secure the dielectric to the drive electrodes to ensure full contact between the dielectric and the drive electrodes. Typically, the sheet of dielectric material can be sufficiently compliant so that it can bend or flex under relatively low forces (e.g., a pressure of 50 kPa or greater). The dielectric sheet can be of any suitable thickness; for example, the sheet can be less than 30 microns thick (e.g., less than 20 microns thick, etc.).

[0029] As will be described in greater detail below, any of these devices can include a microfluidic channel formed in the second side of the top plate, wherein the microfluidic channel extends along the second side of the top plate and at least one opening between the microfluidic channel and the air gap.

[0030] The top plate may be formed from any suitable material, including in particular clear or transparent materials (eg, acrylic, etc.).

[0031] For example, a cartridge for a digital microfluidics (DMF) device may include: a sheet of flexible dielectric material having a first side and a second side, the first side forming an exposed bottom surface on a bottom portion of the cartridge; a first hydrophobic layer on the second side of the sheet of dielectric material; a top plate having a first side and a second side; a ground electrode on the first side of the top plate, wherein the ground electrode comprises a grid pattern of opaque material forming a plurality of open cells along the first side of the top plate; a second hydrophobic layer 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 comprises a spacing greater than 280 microns (e.g., greater than 300 microns or greater, 400 microns or greater, etc.). Typically, the cartridge has a bottom portion and a top portion.

[0032] As mentioned, this article also describes a cartridge in which microfluidic channels are integrated into DMF components, particularly DMF components including the top plate of a DMF device. Applicants have discovered that integrating one or more microfluidic channels into the top plate can allow the cartridge to be more compact, as well as allowing for a higher degree of control and manipulation of processes in the air gap, which can be additionally controlled by electrowetting the DMF system.

[0033] For example, a box for a digital microfluidic (DMF) device (the box having a bottom and a top) may include: a sheet of dielectric material, the 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; a first hydrophobic layer on the second side of the sheet of dielectric material; a top plate, the top plate having a first side and a second side; a ground electrode on the first side of the top plate; a second hydrophobic layer covering the ground electrode on the first side of the top plate; an air gap, the air gap separating the first hydrophobic layer and the second hydrophobic layer; a microfluidic channel formed in the second side of the top plate, wherein the microfluidic channel extends along the second side of the top plate; an opening between the microfluidic channel and the air gap; and a cover covering the microfluidic channel, wherein the cover includes one or more access ports for accessing the microfluidic channel.

[0034] As mentioned, the sheet of dielectric material can be flexible and can form the bottom-most surface of the box. The sheet can generally be flat (flat), although it can be flexible. The outer surface can be protected by a removable (e.g., peelable) cover. The dielectric properties can be generally consistent with those of a DMF (and particularly air-based DMF) device. The dielectric can be coated with a first hydrophobic layer on the inside (second side). The hydrophobic layer can be a coating of a relatively inert (e.g., non-reactive with aqueous droplets moving in the air gap) hydrophobic material.

[0035] The top plate can be flat and can be coextensive with (or larger than) the bottom dielectric material. The top plate can be of any suitable thickness, and in particular can be thick enough so that the microfluidic channels can be engraved into the second side of the top plate. As mentioned above, a ground electrode can be formed on all or some of the first side of the top plate, and a second hydrophobic layer can be coated on the ground electrode and / or the top plate (particularly where the top plate is exposed by an open window in the ground plate). In any of these examples, the thickness of the electrode coating can be minimal so that the electrode can be considered flush with the top plate bottom (first) side of the top plate.

[0036] In any of the devices and methods described herein, the air gap separating the first hydrophobic layer and the second hydrophobic layer (e.g., between the dielectric and the top plate) can be relatively large (e.g., >280 microns, 400 microns or greater, 500 microns or greater, 1 mm or greater, etc.) compared to conventional DMF air gap systems.

[0037] The microfluidic channel formed in the second side of the top plate typically extends through the top plate along the second side of the top plate, and the access opening (access opening) between the microfluidic channel and the air gap can be formed between the microfluidic channel and the air gap to enter the top plate. Any device described herein can also include a lid covering the microfluidic channel. The lid can be made of any suitable material, including acrylic acid. The lid can include one or more ports or openings that enter the microfluidic channel and / or enter the air gap.

[0038] The microfluidic channel can be configured to contain any suitable amount of fluid that is useful for mixing, adding, removing, or otherwise interacting with the droplets in the air gap. For example, the microfluidic channel can be configured to hold 0.2 ml or more (e.g., 0.3 ml or more, 0.4 ml or more, 0.5 ml or more, 0.6 ml or more, 0.7 ml or more, 0.8 ml or more, 0.9 ml or more, 1 ml or more, 1.5 ml or more, 2 ml or more, 3 ml or more, 4 ml or more, 5 ml or more, 6 ml or more, 7 ml or more, 8 ml or more, 9 ml or more, 10 ml or more, etc.) of fluid in the microfluidic channel. The microfluidic channel can be connected to one or more reservoirs (e.g., a waste reservoir, a storage reservoir, etc.) and / or can be connected to one or more additional microfluidic channels.

[0039] For example, the microfluidic channel can include a first microfluidic channel, and the opening between the microfluidic channel and the air gap can include a first opening; the device can also include a second microfluidic channel formed in a second side of the top plate and a second opening between the second microfluidic channel and the air gap, wherein the second microfluidic channel extends along the second side of the top plate, wherein the first opening and the second opening are adjacent to each other. The first opening and the second opening can be a minimum distance apart, which can allow a "bridging droplet" to form in an air gap having a minimum size. For example, the first opening and the second opening can be no more than about 2 cm apart from each other on the surface of the top plate (e.g., no more than about 1 cm apart, no more than about 9 mm apart, no more than about 8 mm apart, no more than about 7 mm apart, no more than about 6 mm apart, no more than about 5 mm apart, no more than about 4 mm apart, no more than about 3 mm apart, no more than about 2 mm apart, no more than about 1 mm apart, etc.).

[0040] Any of these boxes may also include a window from the top of the box to the air gap, through which the air gap is visible. This can allow imaging into the air gap. The imaging can be used to detect outputs (e.g., reaction outputs, such as binding, colorimetric assays, RT-PCR, etc.). The window can be any suitable size; for example, the window can form between 2% and 50% of the top of the box. The window can be on one side of the box and / or at one end of the box. Multiple imaging windows can be used.

[0041] As mentioned, the bottom of the box is formed by the first side of the sheet of dielectric material.The top of the box may include a plurality of openings into the air gap.

[0042] Typically, the cartridge can include one or more reagent reservoirs on the second side of the top plate. For example, the cartridge can include one or more reagents, particularly lyophilized (e.g., "freeze-dried") reagents, in the reservoirs or within the air gap. For example, the cartridge can include one or more freeze-dried reagent reservoirs on the second side of the top plate.

[0043] For example, a box (having a bottom and a top) for a digital microfluidic (DMF) device 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; a first hydrophobic layer on the second side of the sheet of dielectric material; a top plate having a first side and a second side; a ground electrode on the first side of the top plate; a second hydrophobic layer covering the ground electrode on the first side of the top plate; an air gap separating the first hydrophobic layer and the second hydrophobic layer, wherein the air gap comprises a spacing greater than 500 microns; a first microfluidic channel and a second microfluidic channel, wherein the first microfluidic channel and the second microfluidic channel are formed in the second side of the top plate, wherein the first microfluidic channel and the second microfluidic channel extend along the second side of the top plate; a first opening between the first microfluidic channel and the air gap and a second opening between the second microfluidic channel and the air gap, wherein the first opening and the second opening are adjacent to each other within approximately 2 cm; and a cover covering the microfluidic channel, wherein the cover comprises one or more access ports for accessing the microfluidic channel.

[0044] Also described herein is a DMF reader apparatus for use with any of the cartridges described herein. For example, the DMF reader apparatus (device) can be configured to apply a vacuum across the dielectric bottom surface of the cartridge so that the electrodes are in uniform, close contact with the dielectric forming each unit cell form, which allows droplets of fluid to move in the air gap. The applicant has surprisingly discovered that simply bonding the dielectric material to the electrodes is insufficient because it results in unequal contact and variations in the power required to move the droplets, as well as inefficiencies in droplet movement, control, and consistency. Furthermore, even when combined with an adhesive, the use of a vacuum has similar problems, particularly when the dielectric is flexible. Apparatus and methods for using them are described herein, in which a vacuum is used to secure the dielectric bottom of the cartridge through multiple openings in the drive electrodes themselves or around / closely adjacent to the drive electrodes. In variations in which a vacuum is applied through all or some of the drive electrodes (e.g., on a seating surface, such as spaced apart in a pattern at a corner), the dielectric is uniformly and consistently held on the drive electrodes, even when a relatively low negative pressure relative to the vacuum is used. This configuration may also allow for partitions or barriers to be formed in the cartridge by including protrusions on the cartridge-holding surface on which the cartridge is held.

[0045] For example, described herein is a digital microfluidic (DMF) reader apparatus configured to operate with a disposable cartridge having a bottom dielectric surface, a top plate having a ground electrode, and an air gap between the bottom dielectric and the top plate, the apparatus comprising: a base surface for positioning the disposable cartridge; a plurality of drive electrodes on the base surface, wherein each drive electrode comprises an opening therethrough; a vacuum pump for applying a vacuum to a vacuum port; and a control for applying energy to sequentially activate and deactivate one or more selected drive electrodes to move droplets in the air gap of the cartridge along a desired path in the air gap, wherein the DMF reader 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 base surface.

[0046] In certain variations, the apparatus includes a vacuum manifold coupling a vacuum pump to a plurality of vacuum ports for applying a vacuum.

[0047] The DMF reader device described herein can be configured to operate together with any box described herein, and can be suitable for use with such box. However, it should be understood that the box is not an essential part of the DMF reader device. Typically, these devices can operate together with a box (e.g., a reusable box or a disposable box) having a bottom dielectric surface, a top plate with a ground electrode, and a gap (e.g., typically but not necessarily an air gap) between the bottom dielectric and the top plate.

[0048] The DMF device may also typically include a base surface for positioning the disposable cartridge. The base surface may include: a drive electrode, which may be flush or substantially flush with the base surface; and / or any protrusions that may be used to form a partition in the gap region (e.g., an air gap) of the cartridge by predictably deforming the dielectric in the gap region. The plurality of drive electrodes on the base surface may be formed on or milled into the base surface. For example, the base surface may be a substrate, such as a printed circuit board (e.g., an electrically insulating surface), to which the drive electrodes are attached or formed.

[0049] Typically, as mentioned above, all or a majority (e.g., >50%, >60%, >70%, >80%, >90%, >95%, etc.) of the drive electrodes in the electrode array can include openings through the drive electrodes and connected to a vacuum source. The vacuum source can 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 to the device (e.g., wall vacuum). The openings through the electrodes can be the same size, and they can be located anywhere on / through the drive electrodes. For example, they can pass through the center of the drive electrode, and / or through the edge region of the drive electrode, etc. The openings can be of any shape (e.g., circular, oval, square, etc.). In some variations, the size of the openings can be approximately 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.).

[0050] Typically, the vacuum manifold can be coupled to a plurality of vacuum ports and / or can include a plurality of vacuum ports, each vacuum port being 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 surface of the susceptor. For example, the vacuum manifold can be a pipe or other passage below the surface of the susceptor that connects to the openings in the drive electrode.

[0051] DMF device described herein typically includes controller for coordination and driving electrode.The controller may include one or more processors, memory and any other circuit necessary or useful for operating the equipment, and the operation includes applying of coordinated energy to activate / deactivate (inactivate) driving electrode, for vacuum control and / or microfluidic control pump, one or more valves (for example, for microfluidic control, vacuum control), temperature control device (for example, resistance heater, Peltier (Peltier) cooling etc.), motor (for example, for driving opening and closing equipment door, optics etc.), one or more displays etc.

[0052] As mentioned, any of these devices may include one or more protrusions extending from a surface of the base, 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.

[0053] Any of these devices may include an optical reader configured to detect an optical signal from a box placed on the base surface. The optical reader may be movable or fixed. The optical reader may be used to detect (e.g., sense) a feed or a change due to one or more interactions (e.g., binding, enzymatic reaction, etc.) in the droplet. The optical reader may be configured to detect an optical signal from a box placed on the base surface. Thus, the optical sensor may provide detection of a readout from the device. Any of these devices may include one or more motors, e.g., the motor being configured to move the optical reader.

[0054] The device may also include one or more temperature sensors (e.g., thermistors, etc.). For example, the device may include one or more temperature sensors coupled to the base surface. In some variations, the thermistors may protrude from the base surface and form a barrier or chamber in the air gap of the cartridge. Alternatively or in addition, the one or more temperature sensors may be in a substrate on the base surface and in thermal contact with the base surface, for example, via a thermally conductive material (e.g., copper).

[0055] As mentioned, the devices described herein may include one or more heaters, particularly resistive heaters. For example, the device may include a resistive heater beneath at least some of the drive electrodes (or covering at least some of the drive electrodes); this may allow for temperature regulation of 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.).

[0056] The device can also be included in one or more above or below one or more magnets in the driving electrode, and the magnet is configured to be activated to apply a magnetic field. Therefore, magnetic bead can be used for other reactions in binding material or DMF device, and magnetic bead can be selectively maintained in one or more regions of equipment. For example, one or more neodymium magnets can be used, for example, by moving magnet closer or further away from box so that magnetic particles are maintained in place (for example, magnet is moved upwards to electrode 3mm, 4mm, 5mm, 6mm, 7mm, 8mm etc.). Electromagnet can be selectively activated or deactivated to keep / release magnetic particles.

[0057] 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.).

[0058] In addition to the base surface, any one of these DMF reader devices can also include one or more cartridge trays, and box can be loaded into the cartridge tray so that box can be automatically moved to the position in the device. For example, any one of these devices can include a cartridge tray for maintaining box on a predetermined orientation (which can be fixed by the shape of the box and the complementary receiving tray); the cartridge tray can be configured to move the disposable box onto the base surface. Once on the base surface, vacuum can be applied to lock its position. In addition, it is also possible to connect from the top of the box to one or more microfluidic ports, for example, for applying positive pressure and / or negative pressure (for example, vacuum) to drive the fluid in the microfluidic channel on the top of the box and / or drive fluid into / leave gap (for example, air gap) region in the box.

[0059] In some embodiments, the present invention provides the device of the present invention.Usually, any one of these devices can comprise housing, front panel display and one or more input terminals (for example, touch screen display, dial, button, slide block etc.) and / or power switch.The device can be configured to be stackable, and / or can be configured to operate in conjunction with one or more other DMF devices.In some modifications, single housing can enclose a plurality of box base surfaces, and each box base surface has (by single or multiple controllers) individually addressable / controllable drive electrode array, and this allows the parallel processing of a plurality of boxes; In these modifications, all or some of parts (pump, motor, optical subsystem, controller) can be shared between different box base surfaces.

[0060] 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.

[0061] As mentioned, any DMF device described herein may also include one or more microfluidic vacuum ports positioned above the base surface and configured to engage with access ports for accessing the microfluidic channels of the cartridge when the cartridge is positioned on the base surface.

[0062] For example, a digital microfluidics (DMF) reader device is configured to operate with a disposable cartridge having a bottom dielectric surface, a top plate having a ground electrode, and an air gap between the bottom dielectric and the top plate, the digital microfluidics (DMF) reader device may include: a base surface for positioning the disposable cartridge; a plurality of drive electrodes on the base surface, wherein each drive electrode includes an opening therethrough; a plurality of vacuum ports, wherein each vacuum port is coupled to one or more of the openings in the drive electrodes; a vacuum pump for applying a vacuum to the vacuum ports; one or more protrusions extending from the base surface; and a control for applying energy to sequentially activate and deactivate one or more selected drive electrodes to move droplets in the air gap of the cartridge along a desired path in the air gap, wherein the DMF reader is configured to apply a vacuum to the vacuum ports to secure each drive electrode to the bottom dielectric of the disposable cartridge when the disposable cartridge is placed on the base surface, such that the one or more protrusions separate the air gap.

[0063] Also described herein are methods for preventing or reducing evaporation in any of these devices. For example, a method for preventing droplet evaporation in an air-matrix digital microfluidic (DMF) device is described herein, comprising: introducing an aqueous reaction droplet into an air gap of the 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 drive electrodes on or in the first plate to move the aqueous reaction droplet in the air gap of the air-matrix DMF device, such that the aqueous reaction droplet combines with droplets of a non-polar fluid in the air gap of the air-matrix DMF device to form coated reaction droplets, wherein the non-polar fluid coats the aqueous reaction droplet and protects the reaction droplet from evaporation; and sequentially energizing the drive electrodes to move the coated reaction droplet in the air gap of the air-matrix DMF device.

[0064] In some embodiments, the volume of non-polar fluid can be less than the volume of water-containing reaction droplet.Any one of these methods can be included in the droplet of coating and one or more other water-containing droplet combinations in the air gap of air matrix DMF device.Any one of these methods can also comprise by the droplet of coating being withdrawn at least partially in the microfluidic channel from the air gap of air matrix DMF device, the coating of non-polar fluid is removed.The method can also comprise by the opening in the first plate or the second plate, and 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.

[0065] For example, a method for preventing droplet evaporation in an air-matrix digital microfluidics (DMF) device may include: introducing aqueous reaction droplets 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 drive electrodes on or in the first plate to move the aqueous reaction droplets in the air gap of the air-matrix DMF device, such that the aqueous reaction droplets combine with droplets of a non-polar fluid in the air gap of the air-matrix DMF device (although in some variations, the non-polar fluid may be combined with a sample before being loaded into the air gap), which forms coated reaction droplets in which the non-polar fluid coats the aqueous reaction droplets and protects the reaction droplets from evaporation, wherein the non-polar fluid is a liquid between 10 degrees Celsius and 100 degrees Celsius, and further wherein the volume of the non-polar fluid is less than the volume of the aqueous reaction droplets; and sequentially energizing the drive electrodes to move the coated reaction droplets in the air gap of the air-matrix DMF device. Although the volume of the non-polar liquid can be less than the volume of the droplet, the volume of the non-polar liquid housing the droplet can be greater than the volume of the droplet (up to about 3 times the volume of the droplet).

[0066] Method and apparatus described herein can be particularly well suited for use and processing with large volume droplets.Typically, most of the unit droplets of DMF devices and particularly air matrix DMF devices are limited to approximately 4 microlitres or less aqueous fluids, and air gap is limited to a spacing less than approximately 250 microns or 300 microns between drive electrode and ground electrode (top plate and base plate in air gap region).This paper describes the method for operating on larger volumes, wherein the spacing between drive electrode (for example, base plate) and ground electrode (for example, top plate) can be much larger (for example, between approximately 280 microns and 3mm, between approximately 300 microns and 3mm, between approximately 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 greater, 6 microliters or greater, 7 microliters or greater, 8 microliters or greater, 9 microliters or greater, 10 microliters or greater, 11 microliters or greater, 12 microliters or greater, 13 microliters or greater, 14 microliters or greater, 15 microliters or greater, etc., for example, between 5 microliters and 20 microliters, between 5 microliters and 15 microliters, between 7 microliters and 20 microliters, between 7 microliters and 15 microliters, etc.).

[0067] Dispensing large droplets using electrowetting is conventionally done 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 for dispensing a predetermined volume of liquid using electrowetting are described herein. For example, methods are described herein for dispensing a predetermined volume of fluid into an air gap of an air matrix microfluidic (DMF) device, 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 further comprises 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 filled air gap; and when the first electrode is activated, applying suction to withdraw the fluid into the port, which leaves a droplet of the fluid in the air gap adjacent to the activated first electrode.

[0068] 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 withdraw fluid into the port when the first drive electrode is activated includes: withdrawing the fluid when the first drive electrode and the 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.

[0069] The first drive electrode may be separated from the port by a spacing of at least one drive electrode. Any of these methods may further include deactivating one or more drive electrodes adjacent to a second portion of the air gap, the second portion being in the filled portion of the air gap and between the port and the first drive electrode. The air gap may be greater than 500 microns.

[0070] Filling the portion of the air gap may include applying a positive pressure to expel the 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.

[0071] Applying suction to withdraw the fluid into the port when the first electrode is activated may include leaving a droplet of fluid having a volume of 10 microliters or greater in the air gap adjacent the activated first electrode.

[0072] For example, a method for dispensing a predetermined volume of fluid into an air gap of an air-matrix digital microfluidic (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 filled portion of the 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 withdraw the fluid 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.

[0073] This paper also describes the control system for DMF device, such as those described herein. Especially, this paper describes the control system comprising the graphical user interface for operating any one of these devices. These control systems (subsystems) can include software, hardware and / or firmware. Therefore, any one of these devices can be configured to be stored in a non-temporary medium (for example, memory) for carrying out any one of the method and program described herein.

[0074] For example, this document 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 user-selected fluid handling control commands; 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 executing the fluid handling protocol using the DMF device based on the calculated path.

[0075] 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 cartridge to perform each of these steps (e.g., by moving the droplet to a suitable region of the cartridge, including a heater, magnet, microfluidic port, etc.) such that the droplet can be manipulated as needed. For example, receiving a fluid handling protocol may include receiving a series of fluid handling control commands. Calculating a path may include calculating a path based on the arrangement of heating and cooling zones in a DMF device. Calculating a path may include determining the shortest path that does not intersect itself. Typically, executing a fluid handling protocol on a DMF device may include executing the fluid handling protocol in a disposable cartridge coupled to the DMF device.

[0076] Also described herein is a digital microfluidic (DMF) reader device configured to operate with a removable and / or disposable cartridge having a bottom dielectric surface, a top plate having a ground electrode, and an air gap between the bottom dielectric and the top plate, the device comprising: a base surface for seating the disposable cartridge on the top surface; a first plurality of drive electrodes on the base 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 base surface; a plurality of thermal vias; The cartridge includes a thermal via, wherein the thermal via comprises a thermally conductive material and is in thermal communication with a first area of ​​the base surface but is electrically isolated from a subset of the electrodes, and further wherein the thermal via is in thermal communication with 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 move a droplet in an air gap of the cartridge along a desired path in the air gap.

[0077] 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).

[0078] The thermal vias can each be filled with a thermally conductive material; the material can be electrically conductive or electrically insulating. In some variations, the thermally conductive material is a metal. The reader can also include one or more resistive heaters underlying at least some of the drive electrodes.

[0079] The base surface can be formed or at least partially formed on a printed circuit board (PCB), including on an electrode array formed on a PCB. As mentioned above, any reader described herein can include one or more magnets; in some variations, the magnet can be below one or more of the drive electrodes, which are configured to be activated to apply a magnetic field. For example, the magnetic field can pass through an opening in the drive electrode. The reader can 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.

[0080] Also described herein are methods for detecting the location and / or identity of a material within an air gap of a digital microfluidics (DMF) cartridge. The material can include liquid droplets (e.g., aqueous droplets), wax, droplets coated / encapsulated in wax (e.g., liquid wax), oil droplets, droplets with magnetic particles, and the like. The identity of the material can be determined at a specific location within the air gap, such as between the upper and lower surfaces forming the air gap within the cartridge. The cartridge can be divided into cells (e.g., regions above individual drive electrodes).

[0081] For example, a method of detecting position and / or identity may include: disconnecting a reference electrode on a first side of an air gap of a DMF box from a drive circuit; setting a voltage of one or more drive electrodes in an array of drive electrodes on a second side of the air gap to a high voltage while setting all other drive electrodes in the array of drive electrodes to ground; sensing a voltage at the reference electrode; determining a 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; and identifying a material in the air gap adjacent to the one or more drive electrodes based on the determined capacitance.

[0082] The method can 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 can be repeated iteratively to track the movement of the material in the air gap.

[0083] Disconnecting the reference electrode can include allowing the reference electrode to float (e.g., not grounded). The reference electrode can 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 the movement of droplets in the air gap via digital microfluidics) can include connecting the reference electrode to a sensing circuit for detecting a voltage at the reference electrode and, therefore, detecting the capacitance of the air gap. The reference circuit can include one or more reference capacitors arranged to allow measurement of the air gap capacitance.

[0084] Setting the voltage of one or more of the drive electrodes to a high voltage may include setting the voltage of one or more of the drive electrodes to between 10V and 400V (eg, between 100V and 500V, such as approximately 300V, etc.).

[0085] Any of these methods can include determining a total capacitance for the air gap by setting the voltage of all drive electrodes in the array of drive electrodes to a high voltage and sensing the voltage at the reference electrode to determine the total capacitance when the reference electrode is disconnected from the drive circuit. The method can 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 a first side of the air gap and a second side of the air gap based on the voltage sensed at the reference electrode can also include using the total capacitance.

[0086] Identifying the material in the air gap may include identifying the material in the air gap using a reference database based on the determined capacitance, the reference database including a plurality of capacitance ranges.

[0087] Also described herein are cartridges (e.g., disposable and / or removable cartridges) for digital microfluidics (DMF) devices that include a tensioning frame to hold a bottom dielectric material in tension and, therefore, flat. For example, any cartridge described herein can 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 comprises a first hydrophobic surface; a tensioning frame that holds the sheet of dielectric material in tension so that it is substantially flat; a top plate having a first side and a second side and a thickness between the first and second sides; 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 and second hydrophobic layers, wherein the air gap comprises a spacing greater than 280 microns. Any other cartridge features described herein can be included with these cartridges.

[0088] Any of these cartridges may also include a lip that extends at least partially (including completely) around the sheet of dielectric material and protrudes from the sheet of dielectric material. The lip may engage with a channel or groove on the base surface. Alternatively or in addition, the cartridge may include a peripheral channel or groove into which a protrusion on the base surface of the reader engages.

[0089] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] 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:

[0091] Figure 1A is a schematic diagram from a top perspective of one example of an air matrix digital microfluidics (DMF) device.

[0092] Figure 1B Shown through Figure 1A An enlarged view of a cross section of a portion of an air matrix DMF device is shown in FIG, the cross section being taken through a thermal conditioning region (hot zone).

[0093] Figure 1C Shown through Figure 1A A magnified view of a second cross-section of a region of the air-matrix DMF device; the region includes perforations through the base plate and the actuating electrodes and is configured such that replenishing droplets can be delivered from the perforations (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.

[0094] Figure 2 is similar to Figure 1A-1C An example of a DMF surface using a rigid box including electrodes and air gap regions is shown.

[0095] Figure 3A An example of a typical DMF arrangement, for example using a rigid box, is shown; Figure 3B An example of a DMF configuration is shown where the 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).

[0096] Figure 3C is an example of a DMF device configured as a compact driver / reader configured to work with a removable / disposable cartridge. The DMF device includes an array of electrodes (e.g., greater than 500 different electrodes) and multiple independent areas for controlling heating / cooling of the beads (thermal cycling, etc.), pumping microfluidic channels, automated placement and sealing of the cartridge, and optical observation / management.

[0097] Figure 3Dis another example of a DMF device as described herein, which is configured as a compact drive / reader that can include greater than 900 (e.g., greater than 920 different electrodes), independent heaters for the isothermal zone and thermal cycler, magnetic zones that can be independently engaged / disengaged, pumps and valves for operating microfluidics in a disposable cartridge (in addition to DMF control via multiple electrodes), a vacuum manifold coordinated with the multiple electrodes (e.g., with ports that pass through the electrodes to seal the dielectric and secure to the electrodes for precise and reliable DMF control), multiple independent qPCR zones, multiple optical channels, and a pulling mechanism for inserting / removing cartridges that allows access from both the top and bottom of the device. Figure 3C and Figure 3D The apparatus shown in can provide liquid cooling of both the ambient zone and the heated zone.

[0098] Figure 3E yes Figure 3C-3D Another example of a device is shown in , which shows an exemplary arrangement of the following components: a pump (e.g., a vacuum pump that secures the cartridge), a liquid cooler and compressor, one or more motors for actuating a drawer that receives the cartridge and for actuating the optics, a control for opening / closing the drawer, a manifold for operating any microfluidics on the cartridge (in addition to or in lieu of DMF), and an electrode array for driving the DMF in the cartridge. In this example, a disposable cartridge is shown inserted into the device.

[0099] Figure 3F is an exemplary DMF apparatus (e.g. Figure 3C-3E An example of a housing for a device (shown in FIG) configured as a single tray (box) device. Figure 3F , the tray is shown extended. The dimensions shown are for illustrative purposes only and may be larger or smaller by, for example, + / - 5% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 75%, 100%, etc.).

[0100] Figure 3G and Figure 3H Shown Figure 3F Front side of exemplary DMF device ( Figure 3G ) and back ( Figure 3H The tray for loading / unloading cassettes is shown closed.

[0101] Figure 3I Another example of an exemplary DMF apparatus configured to process multiple cartridges is shown. Figure 3Iis a front view of a device configured to handle six cartridges and including six access controls and display panels, which may be color-coded. In the housing shown, components such as pumps, motors, optics, controllers, etc. may be shared, and / or multiple separate components (e.g., electrode arrays, sub-controllers, etc.) may be used. The housing may be configured to allow multiple devices to be stacked.

[0102] Figure 3J yes Figure 3I Front perspective view of .

[0103] Figure 3K The diagram shows Figure 3I-3J Example of a back view of a multiplex device.

[0104] Figure 3L is an enlarged view of the leftmost cartridge drawer, including the cartridge-specific display, input terminals (eg, buttons, touch screen, etc.), and cartridge drawer.

[0105] 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 having thermistors and / or cooling and / or heating). Figure 4A In FIG, 18 rows and 10 columns are shown; larger or smaller arrays may be used.

[0106] Figure 4B A magnified area of ​​the electrodes forming the upper electrode layer is shown showing vacuum openings through most (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.

[0107] Figure 4C The diagram shows that there may be Figure 4B A resistive heating layer is provided below the electrode layer (shown in FIG). 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.

[0108] Figure 5AA 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 cartridge dielectric to the electrodes.

[0109] Figure 5B Examples of fans, radiators, reservoirs, and pumps are shown that can be used for liquid coolant for the cartridge contact surfaces, including electrodes. The pump, piping, fans, radiators, and reservoirs can be used to move water or liquid coolant beneath the electrodes so that the coolant absorbs heat as it passes beneath the electrodes, where it can then be recirculated after being cooled again by the fans and radiators.

[0110] Figure 5C shows a similar Figures 4A-4C Another view of the electrode PCB shown in FIG, which is connected to a vacuum pump and liquid coolant (input and output).

[0111] Figure 5D and Figure 5E The diagram 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 , the vacuum is off and the dielectric is not held 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 a plurality of openings through the electrode, which hold the dielectric uniformly against the electrode and surprisingly produce uniform electrical properties between the removable cartridge and the electrode.

[0112] Figure 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.

[0113] Figure 5G Shown below the electrode Figure 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.

[0114] Figure 6 The figure shows different functional areas that can be formed by electrode arrays and / or removable cartridges. Figure 6 In the example, the removable cartridge has been made transparent (the microfluidic area above the top plate, air gap, and dielectric forming the DMF portion of the cartridge 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 6In FIG, seven of the electrodes are configured as magnetic regions 603 that can exert a local magnetic force (on the electrode) to retain the magnetic beads or particles in the droplet on the electrode. 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 Figure 6 In the figure, the six 16-electrode regions 607 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 spanning five electrodes and the other spanning 32 electrodes) are also included and can thermally cycle over all or regions of the zones. Four optical reading zones 608 (each spanning four electrodes) are spaced apart from each other on the right perimeter of the device. Typically, the heating and / or thermal cycling zone is located in the center, separated from the cooling / storage zone on the periphery. There can be overlap between these zones (e.g., between the magnetic zone and the heating / cooling zone).

[0115] Figure 6 Also shown in transparent view are microfluidic portions that may be formed above the air gap (and in the top plate, as described). For example, 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 , the microfluidic channel also includes a pair of ports 617, 618 through which positive and / or negative pressure can be applied to (along with any valves) regulate the movement of fluid in the microfluidic region and (in some variations) regulate the movement of fluid into or out of the air gap. The microfluidic portion may also include one or more waste chambers 621.

[0116] Figure 7A 1 is a top view of an exemplary box as described herein. In this example, the box includes a DMF portion comprising 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 the channel input and / or output. The 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.

[0117] Figure 7B Show Figure 7A Top perspective view of the box.

[0118] Figure 7C It is from Figure 7A and Figure 7BAn end or side view of the box viewed from the left side showing the upper microfluidic channel and the lower DMF portion (showing the spacing between the top, ground, plate and dielectric forming the air gap).

[0119] Figure 7D yes Figures 7A-7C A top view of the box with the cover for the microfluidic channels removed. Figure 7D The channels are shown.

[0120] Figure 8A is an example of a disposable box that includes a plastic top plate and a dielectric.

[0121] Figure 8B Paper digital microfluidics that can be used as part of a cartridge are shown.

[0122] Figure 9A An example of an open array of electrodes beneath a disposable plastic top plate and dielectric is shown.

[0123] Figure 9B A box is shown over the open array, held in place by a vacuum to keep it rigidly attached over the electrodes.

[0124] Figure 9C 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 cartridge (e.g., bottom, dielectric layer) aligned and secured to the device. Positive pressure can be applied to release the cartridge.

[0125] Figure 10A Schematic diagram illustrating an example of a patterned ground electrode on a top plate as described herein.

[0126] Figure 10B Shown in Figure 10A A side view of the patterned top plate is shown in FIG.

[0127] Figure 11A and Figure 11B A front view and a side view, respectively, are shown of another variation of a top plate including a ground electrode formed of an opaque conductive ink (e.g., silver conductive ink, carbon conductive ink, etc.) formed in a grid pattern including a plurality of window openings forming a grid.

[0128] Figure 12A is an example of applying conductive ink to form a ground electrode on the top plate. Figure 12B An example of a patterned top plate ground electrode including a plurality of openings therethrough is shown.

[0129] Figure 13A and Figure 13B The figure shows an example of a patterned ground electrode (top plate) on a flexible transparent substrate.

[0130] Figures 14A-14C The diagram illustrates the operation of a DMF device using a patterned ground electrode.

[0131] Figures 15A-15C An example of a microfluidic channel interfacing with a DMF air gap region as described herein is shown. Figure 15A In FIG, the microfluidic portion of the cartridge is shown as a pair of channels, each channel being connected to an inlet / outlet, and each channel terminating in a bridge region (in this example, below the microfluidic portion) that forms an opening into the air gap of the DMF portion of the cartridge. Fluids can be removed, added, washed, etc. in and out of the air gap of the DMF portion. Figure 15B and Figure 15C 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. 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.

[0132] Figure 16A An example of a cross section through the top plate to form a microfluidic channel immediately adjacent to (eg, as part of, above, or below) the DMF portion is shown. Figure 16B An example of a top plate in which microfluidic channels have been formed is shown.

[0133] Figure 16C FIG. 5 is another example of a top plate of a DMF device configured as a microfluidic channel. The top plate is shown as an acrylic material in which channels and holes have been formed (e.g., by milling, cutting, rastering, etc.).

[0134] Figure 16D Another example of a microfluidic channel formed in the top plate of the DMF portion of the cartridge is shown.

[0135] Figure 17A and Figure 17B 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. Figure 17AIn the , fluid application and extraction equipment are connected through the top plate. Figure 17B In the example, fluid application and extraction equipment are connected from the side panels.

[0136] Figure 17C is another example of a DMF cartridge configured for mixing, extracting, adding, etc., fluids having one or more droplets in the air gap of the DMF cartridge. Figure 17C 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 Figure 17A ) The air gap in the interface region can be larger than the air gap in other parts of the DMF box. Figure 17D In the example, the interface 1127 for the fluid line is at the edge of the air gap, similar to Figure 17B ;exist Figure 17D In the case of , the air gap area is larger than in other areas of the box. Figures 17A-17D In either, the fluid lines (e.g., 1143, 1145) and reservoirs (1105, 1107) can form part of the DMF apparatus and can be connected to ports on the box (e.g., the top surface of the box) and / or one or more valve interfaces.

[0137] Figures 18A-18C The diagram shows something similar to Figure 17A The operation of the fluid application and extraction apparatus is shown.

[0138] Figures 19A-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.

[0139] Figures 20A-20C It is shown that when a non-polar material (such as liquid paraffin) is used, the Figure 20B ) and two hours later ( Figure 20C )'s resistance to evaporation, which shows little or no evaporation.

[0140] Figures 21A-21D The figure shows the use of non-polar jacket materials in an air-based 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. Figure 21E-Figure 21I The diagram shows adding a large sample to the jacket material and mixing the sample.

[0141] Figures 22A-22D The figure shows the control of the droplet volume 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 cases, the electrowetting force alone may not be sufficient to dispense a droplet of a predetermined volume. Figures 22A-22D As shown in , droplets broken off from a large volume can be used to dispense a predetermined volume. Figure 22A In the distribution electrode is activated, spaced from the distribution port (tube). Figure 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. Figure 22C The reagent is then drawn back into the dispensing port while the dispensing electrode is active, but the electrodes between the dispensing port and the dispensing electrode are not active, which forms a neck that is eventually disconnected (e.g. Figure 22D ), which leaves a droplet of a predetermined volume on the dispensing electrode.

[0142] Figures 23A-23F The diagram shows the use of the above Figures 22A-22D The technique described is an example of dispensing droplets of pre-defined volumes.

[0143] Figure 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.

[0144] Figure 25A and Figure 25B Commonly illustrates visual controls or commands ( Figure 25A ) and scenarios describing the use of these visual controls / commands ( Figure 25B ) instance.

[0145] Figures 26A-26H The figure shows an example of a user interface for controlling a DMF device as described herein.

[0146] Figure 27A and Figure 27B Illustrated are top and bottom perspective views, respectively, of one example of a top portion of a digital microfluidics cartridge as described herein.

[0147] Figure 28 The figure shows an example of a portion of a box, which shows the thermal control area.

[0148] Figure 29is an example of a portion of a reader (e.g., a cartridge base portion) having a reduced thermal mass to increase the rate of temperature adjustment of a cartridge held on the base portion.

[0149] Figure 30 is another example of a portion of a reader (e.g., a cartridge base portion) having a reduced thermal mass to increase the rate of temperature adjustment of a cartridge held on the base portion.

[0150] Figure 31A and Figure 31B The figure shows an example of a reader that includes thermal vias to help control the temperature of a cartridge (eg, of one or more cells of an air gap of a cartridge).

[0151] Figure 32 is an example of a cartridge that includes an opening in the top plate for sampling or adding fluid to the droplets in the cartridge.

[0152] Figure 33A An ITO sensing circuit with switches is shown.

[0153] Figure 33B The figure shows another example of a capacitance sensing circuit including a plurality of reference capacitors.

[0154] Figures 34A-34C One method of identifying and / or locating droplets in an air gap as described herein is illustrated. Figure 34A An 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. Figure 34B is a graph showing exemplary voltage measurements from a sensing electrode (top electrode). Figure 34C is a graph showing an example of changes in the electrical permittivity of water as a function of temperature.

[0155] Figure 35A is a top view of an example of a vacuum chuck.

[0156] Figure 35B yes Figure 35A Cross-sectional view of the vacuum chuck.

[0157] Figure 36 Shown Figures 35A-35B An isometric view of the chuck is shown in FIG.

[0158] Figure 37 Shows something like Figure 35A-Figure 35B A top view of the chuck is shown in FIG.

[0159] Figure 38A Another example of a vacuum chuck is shown.

[0160] Figure 38B A cross-sectional and enlarged view of the chuck is shown.

[0161] Figure 39 Shows something like Figures 35A-35B A bottom view of the chuck is shown in FIG.

[0162] Figure 40 Shows something like Figure 35A An isometric view of the chuck is shown in FIG.

[0163] Figure 41A

[0066] Shown is one example of a heat dissipation system that may be included in any of the reader devices described herein.

[0164] Figure 41B It passes through Figure 41A Cross-sectional view of the chuck.

[0165] Figure 42 A front view of the chuck and fan is shown.

[0166] Figure 43 An example of the arrangement of the chuck, fan and PCB (part of the base surface) is shown.

[0167] Figure 44 is a perspective view of a chuck, which may include a thermal (eg, heat) dissipation system for regulating the temperature of the cartridge.

[0168] Figure 45A yes Figure 44 A top view of the chuck.

[0169] Figure 45B It passes through Figure 45A Cross-sectional view of the chuck.

[0170] Figure 46 A side view of the assembly of the chuck, heat sink, and 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).

[0171] Figures 47A-47C The diagram shows components of a vacuum chuck and cooling subsystem (eg, heat sink block and cooling fan).

[0172] Figure 48 The figure shows an example of components for a reader, which include: 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 to the PCB; and a thermal regulator subsystem, which includes a heat sink / heat block and a pair of cooling fans.

[0173] Figure 49A and Figure 49B A tensioning frame and a film frame are shown respectively for fixing and keeping smooth a film (eg a dielectric film) which may form the bottom of the box.

[0174] Figure 49C is a side view of the assembled tension frame.

[0175] Figure 49D is a perspective view of the assembled tension frame.

[0176] Figure 50A is an instance of an exploded view of a box.

[0177] Figure 50B is another example of an exploded view of a box.

[0178] Figure 51 is an exploded view of an example of the cartridge and cartridge base portions of a reader.

[0179] Figure 52A is a top view of the reader's PCB, onto which the cartridge may be placed.

[0180] Figure 52B yes Figure 52A A side view of the PCB portion is shown in FIG.

[0181] Figure 52C is an example of a side view of a cartridge shown on the base surface of a reader.

[0182] Figure 52D yes Figure 52C Magnified view of .

[0183] Figure 53 is an exploded view of the cartridge and base surfaces / areas of the reader.

[0184] Figure 54A is a top view of the reader's PCB (which may form the base surface).

[0185] Figure 54B It passes through Figure 54A A side cross-sectional view of a portion of the reader is shown in FIG.

[0186] Figure 55 An example of an electrode grid arrangement with independent action zones is shown.

[0187] Figure 56 Four independently controlled 1-plex modules are shown schematically with a console unit that can operate them all. DETAILED DESCRIPTION

[0188] Generally, digital microfluidic devices and methods are described herein. In particular, the air matrix digital microfluidic devices comprising systems and equipment are described herein, and methods for operating the air matrix digital microfluidic devices to process fluid samples are described herein. For example, a DMF device can include a compact DMF driver / reader that is configured to work with a removable box / disposable box. The DMF driver / reader can include an array of drive electrodes that 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 that is open to the environment (for example, for air) via an opening (for example, side (lateral) opening and / or top opening). The air gap can be formed between two dielectric layers. The upper top region can include one or more ground electrodes. The ground electrode can advantageously be formed from an opaque material that is patterned to include one or more windows that allow imaging through the top. The windows may be arranged above the electrodes such that the ground region extends relative to the drive electrodes, around the drive electrodes and / or between the drive electrodes.

[0189] Any of the devices described herein may also include fluid application and extraction components (e.g., fluid application and / or extraction equipment) that are connected to the air gap through the top or through the side of the box. Any of the devices 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 are user interfaces for interfacing with the device, including user interfaces 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.

[0190] Figure 2 The diagram shows something similar to Figure 1A-1C An example of a DMF apparatus is shown in FIG. 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 FIG, four reservoir areas 203, 205, 207, 209 are positioned 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.

[0191] exist Figure 2In the device, the DMF driving electrode 211 is a solid flat electrode. The application of energy between the driving electrode and the ground electrode 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.

[0192] However, it would be beneficial to provide a DMF reader apparatus (eg, 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 electrodes are part of the reader but the box only includes a ground electrode (e.g., top plate), an air gap, and a dielectric bottom. For example, in Figure 3A In 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 the 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 supply board 311.

[0193] 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 therebetween. As will be described in greater detail herein, a vacuum (e.g., a vacuum manifold) can be positioned beneath the electrode 313 to apply 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 fully secure the dielectric, and therefore the remainder of the cartridge, to the reader device. The electrodes 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 (e.g., a vacuum manifold) can be positioned beneath the electrode 313 to apply 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 fully secure the dielectric, and therefore the remainder of the cartridge, to the reader 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 is hydrophobic.

[0194] Figure 3C is an example of a compact DMF drive / reader that can be used with any of the cartridges described herein. Figure 3C, the dimensions (15 cm or 6 inches in height, 20 cm or 8 inches in width) are exemplary only, but illustrate the compact nature of the reader. The reader can include a cartridge base surface 351 below which vacuum components, heating components, cooling components, magnetic components, and other components including control circuitry can be positioned. In this example, microfluidic control components (e.g., valves, pumps, etc.) can be positioned above the cartridge base surface for controlling these elements.

[0195] Figure 3D Another example of a DMF reader device is shown that includes an integrated drive electrode on a portion of the base surface. A drawer (not shown) can be used to insert / remove the cartridge and place the cartridge on the base surface, wherein a vacuum can be used to secure the cartridge in place and provide full electrical contact between the cartridge's drive electrode and the dielectric. Both the microfluidic processing portion 355 and the optical device (e.g., optical reader) can be positioned on the base surface. Figure 3E Shown Figure 3C and Figure 3D Another perspective view of the device of FIG, which shows a drawer 361 holding an exemplary disposable box 363. As shown, the drawer can open / close (for example, by pressing a control, such as button 362) to pull the box into the device and pull it out from the device, and the box is positioned on the base surface, which includes a drive electrode array 365, wherein each of the drive electrodes (in this example, and shown in more detail below) includes an opening for applying vacuum to keep the dielectric on the drive electrode. When the microfluidic part is maintained on the base surface, the microfluidic part can be above the base surface and therefore engage with the box above the box. For example, a microfluidic valve manifold 367 can be included and can be connected to a pump or pumps 369. The same or a separate pump 371 can be used to provide pressure for keeping the dielectric on the base surface by the electrodes. The system can also include an optical device subsystem 373 for imaging by at least a portion of the box to report data on the reaction performed on the device. A motor can also be included for driving the optical device and / or the drawer to open / close. A liquid cooler and compressor 375 may also be included for circulating cooling liquid, for example, beneath the cassette.

[0196] Figure 3F Shown Figure 3E 381 is open. The housing may include feet 383 that can engage with receiving sites 385 on the top surface so that the devices can be easily and safely stacked. Figure 3G and Figure 3H The front and rear views are shown respectively.

[0197] In some variations, the apparatus may include multiple cartridge receiving sites (e.g., base surfaces) for operating on multiple cartridges in parallel. For example, Figure 3I-3K The example of device is illustrated, and wherein six box receiving drawers can be used to operate simultaneously on up to six independent boxes.In this example, each receiving drawer can comprise button, is used for opening / closing drawer, and can comprise independent readout screen 390. Figure 3I and Figure 3J A front view and a front perspective view are shown respectively, and Figure 3K In this variation, internal components (eg, processors and optical sensors) may be shared between the different base surfaces within each sub-area of ​​the device. Figure 3L A detailed view of one example of the front of the device is shown.

[0198] The base surface of the exemplary DMF reader device is Figures 4A-4C and Figures 9A-9C This is shown in more detail in Figure 4A In , the base surface includes an array of drive electrodes 401 (labeled 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 base surface also includes a temperature sensor (thermistor 405) that is positioned between the electrodes in different orientations. Figure 4B A slightly enlarged view of the base 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, a pattern of drive electrodes that is not a monolithic block 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 1A and Figure 2 As shown in .

[0199] Figure 4C An example of a heater is shown which may be positioned beneath some of the drive electrodes, e.g. 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 base surface). In general, resistive heating and thermistors 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 Figures 4A-4CThe current can be regulated, for example, by a PID control loop in combination with a thermistor, and thus the temperature of the drive electrode and / or the adjacent dielectric (and thus any droplets on the cell below the dielectric / drive electrode) is regulated. To cool the dielectric (and the entire susceptor surface), a liquid cooler can be circulated through the substrate, for example on the bottom of the susceptor surface. Figure 4C In the example of FIG, the resistive heater is shown as a continuous trace of low-resistance material (eg, having a resistance between approximately 10-15 ohms).

[0200] Any suitable temperature regulation technique can be used. For example, stirring (e.g., magnetic stirring) can be used. Even small volume droplets can contain local temperature ranges, so the temperature distribution can have a standard deviation. This can be reduced by stirring, for example, via magnetic beads. With sufficient stirring, the droplets can be close to isothermal. In any of these variations, a 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.

[0201] 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 by including a top thermal mass separate from the cassette that engages the top of the cassette when the cassette is on the base surface. For example, the heated and / or cooled top thermal mass can be a manifold that presses down on the cassette.

[0202] 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 3-5 degrees of room temperature, e.g., between 15-35 degrees Celsius). Figure 5A , an example of a base surface 501 is shown removed from the device to illustrate a base plate with liquid coolant coupled to the base surface such that coolant can be pumped through the base surface 501 into 503 and out of 505 .

[0203] Figure 5B A 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.

[0204] 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 against the base 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 consistently 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 base surface having an electrode with an opening through which a vacuum is applied. Figure 5D In , the vacuum is off and the dielectric 555 rests loosely on the base surface in many wrinkles. Figure 5E In the process, a vacuum is applied via electrodes.

[0205] Using vacuum in this way allows for reduced dielectric thickness and, thereby, lower power (e.g., voltage) requirements. Compared to using an adhesive or using a vacuum applied externally to the electrodes, Figures 5A-5E The configuration shown in FIG results in 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).

[0206] exist Figure 5C , a pump 560 is shown connected via tubing to a vacuum manifold configured to pull air through the holes in the electrodes. The dielectric film sits on top and remains rigid as long as the air is pumped out. Additionally, any protrusions in the surface of the dielectric (particularly protrusions around or slightly smaller than the width of the box's air gap) will not interfere with the seal, but will form a shell, channel, barrier, or other structure in the air gap that can help separate the air gap.

[0207] Figure 5F and Figure 5G The diagram shows the upper and middle layers of the base surface showing the connection between the vacuum source (via connector 565) through mechanical and / or plumbing manifolds ( Figure 5G ), and out of the opening through the electrode ( Figure 5F ).

[0208] Figures 9A to 9C The figure shows an example of a base surface 900 on which the cartridge may be held by vacuum ports passing through the electrodes. Figure 9AIn , the base surface is formed on a substrate (e.g., a PCB or other electrically insulating surface) and includes an array of electrodes 901, shown in this example 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 base surface, or may be slightly raised above the base surface. In Figure 9B In the figure, the box 905 is shown as being placed on top of the array of drive electrodes 901 on the base surface 900. The box can be placed on the base surface by a drawer (as described above). Figure 3E and Figure 3F ). When on the base surface, a vacuum can be applied by all or a subset of the drive electrodes (e.g., the drive electrodes over which fluid can be transported in the air gap) to hold the dielectric (and therefore the cartridge) in place. As mentioned above, without applying a vacuum by the electrodes themselves, more energy may be required to reliably drive the fluid in the air gap, and the dielectric must be thicker. Figure 9C An enlarged view of a portion of the susceptor surface 900 is shown showing the electrode 901 having a central opening 909 into the vacuum manifold.

[0209] The base 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, solution addition, cooling, imaging / detection, etc. These areas can be defined in the DMF reader 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 base surface. Figure 6 In 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 the cell (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 cooling zones (e.g., cooled via the Peltier zone below) 605. These zones 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 the droplets. One or more magnetic zones 603 can be used to turn on / off a magnetic field that can be used to immobilize 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 can be possible because the magnet can be positioned directly below the heating zone when resistive heating is used.

[0210] In addition to the zones formed by the configuration of the base 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 is already transparent. Typically, the microchannel can be used to mix, distribute waste and remove waste from the top plate in 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 removing waste.

[0211] box

[0212] In general, a cartridge as described herein can 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 can be a Teflon coating. The cartridge can also include one or more microfluidic channels, particularly microfluidic channels formed directly in the top plate with controlled access to the air gap.

[0213] For example, Figures 7A-7D The figure shows an example of a cartridge 700 that includes a microfluidic region on an upper surface that is covered by a lid 703 having one or more access ports 705, 707 for accessing the microfluidic portion of the device. The lid 703 may also include one or more valves and / or one or more openings 709 that can be used to deliver or remove fluids and / or gases (e.g., air). The cartridge may also include openings through a top plate 713 that include openings connecting microfluidic channels to air gap regions within the channels.

[0214] Any of the cartridges described herein may also include one or more transparent window regions 711 for optically imaging one or more regions in the air gap (readout regions). Figure 7B yes Figure 7A Top perspective view of the box. Figure 7C A side view of the box is shown showing the lowermost bottom dielectric film 751 material. The air gap is Figure 7C It is not visible but may refer to the spacing 753 between the dielectric and the ground electrode. Figure 7D Shown is the top plate with the cover removed. 7A to 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. Figure 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. Figures 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 certain variations it may be beneficial to include more reagents, including freeze-dried reagents that can be rehydrated.

[0215] Figure 8A-8B The figure shows different examples of boxes that can be used. Figure 8A In the exemplary box 800 (similar to Figures 7A-7D The box shown in FIG) is shown on a base surface 803 including electrodes. The box 800 includes an air gap ( Figure 8A 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 access regions for accessing the air gap and / or the microfluidic portion. Figure 8B Three different DMF design configurations on paper are shown in Figure 2. The paper DMF devices were formed by inkjet printing an array of silver drive electrodes and reservoirs connected to contact pads onto a paper substrate.

[0216] In the box, the top plate can be any suitable material, including a transparent material, such as acrylic. The top plate can be formed from one or more conductive polymers (or can include one or more conductive polymers). A ground electrode can be formed on the top plate. In particular, the ground electrode can be formed from a conductive material, which conductive material in particular includes a printed conductive material, such as a conductive ink. In particular, the return electrode can be a pattern with a plurality of window openings forming a grid (such as a grid pattern). The pattern can be selected so that when the box is fixed to the base surface of the reader, the window openings are aligned with the drive electrodes. In Figure 10A, a ground electrode 1001 is shown having a grid pattern comprising a plurality of open square windows 1003. As already mentioned, the window openings forming the grid pattern can be of any suitable shape, including other quadrilateral shapes (e.g., rectangular, etc.), other polygonal shapes, elliptical shapes (e.g., circular, oval, etc.), regular and irregular shapes. An additional layer (e.g., a hydrophobic layer) can cover both the conductive material pattern and the plate. Figure 10B An exemplary side view (thickness not to scale) is shown showing the plate 1005 and the conductive patterned electrode 1001. In general, unless otherwise indicated, none of the figures described herein are necessarily shown to scale.

[0217] Figure 11A and Figure 11B Another example of a ground electrode 1101 formed in a grid pattern is shown, with oval 1103 (circular in this example) window openings formed on a first plate 1105'.

[0218] For example, the electrodes may be formed from a conductive ink (eg, silver ink), such as Figure 8B Although not clear, such a printable ink may have advantages over other conductive materials previously described, such as ITO. The use of silver nanoparticles formed in a grid may result in lower, more repeatable, and more precise energy requirements. Figures 10A-10B In one embodiment, the pattern of electrodes has a minimum thickness of between about 50 microns and 200 microns (e.g., 100 microns). The outline around the open window can be configured to be positioned over the space between adjacent electrodes in the drive electrode array. When the box is aligned and fixed in place over the drive electrodes, the overlapping spaces between the drive electrodes on the base plate are covered, but the central area (particularly, the central area may include an opening for applying a vacuum as described above) may be located at the center of the window. Because many conductive inks (e.g., including silver ink) are not transparent, the open window can allow visualization of the air gap under the ground electrode. Although the minimum thickness can be between 50 microns and 150 microns, in practice, the minimum thickness of the grid pattern can be greater than the 100 micron width; for example, the minimum thickness can be between 100 microns and 200 microns.

[0219] The ground electrode may be formed on the substrate (eg, top plate) in any suitable manner. Figure 12A and Figure 12B The figure shows two methods of forming a ground electrode. Figure 12A In , the top electrode is formed by coating a transparent substrate with a conductive ink and allowing the resulting layer to dry. Figure 12B In the invention, patterns such as those described above are formed by printing techniques (e.g. screen printing, printing, etc.). Figure 12B In the example, the pattern is represented by a Figure 10A The pattern shown in FIG5 is formed by printing conductive silver nanoparticle ink.

[0220] Figure 13A and Figure 13B An example of a top plate with a grid patterned ground electrode is shown. Figure 13A and Figure 13B In the invention, a grid pattern is formed in a second order pattern having areas including reservoirs for storing fluid in the air gap and channels and chambers in which different reactions (heating, mixing, cooling, etc.) can take place. Figures 14A-14C The diagram shows Figures 13A-13B The operation of the ground plate of FIG. 1 shows the driving electrode for driving the droplet movement using this ground plate configuration in the box. Figure 14A In , the droplet 1403 is held on the first unit cell in the air gap. Figure 14A In the embodiment of the present invention, an air gap exists between the dielectric drawn down onto the base surface and the drive electrodes, through which the vacuum is drawn. The pattern of the grid forming the ground electrodes matches the arrangement of the drive electrodes in the base surface. The drive electrodes 1411 each include an opening 1413 that is connected to a vacuum manifold through which a vacuum is applied to hold the dielectric, and therefore the cartridge, in place.

[0221] exist Figure 14A and Figure 14B Between, power is applied to the electrode below the droplet and sequentially to one or more adjacent electrodes, which allows a change in the electrowetting of the droplet, driving the droplet 1405 to the left, as shown Figure 14B The process can be repeated as shown in Figure 14C This moves the droplet to another unit cell in the air gap 1407. Movement using a grid patterned ground electrode is equivalent to or better than movement of a monolithic ground electrode.

[0222] In any of these variations, the return electrode on the top plate of the cartridge can be formed from a material layered on the top plate. For example, the conductive layer forming the return electrode on the top plate can be formed from aluminum and a film of a dielectric material and / or a hydrophobic material. In certain variations, the electrode can be formed from ITO, an adhesive, and a dielectric film and / or a hydrophobic film. In certain variations, the conductor can be formed from an ITO film (including a primer and a Teflon coating).

[0223] 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. This is in Figures 15A-15CAs shown in the figure. For example, two microfluidic channels 1501, 1503 can be formed in the top plate of the air gap, and the openings in the air gap can be positioned to be no more than a fixed distance apart 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 1505 can bridge these two areas. Compared to what is easily done 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.).

[0224] For example, in Figure 15A , 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 withdrawn from the air gap into the first channel 1501 through the opening 1505; alternatively or in addition, by applying positive pressure 1507, fluid can be moved from the first channel 1501 into the air gap through the 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. Figure 15B and Figure 15C As shown in .

[0225] exist Figures 15A-15C In the example shown in FIG, the top plate integrates the microfluidic channels as well as the reservoirs and tubing; alternatively or additionally, 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 along with the 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.

[0226] Figures 16A-16D The figure shows examples of microfluidic channels that can be included. For example, Figure 16A The figure shows the formation of microfluidic channels formed in part by the top plate. Figure 16A In the embodiment of the present invention, a portion of the channel can be formed in the plate (e.g., acrylic plate) itself, wherein a second portion of the channel can be formed from another material, the other side of which has been coated with a conductive material (i.e., indium tin oxide, copper, nickel, chromium, and gold). The layers can be held together by an adhesive and / or can be bonded together.

[0227] For example, the microfluidic channels in any of the cartridges and devices described herein can be formed by laser cutting. Figure 16AIn the embodiment of the present invention, the grating channel can be cut into part B (the acrylic forming the top plate), and the hole can be cut in part B. In addition, one or more pump holes can be cut in part A. Double-sided adhesive (e.g., tape) can be used to fix part A to part B, and a roller can be used to place part A on part B, which avoids air bubbles. Thereafter, the pipette hole can be cut out for dispensing reagents, and the bottom can be coated with Teflon (e.g., hydrophobic), and the entire assembly baked between 80 degrees and 200 degrees (e.g., between 90 degrees and 18 degrees, etc.). The ground electrode can already be formed on the plate.

[0228] Figure 16B Another example of a set of microfluidic channels 1605, 1607 formed in the top plate is shown. A set of reagent inlets 1609 is also shown, which provide openings into the air gap area for loading reagents. Alternatively or in addition, (wet or dry / lyophilized) reagents can be pre-loaded into the cartridge, including pre-loading into one or more reservoirs above or in the top plate, for example, pre-loaded in the microfluidic channels, and / or pre-loaded directly into the air gap area. Figure 16C and Figure 16D The figures show additional examples of microfluidic channels that can be formed in the top plate of the cartridge.

[0229] Figure 17A and Figure 17B Schematic diagrams illustrate examples of methods for applying fluid to and removing fluid from the air gap of the DMF apparatus 1120 (including washing). Figure 17A 1107. In the embodiment of the present invention, 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 for the first fluid channel 1143 and the second fluid channel 1145. These fluid channels can be connected to one or more reservoirs 1105, 1107. As already described above, in some variations, two separate connector interfaces (ports) can be used, with each fluid line (e.g., a fluid line can be a microfluidic channel, as described above) 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 the 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.

[0230] Alternatively, if Figure 17C and Figure 17DAs shown in FIG, the box may include air gaps of varying heights. For example, in Figure 17D , the air gap for the area 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 further from the dielectric 1126. Similarly, in Figure 17D For example, by spacing a portion of the top plate 1117 further 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.

[0231] The diagram shows Figure 17C The principle of the prototype DMF device and the cartridge are 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 cartridge. Figure 18A In FIG, the upper plate of the DMF box includes an opening through the top plate 1801, which is connected to a first fluid line 1843 and a 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. Figure 18A 、 Figure 18B and Figure 18C In this example, as 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., hydrophobically coated dielectric) by the DMF device 1809, which enhances binding and / or washing.

[0232] In any of the air gap devices described herein, evaporation can be controlled or reduced, particularly when the droplets in the air gap are heated. Figures 19A-19C The figure shows the effect of evaporation on the droplet 1903 after only a few minutes. The complete droplet is Figure 19A After one minute at 95 degrees Celsius, the droplet volume has been significantly reduced (e.g., a loss of between 5% and 15% of the droplet volume), as shown in FIG. Figure 19B As shown in . After two minutes ( Figure 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 Figures 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. Figure 20AIn the embodiment, the jacketed droplets 2003 in the liquid paraffin 2005 are heated (e.g., to 65 degrees Celsius or higher). After one hour ( Figure 20B ), the droplet had not evaporated significantly. Similarly, after 2 hours ( Figure 20C ), the droplet remains approximately the same volume.

[0233] In use, the non-polar cover material can be added and removed at any point during the DMF process, e.g. Figures 21A-21I Surprisingly, removal can be accomplished, for example, by drawing the jacketed droplet upward from the air gap, such as through a port into a 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 located at the top or side of the air gap. The lower density liquid paraffin may be the first layer to be drawn upward, leaving the aqueous droplet behind. Previously, it was believed that removing jackets of non-polar liquids was difficult or impossible.

[0234] For example, Figure 21A Shown is a shrouded droplet 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, as Figure 21B , which shows the movement of droplets by altering the electrowetting of aqueous droplets through the coordinated application of energy to drive electrodes. Figure 21B In the embodiment of the present invention, the shrouded droplet has been moved to the right. 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 shrouded 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 shrouded. In some modifications, the shrouded droplet (including small aqueous droplets and relatively large-volume non-polar solutions) can be combined with the target droplet so that the target droplet is shrouded. A small amount of aqueous liquid in the shrouded droplet can be a buffer, a diluent or other solutions that allow the shrouded droplet to move in the air gap. When used together with a DMF box with a larger (e.g., 0.5 mm or larger) gap width, this technology is particularly useful. Larger gap width can additionally make it difficult for larger droplets to maintain the shroud of the typically less dense non-polar shroud material. Figure 21C and Figure 21D The figure shows a droplet 2101 that has been combined with another droplet to form a larger jacketed droplet 2101'. Larger droplets can also be moved by controlled actuation of the drive electrodes, such as Figure 21C and Figure 21D As shown in .

[0235] Figures 21E to 21I The figure shows the use of a non-polar liquid mask in a sample containing magnetic bead material. Figure 21E In the embodiment, the jacketed droplet includes a small amount of aqueous liquid 2121 and a relatively large volume of non-polar jacketing material 2123, which can be combined, for example, by moving the jacketed droplet 2123 into the sample droplet 2121. Figure 21F As shown in , 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.

[0236] Once assembled, the jacketed droplet 2121' can be moved (via DMF) to a port that enters the air gap from which the solution can be extracted, e.g. Figure 21H In this example, the solutions can be mixed by applying positive and negative pressure to move the solutions into and out of the fluid channel 2131. The non-polar solution that jackets the droplet 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 Figure 21I As shown in , the magnetic particles to which the desired analyte has 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 cover 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. Figure 21I In the embodiment, the magnetic particles 2133 are retained in the air gap and a separate wash buffer can be applied by moving washing and / or elution droplets 2135 over the magnetic particles.

[0237] In addition to the techniques discussed above for controlling evaporation (e.g., using a sleeve of a non-polar 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 desirable to adjust the humidity up and down with temperature, such as during hybridization or PCR cycling 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 apparatuses 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 base surface of the DMF apparatus 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 electrodes, the protrusions on the base surface can create chambers or channels in the air gap, which include partially forming an in-wall heating 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.

[0238] 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 of predictable volume because the surface tension of the relatively large droplets can require a large amount of energy to release the smaller droplets from the 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 are described herein for separating a unit droplet from a larger volume in an air gap having a width (e.g., spacer thickness) of 500 μm or greater. 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 cell (corresponding to a drive electrode) in the filled area, then withdrawing the solution into the port (or another port) that is offset from the activated electrode so that the droplet remains on the activated electrode when the solution is withdrawn to 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 to combine with one or more other droplets, etc., where the dispensed droplet can then be driven by the drive electrode.

[0239] For example, an integrated companion pump can be used to drive a large volume of aqueous solution into the DMF apparatus (e.g., into the air gap of a DMF cartridge) and onto an activated electrode. The aqueous solution can then be withdrawn away from the DMF apparatus, dispensing unit droplets onto the activated electrode. Figures 22A-22D An example of this method is shown in the figure. Figure 22A In the embodiment, the port 2201 entering 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. Figure 22A 2203). In this example, a single drive electrode 2207 is activated; alternatively, in some variations, the electrode is not activated until the region 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.

[0240] Next, if Figure 22B As shown in , the air gap region including the activated drive electrode is filled with aqueous solution 2203. Figure 22A The release of a large volume (e.g., 250 μL) from a channel (tube 2209) is shown. In certain variations, as the reagent approaches the distal channel 2209, the drive electrode 2207 is activated (e.g., an AC potential of 390 Vrms, or by using a DC potential to otherwise produce an alternating field effect), 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.

[0241] exist Figure 22C , the aqueous solution (reagent 2203) is then withdrawn from the air gap through the same port 2201 or a separate port, wherein the activated drive electrode is separated from the port into which the solution was drawn by a certain distance (for example, 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 broken from the liquid neck withdrawn into the channel 2209. For example, Figure 22C As shown in , aspiration of the reagent back into the tube can cause the droplet to neck off from the remainder 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 Figure 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. Figures 23A-23EMultiple 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.

[0242] Figures 23A-23F The diagram shows dispensing of various predetermined volumes of solution from the reservoir on the cartridge using the method described above. Figure 23A In the embodiment of the present invention, for 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. Figure 23B The droplet can be moved away from the filled area and the process repeated multiple times to produce a plurality of droplets of approximately uniform volume (e.g., 10 microliters + / - 5%, 10%, 15%, 20%, 25%, etc.). Figure 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 volume of 2× (e.g., 20 microliters). Similarly, Figure 23E A large droplet 2307 (eg, 50 microliters) is shown formed by combining five unit droplets. Figure 23F The diagram illustrates the use of a larger drive electrode 2315 (eg, having approximately 4× the surface area) that can be activated when filling the air gap region to form a larger unit droplet 2311 (eg, a 40 μL unit droplet).

[0243] Thus, by filling or flushing the dispensing area of ​​the air gap with a large volume of aqueous solution, 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 (cartridge), 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 smaller volume 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 dispense smaller volume droplets directly from a larger volume by applying electrowetting energy, the higher force effectively prevents direct dispensing in a larger air gap device via DMF. 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 gaps of at least up to 3 mm have been successfully used.

[0244] 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.

[0245] User Control Interface

[0246] In any device and method described herein, the DMF device can be controlled by the 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 carry out the scheme selected by the user or input by the user. Typically, when converting the treatment scheme for operation by the DMF device, there are many considerations, including preventing contamination during the program. Contamination may occur when moving the sample droplet on the path taken by the earlier steps (or parallel steps) in the program, in which the scheme is being executed. 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 is 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.

[0247] This paper describes the user interface for controlling the operation of DMF device, and this user interface allows the user to more easily input scheme information / step into DMF.This can be achieved in part by the following operation: a group of graphical step representations of the step that can be performed (for example, illustrating mixing, adding, heating, cooling, circulation, washing, etc.) is provided, and the user is allowed to select / input these steps in the mode of the duration of step or the degree (for example temperature, etc.) applied that are equally intuitively provided.After input, this device can then determine effective path, to perform the scheme of input within the predetermined layout constraint of DMF device and / or box, to avoid contamination.For example, any one of these devices can determine the path (pathfind (pathfind)) that prevents or reduces the path intersection in the air gap, and such intersection can cause contamination in the air gap.

[0248] Figure 24 is an exemplary schematic diagram illustrating the steps involved in controlling any DMF device described herein. For example, Figure 24 In the , the user can use the graphical / visual user interface ((herein referred to as "SAM") to enter the scheme. This can be referred to Figures 25A-26B ). The graphical scheme can then be converted into a series of target objectives, and the target scheme can then be used by the device to adapt the scheme to the DMF device. Figure 24In the process, the system can determine the path and obtain control over the drive electrodes, heaters, cooling (e.g., Peltier), magnets, microfluidics (pumps), etc.) 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.

[0249] As mentioned, Figure 25A and Figure 25B An example of a visual interface (e.g., a graphical user interface) for inputting a desired solution is shown. Figure 25A In FIG, 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 plan, such as Figure 25B Each icon may have an associated duration, and thus, the icons may be used to select a processing instruction or step for a sample. In this example, the icons are uniquely identified by one or more of: color, image, and text.

[0250] The user may enter the protocol directly into the device, or into a computer or other processor in communication with the DMF device.

[0251] 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., javascript) 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 desired actions and elements (e.g., mixing, etc.); the device can already be familiar with reagents (e.g., components of equipment). The user can then select action, duration, temperature, etc.

[0252] Figures 26A-26H The figure shows an example of a device for determining a path based on an input scheme. For example, Figure 26A A graphical representation of a specific configuration of the DMF cartridge 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 cartridge. Figure 26Bis 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. Figure 26C In the step of moving the first buffer (e.g., SureSelect QXT buffer) to an appropriate location for future processing, a path can be selected based on both past and future movements and can be recursively modified as future protocol steps are defined. Figure 26D In FIG, the path for moving the DNA sample is shown (black). Figure 26E shows the movement of the enzyme mix from a cooling area where it is stored for combination with the sample; Figure 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 ( Figure 26H ). Additional steps can then be performed as indicated.

[0253] Thermal Control

[0254] 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, a device including a cartridge and reader can be configured to quickly and accurately cycle the temperature of a droplet. Alternatively or additionally, droplet detection can quickly and accurately scan an electrode grid for droplets (including, but not limited to, reagents, wax, water, etc.).

[0255] As described above, the reader can be configured to include one or more thermal control elements, including cooling and / or heating. For example, the reader can include resistive heating in some of the cells 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 in continuous thermal connection with 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 forming part of the PCB in the reader), which can increase the rate of temperature change in the cell.

[0256] 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 less thermal mass is present, less energy needs to be removed from 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 longer liquid cooling the stack). The devices and methods described herein can reduce thermal mass by reducing / removing thermal mass from above the droplets or above the area in the upper (top) plate of the cartridge that holds one or more droplets. For example, when the upper / top plate is formed of an acrylic or polycarbonate material, the thermal mass above the air gap area can be reduced by including one or more cavities in the top plate (e.g., a polycarbonate structure and / or an acrylic structure) and filling the cavities with an insulating material or a material with low thermal conductivity (e.g., air). A cavity can be positioned in the top plate of the cartridge above the thermal controller region so that when a droplet of material is below the cavity, heating / cooling applied by the reader (e.g., by the PCB) can more rapidly change the temperature of the droplet in the air gap region. Removing thermal mass above the droplet can be incorporated into any cartridge design described herein. The cavity can be formed near the bottom surface of the top plate (e.g., immediately on one side of the air gap); the cavity can partially extend through the thickness between the top and bottom surfaces of the top plate. Figure 28 The example of a part of box is shown, which shows the thermal control area in the top plate 2801 of box 2804. Box can be positioned on reader 2803. Droplet 2807 in the air gap area of ​​box (for example, the area defined by the bottom surface of upper plate 2801 and the top surface of lower dielectric material sheet 2809). Therefore, in the variation in which the box body including the top plate is formed by the solid polycarbonate sheet on the top plate, one or more cavities (for example 2805) can be produced and can be sealed or filled with an insulating material with low thermal mass. This can prevent heat from being transferred from the sample to the storage area above it. Void replacement material can be air or a similar material with low thermal conductivity and low thermal mass.

[0257] Alternatively or in addition, thermal mass can be removed from the PCB by removing material (e.g., using precision milling) and / or using materials 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 heat 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.

[0258] Figure 29is an example of a milled area in the PCB of a reader device that has a lower thermal mass in order to increase the response time to temperature changes of a droplet in the air gap of the cartridge. In this schematic example showing a cross-section, the layers of the bottom portion (e.g., PCB) may include one or more layers of, for example, copper, and the dielectric below the droplet (in the PCB of the reader) has been milled to create a cavity or void that can be filled with a thermally insulating material (including air). Thus, heat conduction through the PCB can be reduced. Typically, cavities in the top and / or bottom plates can help thermally isolate droplets in the air gap between the top and bottom plates.

[0259] 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 Figure 30 As shown in . Figure 30 A highly conductive dielectric 3005 is shown between the heater 3003 and the copper region of the electrode 3001.

[0260] In certain variations, the reader (and in particular the PCB portion of the reader) may alternatively 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) in a thermally controlled area, and may be filled with any thermally conductive material. For example, filling the vias with a thermally conductive material (such as, but not limited to, copper, epoxy, resin, etc.) may further increase thermal conductivity and may significantly increase the thermal response time of a droplet or other material in the air gap. As a result, 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 ( Figure 31A and Figure 31B As shown in Figure 31A Shows the milled area with thermal vias, Figure 31B Thermal vias are shown without milled areas). For example, Figure 31A The figure shows a plurality of thermal 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 thermally controlled active areas.

[0261] The through-holes may be filled with any suitable thermally conductive material. In some variations, the through-holes are filled with a non-conductive thermally conductive material (e.g., epoxy, resin, etc.).

[0262] One end of the through-hole can be in thermal contact (e.g., can touch) with the final upper surface of the reader device (e.g., the cartridge 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 a 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 in thermal communication with 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.

[0263] The through-holes can be of any suitable size. For example, the thermally conductive vias (referred to herein as thermal vias or simply vias) can 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 can 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 can be cylindrical, extending from a thermal control unit (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, without contacting the electrode so that they remain electrically isolated from the electrode, but not thermally isolated).

[0264] 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.

[0265] The use of thermal vias may provide significant improvements in the rate of change of heating and / or cooling of a thermally controlled area compared to systems that do not include thermal vias.

[0266] Box Features

[0267] In addition to the features described above, any cartridge may alternatively or additionally include one or more openings into or through some of the upper top plate 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 using optics 3221, such as Figure 32 Alternatively or in addition, the openings may be used to passively distribute fluid from the air gap. For example, Figure 32, an opening 3203 in the top plate of the cartridge 3205 can be used to passively dispense fluid from a droplet 3211 positioned below the opening; the droplet can move below the opening via DMF, as described above. Once 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 cartridge, and / or can be analyzed in place. Alternatively, 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 3203 through the top plate that includes the sampled material. 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 some 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 anti-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 Figure 32 The variation shown in FIG shows imaging through the top plate (using optics 3221), however in some variations, imaging can be performed from the bottom of the box, through the bottom. 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).

[0268] 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. Figure 27A and Figure 27B The figures show one example of a top plate formed from a polymer material (eg, acrylic and / or polycarbonate). Figure 27A The upper area of ​​the top plate is shown (which may be covered by one or more covers, not shown). Figure 27A, including multiple dispensing areas 2704, 2706, 2708 of different sizes. For example, a smaller 2706 (e.g., 2 microliters-20 microliters in size), a medium 2704 (e.g., 100 microliters to 1 mL), and a large 2708 (e.g., 1 mL to 5 mL) are shown, as are a waste and / or mixing area 2710. These chambers can be preloaded with fluid, and each chamber can include an opening into the air gap area. A pressure control can be used to apply pressure to drive the fluid out of the opening in the dispensing area and into the air gap, which can be controlled by a reader or other device holding the box. Thus, the reader can include one or more pressure interfaces that can be used to control the release of fluid from the top plate and the handling of fluid in the top plate. Figure 27B The diagram shows Figure 27A The bottom side of the top plate portion shown in FIG. The bottom side may be coated or covered with electrodes and / or dielectrics and / or hydrophobic coatings as described above. Figure 27B In the embodiment of the present invention, the top plate may also or alternatively include one or more channels 2712 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 some variations, can include a dielectric, a hydrophobic membrane, and / or an electrode layer).

[0269] In any of the boxes described herein, the bottom surface that can be configured to contact the base surface of the reader and in particular contact the drive electrodes in the reader is formed of 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 forming an exposed bottom surface on the bottom of the box). The second side of the sheet of dielectric material 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 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 have the bottom surface be substantially flat. Any of the boxes described herein can be configured to apply tension to the sheet of dielectric material. For example, any of these boxes can include a frame to hold the dielectric material in a tensioned state. Thus, the box can include a tensioning frame that holds the bottom sheet of the box.

[0270] 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 reader base 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 cartridge is to interface with the base surface (e.g., PCB interface) to ensure that the membrane remains in full contact with the electrode grid (e.g., drive electrodes) throughout use of the device.

[0271] In any of the boxes described herein, the bottom of the box can 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 can include a tensioning frame to hold the sheet flat by applying tension. The sheet, when exposed as the bottom of the box, can be slightly recessed compared to the outer perimeter of the box bottom, which can fit into a flange or recess on a reader 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.

[0272] For example, Figures 49A-51 The figure shows an example of a box assembly that includes a frame that stretches / smoothes the bottom of the box (e.g., a dielectric sheet). Figures 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-49B The two parts shown in 49C-49D (and the assembled views in 49C-49D) can include a tensioning frame 4901 and a dielectric film frame and / or hydrophobic film frame 4903. When assembled, the membrane forming the bottom of the box can be adhered to the dielectric film frame and / or hydrophobic film frame 4903. The dielectric film frame and / or hydrophobic film frame 4903 can be inserted into a groove 4911 in the tensioning frame using a connector (e.g., a snap-fit ​​mechanism). After snapping into the tensioning frame, the membrane can be pulled tight (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., cutout) area 4909 that can provide a path to electrically connect the return electrode on the upper plate by bypassing the membrane on the bottom surface of the box.

[0273] An example of a box including a frame for keeping the bottom film flat is Figure 50A is shown in an exploded view. Figure 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 to be 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 film 5005 comprising a dielectric material may also be included, with the film being secured in place by a second portion of the film frame 5006. An air gap 5009 may be formed between the film 5005 and the bottom surface 5003 of the top sheet (which may include a return electrode).

[0274] Figure 50BDepicted are the individual components of the box and film tensioning assembly after assembling the optically clear double-sided adhesive and the dielectric 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.

[0275] 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 reader. Figure 51 shows an isometric exploded view of an example of the components of a cassette, 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 cassette; Figure 51 Also shown is a portion of the reader, including a PCB 5111 that forms the base surface of the box. The base 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 slot 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 it downward in the Z direction at the edge of the film. The film can wrap over the filleted edges of the groove, just slightly outside the boundary of the electrode grid (not shown).

[0276] Figure 52A and Figure 52B Shown are a top view and a cross-sectional view, respectively, of one example of a cartridge including a bottom dielectric (and hydrophobic or hydrophobically coated) film, and a film tensioning frame positioned on the PCB assembly portion of a reader. Figure 52B The cross section in FIG2 highlights how the dielectric and / or hydrophobic membrane can be tensioned across the electrodes and sealed down using vacuum ports for at least some of the electrodes (drive electrodes) passing through the PCB, and also illustrates how the edges (extending from the membrane) are positioned in grooves formed in the PCB base surface to seat the membrane. When fully assembled, these components can allow a secure, fully tensioned, and flat dielectric (and / or hydrophobic) membrane to be secured to the drive electrode grid on the PCB. Figure 53 is an exploded view showing the individual components and their arrangement in an assembly including a cartridge upper body frame 5306, a dielectric film 5305 held in tension by a tensioning frame 5304, a PCB 5302 forming a base surface on the reader, grooves or channels on the base surface around the perimeter of an array of drive electrodes (driving electrodes) on the PCB, and a vacuum chuck 5301.

[0277] Figure 54A and Figure 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 cassette 5413 and the membrane assembly, and on the PCB 5415. Figure 54B The cross section in FIG also highlights some of the different effects of the system. Arrows 5405 depict the flow path of the vacuum from the diaphragm vacuum pump 5407 on the outside of the chuck. This can be similar to the above Figure 35B The same flow path as described in

[15] . The arrows outline the force applied downward to the membrane by the vacuum passing through the through-holes in the PCB. The vacuum chuck and the interface with the PCB firmly adhere the membrane to the electrode and apply a downward force 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.

[0278] Reader Characteristics

[0279] In general, any reader described herein can include a PCB portion that can 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 can be mounted to the PCB. This portion of the reader can form a base surface at the bottom of the box so that it can be securely and in a predetermined orientation on the reader. For example, the box can be keyed to fit onto the base surface in a predetermined manner (e.g., by including one or more directional slots, pins, etc.). The reader can also include one or more control units that include one or more processors that can control the activities of the reader and can be configured to drive droplets and analyze information from the box. The controller can also include a memory, one or more data stores.

[0280] The base surface of the reader can be configured to house the box and also prevent arcing, sparking or short circuiting between multiple electrodes on the base surface. For example, the base surface can be coated with another dielectric (the dielectric bottom surface of the box can be located thereon), such as paralyene and / or an optional or additional material. The dielectric bottom surface can prevent arcing between electrodes in the array of electrodes (drive electrodes) on the base surface. The spacing between the drive electrodes can be between about 50 microns and 120 microns. This close packing between the electrodes on an otherwise flat surface can also easily form arcs / short circuits between the electrodes, so that the use of an external dielectric coating (in addition to the dielectric layer of the box) can limit arcing / short circuits between the electrodes.

[0281] As discussed and described above, some or all of the electrodes may include openings therethrough that can 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.

[0282] Droplet detection

[0283] 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 driven electrodes by monitoring the current through the electrodes. Also described herein are devices (e.g., systems or devices, including readers) in which droplet detection is based on capacitance measurements 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 typically be driven 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) during droplet detection. Figure 33A and Figure 33B It is called "ITO sensing" in this paper 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 Figure 33A The voltage divider shown in . Figure 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.

[0284] exist Figure 33A In

[15] , the voltage at the ITO sense node (ITO sense 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 dielectric constant of the material 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 sense. This enables this droplet detection method to not only detect droplets (e.g., presence / absence of a droplet), but also to distinguish between droplets and identify specific reagents in the electrode grid.

[0285] Due to the variability of base capacitance, two calibration capacitors may be included (e.g., Figure 33BC_REF and C_REF_LARGE). Figure 33B The figure shows another example of a capacitance sensing circuit that 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 point, then the reference capacitor can be increased. The regulation circuit for ITO sensing can isolate the voltage from small leakage currents.

[0286] Figure 34A Exemplary 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 reader, 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 seating area. Figure 34A In the example, "ITO" corresponds to the upper (e.g., return) electrode on the upper plate of the cartridge. In this example, C18, C21, C24, C27, C30 are reference capacitors (e.g., 11.9 pF in this case), and C16, C19, C1, C25, C28 are capacitances measured as described above, corresponding to the capacitance with and without a droplet when measuring different drive electrodes (e.g., set to a high voltage while the other drive electrodes are grounded). Water, wax, and air (no droplet) have very different capacitances, which can be used to identify the presence or absence of a droplet (e.g., capacitance greater than or equal to 0.09 pF, greater than or equal to 0.1 pF, etc.). In this example, a capacitance above the threshold (e.g., above 0.06 pF, 0.07 pF, 0.08 pF, 0.09 pF, 0.1 pF, 0.11 pF, etc.) indicates the presence of material in the air gap, above the threshold value (set to a high voltage, such as 300 V). Additionally, a range of measured capacitance above the threshold value 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.).

[0287] Figure 34Bis 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) relative to no droplets (air) on a single test cell is shown. In Figure 34, when the aqueous droplet is present, the detected voltage is about 3.3V, compared to 0.085V when no droplet is present and 0.176V when wax is present. The measured value for wax is twice that of air (no droplets / material), and water is much higher; 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 a droplet is present, the capacitance can change depending on the temperature. This characteristic can further be used to identify water, and can also be used to estimate the temperature. Therefore, in some variations, the capacitance measurement of a droplet can also be used to estimate its temperature. For example, Figure 34C is a graph showing the static relative dielectric constant of water, which shows the change in relative dielectric constant with temperature change (between 0 degrees Celsius and 300 degrees Celsius).

[0288] Chuck design

[0289] Any device described herein (e.g., a reader) may include a chuck (e.g., a vacuum chuck) that may form part of a base 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 magnets and / or heat dissipation features. Any of these elements or portions of these elements may be included or omitted and may be used in any combination.

[0290] The vacuum chuck design can help ensure that a reliable and effective vacuum adheres the bottom of the cartridge (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 through-holes (e.g., copper through-holes).

[0291] In addition, any reader described herein can include a magnet integrated into a base that includes the chuck and / or base surface. The integrated magnet can be configured to allow the actuable magnet to engage with material in the cartridge (e.g., a magnetic bead in a droplet in an air gap) through the vacuum chuck. The magnet can rest slightly below the PCB forming the base surface of the reader without affecting vacuum performance or functionality.

[0292] Any reader described herein may also or alternatively include one or more thermal regulators comprising one or more heat dissipation elements that can quickly and accurately dissipate heat from a heater in the reader that controls the temperature of one or more cells in the cartridge when the cartridge is positioned and held on a base surface of the reader. For example, two designs of heat dissipation elements are described herein that can be used individually or more closely. One exemplary heat dissipation design is configured to dissipate heat from a thermoelectric heater, while the other design is configured to dissipate heat from an embedded heater.

[0293] Figures 35A-48 The figure shows a vacuum chuck portion of a reader that can be used with any of the reader 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 directed beneath a base surface (e.g., a PCB forming part of the base surface) in an area that is pneumatically isolated, for example, by an O-ring. The base surface can have through-holes (e.g., in the PCB) that allow the negative pressure to act directly on the bottom of a cartridge (e.g., a dielectric film and / or a hydrophobic film) that sits on top of the base surface (e.g., a PCB forming part of the base surface), pulling the cartridge bottom downward in the Z direction and adhering it to the electrode grid.

[0294] The vacuum chuck may include one or more of the following: 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. For example, Figure 35A is a top view of an example of a vacuum chuck 3500, and Figure 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 Figure 35B Path of the arrow shown in: 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 base surface formed by the PCB will be placed) is surrounded by an O-ring 3503.

[0295] For example, Figure 36 Shown Figure 35A-Figure 35B An isometric view of the chuck shown in FIG. . Groove 3509B (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 beneath the electrode grid. The base surface can be formed by securing a PCB with electrodes (not shown) to the chuck. For example, Figure 37As shown in FIG, the chuck may include a plurality of threaded holes 3701 for attachment to a base surface (eg, a PCB). Figure 37 Shows something like Figure 35A-Figure 35B In some embodiments, the chuck includes at least four threaded holes ( Figure 37 Eight are shown in the figure, 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: first, to secure the PCB to the chuck so that the interface of the two components is flat, and second, to apply a downward force in the Z direction about the perimeter of the O-ring, effectively creating a pneumatic seal.

[0296] Figure 38A Shows something like Figure 35A-Figure 35B A top view of the chuck is shown in FIG. 1 , and Figure 38B An enlarged cross-sectional view of the chuck is shown. Figure 38B An enlarged image of section AA is shown showing the boundaries of recesses 3801, 3803 (along the X axis), which can create a 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. Figure 38A In the embodiment, an opening 3805 for the magnet is present on the upper region and can include sufficient space for the magnet to be moved into / out of the cartridge (e.g., by moving up / down within 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 to isolate the magnet region from the vacuum region, similar to an external O-ring.

[0297] As mentioned, any of the devices described herein may include integrated magnets. Figures 35A-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 of varying strength) by the reader's controller.

[0298] Thus, the vacuum chuck may include an integrated magnet and may therefore include one or both of a cutout that allows the magnet to pass through the chuck and a second o-ring groove that isolates the magnet region from the pneumatic flow of the vacuum. Figure 39 Shows something like Figure 35A-Figure 35B. A through-cut region 3905 is shown and can be sized to accommodate the desired magnet and allow for 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.

[0299] Figure 40 Shows something like Figure 35A An isometric view of the chuck is shown in FIG. Groove 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, specifically ensuring that the vacuum is not compromised by the magnet cutout.

[0300] Figure 41A and Figure 41B Figures similar to Figure 35A and Figure 35B 4105. The top and side cross-sectional views of the chuck shown in FIG. 4106, but including a through cutout 4115 area for thermal access to a heating component, such as a heater (e.g., a resistive heater) 4105. The heater 4105 is shown above the through cutout 4115 area in the chuck so that it can be easily thermally regulated (e.g., cooled). The resistive heater can be in the PCB ( Figure 41A and Figure 41B not shown).

[0301] For example, Figure 41A An example of a heat dissipation system is shown that can be included in any reader device described herein. The heat dissipation system can be constructed so that any heat load generated by a heater 4105 in the reader (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 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 heat dissipation of a thermoelectric cooler. Both heat dissipation designs can employ unique features in the vacuum chuck, as well as accompanying components to dissipate heat. The two designs can be used together, also in an assembly, or used separately.

[0302] For example, heat dissipation for embedded heaters in vacuum chucks can be configured as plenum chambers. Figure 41A , a top view of the chuck illustrates the heat dissipation aspect of the chuck; Figure 41BA 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 where the heater is located. Figure 41B , the flow paths of the multiple air elements (channels 4101, 4101') that function in the system are shown. The air drawn in 4101 can be warmed by heat (including residual heat) from a heater in the PCB (e.g., a base 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 ( Figure 41B 4101 ' and 4102 '. The figure (shown in FIG) shows the pneumatic flow of the two air elements (warm air and ambient air) when the fan is turned on. The fan is fastened 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 through-cuts 4101, 4101' 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.

[0303] This article also describes a system for dissipating heat from an embedded heater. For example, Figure 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 the fan 4205 fastened to the bottom of the chuck 4203. Figure 42 Shown is a front view of the chuck 4203 and fan 4205. The first arrow 4221 points toward the vacuum chuck (top structure), and the second set of arrows 4201, 4201' depicts the airflow path. Figure 43 An example of an arrangement is shown of a chuck 4303, a fan 4307, a PCB 4305 forming a base surface (e.g., including an array of electrodes, not shown), and a cartridge 4311. The cartridge may be held down by a vacuum through openings (e.g., in some of the electrodes).

[0304] Figure 44 An example of a heat dissipation system for regulating the temperature of a thermoelectric cooler via a vacuum chuck is shown. An isometric view of the chuck (similar to Figure 35A shown in Figure 45B The chuck is shown to include a recess 4509 designed so that a thermoelectric cooler (TEC) can be slotted into it.

[0305] Figures 45A-45B Shown are similar to Figure 35A A top view and a cross-sectional view of the chuck are shown in FIG. Figure 45B The cross section shown in FIG45 (through AA) emphasizes the heat path of the heat generated by the thermoelectric cooling element 4525. The rectangle represents the TEC 4525, and the arrows in the chuck depict the heat spreading throughout the chuck. The device can include one or more heat sinks of desired size that can be fastened to the bottom of the chuck and below the TEC, which then absorbs the heat. Finally, fastened to the heat sink ( Figure 46 Two fans on either side of the ) can work together to push hot air away from the entire system and flush ambient air into the system.

[0306] Figures 47A-47C The figure shows an assembly of one or more devices configured for heat dissipation in a thermoelectric cooler. For example, Figure 46 A front view of the chuck is shown. Figure 46 The downward arrow 4613 in FIG. Figure 45B The heat path of the heat in the chuck is described in FIG. Arrows 4611, 4611' depict the flow path of the air being pushed into the heat sink by the fan and the path of the air being exhausted from the heat sink by the fan. The fans act simultaneously in the same direction. Figures 47A-47C The assembly process and various components that may be included in the device and its method of use are shown. For example, Figure 47A Shown is a chuck 4701, Figure 47B A chuck 4701 plus a heat sink 4703 is shown, and Figure 47C Shown is a chuck 4701 plus a heat sink 4703 plus two fans 4709, 4709'. Figure 48 An exploded view depicting a partial arrangement of reader components, including Figures 47A-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 base surface of the PCB via vacuum.

[0307] Action Zone

[0308] Any device described herein may include one or more action zones that strategically position different possible actions that a droplet can undergo for protocol execution. The purpose of a plexing strategy is to adapt 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 the board. An action zone can be a fixed area below or above an electrode plate for reactions (i.e., mixing, merging, heating, cooling, thermal cycling, magnet capture, waste, optical detection, etc.).

[0309] Figure 55 An example of an electrode grid is shown with separate active zones for either magnetic capture, a heater or thermocycler that can be isothermal, a Peltier with an active cooling zone to 4°C, a waste connection through a channel to the top plate and into a waste chamber, a mixture connection through a channel to the top plate, and optical detection. Thus, Figure 55 An electrode grid with different action areas is shown.

[0310] In order to be suitable for different user demands and laboratory space better, independently single module can be reused together, and this independently single module has its own power supply, environment, internal computer and with the connection of the console unit that is used for user interface separately.In addition, the console unit of user interface can be integrated, to control different modules, and the function that other laboratories require, for example scan sample ID and box ID and this information is integrated into local laboratory or sample management system.Can be wireless or pass through cable with the connection of console unit. Figure 56 Four independently controlled 1-plex modules with a console unit are schematically shown.

[0311] Any of the methods described herein (including the user interface) 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 processor (e.g., a computer, tablet, smartphone, etc.), which instructions, when executed by the processor, cause the processor to control the performance of any of the steps, including but not limited to: displaying, communicating with a user, analyzing, modifying parameters (including timing, frequency, intensity, etc.), determining, alerting, or the like.

[0312] When a feature or element is referred to herein as "on another feature or element," it can be directly on the other feature or element, or there can 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 the other feature or element, 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 illustrated with respect to one embodiment, the features and elements described or illustrated in this manner can be applied to other embodiments. Those skilled in the art will also recognize that a reference to a structure or feature that is "adjacent" to another feature can have a portion that covers or is located below an adjacent feature.

[0313] 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 the 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 can be abbreviated as " / ".

[0314] Spatially relative terms such as "under," "below," "lower," "over," "upper," and similar terms may be used herein to easily describe the relationship of one element or feature to another, as illustrated in the accompanying drawings. It will be understood that spatially relative terms are intended to encompass different orientations of the device in use and operation in addition to the orientation depicted in the accompanying drawings. For example, if the device in the accompanying drawings is inverted, the elements described as being "below" or "beneath" other elements or features will be oriented "above" the other elements or features. Thus, the exemplary term "below" can encompass both the orientations 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 for explanatory purposes only, unless otherwise specifically indicated.

[0315] 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 indicates otherwise. These terms can be used to distinguish one feature / element from another feature / element. Thus, 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.

[0316] 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) can be used together in methods and articles. For example, the term "comprising" will be understood to imply the inclusion of any stated elements or steps but not the exclusion of any other elements or steps.

[0317] In general, any apparatus and method described herein should be understood to be inclusive, but all or a subset of components and / or steps may alternatively be exclusive and may be expressed as "consisting of" or alternatively "consisting essentially of" a variety of components, steps, subcomponents or substeps.

[0318] As used herein in the specification and claims, including those used in the examples, and unless otherwise expressly specified, all numbers can be considered as if preceded by the word "about" or "approximately", even if the term does not explicitly appear. When describing magnitude and / or position, the wording "about" or "approximately" can be used to indicate that the value and / or position described are within the reasonable expected range of the value and / or position. For example, a numerical value can have a value that is + / -0.1% of the stated value (or range of values), + / -1% of the stated value (or range of values), + / -2% of the stated value (or range of values), + / -5% of the stated value (or range of values), + / -10% of the stated value (or range of values), etc. Any numerical value given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value "10" is disclosed, then "about 10" is also disclosed. Any numerical range listed herein is intended to include all subranges contained therein. It should also be understood that when a value is disclosed, "less than or equal to" the value, "greater than or equal to the value," and possible ranges between the values ​​are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "X" is disclosed, then "less than or equal to X" and "greater than or equal to X" (e.g., where X is a numerical value) are also disclosed. It should also be understood that throughout this application, data is provided in a variety of different formats, and that the data represents endpoints and starting points, as well as ranges for any combination of data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, it should be understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, as well as between 10 and 15, are considered disclosed. It should also be understood that every unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0319] Although various illustrative embodiments have been described above, any of a number of changes may be made to the various embodiments without departing from the scope of the invention as described in the claims. For example, in alternative embodiments, the order in which the various described method steps are performed may often be changed, and in other alternative embodiments, one or more method steps may be skipped together. The optional features of the various device and system embodiments may be included in certain embodiments and not included in other embodiments. Therefore, the foregoing description is primarily provided for exemplary purposes and should not be construed as limiting the scope of the invention as set forth in the claims.

[0320] The examples and diagrams included herein show specific embodiments in which the subject matter can be practiced by way of illustration and not limitation. As mentioned, other embodiments can be utilized and derived therefrom so that structural and logical substitutions and changes can be made without departing from the scope of this disclosure. If in fact more than one is disclosed, it is only for convenience that such embodiments of the subject matter of the invention may be referred to herein individually or collectively by the term "invention," and it is not intended that the scope of this application be voluntarily limited to any single invention or inventive concept. Therefore, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may replace the specific embodiments shown. This disclosure is intended to cover any and all modifications or variations of the various embodiments. After reading the above description, the combination of the above embodiments and other embodiments not specifically described herein will be apparent to those skilled in the art.

[0321] This application provides the following:

[0322] 1). A cartridge for a digital microfluidics (DMF) device, the cartridge having a bottom and a top, the cartridge comprising:

[0323] 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 comprises a first hydrophobic surface;

[0324] a top plate having a first side and a second side and a thickness between the first side and the second side;

[0325] a ground electrode on the first side of the top plate;

[0326] a second hydrophobic surface covering the ground electrode on the first side of the top plate; and

[0327] An air gap separates the first hydrophobic layer and the second hydrophobic layer, wherein the air gap comprises a spacing greater than 280 microns.

[0328] 2) The cartridge according to 1), wherein the ground electrode comprises a grid pattern forming a plurality of open cells.

[0329] 3) The cartridge according to 2), wherein the grid pattern of the ground electrode is formed of an opaque material.

[0330] 4) The cartridge according to 1), wherein the ground electrode is formed of conductive ink.

[0331] 5) The cartridge according to 1), wherein the ground electrode is formed of silver nanoparticles.

[0332] 6). The box according to 2), wherein the minimum width of the grid pattern between the open cells is greater than 50 microns.

[0333] 7) The cartridge according to 2), wherein an open cell of the plurality of open cells comprises a quadrilateral shape or an elliptical shape.

[0334] 8) The box according to 1), wherein the ground electrode extends over more than 50% of the first side of the top plate.

[0335] 9). The box according to 1), wherein the top plate includes a plurality of cavities within the thickness of the top plate, and further wherein the cavities are filled with an insulating material having low thermal mass and low thermal conductivity.

[0336] 10) The box according to 9), wherein the insulating material includes air.

[0337] 11). The box according to 1), wherein the dielectric material sheet is flexible.

[0338] 12). The box according to 1) further includes a microfluidic channel, which is formed on the second side of the top plate or in the second side of the top plate, wherein the microfluidic channel extends along the second side of the top plate and at least one opening between the microfluidic channel and the air gap.

[0339] 13). The box according to 1), wherein the top plate comprises polycarbonate and / or acrylic.

[0340] 14). The cartridge of 1) wherein said sheet of dielectric is less than 30 microns thick.

[0341] 15). The cartridge according to 1), wherein the second side of the dielectric material comprises a hydrophobic coating.

[0342] 16). The cartridge according to 1), wherein the air gap comprises a spacing greater than 400 microns.

[0343] 17) A cartridge for a digital microfluidics (DMF) device, the cartridge having a bottom and a top, the cartridge comprising:

[0344] a sheet of flexible dielectric material having a first side and a second side, the first side forming an exposed bottom surface on the bottom of the box;

[0345] a first hydrophobic layer on the second side of the dielectric material sheet;

[0346] a top plate having a first side and a second side and a thickness between the first side and the second side;

[0347] a ground electrode on the first side of the top plate, wherein the ground electrode comprises a grid pattern formed of an opaque material, the grid pattern forming a plurality of open cells along the first side of the top plate;

[0348] a second hydrophobic layer covering the ground electrode on the first side of the top plate; and

[0349] An air gap separates the first hydrophobic layer and the second hydrophobic layer, wherein the air gap comprises a spacing greater than 400 microns.

[0350] 18) The box according to 17), wherein the top plate includes a plurality of cavities within the thickness of the top plate, and further wherein the cavities are filled with an insulating material having low thermal mass and low thermal conductivity.

[0351] 19) The cartridge according to 17), wherein the grid pattern of the ground electrode is formed of conductive ink.

[0352] 20) The cartridge according to 17), wherein the grid pattern of the ground electrode is formed of silver nanoparticles.

[0353] 21). The box according to 17), wherein the minimum width of the grid pattern between the open cells is greater than 50 microns.

[0354] 22) The cartridge according to 17), wherein an open cell of the plurality of open cells comprises a quadrilateral shape or an elliptical shape.

[0355] 23). The box according to 17), wherein the grid pattern of the ground electrode extends over more than 50% of the first side of the top plate.

[0356] 24). The box according to 17) further includes a microfluidic channel formed in the second side of the top plate, wherein the microfluidic channel extends along the second side of the top plate and at least one opening between the microfluidic channel and the air gap.

[0357] 25). The box according to 17), wherein the top plate comprises polycarbonate and / or acrylic.

[0358] 26) A cartridge for a digital microfluidics (DMF) device, the cartridge having a bottom and a top, the cartridge comprising:

[0359] 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;

[0360] a first hydrophobic layer on the second side of the sheet of dielectric material; a top plate having a first side and a second side and a thickness between the first side and the second side;

[0361] a ground electrode on the first side of the top plate;

[0362] a second hydrophobic layer covering the ground electrode on the first side of the top plate;

[0363] an air gap separating the first hydrophobic layer and the second hydrophobic layer;

[0364] a microfluidic channel formed in or on the second side of the top plate, wherein the microfluidic channel extends along the second side of the top plate;

[0365] an opening between the microfluidic channel and the air gap; and

[0366] A cover covers the microfluidic channel, wherein the cover includes one or more access ports for accessing the microfluidic channel.

[0367] 27). The cartridge according to 26), wherein the microfluidic channel is configured to contain more than 1 ml of fluid within the microfluidic channel.

[0368] 28). The cartridge of 26) wherein the air gap comprises a spacing greater than 500 microns.

[0369] 29). A box according to 26), wherein the microfluidic channel includes a first microfluidic channel and the opening between the microfluidic channel and the air gap includes a first opening, and the box also includes a second microfluidic channel formed in the second side of the top plate and a second opening between the second microfluidic channel and the air gap, wherein the second microfluidic channel extends along the second side of the top plate, and wherein the first opening and the second opening are adjacent to each other.

[0370] 30). The box according to 29), wherein the first opening and the second opening are no more than about 2 cm apart from each other.

[0371] 31). The box according to 26), further comprising a window from the top of the box to the air gap, the air gap being visible through the window.

[0372] 32) The box according to 31), wherein the window is formed between 2% and 50% of the top of the box.

[0373] 33). The box according to 26), wherein the bottom of the box is formed by the first side of the dielectric material sheet.

[0374] 34). The box according to 26) further includes a plurality of openings entering the air gap from the top of the box.

[0375] 35). The box according to 26), wherein the top plate comprises polycarbonate and / or acrylic.

[0376] 36). The box according to 26) further includes one or more reagent reservoirs on the second side of the top plate.

[0377] 37). The box according to 26) further includes one or more freeze-dried reagent reservoirs on the second side of the top plate.

[0378] 38). The box according to 26), wherein the dielectric material sheet is flexible.

[0379] 39). The box according to 26), wherein the top plate includes a plurality of cavities within the thickness of the top plate, and wherein the cavities are filled with an insulating material having low thermal mass and low thermal conductivity.

[0380] 40) A cartridge for a digital microfluidics (DMF) device, the cartridge having a bottom and a top, the cartridge comprising:

[0381] 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;

[0382] a first hydrophobic layer on the second side of the dielectric material sheet;

[0383] a top plate having a first side and a second side and a thickness between the first side and the second side;

[0384] a ground electrode on the first side of the top plate;

[0385] a second hydrophobic layer covering the ground electrode on the first side of the top plate;

[0386] an air gap separating the first hydrophobic layer and the second hydrophobic layer, wherein the air gap comprises a spacing greater than 500 microns;

[0387] a first microfluidic channel and a second microfluidic channel, wherein the first microfluidic channel and the second microfluidic channel are formed in the second side of the top plate, wherein the first microfluidic channel and the second microfluidic channel extend along the second side of the top plate;

[0388] a first opening and a second opening, the first opening being between the first microfluidic channel and the air gap, the second opening being between the second microfluidic channel and the air gap, wherein the first opening and the second opening are adjacent to each other and no more than about 2 cm apart; and

[0389] A cover covers the microfluidic channel, wherein the cover includes one or more access ports for accessing the microfluidic channel.

[0390] 41) A digital microfluidics (DMF) reader device configured to operate with a disposable cartridge having a bottom dielectric surface, a top plate having a ground electrode, and an air gap between the bottom dielectric and the top plate, the device comprising:

[0391] a base surface for seating the disposable cartridge;

[0392] a first plurality of drive electrodes on the base surface, wherein all or some of the drive electrodes include openings therethrough;

[0393] a plurality of vacuum ports, wherein each vacuum port is coupled to one or more of the openings through the drive electrode;

[0394] a vacuum pump for applying a vacuum to the vacuum port; and

[0395] a control 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,

[0396] wherein the DMF reader is configured to apply a vacuum to the vacuum port to secure each drive electrode to the bottom dielectric of the disposable cartridge when the disposable cartridge is placed on the base surface.

[0397] 42). The apparatus according to 41) further comprising one or more protrusions extending from the base surface, wherein the one or more protrusions are configured to form a partition in the air gap of the box when a vacuum is applied through the opening in the drive electrode.

[0398] 43). The apparatus according to 41) further comprising an optical reader configured to detect an optical signal from a cartridge placed on the base surface.

[0399] 44). The apparatus according to 41) further comprising a motor configured to move an optical reader configured to detect an optical signal from a cartridge placed on the base surface.

[0400] 45). The device according to 41) further includes one or more temperature sensors, which are coupled to the base surface.

[0401] 46). The device according to 41) further includes a resistive heater, which is located under at least some of the drive electrodes.

[0402] 47). The apparatus according to 41), wherein the base surface comprises a printed circuit board.

[0403] 48). The device according to 41) further includes a magnet, which is below one or more of the drive electrodes and is configured to be activated to apply a magnetic field.

[0404] 49). The apparatus according to 41) further comprising one or more Peltier coolers below at least some of the drive electrodes, configured to cool to less than 10 degrees Celsius.

[0405] 50). The apparatus according to 41) further comprises a cartridge tray configured to move the disposable cartridge onto the base surface.

[0406] 51). The device according to 41) further includes a shell, which encloses the device, wherein the shell is stackable.

[0407] 52). The device according to 41) further includes an output terminal, which is configured to output a signal detected by the device.

[0408] 53). The device according to 52), wherein the output terminal includes a wireless output terminal.

[0409] 54). The apparatus according to 41) further comprises a first thermal control unit configured to cool the base surface to between 15 degrees Celsius and 25 degrees Celsius.

[0410] 55). The device according to 41) further includes one or more microfluidic vacuum ports, which are positioned on the base surface and are configured to engage with an access port for entering the microfluidic channel of the box when the box is placed on the base surface.

[0411] 56). The apparatus according to 41) further comprises a dielectric coating on the outermost surface of the base surface.

[0412] 57). The apparatus according to 41), wherein each of the first plurality of drive electrodes on the surface of the base is separated from an adjacent electrode of the plurality of electrodes by between 50 microns and 120 microns.

[0413] 58). The apparatus according to 41) further comprises a plurality of thermal vias passing through the surface of the base.

[0414] 59) A digital microfluidics (DMF) reader device configured to operate with a disposable cartridge having a bottom dielectric surface, a top plate having a ground electrode, and an air gap between the bottom dielectric and the top plate, the device comprising:

[0415] a base surface for seating the disposable cartridge;

[0416] a plurality of drive electrodes on the base surface, wherein at least some of the drive electrodes include openings therethrough;

[0417] a plurality of vacuum ports, wherein each vacuum port is coupled to one or more of the openings through the drive electrode;

[0418] a vacuum pump for applying a vacuum to the vacuum port; and

[0419] a control 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,

[0420] wherein the DMF reader is configured to apply a vacuum to the vacuum port to secure each drive electrode to the bottom dielectric of the disposable cartridge to retain the disposable cartridge on the base surface.

[0421] 60). A method for preventing droplet evaporation in an air matrix digital microfluidic (DMF) device, the method comprising:

[0422] introducing aqueous reaction droplets into an air gap of the air matrix DMF device, the air gap being formed between a first plate and a second plate of the air matrix DMF device;

[0423] Sequentially energizing the drive electrodes on or in the first plate to move the aqueous reaction droplets within the air gap of the air-based DMF device, so that the aqueous reaction droplets combine with droplets of a non-polar fluid within the air gap of the air-based DMF device to form coated reaction droplets, wherein the non-polar fluid coats the aqueous reaction droplets and protects the reaction droplets from evaporation; and

[0424] The drive electrodes are sequentially energized to move the coated reactive droplets within the air gap of the air-based DMF device.

[0425] 61). The method according to 60), wherein the volume of the non-polar fluid is smaller than the volume of the aqueous reaction droplets.

[0426] 62). The method according to 60) further comprises combining the coated droplets with one or more additional aqueous droplets within the air gap of the air matrix DMF device.

[0427] 63). The method according to 60) further comprises removing the coating of the non-polar fluid by at least partially withdrawing the coated droplets from the air gap of the air matrix DMF device back into the microfluidic channel.

[0428] 64). The method according to 60) further includes adding droplets of the non-polar fluid into the air gap of the air matrix DMF device through openings in the first plate or the second plate.

[0429] 65). The method according to 60), wherein the droplets of the non-polar fluid are liquid between 10 degrees Celsius and 100 degrees Celsius.

[0430] 66). A method for preventing droplet evaporation in an air matrix digital microfluidic (DMF) device, the method comprising:

[0431] introducing aqueous reaction droplets into an air gap of the air matrix DMF device, the air gap being formed between a first plate and a second plate of the air matrix DMF device;

[0432] Sequentially energizing the drive electrodes on or in the first plate to move the aqueous reaction droplets within the air gap of the air-based DMF device, so that the aqueous reaction droplets combine with droplets of a non-polar fluid within the air gap of the air-based DMF device to form coated reaction droplets, wherein the non-polar fluid coats the aqueous reaction droplets and protects the reaction droplets from evaporation;

[0433] wherein the non-polar fluid is liquid between 10 degrees Celsius and 100 degrees Celsius, and further wherein the volume of the non-polar fluid is less than the volume of the aqueous reaction droplet; and

[0434] The drive electrodes are sequentially energized to move the coated reactive droplets within the air gap of the air-based DMF device.

[0435] 67) A method for dispensing a predetermined volume of fluid into an air gap of an air-matrix digital microfluidic (DMF) device, wherein the air gap is greater than 400 microns wide, and further wherein the DMF device includes a plurality of drive electrodes adjacent to the air gap, the method comprising:

[0436] filling a portion of the air gap with the fluid from a port in communication with the air gap;

[0437] applying energy to activate a first drive electrode adjacent to a portion of the filled air gap; and

[0438] When the first electrode is activated, suction is applied to withdraw the fluid into the port, which leaves a droplet of the fluid having a predetermined volume in the air gap adjacent to the activated first electrode.

[0439] 68). A method according to 67), wherein applying energy to activate the first drive electrode includes applying energy to activate one or more drive electrodes connected to the first drive electrode, and further wherein applying suction to withdraw the fluid into the port when the first drive electrode is activated includes: withdrawing the fluid when the first drive electrode and the one or more drive electrodes connected to the first drive electrode are active, which leaves droplets of the fluid in the air gap adjacent to the activated first drive electrode and the one or more drive electrodes connected to the first drive electrode.

[0440] 69). The method according to 67), wherein the first driving electrode is separated from the port by a gap of at least one driving electrode.

[0441] 70). The method according to 67) further includes deactivating one or more drive electrodes adjacent to a second portion of the air gap, the second portion of the air gap being in the filled portion of the air gap and between the port and the first drive electrode.

[0442] 71). The method according to 67), wherein the air gap is greater than 500 microns.

[0443] 72). The method of 67), wherein filling a portion of the air gap comprises applying positive pressure to expel fluid from the port.

[0444] 73). The method according to 67) further includes sequentially energizing drive electrodes adjacent to the air gap to move the droplet within the air gap of the air matrix DMF device.

[0445] 74). A method according to 67), wherein applying suction to withdraw the fluid into the port when the first electrode is activated includes leaving a droplet of the fluid having a volume of 10 microliters or greater in the air gap adjacent to the activated first electrode.

[0446] 75) A method for dispensing a predetermined volume of fluid into an air gap of an air-matrix digital microfluidic (DMF) device, wherein the air gap is greater than 400 microns wide, and further wherein the DMF device includes a plurality of drive electrodes adjacent to the air gap, the method comprising:

[0447] filling a portion of the air gap with the fluid from a port in communication with the air gap;

[0448] applying energy to activate a first drive electrode or a first group of adjacent drive electrodes adjacent to a portion of the filled air gap, wherein the first drive electrode or the first group of adjacent drive electrodes are separated from the port by one or more unactivated drive electrodes;

[0449] When the first electrode or first set of contacting electrodes is activated, suction is applied to withdraw the fluid into the port, which leaves a droplet of the fluid in the air gap adjacent to the first electrode or first set of contacting electrodes.

[0450] 76) A method for controlling a digital microfluidic (DMF) device, the method comprising:

[0451] providing a graphical user interface comprising a menu of fluid handling control commands, the fluid handling control commands comprising one or more of: move, heat, remove, cycle, wait, interrupt, mix, and dispense;

[0452] receiving a fluid processing plan, the fluid processing plan including a fluid processing control command selected by a user;

[0453] calculating a path for moving fluid within an air gap of the DMF device based on the fluid handling protocol, wherein the path minimizes an amount of overlap in the paths to avoid contamination; and

[0454] Based on the calculated pathway, the fluid processing protocol is executed using the DMF apparatus.

[0455] 77). The method according to 76), wherein the fluid processing control commands include at least: moving, heating, removing, waiting and mixing.

[0456] 78). The method according to 76), wherein receiving the fluid processing scheme includes receiving a string of fluid processing control commands.

[0457] 79). The method according to 76), wherein calculating the path comprises calculating the path based on the arrangement of heating zones and cooling zones in the DMF device.

[0458] 80). The method according to 76), wherein calculating the path includes determining the shortest path that does not intersect itself.

[0459] 81). The method according to 76), wherein performing the fluid processing protocol on the DMF device comprises performing the fluid processing protocol in a disposable box coupled to the DMF device.

[0460] 82) A digital microfluidics (DMF) reader device configured to operate with a disposable cartridge having a bottom dielectric surface, a top plate having a ground electrode, and an air gap between the bottom dielectric and the top plate, the device comprising:

[0461] a base surface for seating the disposable cartridge on an upper surface;

[0462] a first plurality of drive electrodes on the base surface, wherein all or some of the drive electrodes include openings therethrough;

[0463] a thermal control for applying thermal energy to a first area of ​​the base surface;

[0464] a plurality of thermal vias, wherein the thermal vias comprise a thermally conductive material and are in thermal communication with the first region of the base surface but are electrically isolated from a subset of electrodes, and further wherein the thermal vias are in thermal communication with the thermal control element;

[0465] a plurality of vacuum ports, wherein each vacuum port is coupled to one or more of the openings through the drive electrode;

[0466] a vacuum pump for applying a vacuum to the vacuum port; and

[0467] A control 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.

[0468] 83). Apparatus according to 82), wherein the thermal vias each have a diameter between 0.5 mm and 1.5 mm.

[0469] 84). The apparatus according to 82), wherein there are between 5 and 15 thermal vias associated with an area corresponding to a single electrode in the first area.

[0470] 85). The apparatus according to 82), wherein each of the thermal vias is filled with a thermally conductive metal.

[0471] 86). The device according to 82) further includes a resistive heater, which is located under at least some of the drive electrodes.

[0472] 87). The apparatus according to 82), wherein the base surface comprises a printed circuit board.

[0473] 88). The device according to 82) further includes a magnet, which is below one or more of the drive electrodes and is configured to be activated to apply a magnetic field.

[0474] 89). The apparatus according to 82) further comprising one or more Peltier coolers below at least some of the drive electrodes and configured to cool to less than 10 degrees Celsius.

[0475] 90) A method for detecting the position and identity of a material in an air gap of a digital microfluidics (DMF) cartridge, the method comprising:

[0476] Disconnecting a reference electrode on a first side of the air gap of the DMF box from a drive circuit;

[0477] setting a voltage of one or more drive electrodes in an array of drive electrodes on a second side of the air gap to a high voltage while setting all other drive electrodes in the array of drive electrodes to ground;

[0478] sensing a voltage at the reference electrode;

[0479] determining a capacitance between the first side of the air gap and the second side of the air gap based on a voltage sensed at the reference electrode; and

[0480] Based on the determined capacitance, a material in the air gap adjacent to the one or more drive electrodes is identified.

[0481] 91). The method according to 90) further includes 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.

[0482] 92). The method according to 90), wherein disconnecting the reference electrode comprises allowing the reference electrode to float.

[0483] 93). The method according to 90), wherein setting the voltage of one or more of the drive electrodes to a high voltage comprises setting the voltage of the one or more of the drive electrodes to between 10V and 400V.

[0484] 94). The method according to 90) further includes determining the total capacitance of the air gap by setting the voltage of all drive electrodes in the array of drive electrodes to a high voltage when the reference electrode is disconnected from the drive circuit and sensing the voltage at the reference electrode to determine the total capacitance.

[0485] 95). The method according to 94) further includes 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.

[0486] 96). The method of 94), wherein 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 further comprises using the total capacitance.

[0487] 97). The method of claim 94), wherein identifying the material in the air gap comprises using a reference database comprising a plurality of capacitance ranges to identify the material in the air gap based on the determined capacitance.

[0488] 98) A cartridge for a digital microfluidics (DMF) device, the cartridge having a bottom and a top, the cartridge comprising:

[0489] 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 comprises a first hydrophobic surface;

[0490] a tensioning frame that holds the sheet of dielectric material in tension so that the sheet of dielectric material is substantially flat;

[0491] a top plate having a first side and a second side and a thickness between the first side and the second side;

[0492] a ground electrode on the first side of the top plate;

[0493] a second hydrophobic surface covering the ground electrode on the first side of the top plate; and

[0494] An air gap separates the first hydrophobic layer and the second hydrophobic layer, wherein the air gap comprises a spacing greater than 280 microns.

[0495] 99). The box according to 98) further includes a flange, which extends around the dielectric material sheet and protrudes from the dielectric material sheet.

[0496] 100). A box according to 98), wherein the tensioning frame includes an outer frame and an inner frame, and wherein the sheet is held between the outer frame and the inner frame.

[0497] 101). A box according to 98), wherein the ground electrode includes a grid pattern forming a plurality of open cells.

[0498] 102). The box according to 101), wherein the grid pattern of the ground electrode is formed of an opaque material.

[0499] 103). A box according to 98), wherein the ground electrode is formed of conductive ink.

[0500] 104). A box according to 98), wherein the ground electrode is formed of silver nanoparticles.

[0501] 105). The box according to 101), wherein the minimum width of the grid pattern between the open cells is greater than 50 microns.

[0502] 106). The box according to 101), wherein the open cells among the plurality of open cells include a quadrilateral shape or an elliptical shape.

[0503] 107). A box according to 98), wherein the ground electrode extends over more than 50% of the first side of the top plate.

[0504] 108). A box according to 98), wherein the top plate includes a plurality of cavities within the thickness of the top plate, and wherein the cavities are filled with an insulating material having low thermal mass and low thermal conductivity.

[0505] 109). A box according to 108), wherein the insulating material comprises air.

[0506] 110). A box according to 98), wherein the dielectric material sheet is flexible.

[0507] 111). The box according to 98) further includes a microfluidic channel, which is formed on the second side of the top plate or in the second side of the top plate, wherein the microfluidic channel extends along the second side of the top plate and at least one opening between the microfluidic channel and the air gap.

[0508] 112). A box according to 98), wherein the top panel comprises polycarbonate and / or acrylic.

[0509] 113). A box according to 98), wherein the sheet of dielectric is less than 30 microns thick.

[0510] 114). A box according to 98), wherein the second side of the dielectric material includes a hydrophobic coating.

[0511] 115). A box according to 98), wherein the air gap comprises a spacing greater than 400 microns.

Claims

1. A cartridge for a digital microfluidic device, the cartridge having a bottom portion and a top portion, the cartridge comprising: a thin, flexible 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, a tensioning frame configured to hold the flexible sheet of dielectric material in tension so as to be substantially flat on an exposed bottom surface on the bottom portion of the cartridge, thereby securing the flexible sheet of dielectric material against a plurality of drive electrodes on the digital microfluidic device; roof; as well as An air gap separates the flexible dielectric material sheet and the top plate.

2. The cartridge according to claim 1, wherein The second side of the flexible sheet of dielectric material includes a first hydrophobic coating.

3. The cartridge of claim 1 , further comprising a ground electrode on the top plate, the ground electrode comprising a second hydrophobic coating.

4. The cartridge according to claim 1, wherein The flexible sheet of dielectric material is configured to deflect under a vacuum of at least 50 kPa.

5. The cartridge according to claim 4, wherein The vacuum is supplied by the digital microfluidic device.

6. The cartridge of claim 1, further comprising a ground electrode comprising a grid pattern of open cells on the top plate, wherein a minimum width of the grid pattern between the open cells is greater than 50 microns.

7. The cartridge according to claim 6, wherein The open cell includes a quadrangular shape or an elliptical shape.

8. The cartridge of claim 1, further comprising a ground electrode extending over a majority of the first side of the top plate.

9. The cartridge according to claim 1, wherein The top plate includes a plurality of cavities within the interior of the top plate, the plurality of cavities configured to reduce a thermal mass of the top plate.

10. The cartridge according to claim 9, wherein The plurality of cavities are filled with air.

11. The cartridge of claim 1 , further comprising a microfluidic channel formed in the top plate, wherein the microfluidic channel is configured to connect an opening on an upper surface of the top plate to the air gap.

12. The cartridge according to claim 11, wherein The microfluidic channel includes an access port disposed on the top plate.

13. The cartridge according to claim 1, wherein The top panel comprises a polycarbonate and / or acrylic structure.

14. The cartridge according to claim 1, wherein The flexible sheet of dielectric material is less than 20 microns thick.

15. The cartridge according to claim 1, wherein The air gap comprises a pitch greater than 400 microns.

16. A cartridge for a digital microfluidic device, the cartridge having a bottom portion and a top portion, the cartridge comprising: a thin, flexible sheet of material having a first side and a second side, the first side forming an exposed bottom surface on the bottom of the box; a tensioning frame configured to hold the flexible sheet of material in tension so as to be substantially flat on an exposed bottom surface on the bottom portion of the cartridge, thereby securing the flexible sheet of material against a plurality of drive electrodes on the digital microfluidic device; a top plate having a first side and a second side and a thickness between the first side and the second side; an air gap between the flexible material sheet and the top plate; a microfluidic channel formed in or on the second side of the top plate, wherein the microfluidic channel extends along the second side of the top plate; an opening between the microfluidic channel and the air gap; as well as A cover covers the microfluidic channel, wherein the cover includes one or more access ports for accessing the microfluidic channel.

17. The cartridge according to claim 16, wherein The microfluidic channel is configured to contain more than 1 ml of fluid.

18. The cartridge according to claim 16, wherein The microfluidic channel includes a first microfluidic channel, and the opening between the microfluidic channel and the air gap includes a first opening, the box also includes a second microfluidic channel formed in the second side of the top plate and a second opening between the second microfluidic channel and the air gap, wherein the second microfluidic channel extends along the second side of the top plate, and wherein the first opening and the second opening are adjacent to each other.

19. The cartridge according to claim 18, wherein The first opening and the second opening are no more than 2 cm apart from each other.

20. The cartridge of claim 16, further comprising a plurality of openings from the top of the cartridge into the air gap.

21. The cartridge of claim 16, further comprising one or more reagent reservoirs on the second side of the top plate.

22. The cartridge of claim 16, further comprising one or more freeze-dried reagent reservoirs on the second side of the top panel.

23. A cartridge for a digital microfluidic device, the cartridge having a bottom and a top, the cartridge comprising: a flexible 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; a tensioning frame that applies tension to keep the flexible dielectric material sheet flat on the exposed bottom surface of the bottom portion of the cartridge so that the flexible dielectric material sheet can be secured to the digital microfluidic device by vacuum, a top plate having a first side and a second side; as well as an air gap between the flexible dielectric material sheet and the top plate; Wherein, the box does not include a driving electrode on the flexible dielectric material sheet.

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