Digital Microfluidic Device and Method of Using the Same

Abstract

To provide digital microfluidic (DMF) methods and apparatuses (including devices, systems, cartridges, DMF readers, etc.).SOLUTION: Described herein are digital microfluidic (DMF) methods and apparatuses (including devices, systems, cartridges, DMF readers, etc.), and in particular DMF apparatuses and methods adapted for large volume. For example, described herein are methods and apparatuses for DMF using an air gap having a gap width that may be between 0.3 mm and 3 mm. Also described herein are DMF readers for use with a DMF cartridges, including those adapted for use with large air gap / large volume, although smaller volumes may be used as well.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications 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 these applications is hereby incorporated by reference in its entirety.

[0002] Incorporation by Reference All publications and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0003] Field This application generally relates to digital microfluidic (DMF) devices and methods. In particular, the devices and methods described herein relate to a void DMF device that includes a cartridge containing an air matrix and a ground electrode, and a durable component including a drive electrode.

Background Art

[0004] Background In recent years, Lab - on - a - Chip and biochip devices have received significant attention in both scientific research applications and potentially point - of - care applications for performing highly repetitive reaction processes with small reaction volumes to save both materials and time. Conventional biochip - type devices utilize micro - or nano - sized channels and corresponding micropumps, microvalves, and microchannels coupled to the biochip to manipulate the reaction process, but these additional components increase the cost and complexity of the microfluidic device.

[0005] Digital microfluidics (DMF) has emerged as a powerful preparation technology for a wide range of biological and chemical applications. DMF enables accurate and highly flexible real-time control over multiple samples and reagents, including solids, liquids, and aggressive chemicals, without the need for pumps, valves, or arrays of complex tubing. In DMF, separate droplets in the nanoliter to μl range are dispensed from reservoirs onto a planar surface coated with a hydrophobic insulator, where they are manipulated (transported, split, merged, mixed) by applying a series of potentials to an array of electrodes. Complex reaction sequences can be performed using only DMF or in a hybrid system where DMF is integrated with channel-based microfluidics. The hybrid system offers unparalleled flexibility. Conceptually, each reaction step can be carried out in the microfluidic format that best accepts it.

[0006] For many applications, it is highly convenient to perform DMF on an open surface where the matrix surrounding the droplets is the ambient air. FIGS. 1A - C show an example of an air matrix DMF device. FIG. 1A shows an example of an air matrix DMF device 100. Generally, the air matrix DMF device includes a plurality of unit cells 191 adjacent to each other, defined by having one working electrode 106 on the opposite side of the grounded electrode 102. Each unit cell can be of any suitable shape, but generally can have approximately the same surface area. In FIG. 1A, the unit cells are rectangular. Droplets (e.g., reaction droplets) fit within the gap between the first plate 153 and the second plate 151 (shown as the top plate and bottom plate in FIGS. 1A - C). The air matrix DMF device as a whole can have any suitable shape and thickness. FIG. 1B is an enlarged cross - sectional view of the thermal zone of the air matrix DMF shown in FIG. 1A, showing a layer of the DMF device (e.g., the layer forming the bottom plate). Generally, the DMF device (e.g., the bottom plate) includes several layers that can include a layer formed on a printed circuit board (PCB) material. These layers can include a protective coating layer, an insulating layer, and / or a support layer (e.g., a glass layer, a grounded electrode layer, a hydrophobic layer; a hydrophobic layer, a dielectric layer, a working electrode layer, a PCB, a thermal control layer, etc.). Any of these surfaces can be hard (e.g., glass, PCB, polymer material, etc.). The air matrix DMF device described herein also includes both reservoirs for samples and reagents and a mechanism for replenishing the reagents.

[0007] In the examples shown in FIGS. 1A - C, the top plate 101, in this case a glass material (a plastic / polymer material including a PCB can be used), provides support, protects the underlying layers from external particles, and also provides some insulation for the reactions occurring within the DMF device. Thus, the top plate can confine / squeeze the droplets between the plates, which can enhance the electric field compared to an open - air - matrix DMF device (without plates). The upper plate (the first plate in this example) 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 clear plastic. However, although transparent, it may be coated with a conductive material or may include a ground electrode (ground electrode layer 102) adjacent to and beneath the substrate for the DMF circuit. In some examples, the ground electrode is a continuous coating, or alternatively, a plurality of, for example, adjacent ground electrodes may be used. Beneath the ground electrode layer is a hydrophobic layer 103. The hydrophobic layer 103 acts to reduce surface wetting and assists in maintaining the reaction droplets as one agglomeration unit.

[0008] In FIGS. 1A - C, the second plate shown as the lower or bottom plate 151 may include a working electrode that defines a unit cell. In this example, similar to the first plate, the outermost layer facing the gap 104 between the plates also includes a hydrophobic layer 103. The material forming the hydrophobic layer may be the same for both plates or different hydrophobic materials. The gap 104 provides a space where reaction droplets are initially contained in a sample reservoir, perform reaction steps, and are moved to maintain various reagents for various reaction steps. Adjacent to the hydrophobic layer 103 on the second plate, there is a dielectric layer 105 that can increase the capacitance between the droplet and the electrode. Adjacent and below the dielectric layer 105, there is a PCB layer (working electrode layer 106) containing the working electrode. The working electrode may form each unit cell. When a voltage is applied, the working electrode can move the droplets in the DMF device to various regions so as to perform various reaction steps under various conditions (such as temperature, combined with various reagents, magnetic regions, pump inlet regions, etc.). A support substrate 107 (such as a PCB) is adjacent and below the working electrode layer 106 (FIGS. 1B and 1C), and can provide support and electrical connections for these components, such as working electrodes, traces connecting them (which may be insulated) and / or additional control elements, such as a thermal regulator 155 (shown as a TEC), temperature sensors, optical sensors, magnets, pumps, etc. One or more control devices 195 for controlling the operation of the working electrode and / or for controlling the application of refill droplets to the reaction droplets are connected, but may be separated from the first plate 153 and the second plate 151, may be formed on the second plate, or may be supported thereby. In FIGS. 1A - C, the first plate is shown as the top plate and the second plate is the bottom plate, but this arrangement may be reversed. Also, a source or reservoir 197 of solvent (refill fluid) is shown connected to an opening in the second plate by a pipe 198.

[0009] As described above, the void 104 provides a space in which reaction processes can occur, holds reagents, and provides an area that can be processed, for example, by mixing, heating / cooling, and combination with reagents (enzymes, labels, etc.). In FIG. 1A, the void 104 includes a sample reservoir 110 and a series of reagent reservoirs 111. The sample reservoir may further include a sample loading function for introducing the initial reaction droplet into the DMF device. The sample loading can be performed from above, below, or laterally and can be specific based on the requirements of the reaction being carried out. The sample DMF device shown in FIG. 1A includes six sample reagent reservoirs, each including an opening or port for introducing each reagent into its respective reservoir. The number of reagent reservoirs can vary depending on the reaction being carried out. The sample reservoir 110 and the reagent reservoirs 111 are in fluid communication via a reaction zone. The reaction zone 112 is in electrical communication with the working electrode layer 106 where the working electrode layer 106 is located below the reaction zone 112.

[0010] The working electrode 106 is shown as a grid or unit cell in FIG. 1A. In other examples, the working electrode may be a completely different pattern or arrangement based on the requirements of the reaction. The working electrode is configured to move droplets from one region of the DMF device to another region. The movement and to some extent the shape of the droplets can be controlled by switching the voltage of the working electrode. One or more droplets can be moved along the path of the working electrode by sequentially applying and de-energizing the voltage to the electrode in a controlled manner. In the example of the DMF device shown, 100 working electrodes (forming approximately 100 unit cells) are connected to seven reservoirs (one sample reservoir and six reagent reservoirs). The working electrode can be made of any suitable conductive material, such as copper, nickel, gold, or combinations thereof.

[0011] In the exemplary devices shown in FIGS. 1A - C, the DMF device is typically incorporated such that the electrodes (e.g., the working electrode and the ground electrode) are part of the same structure into which the sample and / or fluid can be loaded. The electrodes can be part of a cartridge that can be removable. Although cartridges are described (see, e.g., US20130134040), such cartridges have been found to be difficult to use, particularly when imaging through the device and when operating in an air matrix device.

[0012] It would be highly advantageous to have an air matrix DMF device that includes a cartridge that is easy to use and can be fabricated reliably and inexpensively. What is described herein are methods and devices, including systems, that can address these problems. SUMMARY OF THE INVENTION

[0013] SUMMARY OF THE DISCLOSURE What is described herein are digital microfluidic (DMF) methods and devices (including devices, systems, cartridges, DMF readers, etc.). The methods and devices described herein may specifically be adapted for pneumatic DMF devices (also referred to herein as void DMF devices), although these methods and devices may be configured for use in other DMF devices (such as oil gaps, for example). The methods and devices described herein are, in part, used to handle a relatively large amount that was possible with conventional DMF devices because the separation distance between the plates forming the voids of the DMF device can be relatively large (e.g., greater than 280 μm, 300 μm or more, 350 μm or more, 400 μm or more, 500 μm or more, 700 μm or more, 1 mm or more, etc.). Additionally, any of the devices and methods described herein may be configured to include a disposable cartridge having a dielectric layer for forming the bottom of the disposable cartridge; the drive electrodes need not be part of the cartridge; and these devices may be adapted to allow the dielectric to be held firmly to the electrodes during operation (which has been found to be very difficult, especially when the dielectric layer is slightly flexible).

[0014] Any of the devices and methods described herein may include a cartridge that includes a ground electrode as part of the cartridge. In some variations, the ground electrode may be formed into a grid pattern that forms a plurality of cells. The grid pattern may create a clear window that allows visualization through the ground electrode, even when an opaque ground electrode (e.g., an opaque or translucent material, such as a metal coating, such as silver conductive ink) is used to form the ground electrode. The grid pattern may mimic the arrangement of drive electrodes in a DMF device in which the cartridge may be placed. For example, the grid pattern may cover the space between adjacent electrodes when the ground electrode is adjacent to a drive electrode with a gap therebetween. Alternatively, the ground electrode may be formed of a material that is transparent or transparent enough to image through it. In some variations, the ground electrode is a conductive coating. The ground electrode may be electrically continuous (e.g., in electrical contact), but may include one or more openings, for example, through which droplets within the gap may be visualized. Thus, in any of these variations, the top plate of the cartridge may be transparent or transparent enough to be visualized through at least one or more regions.

[0015] For example, a cartridge for a digital microfluidic (DMF) device may have a bottom and a top, have a first surface and a second surface, where the first surface forms an exposed bottom surface of the bottom of the cartridge, and at least the second surface includes a first hydrophobic surface, a sheet of dielectric material; a top plate having a first surface and a second surface; and a ground electrode on the first surface of the top plate. The ground electrode may include a grid pattern that forms a plurality of open cells. The cartridge may also include a second hydrophobic surface on the first surface of the top plate that covers the ground electrode; and a gap that includes a separation distance greater than 280 μm that separates the first hydrophobic layer and the second hydrophobic layer.

[0016] In any of the cartridges described herein, the top plate may include a plurality of cavities within the thickness of the top plate; these cavities may be closed (e.g., sealed) and / or filled with a thermal insulation material having a low thermal mass and a low thermal conductivity. In some variations, the thermal insulation material includes air. The cavities may be disposed over void regions corresponding to heating and / or cooling regions (e.g., thermal control regions); the lower thermal mass in these regions may enable significantly faster heating / cooling of droplets in the voids below the cavities. Thus, the thickness of the top plate in these regions may include cavities; the lower part of the cavity (corresponding to the lower surface of the top plate) may be less than 1 mm thick (e.g., 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, less than 90 microns, 80 microns, 70 microns, 60 microns, 50 microns, 40 microns, 30 microns, etc.). The lower part of the cavity may preferably be as thin as possible while providing structural support for electrodes and any dielectric coating on the lower surface of the top plate. The upper surface of the cavity may be substantially thicker than the lower surface of the cavity (e.g., 1.5×, 2×, 3×, 4×, 5×, etc.).

[0017] The dielectric material forming the lower surface may be made hydrophobic (e.g., by coating, such as dip coating, impregnation with a hydrophobic material, etc.) or may itself be hydrophobic. For example, the lower surface (e.g., the lower surface of the cartridge) may be formed of a film that is a dielectric and hydrophobic material. For example, the lower surface may be a Teflon film that is hydrophobic and acts as a dielectric (which may include an adhesive or adhesive portion, such as a Teflon tape). Other films may include plastic paraffin films (e.g., "Parafilm" such as PARAFILM M). However, in particular, a film (e.g., a Teflon film) that can withstand high temperatures (e.g., 100 °C or higher) is preferred.

[0018] A cartridge for a digital microfluidic (DMF) device can generally include a lower part and an upper part, have a first surface and a second surface, with the first surface forming the exposed lower surface of the lower part of the cartridge, a sheet of dielectric material; a first hydrophobic layer on the second surface of the sheet of dielectric material; a top plate having a first surface and a second surface; a ground electrode on the first surface of the top plate, including a lattice pattern forming a plurality of open cells; a second hydrophobic layer on the first surface of the top plate covering the ground electrode; and may include a void including a separation distance greater than 280 μm (e.g., greater than 300 μm, greater than 400 μm, etc.) separating the first hydrophobic layer and the second hydrophobic layer.

[0019] The term "cartridge" can refer to a container forming a void and can be inserted into a DMF reading / driving device. The cartridge can be disposable (e.g., single-use or limited-use). The cartridge can be configured to enable visualization of the fluid (droplets) in the void. The lattice pattern can be particularly useful for providing an appropriate ground reference to the driving electrodes while enabling visualization. The entire lattice may be electrically coupled to form one return (ground) electrode, or multiple ground electrodes may be arranged on the top plate (either separately and / or via adjacent lattices).

[0020] As described above, the lattice pattern of the ground electrode is formed of an opaque material.

[0021] As used herein, the term "lattice" refers to a repeating pattern of open cells ("windows") of any suitable shape and size, where the boundary lines forming the open cells are formed by an incorporated (and electrically conductive) material, such as conductive ink, a metal coating, etc. The lattice used herein is not limited to a network of lines intersecting each other to form a series of squares or rectangles; the lattice pattern can be formed by forming openings into a plane of a continuous conductive material in other ways to form the ground electrode.

[0022] Therefore, generally, the grid pattern of the grounding electrode can be formed of a conductive ink. For example, the grid pattern of the grounding electrode can be formed of silver nanoparticles. The grid pattern can be laminated on the top plate by printing, screening, spraying or other means.

[0023] Generally, the boundary lines between the open cells forming the grid pattern can have a minimum width. The minimum width between the open cells of the grid pattern can be 50 μm or more (for example, 0.1 mm or more, 0.2 mm or more, 0.3 mm or more, 0.4 mm or more, 0.5 mm or more, 0.6 mm or more, 0.7 mm or more, 0.8 mm or more, 0.9 mm or more, 1 mm or more, etc.). As described above, the open cells (for example, "windows") formed by the grid pattern can be of any shape including quadrilaterals (for example, squares, rectangles, etc.) or ellipses (for example, ovals, circles, etc.) and / or other shapes (+ shape, H-shaped, etc.).

[0024] Generally, the grid pattern of the grounding electrode can extend over most of the top plate (and / or most of the cartridge). For example, the grid pattern of the grounding electrode can extend over 50% or more (for example, 55% or more, 60% or more, 65% or more, 70% or more, 80% or more, 90% or more, etc.) of the first surface of the top plate.

[0025] In any of the cartridges described herein, the sheet of dielectric material can be flexible. This flexibility can help to fix the dielectric to the drive electrode to ensure complete contact between the dielectric and the drive electrode. Typically, the sheet of dielectric material can have sufficient compliance to bend or flex under a relatively low force (for example, a pressure of 50 kPa or more). The sheet of dielectric can be of any suitable thickness. For example, the sheet can be less than 30 microns thick (for example, less than 20 microns thick, etc.).

[0026] As will be described in further detail below, any of these devices may include a microfluidic channel formed on a second surface of the top plate and extending along the second surface of the top plate, and at least one opening between the microfluidic channel and the void.

[0027] The top plate may be formed of any suitable material, particularly a clear or transparent material (such as acrylic).

[0028] For example, a cartridge for a digital microfluidic (DMF) device has a first surface and a second surface, the first surface forming an exposed lower surface of the cartridge, a sheet of flexible dielectric material; a first hydrophobic layer on the second surface of the sheet of dielectric material; a top plate having a first surface and a second surface; a ground electrode on the first surface of the top plate including a lattice pattern formed of an opaque material, the lattice pattern forming a plurality of open cells along the first surface of the top plate; a second hydrophobic layer on the first surface of the top plate covering the ground electrode; and a void including a separation distance greater than 280 μm (such as 300 μm or more, 400 μm or more, etc.) separating the first hydrophobic layer and the second hydrophobic layer. Typically, the cartridge has a lower part and an upper part.

[0029] As described above, also described herein is a cartridge in which the microfluidic channel is incorporated into a DMF component, particularly the top plate of a DMF device. The Applicants have found that incorporating one or more microfluidic channels into the top plate can make the cartridge more compact and allow for a higher degree of control and operation of processes within the voids controlled by electro-wetting in a DMF system than would otherwise be possible.

[0030] For example, a cartridge (a cartridge having a lower part and an upper part) for a digital microfluidic (DMF) device has a first surface and a second surface, the first surface forming an exposed lower surface of the lower part of the cartridge, a sheet of dielectric material; a first hydrophobic layer on the second surface of the sheet of dielectric material; a top plate having a first surface and a second surface; a ground electrode on the first surface of the top plate; a second hydrophobic layer on the first surface of the top plate covering the ground electrode; a void separating the first hydrophobic layer and the second hydrophobic layer; a microfluidic channel formed on the second surface of the top plate and extending along the second surface of the top plate; an opening between the microfluidic channel and the void; and a cover covering the microfluidic channel and including one or more access ports for accessing the microfluidic channel.

[0031] As described above, the sheet of dielectric material can be flexible and can form the lowermost surface of the cartridge. The sheet can generally be flat (planar), but can also be flexible. The outer surface can be protected by a removable (e.g., peel-off) cover. The dielectric properties can generally match those of a DMF (particularly air matrix DMF) device. The dielectric can have its inner (second) side surface coated with the first hydrophobic layer. The hydrophobic layer can be a coating of a hydrophobic material that is relatively inert (e.g., does not react with aqueous droplets moved through the void).

[0032] The top plate can be planar and can have the same extent as (or be larger than) the lower dielectric material. The top plate can be of any suitable thickness, and in particular, can have a sufficient thickness such that microfluidic channels can be etched into the second face of the top plate. The ground electrode can be formed on all or part of the first face of the top plate as described above, and a second hydrophobic layer can be coated on the ground electrode and / or the top plate (in particular, where an open window passing through the ground plate exposes the top plate). In any of these examples, the thickness of the electrode coating can be minimal such that the electrode can be considered coplanar with the top plate bottom (first) face of the top plate.

[0033] In any of the devices and methods described herein, the gap separating the first hydrophobic layer and the second hydrophobic layer (such as the dielectric and the top plate) can be relatively large (e.g., >280 μm, 400 μm or more, 500 μm or more, 1 mm or more, etc.) compared to conventional gap DMF systems.

[0034] The microfluidic channels formed in the second face of the top plate typically extend through the top plate along the second face of the top plate, and an access opening between the microfluidic channels and the gap can be formed in the top plate between the microfluidic channels and the gap. Also, any of the devices described herein can include a cover covering the microfluidic channels. The cover can be formed of any suitable material, such as acrylic. The cover can include one or more ports or openings leading to the microfluidic channels and / or the gap.

[0035] The microfluidic channel can be configured to contain any suitable amount of fluid that can be useful for mixing, adding, removing, or otherwise interacting with droplets in the void. For example, the microfluidic channel can be configured to hold 0.2 ml or more of fluid (such as 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 of fluid, 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.) within the microfluidic channel. The microfluidic channel may be connected to one or more reservoirs (such as a waste reservoir, a storage reservoir, etc.), or may be connected to one or more additional microfluidic channels.

[0036] For example, the microfluidic channel may include a first microfluidic channel, and the opening between the microfluidic channel and the void may include a first opening; the apparatus may further include a second microfluidic channel formed on a second surface of the top plate and extending along the second surface of the top plate, and a second opening between the second microfluidic channel and the void, and the first and second openings may be adjacent to each other. The first and second openings may be separated by a minimum distance, which may enable the formation of a "bridge droplet" having a minimum size in the void. For example, the first and second openings may be within about 2 cm of each other (such as within about 1 cm of each other, within about 9 mm of each other, within about 8 mm of each other, within about 7 mm of each other, within about 6 mm of each other, within about 5 mm of each other, within about 4 mm of each other, within about 3 mm of each other, within about 2 mm of each other, within about 1 mm of each other, etc.) on the surface of the top plate.

[0037] Also, any of these cartridges may include a window from the top of the cartridge to the void, through which the void can be seen. This may enable imaging into the void. This imaging can be used to detect an output (e.g., a reaction output, e.g., binding, colorimetric assay, RT-PCR, etc.). The window can be of an appropriate size. For example, the window can form 2 to 50% of the top of the cartridge. The window may be on one side of the cartridge and / or at one end of the cartridge. Multiple imaging windows may be used.

[0038] As described above, the lower part of the cartridge is formed by the first surface of a sheet of dielectric material. The upper part of the cartridge may include a plurality of openings leading to the void.

[0039] Generally, the cartridge may include one or more reagent reservoirs on the second surface of the top plate. For example, the cartridge may contain one or more reagents, particularly lyophilized (e.g., "freeze-dried") reagents, either in the reservoir or in the void. For example, the cartridge may include one or more freeze-dried reagent reservoirs on the second surface of the top plate.

[0040] For example, a cartridge (including a lower and an upper part) for a digital microfluidic (DMF) device has a first surface and a second surface, where the first surface forms an exposed lower surface of the lower part of the cartridge, a sheet of dielectric material; a first hydrophobic layer on the second surface of the sheet of dielectric material; a top plate having a first surface and a second surface; a ground electrode on the first surface of the top plate; a second hydrophobic layer on the first surface of the top plate covering the ground electrode; a void including a separation distance greater than 500 μm separating the first hydrophobic layer and the second hydrophobic layer; a first microfluidic channel and a second microfluidic channel formed on the second surface of the top plate and extending along the second surface of the top plate; a first opening between the first microfluidic channel and the void and a second opening between the second microfluidic channel and the void adjacent to each other within a range of about 2 cm; and a cover covering the microfluidic channel including one or more access ports for accessing the microfluidic channel may be included.

[0041] Also described herein is a DMF reading device for use with any of the cartridges described herein. For example, the DMF reading device (device) can be configured to apply a vacuum over the entire lower dielectric surface of the cartridge to form each of the unit cells that drive the electrodes to be in intimate contact with the dielectric and move the fluid droplets within the voids uniformly. The Applicants have surprisingly found that simply adhering and fixing the dielectric material to the electrodes is insufficient. This is because it causes non-uniform contact and fluctuations in the power required to move the droplets, as well as inefficiencies in droplet movement, control, and consistency. Further, the use of a vacuum has similar problems even when used in combination with an adhesive, especially when the dielectric is flexible. Described herein are devices and methods of using a vacuum to secure the lower dielectric of the cartridge through a plurality of openings within the drive electrode itself or surrounding / adjacent to the drive electrode. In a variant where a vacuum is applied through all or some of the drive electrodes (e.g., spaced apart in a regular pattern on the mounting surface, e.g., at the corners), the dielectric is consistently held on the drive electrodes in a uniform manner even when using a relatively low negative pressure as the vacuum. This configuration can also enable the formation of partitions or barriers within the cartridge by including protrusions on the cartridge holding surface (on which the cartridge is held).

[0042] For example, what is described herein is a digital microfluidic (DMF) reading device configured to operate with a disposable cartridge having a lower dielectric surface, a top plate having a ground electrode, and a gap between the lower dielectric and the top plate, an installation surface for installing the disposable cartridge; a plurality of drive electrodes on the installation surface (each drive electrode includes an opening therethrough); a vacuum pump for applying a vacuum to a vacuum port; and a control unit for applying energy to one or more selected drive electrodes to sequentially activate and deactivate them to move droplets within the cartridge gap along a desired path within the gap, the disposable cartridge being configured to apply a vacuum to a vacuum manifold to fix each drive electrode to the lower dielectric of the disposable cartridge when the disposable cartridge is placed on the installation surface.

[0043] In some variations, the device includes a vacuum manifold that couples a vacuum pump to a plurality of vacuum ports for applying a vacuum.

[0044] The DMF reading devices described herein can be configured to operate with any of the cartridges described herein and can be adapted for use with such cartridges. However, it should be understood that the cartridge is not an essential part of the DMF reading device. Generally, these devices can operate with a cartridge (e.g., a reusable or disposable cartridge) having a lower dielectric surface, a top plate having a ground electrode, and a gap (e.g., typically a void, although not necessarily) between the lower dielectric and the top plate.

[0045] The DMF device may also generally include an installation surface for installing a disposable cartridge. The installation surface may include any protrusions that can be used to form a partition within the gap region (e.g., void) of the cartridge by predictably deforming a drive electrode and / or a dielectric that is in the same plane or substantially the same plane as the installation surface into the gap region. The plurality of drive electrodes on the installation surface may be formed on the installation surface or milled into the installation surface. For example, the installation surface may be a substrate (e.g., an electrically insulating surface) such as a printed circuit board on which or in which the drive electrodes are attached or formed.

[0046] Generally, as described above, all or most of the drive electrodes in the electrode array (e.g., >50%, >60%, >70%, >80%, >90%, >95%, etc.) may include an opening that penetrates the drive electrode and is connected to a vacuum source. The vacuum source may be a vacuum manifold that connects these openings penetrating the drive electrodes to a vacuum source, such as a vacuum pump that is part of the device or a separate vacuum pump (e.g., wall vacuum) connected to the device. The openings penetrating the electrodes may be of the same size and may be located at any position on / within the drive electrode. For example, the opening may pass through the center of the drive electrode and / or the edge region of the drive electrode, etc. The opening may be of any shape (e.g., circular, oval, square, etc.). In some variations, the size of the opening may be about 1 mm in diameter (e.g., 1.2 mm in diameter, 1.1 mm in diameter, 1.0 mm in diameter, 0.9 mm in diameter, 0.8 mm in diameter, etc.).

[0047] Typically, the vacuum manifold may be connected to and / or include a plurality of vacuum ports, each of which is coupled to one (or, in some variations, two or more) of the openings in the drive electrodes. The vacuum manifold may be located below the installation surface. For example, the vacuum manifold may be a pipe or other channel below the installation surface that connects to the openings in the drive electrodes.

[0048] The DMF devices described herein typically include a control device for associating and driving electrodes. This control device includes one or more processors, memories, and any other circuitry necessary or useful for operating the device, including associating energy application to operate / stop a drive electrode, a pump for vacuum and / or microfluidic control, one or more valves (e.g., for microfluidic control, vacuum control), a temperature control device (e.g., a resistive heater, a Peltier cooler, etc.), a motor (e.g., for driving the opening and closing of a device door, optical components, etc.), one or more displays, etc.

[0049] As described above, any of these devices may include one or more protrusions extending from the installation surface, and the one or more protrusions are configured to form a partition in the air of the cartridge when vacuum is applied through an opening in the drive electrode.

[0050] Any of these devices may include an optical reading device configured to detect an optical signal from a cartridge installed on the installation surface. The optical reading device can be movable or fixed. The optical reading device can be used to detect (e.g., sense) a feed or change due to one or more interactions (e.g., binding, enzymatic reaction, etc.) in a droplet. The optical reading device can be configured to detect an optical signal from a cartridge installed on the installation surface. Thus, the optical sensor can provide detection of a readout from the device. Any of these devices may include one or more motors (e.g., configured to move the optical reading device).

[0051] The device may also include one or more temperature sensors (such as a thermistor, etc.). For example, the device may include one or more temperature sensors coupled to the installation surface. In some variations, the thermistor may protrude from the installation surface and form a barrier or chamber within the void of the cartridge. Alternatively or additionally, one or more temperature sensors may be within the substrate of the installation surface and may be in thermal contact with the installation surface via a heat-conductive material such as copper.

[0052] As described above, the devices described herein may include one or more heaters, particularly resistive heaters. For example, the device may include a resistive heater located under (or above) at least some of the drive electrodes; this may enable a temperature-regulated sub-region of the device. Also, the entire surface of the drive electrode may be cooled to a temperature slightly lower than room temperature (such as 15°C - 25°C, 15°C - 22°C, 15°C - 20°C, 15°C - 18°C, etc.) (such as by circulation of a cooling fluid).

[0053] The device may also include one or more magnets above or below one or more of the drive electrodes configured to apply a magnetic field when activated. Thus, magnetic beads may be used to confine substances or other reactions within the DMF device, and the magnetic beads may be selectively retained within one or more regions of the device. For example, one or more neodymium magnets may be used to hold magnetic particles in place (such as by moving the magnet 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, etc. above the electrode) by bringing the magnet closer to or farther from the cartridge. The electromagnet may be selectively activated or deactivated to hold / release the magnetic particles.

[0054] Also, any of the devices described herein may include one or more Peltier coolers located under at least some of the drive electrodes configured to cool down to 10°C or below (such as 5°C or below, 7°C or below, 11°C or below, 12°C or below, 15°C or below, 20°C or below, etc.).

[0055] In addition to the installation surface, any of these DMF reading devices may include one or more cartridge trays, and when a cartridge is loaded into the tray, the cartridge can be automatically moved to a fixed position within the device. For example, any of these devices may include a cartridge tray for holding the cartridge in a predetermined orientation (which may be fixed by the complementary shapes of the cartridge and the receiving tray); the cartridge tray may be configured to move a disposable cartridge onto the installation surface. Once on the installation surface, a vacuum can be applied to fix it in place. Additionally, for example, connections may be formed from the top of the cartridge to one or more microfluidic ports to apply a positive or negative pressure (such as a vacuum) to drive fluid within the microfluidic channels into and / or out of a gap (such as an air gap) region within and / or above the cartridge.

[0056] Generally, any of these devices may include an outer housing, a front panel display, and one or more inputs (such as a touch screen display, dial, button, slider, etc.) and / or a power switch. The device may be configured to be stackable and / or may be configured to operate with one or more other DMF devices. In some variations, one housing may enclose a plurality of cartridge installation surfaces, each having a separately addressable / controllable (e.g., by one or more control devices) drive electrode array that enables parallel processing of a plurality of cartridges. In these variations, all or some of the components (pumps, motors, optical subsystems, control devices, etc.) may be shared between different cartridge installation surfaces.

[0057] Any of these devices may include an output configured to output signals detected by the device. The output may be on one or more displays / screens and / or may be an electronic output transferred to a memory or a remote processor for storage / processing and / or display. For example, any of these devices may include a wireless output.

[0058] Also, as described above, any of the DMF devices described herein may include one or more microfluidic vacuum ports disposed above the installation surface configured to engage an access port for accessing the microfluidic channels of the cartridge when the cartridge is placed on the installation surface.

[0059] For example, a digital microfluidic (DMF) reading device configured to operate with a disposable cartridge having a lower dielectric surface, a top plate having a ground electrode, and a gap between the lower dielectric and the top plate may include an installation surface for installing the disposable cartridge; a plurality of drive electrodes on the installation surface (each drive electrode including an opening therethrough); a plurality of vacuum ports each 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 installation surface; and a control unit for applying energy to the one or more selected drive electrodes to sequentially activate and deactivate them to move droplets within the gap of the cartridge along a desired path within the gap. The DMF reading device may be configured to apply a vacuum to the vacuum ports to fix each drive electrode to the lower dielectric of the disposable cartridge when the disposable cartridge is placed on the installation surface, and the one or more protrusions are configured to partition the gap.

[0060] Also described herein are methods of preventing or reducing evaporation in any of these devices. For example, described herein is a method of preventing droplet evaporation in an air matrix digital microfluidic (DMF) device, the method comprising: introducing an aqueous reaction droplet into the air matrix DMF device void formed between a first plate and a second plate of the air matrix DMF device; sequentially applying a voltage to drive electrodes on or in the first plate to move the aqueous reaction droplet within the air matrix DMF device void such that it combines with a droplet of nonpolar fluid within the air matrix DMF device void to form a coated reaction droplet in which the nonpolar fluid coats the aqueous reaction droplet to protect the reaction droplet from evaporation; and sequentially applying a voltage to the drive electrodes to move the coated reaction droplet within the air matrix DMF device void.

[0061] The volume of the nonpolar fluid can be less than that of the aqueous reaction droplet. Any of these methods can include combining the coated droplet with one or more additional aqueous droplets within the air matrix DMF device void. Any of these methods can also include removing the nonpolar fluid coating by at least partially recovering the coated droplet from the air matrix DMF device void into a microfluidic channel. The method can also include adding a droplet of nonpolar fluid into the air matrix DMF device void through an opening in the first or second plate. Generally, the droplet of nonpolar fluid can be liquid at 10 °C to 100 °C.

[0062] For example, a method for preventing droplet evaporation in an air matrix digital microfluidic (DMF) device includes introducing an aqueous reaction droplet into the void of the air matrix DMF device formed between a first plate and a second plate of the air matrix DMF device; sequentially applying a voltage to a driving electrode on or in the first plate to move the aqueous reaction droplet within the void of the air matrix DMF device such that it combines with a droplet of a nonpolar fluid within the void of the air matrix DMF device (in some variations, the nonpolar fluid may be combined with the sample before being loaded into the void), forming a coated reaction droplet in which the nonpolar fluid coats the aqueous reaction droplet to protect the reaction droplet from evaporation (the nonpolar fluid is liquid at 10°C to 100°C, and further, the volume of the nonpolar fluid is less than the volume of the aqueous reaction droplet); and sequentially applying a voltage to the driving electrode to move the coated reaction droplet within the void of the air matrix DMF device. The volume of the nonpolar liquid may be less than the volume of the droplet, but the volume of the nonpolar liquid coating the droplet may also be more than the volume of the droplet (up to about 3×).

[0063] The methods and apparatuses described herein may be particularly suitable for use with large quantities of droplets and processing. Typically, most unit droplets of a DMF device, particularly an air matrix DMF device, are limited to an aqueous fluid of about 4 μl or less, and the void is limited to a separation distance of less than about 250 or 300 μm between the drive electrode and the ground electrode (between the top and bottom plates of the void region). What is described herein is a method of manipulating a larger quantity where the separation distance between the drive electrode (e.g., bottom plate) and the ground electrode (e.g., top plate) can be much larger (e.g., about 280 μm to 3 mm, about 300 μm to 3 mm, about 400 μm to 1.5 mm, e.g., 400 μm to 1.2 mm, etc., or 400 μm or more, 500 μm or more, 1 mm or more, etc.). Thus, the unit droplet size (the droplet on one unit cell driven by one drive electrode) can be much larger, e.g., 5 μl or more, 6 μl or more, 7 μl or more, 8 μl or more, 9 μl or more, 10 μl or more, 11 μl or more, 12 μl or more, 13 μl or more, 14 μl or more, 15 μl or more, e.g., 5 to 20 μl, 5 to 15 μl, 7 to 20 μl, 7 to 15 μl, etc.).

[0064] The dispensing of large droplets using electro-wetting is conventionally carried out in relatively small amounts (e.g., less than 5 μl), but it has been found that dispensing a larger amount as one unit is difficult, especially with high accuracy and precision. What is described herein is a method of dispensing a predetermined amount of liquid using electro-wetting. For example, what is described herein is a method of dispensing a predetermined amount of fluid into the void of an air matrix digital microfluidic (DMF) device, where the void is larger than 280 μm in width (e.g., 300 μm or more, 400 μm or more, etc.), and further, the DMF device includes a plurality of drive electrodes adjacent to the void, the step of filling a portion of the void with fluid from a port communicating with the void; the step of applying energy to a first drive electrode adjacent to the filled portion of the void to activate it; and the step of applying suction to the fluid while the first electrode is activated to draw the fluid back into the port, leaving a droplet of the fluid in the void adjacent to the activated first electrode.

[0065] The step of applying energy to the first drive electrode to activate it may include applying energy to one or more drive electrodes adjacent to the first drive electrode to activate them. Further, the step of applying suction to the fluid to draw it back into the port while the first drive electrode is activated may include drawing in the fluid while the first drive electrode and one or more drive electrodes adjacent to the first drive electrode are active, and leaving droplets of the fluid in a gap adjacent to the activated first electrode and one or more drive electrodes adjacent to the first drive electrode.

[0066] The first drive electrode may be spaced from the port by at least the spacing of one drive electrode. Any of these methods may further include the step of deactivating one or more drive electrodes adjacent to a second portion of the gap that is within the filled portion of the gap and between the port and the first drive electrode. The gap may be greater than 500 μm.

[0067] The step of filling a portion of the gap may include applying a positive pressure to extrude the fluid from the port. The method may further include the step of sequentially applying a voltage to drive electrodes adjacent to the gap to move droplets within the gap of the air matrix DMF device.

[0068] The step of applying suction to the fluid to draw it back into the port while the first electrode is activated may include leaving droplets of the fluid having a volume of 10 μl or more in a gap adjacent to the activated first electrode.

[0069] For example, a method of dispensing a predetermined amount of fluid into the voids of an air matrix digital microfluidic (DMF) device, the voids being larger than 280 μm in width (e.g., 300 μm or more, 400 μm or more, etc.), and further, the DMF device including a plurality of drive electrodes adjacent to the voids, the step of filling a portion of the voids with fluid from a port communicating with the voids; the step of applying energy to a first drive electrode or a group of first continuous drive electrodes adjacent to the filled portion of the voids to activate it (the first drive electrode or the group of first continuous drive electrodes being spaced from the port by one or more non-activated drive electrodes); and the step of applying suction to the fluid while the first electrode or the group of first continuous electrodes is activated to draw the fluid back into the port, leaving a droplet of the fluid in the voids adjacent to the first electrode or the group of first continuous electrodes.

[0070] Also described herein is a control system for a DMF device as described herein. In particular, described herein is a control system including a graphical user interface for operating any of these devices. These control systems (subsystems) may include software, hardware, and / or firmware. Accordingly, any of these devices may be configured as instructions stored in a non-transitory medium (e.g., memory) for performing any of the methods and procedures described herein.

[0071] For example, what is described in this specification is a method for controlling a digital microfluidic (DMF) device, comprising the steps of: providing a graphical user interface including a menu of fluid handling control commands including one or more of moving, heating, removing, cycling, waiting, separating, mixing, and dispensing; receiving a fluid handling protocol including the fluid handling control commands selected by a user; calculating a path for moving fluid within the voids of the DMF device based on the fluid handling protocol (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.

[0072] The fluid handling commands may include at least one of moving, heating, removing, waiting, and mixing. For example, the fluid handling commands may include all of moving, heating, removing, waiting, and mixing. The user may select the icons corresponding to each of these commands, enter them in a certain order, and / or indicate incubation timing and temperature conditions. The device may automatically determine the optimal path within the void region of the cartridge for performing each of these steps (e.g., by moving droplets to the appropriate regions of the cartridge including heaters, magnets, microfluidic ports, etc. so that the droplets can be manipulated as needed. For example, the step of receiving a fluid handling protocol may include receiving a series of fluid handling control commands. The step of calculating the path may include calculating the path based on the arrangement of heating zones and cooling zones in the DMF device. The step of calculating the path may include determining the shortest path that does not cross itself. Generally, the step of executing a fluid handling control protocol on the DMF device may include executing the fluid handling protocol in a disposable cartridge coupled to the DMF device.

[0073] Also described herein is a digital microfluidic (DMF) reading device configured to operate with a removable and / or disposable cartridge having a lower dielectric surface, a top plate having a ground electrode, and a void between the lower dielectric and the top plate, an installation surface for installing the disposable cartridge on the top surface; a first plurality of drive electrodes on the installation surface (all or some of the drive electrodes including an opening therethrough); a thermal control unit for applying thermal energy to a first region of the installation surface; a plurality of thermal vias including a thermally conductive material, in thermal communication with the first region of the installation surface, but electrically isolated from a subset of the electrodes, and further in thermal communication with the thermal control unit; a plurality of vacuum ports each 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 unit for applying energy to the one or more selected drive electrodes to sequentially activate and deactivate them to move droplets within the void of the cartridge along a desired path within the void.

[0074] The thermal vias can have any suitable dimensions. For example, each thermal via can have a diameter of about 0.5 to about 2 mm (such as about 0.5 mm to about 1.8 mm, about 0.5 mm to about 1.5 mm, about 0.5 mm to 1.2 mm, about 0.8 mm to 1.2 mm, etc.). Any number of thermal vias can be used per cell (for example, there can be about 5 to 15 thermal vias associated with the region corresponding to one electrode in the first region).

[0075] Each thermal via can be filled with a thermally conductive metal. The material can be conductive or electrically insulating. In some variations, the thermally conductive material is a metal. The reading device can further include one or more resistive heaters located under at least some of the drive electrodes.

[0076] The setting surface can be formed or at least partially formed on a printed circuit board (PCB), including on an array of electrodes formed on the printed circuit board (PCB). As described above, any of the reading devices described herein may include one or more magnets; in some variations, the magnets are configured to apply a magnetic field when activated and may be under one or more of the drive electrodes. For example, the magnetic field can pass through an opening in the drive electrode. The reading device may include one or more Peltier coolers located under at least some of the drive electrodes, configured to cool to below 10°C.

[0077] Also described herein is a method for detecting the position and / or identity of a substance in the voids of a digital microfluidic (DMF) cartridge. The substance can include droplets (e.g., aqueous droplets), wax, droplets coated / covered with wax (e.g., liquid wax), oil droplets, droplets having magnetic particles, etc. The identity can be determined with respect to a specific position in the void, e.g., a substance between the upper and lower surfaces forming the void in the cartridge. The cartridge can be divided into cells (e.g., regions over individual drive electrodes).

[0078] For example, the method for detecting the position and / or identity can include the steps of disconnecting a reference electrode on a first surface of the void of the DMF cartridge from a drive circuit; setting the voltage of one or more drive electrodes of an array of drive electrodes on a second surface of the void to a high voltage and setting all other drive electrodes of the array of drive electrodes to ground; sensing the voltage of the reference electrode; determining the capacitance between the first surface of the void and the second surface of the void based on the voltage sensed at the reference electrode; and identifying the substance in the void adjacent to one or more of the drive electrodes based on the determined capacitance.

[0079] The method may also include the step of reconnecting the reference electrode to the drive circuit and the step of driving the droplets in the void by applying a voltage between the reference electrode and one of the drive electrodes. These steps may be repeatedly performed to track the movement of the substances in the void.

[0080] The step of disconnecting the reference electrode may include floating the reference electrode (e.g., not grounding it). The reference electrode may be the entire upper electrode (on the first surface of the void, opposite the array of drive electrodes). The step of disconnecting the reference electrode from the drive circuit (e.g., from a control device that drives the movement of droplets in the void by digital microfluidics) may include connecting the reference electrode to a sensing circuit for detecting the voltage of the reference electrode and thus the capacitance of the void. The reference circuit may include one or more reference capacitors arranged to enable measurement of the void capacitance.

[0081] The step of setting the voltage of one or more of the drive electrodes to a high voltage may include setting one or more of the drive electrodes to 10 - 400 V (e.g., 100 V - 500 V, e.g., about 300 V, etc.).

[0082] Any of these methods may include the step of determining the total capacitance of the void by setting the voltages of all the drive electrodes of the drive electrode array to a high voltage and sensing the voltage of the reference electrode to determine the total capacitance when the reference electrode is disconnected from the drive circuit. The method may further include the step of 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, the step of determining the capacitance between the first surface and the second surface of the void based on the voltage sensed at the reference electrode may further include using the total capacitance.

[0083] The step of identifying the substances in the void may include identifying the substances in the void based on the determined capacitance using a reference database that includes a plurality of ranges of capacitance.

[0084] Also described herein is a cartridge (e.g., a disposable and / or removable cartridge) for a digital microfluidic (DMF) device that includes a tension frame to maintain tension, and thus flatness, of a lower dielectric material. For example, any of the cartridges described herein may include: a sheet of dielectric material having a first surface and a second surface, the first surface forming an exposed lower surface of the cartridge, at least a second surface of the sheet of dielectric material including a first hydrophobic surface; a tension frame that holds the tension of the sheet of dielectric material so that the sheet of dielectric material is substantially flat; a top plate having a first surface, a second surface, and a thickness therebetween; a ground electrode on the first surface of the top plate; a second hydrophobic surface on the first surface of the top plate that covers the ground electrode; and a void including a separation distance greater than 280 μm that separates the first hydrophobic layer and the second hydrophobic layer. Any of the other cartridge functions described herein may be included in these cartridges.

[0085] Also, any of these cartridges may include a lip that at least partially (including completely) extends around and protrudes from the sheet of dielectric material. This lip may engage a channel or trough on the installation surface. Alternatively or additionally, the cartridge may include a peripheral channel or trough for engagement by a protrusion of the installation surface of the reading device.

[0086] The tension frame may include an outer frame and an inner frame. The sheet may be held between the outer and inner frames. These cartridges may include any of the other cartridge functions described herein. [Invention 1001] A cartridge for a digital microfluidic (DMF) device, having a lower and an upper part, A sheet of dielectric material having a first surface and a second surface, wherein the first surface forms the exposed lower surface of the cartridge, and at least the second surface of the sheet of dielectric material includes a first hydrophobic surface; A top plate having a first surface, a second surface, and a thickness therebetween; A ground electrode on the first surface of the top plate; A second hydrophobic surface on the first surface of the top plate covering the ground electrode; and A void separating the first hydrophobic layer and the second hydrophobic layer, the void including a separation distance exceeding 280 μm A cartridge comprising. [Invention 1002] The cartridge of Invention 1001, wherein the ground electrode includes a grid pattern forming a plurality of open cells. [Invention 1003] The cartridge of Invention 1002, wherein the grid pattern of the ground electrode is formed of an opaque material. [Invention 1004] The cartridge of Invention 1001, wherein the ground electrode is formed of a conductive ink. [Invention 1005] The cartridge of Invention 1001, wherein the ground electrode is formed of silver nanoparticles. [Invention 1006] The cartridge of Invention 1002, wherein the minimum width between the open cells of the grid pattern is greater than 50 μm. [Invention 1007] The cartridge of Invention 1002, wherein the open cells of the plurality of open cells include quadrilaterals or ellipses. [Invention 1008] The cartridge of Invention 1001, wherein the ground electrode extends over more than 50% of the first surface of the top plate. [Invention 1009] The cartridge of Invention 1001, wherein the top plate includes a plurality of cavities within the thickness of the top plate, and further, the cavities are filled with a heat insulating material having a low heat mass and a low thermal conductivity. [The present invention 1010] The cartridge of the present invention 1009, wherein the heat insulating material contains air. [The present invention 1011] The cartridge of the present invention 1001, wherein the sheet of the dielectric material is flexible. [The present invention 1012] The cartridge of the present invention 1001, further comprising a microfluidic channel formed on or in the second surface of the top plate and extending along the second surface of the top plate, and at least one opening between the microfluidic channel and the void. [The present invention 1013] The cartridge of the present invention 1001, wherein the top plate contains polycarbonate and / or acrylic. [The present invention 1014] The cartridge of the present invention 1001, wherein the thickness of the sheet of the dielectric is less than 30 microns. [The present invention 1015] The cartridge of the present invention 1001, wherein the second surface of the dielectric material contains a hydrophobic coating. [The present invention 1016] The cartridge of the present invention 1001, wherein the void contains a separation distance exceeding 400 μm. [The present invention 1017] A cartridge for a digital microfluidic (DMF) device, having a lower part and an upper part, A flexible sheet of dielectric material having a first surface and a second surface, wherein the first surface forms the exposed lower surface of the lower part of the cartridge; A first hydrophobic layer on the second surface of the sheet of the dielectric material; A top plate having a first surface, a second surface, and a thickness therebetween; A ground electrode on the first surface of the top plate, including a lattice pattern formed of an opaque material, the lattice pattern forming a plurality of open cells along the first surface of the top plate; A second hydrophobic layer on the first surface of the top plate that covers the ground electrode; and A void that separates the first hydrophobic layer and the second hydrophobic layer and includes a separation distance exceeding 400 μm A cartridge including the void. [Invention 1018] The cartridge of Invention 1017, wherein the top plate includes a plurality of cavities within the thickness of the top plate, and further, the cavities are filled with a heat insulating material having a low heat mass and a low thermal conductivity. [Invention 1019] The cartridge of Invention 1017, wherein the grid pattern of the ground electrode is formed of a conductive ink. [Invention 1020] The cartridge of Invention 1017, wherein the grid pattern of the ground electrode is formed of silver nanoparticles. [Invention 1021] The cartridge of Invention 1017, wherein the minimum width between open cells of the grid pattern is greater than 50 μm. [Invention 1022] The cartridge of Invention 1017, wherein the open cells of the plurality of open cells include a quadrilateral or an ellipse. [Invention 1023] The cartridge of Invention 1017, wherein the grid pattern of the ground electrode extends over more than 50% of the first surface of the top plate. [Invention 1024] The cartridge of Invention 1017, further including a microfluidic channel formed within the second surface of the top plate and extending along the second surface of the top plate, and at least one opening between the microfluidic channel and the void. [Invention 1025] The cartridge of Invention 1017, wherein the top plate includes polycarbonate and / or acrylic. [Invention 1026] A cartridge for a digital microfluidic (DMF) device, Having a lower part and an upper part, A sheet of dielectric material having a first surface and a second surface, wherein the first surface forms the exposed lower surface of the cartridge, the sheet of dielectric material; A first hydrophobic layer on the second surface of the sheet of dielectric material; A top plate having a first surface, a second surface, and a thickness therebetween; A ground electrode on the first surface of the top plate; A second hydrophobic layer on the first surface of the top plate covering the ground electrode; A void separating the first hydrophobic layer and the second hydrophobic layer; A microfluidic channel formed in or on the second surface of the top plate and extending along the second surface of the top plate; An opening between the microfluidic channel and the void; and A cover covering the microfluidic channel including one or more access ports for accessing the microfluidic channel A cartridge including. [Invention 1027] The cartridge of Invention 1026, wherein the microfluidic channel is configured to accommodate a fluid of more than 1 ml within the microfluidic channel. [Invention 1028] The cartridge of Invention 1026, wherein the void includes a separation distance of more than 500 μm. [Invention 1029] The microfluidic channel includes a first microfluidic channel, the opening between the microfluidic channel and the void includes a first opening, the cartridge further includes a second microfluidic channel formed in the second surface of the top plate and extending along the second surface of the top plate, and a second opening between the second microfluidic channel and the void, and the first and second openings are adjacent to each other, the cartridge of Invention 1026. [Invention 1030] The cartridge of Invention 1029, wherein the first and second openings are within about 2 cm of each other. [The present invention 1031] The cartridge according to the present invention 1026, further comprising a window from the upper part of the cartridge to the void, through which the void is visible. [The present invention 1032] The cartridge according to the present invention 1031, wherein the window forms 2 to 50% of the upper part of the cartridge. [The present invention 1033] The cartridge according to the present invention 1026, wherein the lower part of the cartridge is formed by the first surface of the sheet of dielectric material. [The present invention 1034] The cartridge according to the present invention 1026, further comprising a plurality of openings leading from the upper part of the cartridge into the void. [The present invention 1035] The cartridge according to the present invention 1026, wherein the top plate comprises polycarbonate and / or acrylic. [The present invention 1036] The cartridge according to the present invention 1026, further comprising one or more reagent reservoirs on the second surface of the top plate. [The present invention 1037] The cartridge according to the present invention 1026, further comprising one or more freeze-dried reagent reservoirs on the second surface of the top plate. [The present invention 1038] The cartridge according to the present invention 1026, wherein the sheet of dielectric material is flexible. [The present invention 1039] The cartridge according to the present invention 1026, wherein the top plate comprises a plurality of cavities within the thickness of the top plate, and further, the cavities are filled with a heat insulating material having a low heat mass and a low thermal conductivity. [The present invention 1040] A cartridge for a digital microfluidics (DMF) device, having a lower part and an upper part, a sheet of dielectric material having a first surface and a second surface, the first surface forming the exposed lower surface of the lower part of the cartridge; A first hydrophobic layer on the second surface of the sheet of the dielectric material; A top plate having a first surface and a second surface and a thickness therebetween; A ground electrode on the first surface of the top plate; A second hydrophobic layer on the first surface of the top plate covering the ground electrode; A void including a separation distance greater than 500 μm separating the first hydrophobic layer and the second hydrophobic layer; A first microfluidic channel and a second microfluidic channel formed in the second surface of the top plate and extending along the second surface of the top plate; A first opening between the first microfluidic channel and the void and a second opening between the second microfluidic channel and the void, the first opening and the second opening being adjacent to each other within a range of about 2 cm; and A cover covering the microfluidic channel including one or more access ports for accessing the microfluidic channel A cartridge including [Invention 1041] A digital microfluidic (DMF) reading device, Configured to operate with a disposable cartridge having a lower dielectric surface, a top plate having a ground electrode, and a void between the lower dielectric and the top plate, An installation surface for installing the disposable cartridge; A first plurality of drive electrodes on the installation surface, all or some of the drive electrodes including an opening therethrough; A plurality of vacuum ports each coupled to one or more of the openings passing through the drive electrodes; A vacuum pump for applying a vacuum to the vacuum ports; and A control unit for applying energy to one or more selected drive electrodes to sequentially activate and deactivate them to move droplets in the void of the cartridge along a desired path in the void Including, When the disposable cartridge is disposed on the installation surface, it is configured to apply the vacuum to the vacuum port to fix each drive electrode to the lower dielectric of the disposable cartridge. Device. [Inventive Concept 1042] The device of Inventive Concept 1041, further comprising one or more protrusions extending from the installation surface, the one or more protrusions being configured to form a partition in the void of the cartridge when a vacuum is applied through an opening in the drive electrode. [Inventive Concept 1043] The device of Inventive Concept 1041, further comprising an optical reader configured to detect an optical signal from the cartridge disposed on the installation surface. [Inventive Concept 1044] The device of Inventive Concept 1041, further comprising a motor configured to move an optical reader configured to detect an optical signal from the cartridge disposed on the installation surface. [Inventive Concept 1045] The device of Inventive Concept 1041, further comprising one or more temperature sensors coupled to the installation surface. [Inventive Concept 1046] The device of Inventive Concept 1041, further comprising a resistive heater located under at least some of the drive electrodes. [Inventive Concept 1047] The device of Inventive Concept 1041, wherein the installation surface includes a printed circuit board. [Inventive Concept 1048] The device of Inventive Concept 1041, further comprising one or more magnets located under one or more of the drive electrodes, configured to apply a magnetic field when activated. [Inventive Concept 1049] The device of Inventive Concept 1041, further comprising one or more Peltier coolers located under at least some of the drive electrodes, configured to cool to below 10°C. [Inventive Concept 1050] The device of the present invention 1041 further includes a cartridge tray configured to move the disposable cartridge onto the installation surface. [The present invention 1051] The device of the present invention 1041 further includes a housing surrounding the device, and the housing is stackable. [The present invention 1052] The device of the present invention 1041 further includes an output unit configured to output a signal detected by the device. [The present invention 1053] The device of the present invention 1052, wherein the output unit includes a wireless output unit. [The present invention 1054] The device of the present invention 1041 further includes a first thermal control unit configured to cool the installation surface to 15 to 25°C. [The present invention 1055] One or more microfluidic vacuum ports disposed above the installation surface, configured to engage with an access port for accessing the microfluidic channel of the cartridge when the cartridge is installed on the installation surface. The device of the present invention 1041 further includes such ports. [The present invention 1056] The device of the present invention 1041 further includes a dielectric coating on the outermost surface of the installation surface. [The present invention 1057] The device of the present invention 1041, wherein a plurality of first drive electrodes on the installation surface are each separated from an adjacent electrode in the plurality of electrodes by 50 to 120 μm. [The present invention 1058] The device of the present invention 1041 further includes a plurality of thermal vias penetrating the installation surface. [The present invention 1059] A digital microfluidic (DMF) reading device, configured to operate with a lower dielectric surface, a top plate having a ground electrode, and a disposable cartridge having a gap between the lower dielectric and the top plate, an installation surface for installing the disposable cartridge; A plurality of drive electrodes on the installation surface, at least some of the drive electrodes including openings that penetrate them; A plurality of vacuum ports, each coupled to one or more of the openings that penetrate the drive electrodes; A vacuum pump for applying a vacuum to the vacuum ports; and A control unit for applying energy to one or more selected drive electrodes to sequentially activate and deactivate them to move droplets in the void of the cartridge along a desired path in the void comprising, configured to apply the vacuum to the vacuum ports to fix each drive electrode to the lower dielectric of the disposable cartridge and hold the disposable cartridge on the installation surface, device. [Invention 1060] A method for preventing droplet evaporation in an air matrix digital microfluidic (DMF) device, comprising: introducing an aqueous reaction droplet into a void of the air matrix DMF device formed between a first plate and a second plate of the air matrix DMF device; sequentially applying a voltage to drive electrodes on or in the first plate to move the aqueous reaction droplet within the void of the air matrix DMF device such that it combines with a droplet of a nonpolar fluid within the void of the air matrix DMF device, and the nonpolar fluid coats the aqueous reaction droplet to protect the reaction droplet from evaporation, thereby forming a coated reaction droplet; and sequentially applying a voltage to the drive electrodes to move the coated reaction droplet within the void of the air matrix DMF device comprising, [Invention 1061] The method of Invention 1060, wherein the volume of the nonpolar fluid is less than the volume of the aqueous reaction droplet. [Invention 1062] The method of Invention 1060, further comprising combining the coated droplet with one or more additional aqueous droplets within the void of the air matrix DMF device. [The present invention 1063] The method of the present invention 1060 further comprising the step of removing the coating of the non-polar fluid by at least partially recovering the coated droplets from the voids of the air matrix DMF device into the microfluidic channel. [The present invention 1064] The method of the present invention 1060 further comprising the step of adding droplets of the non-polar fluid into the voids of the air matrix DMF device through openings in the first or second plate. [The present invention 1065] The method of the present invention 1060, wherein the droplets of the non-polar fluid are liquid at 10°C to 100°C. [The present invention 1066] A method for preventing droplet evaporation in an air matrix digital microfluidic (DMF) device, comprising: introducing aqueous reaction droplets into the voids of the air matrix DMF device formed between a first plate and a second plate of the air matrix DMF device; sequentially applying voltages to drive electrodes on or in the first plate to move the aqueous reaction droplets within the voids of the air matrix DMF device such that they combine with droplets of a non-polar fluid within the voids of the air matrix DMF device, thereby coating the non-polar fluid with the aqueous reaction droplets to protect the reaction droplets from evaporation, to form coated reaction droplets, wherein the non-polar fluid is liquid at 10°C to 100°C and further wherein the volume of the non-polar fluid is less than the volume of the aqueous reaction droplets; and sequentially applying voltages to the drive electrodes to move the coated reaction droplets within the voids of the air matrix DMF device comprising the method. [The present invention 1067] A method for dispensing a predetermined amount of fluid into the voids of an air matrix digital microfluidic (DMF) device, wherein the width of the voids is greater than 400 μm, and further wherein the DMF device includes a plurality of drive electrodes adjacent to the voids, the method comprising: filling a portion of the voids with the fluid from a port in communication with the voids; Applying energy to a first drive electrode adjacent to the filled portion of the gap to activate it; and While the first electrode is activated, applying suction to the fluid to draw the fluid back into the port and leaving a droplet having a predetermined amount of the fluid in the gap adjacent to the activated first electrode. A method comprising. [Invention 1068] The step of applying energy to the first drive electrode to activate it includes applying energy to one or more drive electrodes adjacent to the first drive electrode to activate them. Further, the step of applying suction to the fluid to draw the fluid back into the port while the first drive electrode is activated includes drawing in the fluid while the first drive electrode and one or more drive electrodes adjacent to the first drive electrode are active, and leaving droplets of the fluid in the gap adjacent to the activated first drive electrode and one or more drive electrodes adjacent to the first drive electrode. The method of Invention 1067. [Invention 1069] The method of Invention 1067, wherein the first drive electrode is spaced from the port by at least the spacing of one drive electrode. [Invention 1070] The method of Invention 1067, further comprising the step of stopping one or more drive electrodes adjacent to a second portion of the gap, the second portion being within the filled portion of the gap and between the port and the first drive electrode. [Invention 1071] The method of Invention 1067, wherein the gap is greater than 500 μm. [Invention 1072] The method of Invention 1067, wherein the step of filling a portion of the gap includes applying positive pressure to extrude the fluid from the port. [Invention 1073] The method of Invention 1067, further comprising the step of sequentially applying voltage to drive electrodes adjacent to the gap to move droplets within the gap of the air matrix DMF device. [Invention 1074] A method according to the present invention 1067, comprising a step of applying suction to the fluid while the first electrode is operating to draw the fluid back into the port, leaving a droplet of the fluid having a volume of 10 μl or more in a void adjacent to the operating first electrode. [The present invention 1075] A method of dispensing a predetermined amount of fluid into a void of an air matrix digital microfluidic (DMF) device, wherein the width of the void is greater than 400 μm, and further wherein the DMF device includes a plurality of drive electrodes adjacent to the void, the method comprising: filling a portion of the void with the fluid from a port in communication with the void; applying energy to a first drive electrode or a group of first continuous drive electrodes adjacent to the filled portion of the void to activate it, wherein the first drive electrode or the group of first continuous drive electrodes is spaced from the port by one or more non-operating drive electrodes; and applying suction to the fluid while the first electrode or the group of first continuous drive electrodes is operating to draw the fluid back into the port, leaving a droplet of the fluid in a void adjacent to the first electrode or the group of first continuous electrodes comprising a method. [The present invention 1076] A method of controlling a digital microfluidic (DMF) device, comprising: providing a graphical user interface including a menu of fluid handling control commands including one or more of move, heat, retrieve, cycle, wait, separate, mix, and dispense; receiving a fluid handling protocol including a fluid handling control command selected by a user; calculating a path for moving fluid within a void of the DMF device based on the fluid handling protocol, the path minimizing an amount of overlap in the path to avoid contamination; and executing the fluid handling protocol using the DMF device based on the calculated path comprising a method. [The present invention 1077] The method of the present invention 1076, wherein the fluid handling control command includes at least movement, heating, extraction, standby, and mixing. [The present invention 1078] The method of the present invention 1076, wherein the step of receiving the fluid handling protocol includes receiving a series of fluid handling control commands. [The present invention 1079] The method of the present invention 1076, wherein the step of calculating the path includes calculating the path based on the arrangement of the heating zone and the cooling zone in the DMF device. [The present invention 1080] The method of the present invention 1076, wherein the step of calculating the path includes determining the shortest path that does not cross itself. [The present invention 1081] The method of the present invention 1076, wherein the step of executing the fluid handling protocol in the DMF device includes executing the fluid handling protocol in a disposable cartridge coupled to the DMF device. [The present invention 1082] A digital microfluidic (DMF) reading device, configured to operate with a disposable cartridge having a lower dielectric surface, a top plate having a ground electrode, and a gap between the lower dielectric and the top plate, an installation surface for installing the disposable cartridge on the upper surface; a first plurality of drive electrodes on the installation surface, wherein all or some of the drive electrodes include openings therethrough; a thermal control unit for applying thermal energy to a first region of the installation surface; a plurality of thermal vias including a thermally conductive material, being in thermal communication with the first region of the installation surface, being electrically isolated from a subset of the electrodes, and further being in thermal communication with the thermal control unit; a plurality of vacuum ports each 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 unit for applying energy to one or more selected drive electrodes to sequentially activate and deactivate them in order to move droplets within the void of the cartridge along a desired path within the void A device comprising [Invention 1083] The device of Invention 1082, wherein each of the thermal vias has a diameter of 0.5 to 1.5 mm [Invention 1084] The device of Invention 1082, having 5 to 15 thermal vias associated with the region corresponding to one electrode in the first region [Invention 1085] The device of Invention 1082, wherein each of the thermal vias is filled with a thermally conductive metal [Invention 1086] The device of Invention 1082, further comprising a resistive heater located under at least some of the drive electrodes [Invention 1087] The device of Invention 1082, wherein the mounting surface includes a printed circuit board [Invention 1088] The device of Invention 1082, further comprising a magnet under one or more of the drive electrodes configured to apply a magnetic field when activated [Invention 1089] The device of Invention 1082, further comprising one or more Peltier coolers located under at least some of the drive electrodes configured to cool to below 10°C [Invention 1090] A method for detecting the position and identity of a substance in the void of a digital microfluidic (DMF) cartridge, comprising: Disconnecting a reference electrode on a first surface of the void of the DMF cartridge from a drive circuit; Setting the voltage of one or more drive electrodes of an array of drive electrodes on a second surface of the void to a high voltage, wherein all other drive electrodes of the array of drive electrodes are set to ground; Sensing the voltage of the reference electrode Determining the capacitance between the first surface and the second surface of the gap based on the voltage sensed by the reference electrode; and Identifying the substance in the gap adjacent to the one or more drive electrodes based on the determined capacitance A method comprising: [Inventive Concept 1091] The method of Inventive Concept 1090, further comprising reconnecting the reference electrode to the drive circuit and driving the droplet in the gap by applying a voltage between the reference electrode and one of the drive electrodes. [Inventive Concept 1092] The method of Inventive Concept 1090, wherein disconnecting the reference electrode includes floating the reference electrode. [Inventive Concept 1093] The method of Inventive Concept 1090, wherein setting the voltage of one or more of the drive electrodes to a high voltage includes setting the one or more of the drive electrodes to 10 to 400 V. [Inventive Concept 1094] The method of Inventive Concept 1090, further comprising determining the total capacitance of the gap by setting the voltages of all the drive electrodes of the drive electrode array to a high voltage and sensing the voltage of the reference electrode to determine the total capacitance when the reference electrode is disconnected from the drive circuit. [Inventive Concept 1095] The method of Inventive Concept 1094, further comprising 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. [Inventive Concept 1096] The method of Inventive Concept 1094, wherein determining the capacitance between the first surface and the second surface of the gap based on the voltage sensed by the reference electrode further includes using the total capacitance. [Inventive Concept 1097] The method of Inventive Concept 1094, wherein identifying the substance in the gap includes identifying the substance in the gap based on the determined capacitance using a reference database including a plurality of ranges of capacitance. [The present invention 1098] A cartridge for a digital microfluidic (DMF) device, having a lower part and an upper part, a sheet of dielectric material having a first surface and a second surface, the first surface forming the exposed lower surface of the lower part of the cartridge, and at least the second surface of the sheet of dielectric material including a first hydrophobic surface; a tension frame for holding the tension of the sheet of dielectric material so that the sheet of dielectric material is substantially flat; a top plate having a first surface, a second surface, and a thickness therebetween; a ground electrode on the first surface of the top plate; a second hydrophobic surface on the first surface of the top plate covering the ground electrode; and a void separating the first hydrophobic layer and the second hydrophobic layer, the void including a separation distance exceeding 280 μm A cartridge including. [The present invention 1099] The cartridge of the present invention 1098, further including a lip extending around and protruding from the periphery of the sheet of dielectric material. [The present invention 1100] The cartridge of the present invention 1098, wherein the tension frame includes an outer frame and an inner frame, and the sheet is held between the outer frame and the inner frame. [The present invention 1101] The cartridge of the present invention 1098, wherein the ground electrode includes a grid pattern forming a plurality of open cells. [The present invention 1102] The cartridge of the present invention 1101, wherein the grid pattern of the ground electrode is formed of an opaque material. [The present invention 1103] The cartridge of the present invention 1098, wherein the ground electrode is formed of a conductive ink. [The present invention 1104] The cartridge of the present invention 1098, wherein the ground electrode is formed of silver nanoparticles. [The present invention 1105] The cartridge of the present invention 1101, wherein the minimum width between the open cells of the grid pattern is greater than 50 μm. [The present invention 1106] The cartridge of the present invention 1101, wherein the open cells of the plurality of open cells include a quadrilateral or an ellipse. [The present invention 1107] The cartridge of the present invention 1098, wherein the ground electrode extends over more than 50% of the first surface of the top plate. [The present invention 1108] The cartridge of the present invention 1098, wherein the top plate includes a plurality of cavities within the thickness of the top plate, and further, the cavities are filled with a heat insulating material having a low heat mass and a low thermal conductivity. [The present invention 1109] The cartridge of the present invention 1109, wherein the heat insulating material includes air. [The present invention 1110] The cartridge of the present invention 1098, wherein the sheet of the dielectric material is flexible. [The present invention 1111] The cartridge of the present invention 1098, further including a microfluidic channel formed on or in the second surface of the top plate and extending along the second surface of the top plate, and at least one opening between the microfluidic channel and the void. [The present invention 1112] The cartridge of the present invention 1098, wherein the top plate includes polycarbonate and / or acrylic. [The present invention 1113] The cartridge of the present invention 1098, wherein the thickness of the sheet of the dielectric is less than 30 microns. [The present invention 1114] The cartridge of the present invention 1098, wherein the second surface of the dielectric material includes a hydrophobic coating. [The present invention 1115] The cartridge of the present invention 1098, wherein the void includes a separation distance exceeding 400 μm. [Brief Description of the Drawings]

[0087] The novel features of the present invention are described in detail in the following claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description and the accompanying drawings that illustrate exemplary embodiments in which the principles of the present invention are utilized.

[0088]

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DETAILED DESCRIPTION OF THE INVENTION

[0089] Detailed Description Generally, what is described herein are digital microfluidic devices and methods. In particular, what is described herein are air matrix digital microfluidic devices including systems and devices, and methods of operating the same to process fluid samples. For example, a DMF device may include a compact DMF drive / reader device configured to operate with a removable / disposable cartridge. The DMF drive / reader device may include an array of drive electrodes adapted to align and fix the cartridge in place by applying negative and / or positive pressure to a plurality of locations on the cartridge, specifically electrode contact points. The cartridge may include voids that communicate with the environment (e.g., air) through openings such as lateral (side) openings and / or upper openings. The voids may be formed between two dielectric layers. The upper, i.e., the upper region, may include one or more ground electrodes. The ground electrodes may advantageously be formed of an opaque material patterned to include one or more windows that allow imaging through the top. These windows may be disposed on the electrodes such that the ground region extends around and / or between the drive electrodes, opposite the drive electrodes.

[0090] Also, any of the devices described herein may include fluid application / extraction components (e.g., fluid application and / or extraction devices) connected to the voids through the top or side of the cartridge. Any of the devices described herein may include or use a nonpolar coating material (e.g., a nonpolar liquid, e.g., room temperature wax) that forms a protective coating around aqueous droplets in the device and can be moved with the droplets. Also described herein is an interface for interacting with the device, including a user interface for controlling the device to move, mix, combine, wash, magnetically concentrate, heat, cool, etc. These user interfaces may enable manual, automatic, or semi-automatic protocol input, control, and / or execution.

[0091] Figure 2 shows an example of a DMF device similar to those shown in FIGS. 1A - C. In FIG. 2, the DMF device includes a plurality of drive electrodes 201 (formed into non - square / non - rectangular shapes and arranged adjacent to each other in rows or columns). In FIG. 2, four reservoir regions 203, 205, 207, 209 are arranged on the right side and can be pre - loaded with droplets of substances to be added or otherwise held during the operation of the DMF device. Some or all of the electrodes can be heated or cooled.

[0092] In the device of FIG. 2, the DMF drive electrode 211 is a rigid planar electrode. The application of energy between the drive electrode and the ground or reference electrode causes the movement of aqueous (e.g., polar) droplets. In FIG. 2, the ground or reference electrode is formed as a transparent conductive coating (e.g., ITO) on the upper plate which is also clear (transparent). This enables the device to be monitored from above the air matrix / void, including monitoring of any cell, e.g., a unit cell.

[0093] However, it would be beneficial to provide a DMF reading device (e.g., a device, a system, etc.) that can be used with a disposable cartridge that does not include drive electrodes. FIGS. 3A and 3B show different structures of a DMF system that includes built - in drive electrodes (FIG. 3A), and a system where the drive electrodes are part of the reading device, but the cartridge includes only a ground electrode (e.g., a top plate), a void, and a dielectric bottom. For example, in FIG. 3A, the void is formed between a grounded top plate 303 and drive electrodes and a dielectric film 305 (e.g., a Teflon (registered trademark) film). The drive electrodes and the dielectric film are part of the cartridge that includes the top plate and can be separately attached to a substrate (switch board 307) that connects to a main processor 309 and a power board 311.

[0094] In contrast, in FIG. 3B, the cartridge does not include the drive electrode 313. Instead, it includes the top plate / ground electrode, the dielectric, and the gap therebetween 315. As will be described in more detail herein, a vacuum (e.g., a vacuum manifold) is disposed under the electrode 313 to apply a pressure (e.g., 50 kPa to 250 kPa, 50 kPa or more, 60 kPa or more, 70 kPa or more, 80 kPa or more, 90 kPa or more, 100 kPa or more, 110 kPa or more, etc.) to completely fix the dielectric and thus the rest of the cartridge to the reading device. The electrode can be supported on a substrate, such as a printed circuit board or a switchboard 317, which can also be connected to the main processor 319 and the power supply 321. As shown in FIG. 3B, the dielectric film can be hydrophobic (e.g., a Teflon® film can be used) or can be treated, coated, sprayed, immersed, etc. with a hydrophobic material to make at least the side facing the gap hydrophobic.

[0095] FIG. 3C is an example of a compact DMF drive device / reading device that can be used with any of the cartridges described herein. In the perspective view shown in FIG. 3C, the dimensions (height 15 cm or 6 inches, width 20 cm or 8 inches) are merely examples and illustrate the compactness of the reading device. The reading device can include a cartridge installation surface 351, under which a vacuum, heating, cooling, magnetic, and other components, such as control circuits, can be disposed. In this example, microfluidic control components (e.g., valves, pumps, etc.) can be disposed on the cartridge installation surface for the control of these elements.

[0096] Figure 3D shows another example of a DMF reading device that includes drive electrodes incorporated into a portion of the installation surface. A drawer (not shown) can be used to insert / remove the cartridge and place it on the installation surface, and a vacuum can be used to fix the cartridge in place and form a complete electrical contact between the drive electrodes and the dielectric of the cartridge. The microfluidic handling portion 355 and optical components (such as an optical reading device) can be arranged on the installation surface. Figure 3E shows another perspective view of the device of Figures 3C and 3D, showing a drawer 361 that holds an exemplary disposable cartridge 363. The drawer can draw the cartridge into and out of the device and open and close to place the cartridge on an installation surface that includes a drive electrode array 365, where each drive electrode (shown in more detail below) includes an opening for the application of a vacuum to hold the dielectric on the drive electrode. On the installation surface, and thus on the cartridge, the microfluidic portion can engage the cartridge held on the installation surface. For example, a microfluidic valve manifold 367 can be included and can be connected to a pump 369. The same or a separate pump 371 can be used to provide pressure to hold the dielectric on the installation surface via the electrodes. The system can also include an optical component subsystem 373 for imaging through at least a portion of the cartridge to report out data regarding the reactions being performed on the device. Also, motors for driving the optical components and / or opening and closing the drawer can be included. Also, a liquid cooler and compressor 375 can be included, for example, to circulate a coolant under the cartridge.

[0097] Figure 3F shows a perspective view of the device of Figure 3E with the drawer 361 open and a cover 381 attached. The housing can include feet 383 that can engage a receiving site 385 on the upper surface so that these devices can be stacked easily and securely. Figures 3G and 3H show a front view and a rear view, respectively.

[0098] In some embodiments, the apparatus may include a plurality of cartridge receiving sites (e.g., mounting surfaces) for operating on a plurality of cartridges in parallel. For example, FIGS. 3I - 3K show an example of an apparatus that can operate simultaneously on six separate cartridges using six cartridge receiving drawers. In this example, each receiving drawer may include a button for opening and closing the drawer, and may include a separate read screen 390. FIGS. 3I and 3J show a front view and a front perspective view, respectively, and FIG. 3K is a rear view. In this embodiment, internal components, such as a processor and an optical sensor, may be shared between different mounting surfaces within each sub-region of the apparatus. FIG. 3L shows a detailed view of an example of the front of the apparatus.

[0099] The mounting surfaces of an exemplary DMF reading device are shown in detail in FIGS. 4A - 4C and FIGS. 9A - 9C. In FIG. 4A, the mounting surface includes an array of drive electrodes 401 (labeled rows 0 - 9 and columns A - R). 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. In FIG. 4A, the mounting surface also includes temperature sensors (thermistors 405) arranged in different orientations between the electrodes. FIG. 4B shows an enlarged view of the mounting surface including the drive electrodes, showing the thermistors 405 between the drive electrodes. The vacuum openings 407 are more clearly visible in FIG. 4B. Drive electrodes of any shape and size may be used, including a grid pattern of drive electrodes. Additionally, a non-monolithic pattern of drive electrodes may be formed. For example, the electrode pattern may include open areas (e.g., areas surrounding the drive electrodes) that do not include drive electrodes, as shown in FIGS. 1A and 2.

[0100] Figure 4C shows an example of a heater that can be placed under some of the drive electrodes, for example, a subset of the drive electrodes shown in Figure 4B. In this example, a resistive heating circuit 409 can be located under the drive electrodes (e.g., can be embedded in any layer of the PCB forming the mounting surface). Generally, resistive heating and thermistors can be embedded in any layer of the electrode PCB. The heater can be part of the PCB having the electrodes and thermistors, as shown in Figures 4A - 4C. The current, and thus the temperature of the drive electrodes and / or the adjacent dielectric (and thus any droplets on the cell under the dielectric / drive electrode), can be adjusted, for example, by a PID control loop in combination with a thermistor. To cool the dielectric (and the entire mounting surface), a coolant can be passed through the substrate and circulated, for example, on the lower part of the mounting surface. In the example of Figure 4C, the resistive heater is shown as a continuous trace of a low - resistance material (e.g., having a resistance value of about 10 - 15 ohms).

[0101] Any suitable temperature - adjustment technique can be used. For example, agitation (e.g., magnetic stirring) can be used. Even a small number of droplets can contain a certain range of local temperatures, and thus the temperature distribution can have a standard deviation. This can be reduced, for example, by agitation using magnetic beads. With sufficient agitation, the droplets can be brought close to an isothermal state. In any of these variations, a top plate can be used to assist in temperature adjustment. For example, the top plate can be used as a heat sink. A heat conductor (e.g., a steel block) on the top plate can significantly reduce the time required for the top plate to cool. If the top plate has a large heat mass or mass is added to it, the time required for a certain number of heat cycles can be reduced.

[0102] The temperature difference between the top plate and the lower heater (e.g., an embedded heater) can help determine the temperature standard deviation. Heating the top plate simultaneously with the electrodes can reduce the time required to raise the temperature. For example, the top plate can include a local resistance heater similar to that shown in FIG. 4C. The top plate to be heated / cooled can be achieved separately from the cartridge by including an upper thermal mass that engages the top of the cartridge when on the installation surface. For example, the upper thermal mass to be heated and / or cooled can be a manifold that is pressed against the cartridge.

[0103] As described above, a coolant can be applied to the lower and / or upper part of the cartridge. In particular, a circulating coolant can be used. In some variations, the entire lower part of the cartridge can be cooled (e.g., within a range from room temperature to 3 - 5°C, e.g., to 15 - 35°C). FIG. 5A shows an example of the installation surface 501 removed from the device to show the coolant coupled to the substrate of the installation surface so that the coolant can be pumped through the installation surface 501 and into 503 and out of 505.

[0104] FIG. 5B shows a pump 511, tubing 517, fan 515, heat sink 516, and reservoir 513 used to move cooling water or coolant under the electrodes. The coolant absorbs heat while passing under the electrodes and is cooled again while passing through the fan and heat sink.

[0105] As described above, the vacuum applied through the openings in the electrodes by the device enables the dielectric of the cartridge to be securely and releasably held. Openings that do not penetrate the electrodes do not hold the dielectric smoothly on the installation surface. However, when vacuum is applied through all of the operable drive electrodes, the dielectric is held flat against the drive electrodes and consistently lower energy can be applied. For example, FIGS. 5D and 5E show the fixing of a dielectric (shown not attached to the cartridge for illustration purposes) on an installation surface having electrodes with openings through which vacuum is applied when applied. In FIG. 5D, the vacuum is off and the dielectric 555 lies loosely on the installation surface with many wrinkles gathered. In FIG. 5E, vacuum is being applied through the electrodes.

[0106] The use of vacuum in this way enables a reduction in dielectric thickness and thus a reduction in the required power (e.g., voltage). The configuration shown in FIGS. 5A - 5E reduces the required power for DMF by half compared to the use of adhesives or the use of vacuum applied externally to the electrodes. In the illustrated example, the thickness of the dielectric can be 7 - 13 microns. When an adhesive is used, the dielectric is approximately twice as thick (e.g., 25 microns).

[0107] FIG. 5C shows a pump 560 connected via a pipe to a vacuum manifold configured to draw air through holes in the electrodes. The dielectric film is positioned on top and remains firmly in place as long as the pump draws air. Additionally, any protrusions on the surface of the dielectric (especially those around or slightly smaller than the width of the void of the cartridge) can form enclosures, channels, barriers, or other structures within the void that do not interfere with the seal and help partition the void.

[0108] FIGS. 5F and 5G show the connection passing between the installation surface and the intermediate layer, through a mechanical and / or pipe manifold (FIG. 5G), and out of the openings penetrating the electrodes (FIG. 5F) from a vacuum source (via connector 565).

[0109] Figures 9A - 9C show examples of an installation surface 900 on which a cartridge can be held above by a vacuum port passing through an electrode. In FIG. 9A, the installation surface is formed on a substrate (e.g., a PCB or other electrically insulating surface) and includes an array 901 of electrodes shown as a quadrilateral (e.g., a square) in this example. Any other suitable shape may be used. The drive electrodes 901 are thin conductive surfaces that may be in the same plane or substantially in the same plane as the installation surface, or may protrude slightly above the installation surface. In FIG. 9B, the cartridge 905 is shown positioned on the installation surface 900, on top of the array of drive electrodes 901. This cartridge can be positioned on the installation surface by a drawer (such as those shown previously in FIGS. 3E and 3F). When placed on the installation surface, a vacuum can be applied through all or a subset of the drive electrodes (e.g., such as when fluid is being carried through the void) to hold the dielectric (and thus the cartridge) in place. As described above, if no vacuum is applied through the electrodes, more energy may be required to reliably drive the fluid within the void, and the dielectric would have to be thicker. FIG. 9C shows an enlarged view of a portion of the installation surface 900, showing the electrode 901 having a central opening 909 leading to a vacuum manifold.

[0110] The installation surface of the device can be divided into functional regions that control the position and operation of various parts, including heating, magnetic bead control, washing, solution addition, cooling, imaging / detection, etc. These regions can be defined within the DMF reader device. For example, referring to FIG. 6 below, FIG. 6 shows various functional regions defined based on connections within and / or under (or, in some variations, above) the installation surface. For example, in FIG. 6, the solution can be dispensed through one or more holes through the top of the cartridge (e.g., the top plate). Thus, the drive electrodes under the fixed dielectric can form multiple unit cells (one drive electrode per unit cell), and the region of each cell or cells can be controlled to perform a specified function. For example, in FIG. 6, the DMF device includes the arrangement of zones or unit cells disposed around the cartridge, such as a cooling zone (e.g., cooling by a lower located Peltier zone). These regions can also be used to store the solution and can be maintained at 3°C to 20°C (e.g., less than 10°C, about 2°C to 25°C). The 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 useful for immobilizing magnetic particles (e.g., for removing substances, etc.). Any of the zones may overlap. For example, at least one unit cell in the heating zone may be a magnetic zone. Other functional zones include an imaging / optical zone. In this case, a dual function may be possible because when using resistive heating, the magnet can be placed directly under the heating zone.

[0111] In addition to the zones formed by the structure of the installation surface of the DMF device, functional zones for providing an aliquot of the solution, mixing the solution, and / or removing the solution can be formed in the cartridge, for example, by cutting into the top plate, to provide close access to the voids. In FIG. 6, the upper (top plate) microfluidic region is made transparent. Generally, microchannels can be used for mixing, dispensing, and discarding from the void region onto the top plate. In addition, any of these cartridges can include a reagent reservoir in the top plate. The fluid can be controlled by one or more valves (e.g., by valve control) for dispensing, mixing, and discarding.

[0112] Cartridge Generally, the cartridges described herein can include a dielectric, a first hydrophobic coating on the dielectric, a second hydrophobic coating on the ground electrode (and / or top plate), and a top plate to which the ground electrode is coupled. The hydrophobic coating can be, for example, a Teflon® coating. The cartridge can also include one or more microfluidic channels, particularly those directly molded in the top plate that provide controlled access to the voids.

[0113] For example, FIGS. 7A-7D show an example of a cartridge 700 that includes a microfluidic region 703 on the upper surface, covered by a cover 703 having one or more access ports 705, 707 for accessing the microfluidic portion of the device. The cover 703 can also include one or more valves and / or one or more openings 709 that can be used to deliver / remove fluid and / or gas (e.g., air). The cartridge can also include an opening 713 that penetrates the top plate and includes an opening that connects the microfluidic channel to the void region within the channel.

[0114] Also, any of the cartridges described herein may include one or more transparent window regions 711 for optically imaging one or more regions (reading regions) within the void. FIG. 7B is a top perspective view of the cartridge of FIG. 7A. FIG. 7B shows a side view of the cartridge showing the lowermost dielectric film 751 material. The void, although not visible in FIG. 7C, may refer to the space 753 between the dielectric and the ground electrode. FIG. 7D shows the top plate with the cover removed. Comparing FIG. 7A with FIG. 7D with the top removed, first and second microfluidic channels are shown, each having an opening leading from the microfluidic channel to the void. In FIG. 7D, the two channels can be used simultaneously by pushing / pulling fluid through one channel and into the cells located thereunder for washing, mixing, waste removal, etc. In FIGS. 7A-7D, there are via holes that penetrate the top plate and lead to air. The top plate may be thicker, but in some variations, it may be beneficial to include more reagents, including freeze-dried reagents that can be rehydrated.

[0115] FIGS. 8A-8B show different examples of cartridges that can be used. In FIG. 8A, an exemplary cartridge 800 (similar to that shown in FIGS. 7A-7D) is shown on an installation surface 803 that includes electrodes. Cartridge 800 includes a microfluidic portion 805 formed over a void (not visible in FIG. 8A) at one end of the cartridge. The other end of the cartridge includes a window region 807 through which a portion of the void can be imaged. Both the front (window) region and the rear (microfluidic) region of the cartridge may include access regions for accessing the void and / or the microfluidic portion. FIG. 8B shows three different DMF design configurations on paper. The paper DMF device is formed by inkjet printing an array of silver drive electrodes and an array of reservoirs connected to contact pads on a paper substrate.

[0116] Within the cartridge, the top plate can be any suitable material, including a transparent material such as acrylic. The top plate can be formed of (or can include) one or more conductive polymers. The ground electrode can be formed on the top plate. In particular, the ground electrode can be formed of a particularly printed conductive material, such as a conductive material including conductive ink. The return electrode can particularly be a pattern (such as a grid pattern) having a plurality of window openings forming a grid. The pattern can be selected such that the window openings align with the drive electrodes when the cartridge is fixed to the installation surface of the reading device. FIG. 10A shows a ground electrode 1001 having a grid pattern including a plurality of open square windows 1003. As already described, the window openings forming the grid pattern can be of any suitable shape, including other quadrilaterals (such as rectangles), other polygons, ellipses (such as circles, ovals, etc.), regular and irregular shapes. A further layer, such as a hydrophobic layer, may cover both the conductive material pattern and the plate. FIG. 10B shows an exemplary side view (not to scale in terms of thickness) showing the plate 1005 and the conductive patterned electrode 1001. Generally, none of the drawings described herein are necessarily drawn to scale unless otherwise indicated.

[0117] FIGS. 11A and 11B show another example of a ground electrode 1101 formed in a grid pattern having oval (circular in this example) window openings 1103 formed on a first plate 1105.

[0118] For example, the electrodes can be formed of a conductive ink such as silver ink, as shown in FIG. 8B. Such printable inks, although not well defined, may have advantages over the other conductive materials such as ITO. The use of silver nanoparticles formed into a grid can result in lower, more repetitive, and more accurate required energy. In FIGS. 10A - 10B, the electrode pattern has a minimum thickness of about 50 - 200 microns (e.g., 100 microns). The contour around the open window can be configured to be disposed over the space between adjacent electrodes in the drive electrode array. When the cartridge is aligned and fixed in place on the drive electrodes, the overlapping spacing between the drive electrodes on the bottom plate is covered, but the central region (which may particularly include an opening for applying a vacuum as described above) can be centered within the window. Since many conductive inks (e.g., silver ink) are not transparent, the open window can enable visualization of the void under the ground electrode. The minimum thickness can be 50 - 150 microns, but in practice, the minimum thickness of the grid pattern may be greater than 100 microns wide. For example, the minimum thickness can be 100 - 200 microns.

[0119] The ground electrode can be formed on the substrate (e.g., top plate) in any suitable manner. For example, FIGS. 12A and 12B show two methods of forming the ground electrode. In FIG. 12A, a clear substrate is coated with a conductive ink and the resulting layer is dried to form the upper electrode. In FIG. 12B, a pattern as described above is formed by printing techniques (e.g., screening, printing, etc.). In FIG. 12B, the pattern is formed by printing a conductive silver nanoparticle ink in a pattern similar to that shown in FIG. 10A.

[0120] Figures 13A and 13B show examples of top plates having a grid pattern ground electrode. In FIGS. 13A and 13B, the grid pattern is formed into a secondary pattern having a reservoir for storing fluid in the voids and a region including passages and chambers in which various reactions (heating, mixing, cooling, etc.) can be carried out. FIGS. 14A - 14C show the operation of the ground plates of FIGS. 13A - 13B and show drive electrodes for driving the movement of droplets using this ground plate structure in the cartridge. In FIG. 14A, droplet 1403 is held in the void on the first unit cell. In FIG. 14A, the void is between the dielectric pressed against the installation surface by the vacuum applied through the drive electrode and the drive electrode. The pattern of the grid forming the ground electrode coincides with the arrangement of the drive electrodes in the installation surface. Each of the drive electrodes 1411 includes an opening 1413 connected to the vacuum manifold, through which vacuum is applied to hold the dielectric, and thus the cartridge, in place.

[0121] Between FIGS. 14A and 14B, power is sequentially applied to the electrode located under the droplet and one or more adjacent electrodes to enable a change in the electro-wetting of the droplet and drive droplet 1405 to the left as shown in FIG. 14B. Repeating this process can move the droplet to another unit cell 1407 in the void as shown in FIG. 14C. Movement using the grid pattern ground electrode is equivalent to or better than the movement of the monolithic ground electrode.

[0122] In any of these variations, the return electrode on the top plate of the cartridge can be formed of a material laminated on the top plate. For example, the conductive layer forming the return electrode on the top plate can be formed of aluminum and a film of dielectric and / or hydrophobic material. In some variations, the electrode can be formed of ITO, an adhesive, and a dielectric and / or hydrophobic film. In some variations, the conductor can be formed of an ITO film (including a primer and a Teflon® coating).

[0123] As already described above, any of these devices and methods may include one or more microfluidic channels incorporated into a cartridge. In particular, the device may include a microfluidic mixing / extraction region. This is shown in FIGS. 15A - 15C. For example, two microfluidic channels 1501, 1503 may be formed in the top plate of the void, and the openings leading to the void may be arranged within a certain distance from each other. Fluid may be sent through the void from one microfluidic channel to another microfluidic channel. The region of the void between these openings may bridge these two regions 1505. This configuration can be used to mix larger droplets (e.g., larger than 5 μl, larger than 7 μl, larger than 10 μl, larger than 15 μl, larger than 20 μl, larger than 25 μl, larger than 30 μl, larger than 1 ml, etc.) that can be more easily mixed within the void.

[0124] For example, in FIG. 15A, a first pressure source 1507 (negative pressure and / or positive pressure) is shown attached to one end of the microfluidic channel, and a second pressure source 1509 (positive pressure and / or negative pressure) is shown attached to another microfluidic channel. Fluid may be drawn into the first channel 1501 from the void through the opening 1505. Alternatively or additionally, by applying a positive pressure 1507, the fluid may be moved from the first channel 1501 through the opening 1505 into the void. Simultaneously and in parallel, by applying a negative pressure 1509 within the second channel, the fluid may be drawn from the void into the second channel at the same opening 1505 or near it. As shown in FIGS. 15B and 15C, by alternately applying positive and negative pressures, a relatively large amount of solution can be passed between the two microfluidic channels in and out of the void.

[0125] In the example shown in FIGS. 15A - 15C, the top plate incorporates microfluidic channels as well as reservoirs and tubing. Alternatively or additionally, one or more ports (e.g., for connection to a pressure source, valve, etc.) may be included. For example, a cover covering the microfluidic channels may be included along with ports and / or valves, etc. For example, by reversing the polarity of a peristaltic pump, positive and negative pressures can be applied within the microfluidic channels.

[0126] FIGS. 16A - 16D show examples of microfluidic channels that may be included. For example, FIG. 16A shows the formation of a microfluidic channel partially formed by the top plate. In FIG. 16A, a portion of the channel is formed within the plate itself (e.g., an acrylic plate), and a second portion of the channel may be formed from another material that is coated on one side with a conductive material (i.e., indium tin oxide, copper, nickel, chromium, and gold). The layers may be held together by an adhesive or bonded together.

[0127] For example, the microfluidic channels in any of the cartridges and devices described herein can be formed by laser cutting. For example, in FIG. 16A, a raster channel is cut into portion B (the acrylic that forms the top plate), and holes can be cut into portion B. Additionally, one or more pump holes can be cut into portion A. 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 while avoiding air bubbles. Thereafter, pipette holes for dispensing reagents can be punched out, the bottom can be coated with Teflon® (e.g., hydrophobic), and the entire assembly can be baked at 80 - 200 degrees (e.g., 90 - 180 degrees, etc.). The ground electrode may already be formed on the plate.

[0128] FIG. 16B shows another example of a set of microfluidic channels 1605, 1607 formed in the top plate. Also shown is a set of reagent inlets 1609 that provide openings leading to void regions for loading reagents. Alternatively or additionally, the reagents may be pre-loaded into the cartridge, including, for example, into one or more reservoirs on or in the top plate, such as in the microfluidic channels, and / or directly into the void regions (wet or dry / lyophilized). FIGS. 16C and 16D show further examples of microfluidic channels that may be formed in the top plate of the cartridge.

[0129] FIGS. 17A and 17B schematically show examples of methods for applying and removing (including washing) fluid to / from the voids of the DMF device 1120. In FIG. 17A, for example, the void 1121 of the cartridge is formed between the top plate 1117 and the lower 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 may be connected to one or more reservoirs 1105, 1107. As already described above, in some variations, two separate connector interfaces (ports) may be used, with one connected to each fluid line (which may be, for example, a microfluidic channel as described above). The bridging droplet in the void region 1121 connects to both the inlet line and the outlet line, and fluid is drawn into and then withdrawn from the fluid lines 1143, 1145 to mix the droplets, add fluid to the droplets, remove fluid from the droplets, repeatedly expose solid-phase capture elements (such as magnetic beads, non-magnetic beads, etc.) to the same fluid, deplete the fluid from the analyte of interest, and concentrate the analyte, for example, on a solid phase or other surface.

[0130] Alternatively, as shown in FIGS. 17C and 17D, the cartridge may include voids of different heights. For example, in FIG. 17D, a portion of the top plate 1115 (or a separate top plate 1115 connected to another top plate 1117) may be further spaced from the dielectric 1126, so that the void in the region around the connector interface 1127 may be larger (e.g., 0.5 to 2 mm) than the void between the other region of the top plate and the dielectric 1121. Similarly, in FIG. 17D, by further spacing a portion of the top plate 1117, for example, from the bottommost layer of the dielectric 1126, the void 1119 near the connector interface at the edge of the device may be larger than the void 1121 in other regions.

[0131] A prototype DMF device and cartridge for explaining the principle shown in FIG. 17C are shown in FIGS. 18A - 18C and were used to demonstrate proof of principle for mixing a larger amount of solution in the voids of the DMF cartridge. In FIG. 18A, the upper plate of the DMF cartridge included an opening passing through the top plate 1801 connected to the first fluid line 1843 and the second fluid line 1845. By alternately applying a negative pressure (suction) between the first and second fluid lines, the fluid was made to flow back and forth between the first reservoir 1805 and the second reservoir 1807 as shown in the sequence of FIGS. 18A, 18B, and 18C. In this example, magnetic particles holding the analyte of interest were magnetically held within the voids by the DMF device 1809 (e.g., against the lower, e.g., hydrophobic-coated dielectric) while the fluid was being exchanged between the reservoirs to enhance binding and / or washing.

[0132] In any of the void devices described herein, evaporation can be suppressed or reduced, particularly when heating droplets within the voids. FIGS. 19A - 19C show the effect of evaporation on droplet 1903 after just a few minutes. In FIG. 19A, the intact droplet is shown. After 1 minute at 95° C., the volume of the droplet decreased significantly (e.g., losing 5 - 15% of the droplet volume as shown in FIG. 19B). After 2 minutes (FIG. 19C), the droplet is 20 - 34% smaller. To prevent this loss due to evaporation, as shown in FIGS. 20A - 20C, the droplet within the void can be coated within a non - polar coating. For example, a liquid paraffin material (e.g., a non - polar material that is liquid within the operating range described herein, e.g., 10° C. - 99° C.) can be used. In FIG. 20A, the droplet 2003 coated with liquid paraffin 2005 is heated (e.g., to 65° C. or above). After 1 hour (FIG. 20B), the droplet has not evaporated appreciably. Similarly, after 2 hours (FIG. 20C), the droplet remained approximately the same amount.

[0133] During use, as shown in FIGS. 21A - 21I, a non - polar coating material can be added and removed at any point during the DMF procedure. Surprisingly, removal can be achieved, as described above, for example, by pulling the coated droplet out of the void, e.g., from the port entering the microfluidic channel. For example, liquid paraffin can be removed into a waste reservoir by applying a negative pressure to the droplet through a port passing through the upper or side of the void. The less - dense liquid paraffin can be the first layer to be sucked up, leaving the aqueous droplet behind. Previously, it was thought to be difficult or impossible to remove the coating of non - polar liquids.

[0134] For example, FIG. 21A shows a coated droplet in which an aqueous droplet 2101 is surrounded by a nonpolar liquid 2103 (e.g., liquid paraffin). Also, in this example, small bubbles are formed in the liquid paraffin. As shown in FIG. 21B, the droplet can be easily moved, showing droplet movement by changing the electro-wetting of the aqueous droplet by the coupled application of energy to the drive electrode. In FIG. 21B, the coated droplet is moving to the right. Initially, the nonpolar liquid can be applied into the void either directly above the droplet or in the region of the void into which the droplet can move, so as to combine the aqueous droplet with the nonpolar liquid. The coated droplet can also contain its own nonpolar droplet or can be combined with one or more additional droplets which can be in an uncoated state. In some variations, a coating droplet (containing a small aqueous droplet and a relatively large amount of nonpolar solution) can be combined with a target droplet to coat the target droplet. The small amount of aqueous liquid in the coating droplet can be a buffer, a diluent, or other solution that enables the coating droplet to be moved into the void. This technique is particularly useful when used in a DMF cartridge having a relatively large (e.g., 0.5 mm or more) gap width. A relatively large gap width can otherwise make it difficult for relatively large droplets to maintain a coating of a typically lower density nonpolar coating material. FIGS. 21C and 21D show a droplet 2101 that has been combined with another droplet to form a larger coated droplet 2101'. As shown in FIGS. 21C and 21D, the controlled operation of the drive electrode can also move relatively large droplets.

[0135] FIGS. 21E - 21I show the use of nonpolar liquid coatings in a sample containing magnetic bead material. In FIG. 21E, the coating droplet contains a small amount of aqueous liquid 2121 and a relatively large amount of nonpolar coating material 2123, and these two can be combined, for example, by moving the coating droplet 2123 into the sample droplet 2121 as shown in FIG. 21F, enabling them to combine and the coating material now coats the sample droplet. In this case, the sample droplet is quite large and contains a sample-absorbing magnetic bead at a certain concentration.

[0136] When combined, the coated droplets 2121' can be moved (by DMF) to a port leading to the void, from which the solution can be extracted as shown in FIG. 21H. In this example, the solution can be mixed by applying positive and negative pressures to move the solution in and out of the fluid channel 2131. By applying a negative pressure to draw the solution from the void through the upper port, the nonpolar solution coating the droplets can be removed. The first solution removed is the coating material. Thereafter, as shown in FIG. 21I, the magnetic particles to which the desired analyte is bound are held (e.g., by applying a magnetic field) at the bottom side of the void, and the droplet solution can be removed and / or washed in the absence of a nonpolar coating solution (which may potentially interfere with the binding or release of the analyte from the magnetic particles if present). In FIG. 21I, the magnetic particles 2133 remain in the void, and a separate wash buffer can be applied by moving the wash and / or elution droplets 2135 over the magnetic particles.

[0137] In addition to the techniques described above for suppressing evaporation (e.g., using a coating of a non-polar liquid), any of the methods and apparatuses described herein may also include creating a "zero evaporation" condition by controlling the partial pressure of water vapor within the cartridge, for example, by balancing the rate of water molecules leaving the water surface with the rate of water molecules entering the water surface. The equilibrium need not be perfect, but can be adjusted by regulating the temperature and pressure to stay as close as possible to the zero evaporation condition. This can vary with temperature. For example, if the relative humidity is controlled, it may be optimal to adjust the humidity up and down with the temperature, for example, during hybridization or PCR cycling using the apparatus. Alternatively or additionally, any of these apparatuses may regulate evaporation by using local replenishment to slightly move the droplet and recapture nearby condensate (see, for example, FIGS. 19B - 19C showing evaporating droplets surrounding the main droplet). Any of these methods and apparatuses may additionally or alternatively use a heated zone surrounded by a wall to reduce the surface area where evaporation can occur. For example, as described above, in some variations, the mounting surface of the DMF device may include protrusions that form a local region within the cartridge. Since a precise vacuum can be applied to control the contact between the flexible dielectric and the electrode, the protrusions on the mounting surface can create a chamber or channel within the void, including forming a partially wall - surrounded heated zone that can reduce the evaporation surface area. In some variations, the top plate may be spaced differently across the cartridge; the evaporation rate may be lower for thinner droplets compared to thicker droplets. Thus, any of the heated regions may have a relatively narrow void width to reduce evaporation.

[0138] In any of the large-volume droplet DMF cartridges, for example, in a DMF cartridge having a gap separation distance of 0.5 mm or more (e.g., 0.6 mm or more, 0.7 mm or more, 0.8 mm or more, 0.9 mm or more, 1 mm or more, e.g., 0.4 mm to 2 mm, 0.5 mm to 2 mm, 0.5 mm to 1.8 mm, 0.5 mm to 1.7 mm, etc.), it has been found that it is particularly difficult to dispense droplets having a predictable amount because the surface tension of relatively large droplets may require a greater amount of energy to release smaller droplets from larger droplets. Generally, in a DMF system, the ratio of the spacer (void) thickness to the electrode size determines the droplet dispensing volume. In conventional digital microfluidic techniques, a spacer thickness of less than about 500 micrometers (0.5 mm) enables the electrowetting force to divide a unit droplet from a larger amount of liquid. This was not possible with a larger spacer thickness (e.g., greater than 500 micrometers). What is described herein is a method of dividing a unit droplet from a larger amount in a void having a width (e.g., spacer thickness) of 500 μm or more. In some variations, this can be done, for example, by filling the void region with a solution dispensed from a port (which can be a side port, top port, or bottom port), then selectively actuating cells (corresponding to drive electrodes) in the filled region, and then withdrawing the solution into a port (or another port) offset from the actuated electrode so that the droplet remains on the actuated electrode as the solution is drawn into the port. The droplet on the actuated electrode separates from the larger amount (e.g., by constricting and breaking off) leaving behind the dispensed droplet, which can then be combined with one or more other droplets driven by drive electrodes, etc.

[0139] For example, an incorporated companion pump can be used to pump a large amount of aqueous solution into a DMF device (e.g., into the cavity of a DMF cartridge) and apply it to an activated electrode. Subsequently, the aqueous solution can be recovered from the DMF device and dispensed onto the activated electrode, following a unit droplet. FIGS. 22A - 22D show examples of this method. In FIG. 22A, port 2201 to void 2205 of the DMF cartridge is connected to a fluid channel (e.g., the microfluidic channel) shown in FIG. 22A as tube 2209 holding an aqueous solution (reagent 2203). In this example, one drive electrode 2207 is activated. Alternatively, in some variations, the electrode does not activate until the region of the void containing the activated drive electrode is filled. Activating it beforehand can help distribute a predetermined amount into 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 number of droplets.

[0140] Next, as shown in FIG. 22B, the region of the void containing the activated drive electrode is filled with the aqueous solution 2203. FIG. 22A shows the release of a large amount (e.g., 250 μL) from the channel (tube 2209). In some variations, as the reagent approaches the distal channel 2209, the drive electrode 2207 activates (e.g., using an AC potential of 390 Vrm or creating an alternating electric field effect in another way), which generates an electrowetting force that further facilitates the transfer of the reagent from tube 2209 to the activated drive electrode 2207; additional flow occurs from the channel, and as a result, the droplet grows to completely cover the activated drive electrode.

[0141] Next, in FIG. 22C, the aqueous solution (reagent 2203) is recovered from the void through the same port 2201 or a separate port. The activated drive electrode is separated by a certain distance (for example, a distance that can be approximately equal to the width of the activated electrode) from the port into which the solution is drawn. This distance is sufficient for the droplet on the activated drive electrode to constrict and break away from the liquid being drawn back into channel 2209. For example, when the reagent is drawn back into the tube as shown in FIG. 22C, constriction of the droplet from the remaining portion of the solution can occur. The constriction region continuously shrinks so that, as shown in FIG. 22D, a unit droplet (for example, 10 μL) remains on the activated drive electrode. As shown in FIGS. 23A-23E, the same process can be repeated by activating two, three, and five electrodes to dispense approximately multiples (for example, 20, 30, and 50 μL) of that unit droplet, respectively. As described above, multiple droplets can be dispensed individually and combined, or a larger amount can be dispensed at once using multiple electrodes. The size of the droplet (the volume of the droplet) can be based in part on the size of the drive electrode and the spacing of the voids.

[0142] Figures 23A - 23F show the dispensing of various predetermined amounts of solution from a reservoir above a cartridge using the above method. In Figure 23A, for example, the region of the void containing a port connected to a channel holding the solution above a larger void (e.g., 0.5 mm wide) is filled with solution 2301 as shown, and as shown in Figure 23B, a predetermined amount of solution (e.g., 10 μl) is separated using one activated electrode. This droplet moves away from the filled region, and repeating the process multiple times can generate a plurality of droplets of a substantially uniform amount (e.g., 10 μl ± 5%, 10%, 15%, 20%, 25%, etc.). In Figure 23D, a first unit droplet 2303 (e.g., having an amount of 10 μl) is shown adjacent to two combined unit droplets 2305 that form a second droplet having twice the amount (e.g., 20 μl). Similarly, Figure 23E shows a large droplet 2307 (e.g., 50 μl) formed by combining five unit droplets. Figure 23F illustrates the use of a larger drive electrode 2315 (e.g., having approximately four times the surface area) that can be activated when filling the void region to form a larger unit droplet 2311 (e.g., 40 μL unit droplet).

[0143] Thus, by filling or flushing the dispensing region of the void with a large amount of aqueous solution, activating the drive electrode (or on an already activated drive electrode), and then removing the solution (e.g., pumping it out), a relatively accurate amount of droplets can be left. As described above, when using a large - volume DMF device (cartridge) having an interval between 0.4 or 0.5 to 3 mm, this technique can be used to dispense a smaller amount of droplets from a larger reservoir with reasonable force. Different from a void DMF device with smaller voids that can directly dispense smaller droplets from a larger amount by applying electro - wetting energy, a larger force effectively prevents direct dispensing by DMF in a device with larger voids. In many of the examples provided herein, the gap interval of the void is 1 mm - 1.3 mm (e.g., about 1.14 mm), although intervals up to at least 3 mm are being used normally.

[0144] The dispensing of the solutions described herein can be particularly important in processing (e.g., mixing) the samples and replenishing the solutions lost by evaporation in such a system.

[0145] User control interface In any of the devices and methods described herein, the DMF device can be controlled by the user such that one or more protocols (e.g., laboratory procedures) can be performed on the samples inserted into the DMF device (e.g., cartridge). For example, the DMF device can include a user interface that enables the user to dynamically and flexibly control the operation of the DMF device to perform the protocol selected by the user or the protocol input by the user. Generally, when converting the processing protocol for the operation by the DMF device, there are numerous considerations, including preventing contamination during the procedure. There is a risk of contamination when moving the sample droplets on which the protocol is being executed along the path traced by the previous (or parallel) steps in the procedure. Typically, one or more reaction droplets being processed may need to be moved to different locations within the voids of the DMF cartridge or temporarily removed from the void region. Otherwise, it would be difficult for the user to coordinate these movements to avoid previous or future paths (e.g., contamination) and remember the locations suitable for heating, cooling, mixing, adding, removing, thermal cycling, etc.

[0146] What is described herein is a user interface for controlling the operation of a DMF device that enables a user to easily input protocol information / steps via a DMF. This can be achieved in part by providing a set of graphical process representations of the steps that can be executed (e.g., mixing, adding, heating, cooling, cycling, washing, etc.) and enabling the user to select / input these steps in an intuitive way that provides the duration of the step or the degree to which it is applied (e.g., temperature, etc.). And once input, the device can determine an efficient path for executing the protocol within the predefined layout constraints of the DMF device and / or cartridge to avoid contamination. For example, any of these devices can determine a path that prevents or reduces path intersections in the voids (such intersections can cause contamination) (path finding).

[0147] FIG. 24 is an exemplary schematic diagram showing the steps involved in controlling any of the DMF devices described herein. For example, in FIG. 24, a user can input a protocol using a graphical / visual user interface (referred to herein as "SAM"). This can be described in more detail with reference to FIGS. 25A - 26B. The graphical protocol is then converted into a series of target goals, and this target protocol can be used by the device to adapt this protocol to the DMF device. In FIG. 24, the system can determine a path and induce the control of drive electrodes, heaters, cooling (e.g., Peltier), magnetic, microfluidics (pumps, etc.) to achieve the protocol. The path can be optimized to require the shortest path, but is compelled to limit or reduce overlaps in the path to prevent contamination, loss of materials (including reagents and / or Teflon®), heat dissipation, etc.

[0148] As described above, FIGS. 25A and 25B show an example of a visual interface (e.g., a graphical user interface) for entering a desired protocol. FIG. 25A shows a series of control icons (“move,” “heat,” “retrieve,” “cycle,” “mix,” “separate,” “dispense,” and “wait”). As shown in FIG. 25B, the user can select or arrange these icons to provide a graphical representation of the processing protocol. Each icon can have a corresponding duration, and thus these icons can be used to select processing instructions or steps for the sample. In this example, the icons are uniquely identified by one or more of color, image, and text.

[0149] The user may enter the protocol directly into the device or into a computer or other processor that communicates with the DMF device.

[0150] Once entered, the protocol can be converted into a data structure format (e.g., a JSON format that indicates the name of the protocol and sample, where to send the sample, what amounts to use, etc.). This data structure can then be used directly or converted into a format (e.g., Java script) to enable the device to determine a path for loading the cartridge to achieve the desired protocol. The path finding may be performed locally (e.g., within the DMF device) or remotely and sent to the DMF device. The path finding can be configured to maximize based on the shortest path length that avoids intersections or some intersections to prevent contamination. Thus, the device can determine the shortest route to avoid contamination. Generally, the user interface can enable the user to easily select the desired operations and elements (e.g., mixing, etc.). The device may already be associated with reagents (e.g., elements of the device). In that case, the user can select operations, durations, temperatures, etc.

[0151] Figures 26A - 26H illustrate an example of an apparatus for determining a path from an input protocol. For example, Figure 26A shows an illustration of a particular configuration of the DMF cartridge voids for planning the first set of steps, such as sample preparation. The apparatus can know the distribution of cells in the voids and the configuration of the functional zones (heater, cooler, mixing / microfluidics, waste removal, dispensing, etc.) in the DMF cartridge. Figure 26B is an illustration of an apparatus for determining a path for labeling a sample having genomic DNA (or a fragment of DNA) with adapter tags. In Figure 26C, a step is performed to move the first buffer (e.g., SureSelect QXT buffer) to an appropriate location for future processing. The path is selected in light of both past and future movements and can be recursively changed when future protocol steps are determined. In Figure 26D, the path for moving the DNA sample is shown (in black). Figure 26E shows the movement of an enzyme mix from a cooling region where the enzyme mix is stored for binding to the sample. Figure 26F shows a user mixing the sample with the buffer and the enzyme mix. The mixed sample (Figure 26G) is then moved (Figure 26H) to a heating / cooling zone for cycling along the calculated path. Further steps can then be performed as instructed.

[0152] Thermal control Any of the apparatuses described herein may include functions for thermal control (e.g., heating and / or cooling) and / or droplet detection (e.g., tracking and / or identification). For example, an apparatus including a cartridge and a reader device may be configured to rapidly and accurately cycle the temperature of the droplets. Alternatively or additionally, droplet detection may rapidly and accurately scan an electrode grid with respect to droplets (including, but not limited to, reagents, wax, water, etc.).

[0153] As described above, the reading device may be configured to include one or more thermal control elements including cooling and / or heating. For example, the reading device may include resistive heating in some of the cells to heat the droplets in the void. For example, in some variations, the resistive heater may be included in a second layer of a printed circuit board (PCB), such as a portion of the first copper layer beneath the surface of the PCB. The device may also include a heat sink or cooling element, such as a liquid cooler (chiller) that is constantly thermally connected to the PCB. Also, any of these variations may include one or more of a reduction in thermal mass and / or heat conduction through the PCB (e.g., through electrodes forming a part of the PCB in the reading device) that increase the rate of temperature change in the cell.

[0154] Thermal mass reduction may refer to the reduction or removal of thermal mass from a device (such as a system, device, etc.) for reducing the total amount of energy required to reach a certain temperature or temperature range. Ideally, when the thermal mass is low, the energy that needs to be removed from the system to lower the sample temperature during thermal cycling is reduced, enabling a faster cycle speed without the need for a very large heating and cooling system (i.e., without additional liquid cooling to the stack-up). The devices and methods described herein can reduce the thermal mass by reducing / removing the thermal mass from above the droplet or the region holding one or more droplets in the top plate of the cartridge. For example, when the top plate is formed of an acrylic or polycarbonate material, one or more cavities can be included in the top plate (such as a polycarbonate and / or acrylic structure), and by filling the cavities with a thermal insulation material or a material with low thermal conductivity (such as air), the thermal mass above the void region can be reduced. The cavities can be disposed in the top plate of the cartridge above the thermal control device region, such that when a droplet of a substance is below the cavity, heating / cooling applied, for example, by a reading device from a PCB can more rapidly change the temperature of the droplet in the void region. Removing the thermal mass above the droplet can be incorporated into any design of the cartridges described herein. The cavities can be formed near the lower surface of the top plate (e.g., directly on one side of the void). The cavities can partially penetrate the thickness between the upper and lower surfaces of the top plate. FIG. 28 shows an example of a portion of a cartridge showing the thermal control region in the top plate 2801 of the cartridge 2804. The cartridge can be disposed on the reading device 2803. A droplet 2807 within the void region of the cartridge (e.g., the region defined by the lower surface of the upper plate 2801 and the upper surface of the lower dielectric material sheet 2809). Thus, in a variant where the cartridge body including the top plate is formed of a rigid polycarbonate component on the top plate, one or more cavities can be created (e.g., FIG. 29) and can be surrounded or filled with an insulating material having a low thermal mass. This can prevent heat from the sample from conducting to the storage region above it.The void replacement material can be air or a similar substance having low thermal conductivity and low thermal mass.

[0155] Alternatively or additionally, the thermal mass may be removed from the PCB by removing material (e.g., by precision milling) and / or using materials having a very low thermal mass. For example, to reduce the thermal mass, one or more layers of the PCB in a heater zone (e.g., a heating or thermal control region) may be removed. This can be done from the bottom side of the substrate so as not to damage the surface finish of the electrodes.

[0156] Figure 29 is an example of a milled area in a PCB of a reader having a lower thermal mass to increase the response time to temperature changes of droplets in the void of the cartridge. In this schematic example showing a cross-section, the lower (e.g., PCB) layer may include one or more layers of copper, for example, and the dielectric (in the PCB of the reader) under the droplet is milled to create a cavity or void that can be filled with a thermal insulator such as air. Thus, heat conduction through the PCB can be reduced. Generally, voids in the top and / or bottom plates can help thermally isolate the droplets in the void between the top and bottom plates.

[0157] Any of the methods and apparatuses described herein can improve performance by increasing the thermal conductivity between the heater source and the electrodes in addition to accelerating the temperature change of the droplets by reducing the thermal mass. For example, if the heater layer on the PCB is in the second layer, as shown in Figure 30, using a high thermal conductivity dielectric layer increases heat transfer from the heater layer to the electrodes. Figure 30 shows a high conductivity dielectric 3005 between the heater 3003 and the copper region of the electrode 3001.

[0158] In some embodiments, the reading device (particularly the PCB portion of the reading device) may alternatively or additionally be configured to increase the thermal conductivity by including one or more thermal vias near each active (e.g., driving) electrode / cell. The thermal via is a channel or passage that thermally contacts an area near the electrode, including an area under the electrode, such as the area of the PCB material, in the thermal control region, and can be filled with any thermally conductive material. For example, filling the via with a thermally conductive material (such as copper, epoxy, resin, etc.) can further increase the thermal conductivity and dramatically increase the thermal response time of droplets or other substances in the void. Therefore, heating and / or cooling can be much faster than without the vias. The thermally conductive vias can be implemented regardless of the presence or absence of a milling area in the PCB (shown in FIGS. 31A showing the milling area with the thermally conductive vias and 31B showing the thermally conductive vias without the milling area). For example, FIG. 31A shows a plurality of thermally conductive vias 3105 in an example of a bottom plate (such as a PCB) milled to provide a thermally isolated area around a thermally controlled active area.

[0159] The via can be filled with any suitable thermally conductive material. In some embodiments, the via is filled with a non-conductive thermally conductive material (such as epoxy, resin, etc.).

[0160] One end of the via can be in thermal contact (e.g., can contact) with the final upper surface of the reading device device (such as the cartridge contact surface) and / or an area adjacent to the electrode. In particular, when the thermal via is filled with a conductive material (such as copper), the thermally conductive via can contact the area adjacent to the electrode but cannot be in electrical contact with the electrode. Another portion of the thermal via can be in thermal contact with a heat sink under the upper surface (such as the side and / or bottom surface). In some embodiments, the opposite end of the via can contact a temperature-controlled surface (such as a cooled surface, a heated surface, etc.). In some embodiments, the via can have an area at one end in thermal communication with a thermal control device (such as a heater, a cooler, a heat sink, etc.). The via can penetrate the vacuum chuck where the PCB is located.

[0161] The vias can be of any suitable dimension. For example, a thermally conductive via (referred to herein as a thermal via or simply a via) can have a diameter of 0.1 mm to 3 mm, 0.1 mm to 2 mm, 0.5 mm to 1.5 mm, about 0.8 mm, about 1 mm, about 1.2 mm, about 1.4 mm, etc. The thermal via can have a circular, oval, rectangular, square, triangular or any other cross-section and extend through the printed circuit board from a thermally controlled section (such as one or more of a heater, cooler, heat sink, etc.) to an area directly under or adjacent to the electrode (in some variations, without contacting the electrode such that it remains thermally but not electrically isolated from the electrode).

[0162] As described above, any suitable number of vias can be formed per cell (e.g., corresponding to each electrode driving the movement of fluid in the void of the cartridge). For example, each cell in a thermally controlled area (which may include multiple thermally controlled cells) can contact 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc., or more vias. For example, each thermally controlled cell can contact more than 8 vias.

[0163] The use of thermal vias can provide a dramatic improvement in the heating and / or cooling rate variability of a thermally controlled area compared to a system without thermal vias.

[0164] Cartridge Features In addition to the above features, any of the cartridges may alternatively or additionally include one or more openings in the top plate over some of the cells (e.g., the regions corresponding to one or more drive electrodes). As shown in FIG. 32, these openings may be open and allow direct imaging 3221. Alternatively or additionally, the openings may be used for passive dispensing of fluid from the voids. For example, in FIG. 32, the opening 3203 in the top plate of the cartridge 3205 may be used to passively dispense fluid from the droplet 3211 disposed below the opening. The droplet may be moved below the opening by DMF as described above. Once placed, a predetermined amount of fluid may be passively dispensed from the droplet into the opening, for example by capillary action, and the droplet may move away from the opening. The sampled substance may then be analyzed or processed using microfluidics in the upper part of the cartridge or may be analyzed at a predetermined location. Alternatively, the sampled substance may be added to another droplet 3219 after the first droplet 3211 has moved away. The second droplet is placed below the opening in the top plate containing the sampled substance 3203. This sampled substance (fluid) from the first droplet may be an amount that has been metered based on the dimensions of the opening 3203. The top plate may include a hydrophilic surface or surface coating. In some variations, the openings in the top plate may be pre-loaded with a substance such as liquid wax or other coating material, which may combine with the droplet as the droplet moves below the opening (e.g., to dispense an anti-evaporation coating such as a coating material, such as liquid paraffin, oil, etc.). The openings in the top plate also act as heat insulators. The opening may extend over a portion of the cell such that the return electrode may be at the edge of the opening. The opening may be of any size and dimension (e.g., circular, square, etc.). The variation shown in FIG. 32A shows imaging through the top plate (using optical component 3221), but in some variations, the imaging may be performed from below through the lower part of the cartridge. For example, the region in the lower part of the cartridge (e.g., the dielectric film) may be transparent or light transmissive for imaging (e.g., fluorescence).

[0165] In any of the cartridges described herein, the top plate may include a plurality of manifolds for delivering one or more materials into the voids. FIGS. 27A and 27B show an example of a top plate formed of a polymeric material (e.g., acrylic and / or polycarbonate). FIG. 27A shows the upper region of the top plate (which may be covered by one or more covers not shown). In FIG. 27A, a plurality of dispensing regions 2704, 2706, 2708 of different sizes are included. For example, small ones 2706 (e.g., 2 - 20 μl size), medium ones 2704 (e.g., 100 μl - 1 mL), and large ones 2708 (e.g., 1 mL - 5 mL) are shown, similar to the waste and / or mixing region 2710. These chambers may be pre - loaded with fluid and each may include an opening leading to the void region. Pressure control may be used to apply pressure to drive the fluid from the opening of the dispensing region into the voids, which may be controlled by a reader or other device holding the cartridge. Thus, the reader may include one or more pressure interfaces that can be used to control the release of fluid from the top plate and fluid handling in the top plate. FIG. 27B shows the underside of the top - plate portion shown in FIG. 27A. The underside may be coated or covered with an electrode and / or dielectric and / or hydrophobic coating as described above. In FIG. 27B, the top plate may additionally or alternatively include one or more channels 2712 on the surface of the plate that may allow mixing as described above. The underside of these channels may be formed by an upper dielectric and / or return electrode (which may include a dielectric, hydrophobic film, and / or electrode layer in some variations).

[0166] In any of the cartridges described herein, the installation surface of the reading device, particularly the lower surface that can be configured to contact the drive electrodes of the reading device, is formed of a dielectric material as described above. The lower surface can be a sheet of dielectric material having a first surface and a second surface (the first surface forms the lower surface exposed on the lower surface of the cartridge). The second surface of the sheet of dielectric material can include a hydrophobic surface and form one side surface of the void. The lower surface can be, for example, a film that is itself a dielectric and / or coated with a dielectric material. For example, in some variations, the film is a dielectric and / or hydrophobic film. It can be beneficial for this lower surface to be substantially flat. Any of the cartridges described herein can be configured to apply tension to the sheet of dielectric material. For example, any of these cartridges can include a frame for holding the tension of the dielectric material. Thus, the cartridge can include a tension frame that holds the lower sheet of the cartridge.

[0167] The dielectric and / or hydrophobic film tension design can pre-apply tension to the sheet (such as a dielectric and / or hydrophobic film) so that the surface of the sheet becomes planar and remains planar during the interface with the installation surface of the reading device (such as a PCB) and during the use of the DMF device. The purpose of the tension frame that holds the film (such as a dielectric and / or hydrophobic film) in the cartridge is to interface with the installation surface (such as a PCB interface, etc.) to ensure that the film remains in full contact with the electrode grid (such as the drive electrodes) during the use of the device.

[0168] In any of the cartridges described herein, the lower portion of the cartridge may include a sheet of dielectric material having a first surface and a second surface, with the first surface forming the exposed lower surface of the lower portion of the cartridge, as described above. Any of the cartridges described herein may include a tension frame for holding the sheet flat by applying tension. The sheet may be slightly recessed compared to the outer perimeter of the lower portion of the cartridge that can fit into a lip or recess of a reading device when the lower portion of the cartridge is exposed, as described in more detail below. Thus, the sheet of dielectric material at the lower portion of the cartridge need not be the bottommost surface.

[0169] For example, FIGS. 49A - 51 show an example of a cartridge assembly that includes a frame for stretching / smoothing the lower portion of the cartridge (e.g., a dielectric sheet). FIGS. 49A - 49D show an example of a tension frame. In this example, the cartridge body includes a two - part film tensioning mechanism. The two parts shown in FIGS. 49A - 49B (and the assembled view of 49C - 49D) may include a tension frame 4901 and a dielectric and / or hydrophobic film frame 4903. When assembled, the film forming the lower portion of the cartridge may be adhered to the dielectric and / or hydrophobic film frame 4903. The film and film frame 4903 assembly may be inserted into a groove of the tension frame 4911 by using a connector (e.g., a snap - fit mechanism). When snap - fitted to the tension frame, the film is firmly stretched in all directions in the X - Y plane. This frame assembly may then be fixed to the cartridge body. The assembled frame may include a lower - profile (e.g., cut - out) region 4909 that provides access for electrically connecting a return electrode on an upper plate by bypassing the film on the lower surface of the cartridge.

[0170] An example of a cartridge including a frame for holding the lower membrane flat is shown in the exploded view of FIG. 50A. FIG. 15A shows the individual components in the cartridge and film tension assembly. This figure also shows an overview of their arrangement in the assembly. The first two components to be assembled can include, for example, an optically clear double-sided adhesive 5002 and a sheet 5003 of dielectric material (coated on a conductive material). Also included can be a frame (such as tension frame 5004) and a sheet 5005 including the dielectric material, and the film is fixed in position by a second portion of film frame 5006. A void 5009 can be formed between the film 5005 and the lower surface 5003 of the upper part (which can include a return electrode).

[0171] FIG. 50B shows the individual components of the cartridge and film tension assembly after assembling an optically clear double-sided adhesive and a dielectric and / or hydrophobic material coated on a conductive material. The conductive material can be any conductive material such as ITO, aluminum film, copper, etc.

[0172] The film / cartridge and PCB interface may include the film tension frame, and a groove (trough) drilled out on the upper surface of the PCB may form a boundary around the electrode grid of the reading device. FIG. 51 shows an isometric view of an example of an assembly of a cartridge including a film 5120 and a film tension frame (outer frame 5121 and inner frame 5123) and the upper (top) portion 5109 of the cartridge. FIG. 51 also shows a part of the reading device including a PCB 5111 that forms the mounting surface of the cartridge. The mounting surface also includes a trough 5105 for receiving a lip (formed by the tension frame 5103 in this example) around the lower film of the cartridge. The trough may be a groove drilled out around the electrode grid. As shown by the assembly arrangement in this aspect, the film tension frame 5103 may fit into this trough 5105 around the electrode grid. When assembled, the film tension frame 5103 may tension the film in the X and Y directions, but may also pull it downwards in the Z direction at the edge of the film. The film may cover the filleted ends of the trough slightly outside the boundary of the electrode grid (not shown).

[0173] Figures 52A and 52B show a plan view and a cross-sectional view, respectively, of an example of a cartridge that includes a lower dielectric (and hydrophobic or hydrophobically coated) film and a film tension frame installed in the PCB assembly portion of a reading device. The cross-sectional view of FIG. 52B emphasizes a method by which a dielectric and / or hydrophobic film can be firmly stretched and sealed onto electrodes using vacuum ports that penetrate at least some of the electrodes (drive electrodes) of the PCB, and also shows the installation of an edge (extending protruding from the film) in a trough formed in the PCB installation surface for installing the film. When fully assembled, these components may enable a reliable and fully stretched planar dielectric (and / or hydrophobic) film to be fixed to the drive electrode grid of the PCB. FIG. 53 is an exploded view showing individual components and their arrangement in an assembly including a cartridge upper body frame 5306, a dielectric film 5305 held under tension by a tension frame 5304, a PCB 5302 forming an installation surface on the reading device, a groove or channel on the installation surface around an array of drive electrodes on the PCB, and a vacuum chuck 5301.

[0174] Figures 54A and 54B show a plan view and a cross-sectional view, respectively, of the assembly. The cross-sectional view emphasizes the relationship of the cartridge 5413 and the film assembly and the vacuum chuck 5411 on the PCB 5415. The cross-section of FIG. 54B also emphasizes some different effects of this system. Arrow 5405 indicates a channel for the vacuum originating from a diaphragm vacuum pump 5407 outside the chuck. This may be the same channel described previously in FIG. 35B. The arrow depicts a downward force applied to the film by the vacuum through via holes in the PCB. The interface between the vacuum chuck and the PCB firmly adheres the film to the electrodes and applies a downward force in the Z direction. The film tension mechanism and the PCB trough ensure that the film remains planar by applying forces in the X and Y directions while maintaining contact around the edge by means of a fillet along the inner edge of the trough.

[0175] Reading device features Generally, any of the reading devices described herein may include a PCB portion that can include an electrode array, an active thermal control unit (such as a heater, a cooler, etc.), a magnetic field applicator, etc., and a chuck (vacuum chuck) that can be attached to the PCB. This portion of the reading device can form an installation surface for the lower part of the cartridge, and the cartridge can be installed firmly in a predetermined orientation in the reading device. For example, the cartridge may have a key to fit onto the installation surface in a predetermined manner (such as by including one or more orientation slots, pins, etc.). The reading device may also include one or more control units that can control the activities of the reading device, drive droplets, and analyze information from the cartridge, and include one or more processors configured to do so. The control device may also include a memory and one or more data stores.

[0176] The installation surface of the reading device can be configured to install the cartridge and to prevent arc discharge, spark generation, or short circuit between a plurality of electrodes on the installation surface. For example, the installation surface may be coated with a further dielectric such as parylene and / or an alternative or additional material (on which the dielectric lower surface of the cartridge can be located). The dielectric lower surface can prevent arc discharge between the electrodes in an array of electrodes (drive electrodes) on the installation surface. The spacing between the drive electrodes can be about 50 - 120 micrometers. This dense packing between the electrodes on an otherwise flat surface would be susceptible to arc discharge / short circuit between the electrodes in other ways, and thus the use of an outer dielectric coating (in addition to the dielectric layer of the cartridge) can limit spark generation / arc discharge between the electrodes.

[0177] As described in detail above, some or all of the electrodes may include an opening through which they may be connected to a vacuum source for placing the electrodes on the device. For example, in some variations, all of the electrodes of the array include an opening therethrough. In other variations, every other electrode may include an opening (e.g., alternately). In some variations, every third electrode, every fourth electrode, etc. In some variations, only the corner electrodes may include an opening.

[0178] Droplet detection Any of the devices described herein may include droplet detection. As described above, droplet detection may be performed based on the capacitance of the electrodes in an array of drive electrodes by monitoring the current through the electrodes. Also described herein are devices (e.g., systems or devices including a reader) in which droplet detection is based on capacitance measurements by creating a capacitor divider. In this example, the top plate forms a reference frame (e.g., a reference electrode such as an ITO electrode) and may be driven at typically 0 - 300V to generate an AC signal. During droplet detection, the reference electrode (upper electrode) may be disconnected from the drive signal and its voltage sensed by a control device (e.g., a microprocessor) (referred to as "ITO sense" in FIGS. 33A and 33B as it can act as a sense electrode) and electrically coupled to one or more reference capacitors. One or a group of electrodes may operate at a higher known voltage (e.g., 300V DC) while all other electrodes are grounded. This creates a divider as shown in FIG. 33A. FIG. 33A shows an ITO sense circuit having a switch for toggling between sensing (e.g., capacitance sensing from the reference / top plate) and driving (e.g., for moving one or more droplets).

[0179] In FIG. 33A, the voltage of the ITO sensing node (ITO sensing electrode) is driven by the ratio of C_A to the total capacitance (C_A + C_B). The capacitance of C_A changes based on the material dielectric constant between the plates of the capacitor (between the electrode and the ITO). The capacitance of C_B also changes with respect to what exists between the ITO and the remaining electrodes. Air, wax, water, and reagents have different dielectric constants and thus change the capacitance and voltage in the ITO sense. This enables this droplet detection method not only to detect droplets (e.g., the presence / absence of droplets), but also to identify the droplets and identify specific reagents within the electrode grid.

[0180] Due to the variability of the base capacitance, two calibration capacitors may be included (e.g., C_REF and C_REF_LARGE in FIG. 33B). FIG. 33B 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 by using the reference capacitor. If there is a C_Total large enough to saturate the voltage of the ITO sense, the reference capacitance can be increased. The conditioning circuit for the ITO sense can isolate the voltage from small leakage currents.

[0181] FIG. 34A shows exemplary values of capacitance that can indicate the presence or absence (and / or the identity of the substance) of droplets in one or more cells within the void. As described above, a "cell" within the void can correspond to the area above a drive electrode when a cartridge containing the void is placed within a DMF reader that can have an array of drive electrodes over a cartridge placement area. In FIG. 34A, "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 the capacitances determined as described above and correspond to the capacitances when different drive electrodes are measured with or without droplets (e.g., set to a high voltage while grounding other drive electrodes). Water, wax, air (without droplets) have very different capacitances, and using these, the presence or absence of droplets can be identified (e.g., capacitance of 0.09 pF or more, 0.1 pF or more, etc.). In this example, a capacitance exceeding this threshold (e.g., exceeding 0.06 pF, 0.07 pF, 0.08 pF, 0.09 pF, 0.1 pF, 0.11 pF, etc.) indicates the presence of a substance in the void being inspected (set to a high voltage, e.g., 300 V). Further, the range of measured capacitance exceeding this threshold can indicate the composition of the droplet, e.g., aqueous (water) and / or wax / oil. For example, a capacitance exceeding 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 of about 0.09 pF to about 3 pF can indicate that the droplet is wax or oil (e.g., about 0.07 pF to about 3.3 pF, about 0.09 pF to about 3.0 pF, etc.).

[0182] Figure 34B is a graph showing an example of the voltage measured using this technique, showing the difference between the various voltages measured with various droplets (water, wax) on one test cell and the voltage measured without droplets (air). In FIG. 34, the voltage detected when an aqueous droplet is present is about 3.3 V, compared to 0.085 V when no droplet is present and 0.176 V when wax is present. The measured value of wax is twice that of air (no droplet / substance), and water is much higher. In this example, the circuit limits the value to 3.3 V. Different substances can be detected by their different dielectric constants. The dielectric constant of water can also be a function of temperature. Thus, in some variations, when a droplet is present, the capacitance can vary as a function of temperature. This property can be further used to identify water and also to estimate the temperature. Thus, in some variations, the temperature of the droplet may be estimated using capacitance measurements of the droplet. For example, FIG. 34C is a graph showing the static relative dielectric constant of water, showing the change in relative dielectric constant with temperature change (0 to 300 °C).

[0183] Chuck design Any of the devices described herein, such as a reading device, may include a chuck (e.g., a vacuum chuck) that can form part of the mounting surface, as described 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) or integrated with a magnet and / or a heat dissipation mechanism. Any of these elements or portions of these elements may be included or omitted and may be used in any combination.

[0184] The vacuum chuck design can help ensure that a reliable and effective vacuum adheres the lower part 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., manifolds) vias (e.g., copper vias).

[0185] In addition, any of the reading devices described herein may include a magnet incorporated in a base, including a chuck and / or an installation surface. The incorporated magnet may be configured such that an operative magnet can engage with a substance in a cartridge (e.g., magnetic beads in droplets in a void) through a vacuum chuck. The magnet may be located slightly below the PCB forming the installation surface of the reading device without affecting the vacuum performance or function.

[0186] Any of the reading devices described herein may additionally or alternatively include one or more thermal regulators including one or more heat dissipation elements that can rapidly and accurately dissipate heat from a heater in the reading device that controls the temperature of one or more cells in the cartridge when the cartridge is installed and held on the installation surface of the reading device. For example, those described herein are two designs for heat dissipation elements that can be used separately or more tightly. One exemplary heat dissipation design is configured to dissipate heat from a thermoelectric heater, and another design is configured to dissipate heat from an embedded heater.

[0187] Figures 35A - 48 show the vacuum chuck portion of a reading device that can be used with any of the reading devices described herein. Generally, the vacuum chuck may be configured such that a negative pressure is applied through the chuck (e.g., from a vacuum pump) and sent under an installation surface (e.g., a PCB forming part of the installation surface) of an area that is air - isolated (e.g., by an O - ring). The installation surface may have via holes (e.g., in a PCB), and these via holes allow the negative pressure to act directly on the lower part of a cartridge (e.g., a dielectric and / or hydrophobic film) installed on the upper surface of the installation surface (e.g., the PCB forming the installation surface), pulling the lower part of the cartridge in the Z - direction and adhering it to the electrode grid.

[0188] The vacuum chuck may include one or more of a vacuum channel having ports at both ends, a groove for an O-ring, screw holes for attaching a PCB, and a recess under the electrode grid. For example, FIG. 35A is a plan view of an example of a vacuum chuck 3500, and FIG. 35B is a cross-sectional view. Cross-section A-A emphasizes the vacuum channel and its associated ports. The air flow 3505 follows the path of the arrow shown in FIG. 35B. First, it passes through at least one inlet port, then flows through channel 3507, and finally exits through side port 3509. A portion of the chuck (on which the mounting surface formed by the PCB is disposed) is surrounded by an O-ring 3503.

[0189] For example, FIG. 36 shows an isometric view of the chuck shown in FIGS. 35A - 35B. The groove 3509 (which may be designed using, for example, the Parker O-Ring design standard) is configured to fit an O-ring. When positioned in place, with the chuck fixed to the PCB, the O-ring can air-tightly isolate the vacuum directly under the electrode grid. The mounting surface may be formed by fixing a PCB having electrodes (not shown) to the chuck. For example, as shown in FIG. 37, the chuck may include a plurality of screw holes 3701 for attaching the mounting surface (e.g., a PCB). FIG. 37 shows a plan view of a chuck similar to that shown in FIGS. 35A - 35B. In some variations, the chuck includes at least four screw holes (eight are shown in FIG. 37), each being equidistant at least in the X or Y direction and centered about the origin of the chuck. The screw holes can serve two purposes. First, to fix the PCB to the chuck such that the interface of the two components is planar, and second, to apply a downward force in the Z direction around the O-ring to effectively create an air seal.

[0190] FIG. 38A shows a plan view of a chuck similar to that shown in FIGS. 35A - 35B, and FIG. 38B shows an enlarged cross - sectional view of this chuck. FIG. 35B shows an enlarged image of cross - section A - A, showing the boundary lines 3801, 3803 (along the X - axis) of recesses that can create a space between the PCB and the surface of the chuck (only in the isolated areas where the vacuum is active). This space can optimize the air flow of the vacuum as described herein. In FIG. 38, an opening 3805 for the magnet is in the upper region and can include sufficient space to move the magnet into / from the cartridge (e.g., up and down within the space or, in some variations, laterally). The area around the magnet opening can include a gasket or sealing ring (e.g., an O - ring) 3809 similar to an outer O - ring to isolate the magnet area from the vacuum area.

[0191] As described above, any of the devices described herein may include an embedded magnet. In FIGS. 35A - 39, a recessed area 3905 can be used to hold an embedded magnet that can be moved up and down by the system to actuate / de - actuate a magnetic field. Or, in some variations, the magnet may be fixed and toggled (on / off and / or strength change) by the control device of the reader.

[0192] Thus, the vacuum chuck may include an embedded magnet and thus may include one or both of a cut - out that allows the magnet to pass through the chuck and a second O - ring groove that isolates the magnet zone from the air flow of the vacuum. FIG. 39 shows a bottom view of a chuck similar to that shown in FIGS. 35A - 35B. A through - cut area 3905 is shown and can be sized to fit the desired magnet, allowing unimpeded movement of the operative magnet. The magnet can be passed through the cut - out and placed directly under the PCB when in use or removed from the cut - out when not in use.

[0193] Figure 40 shows an isometric view of a chuck similar to that shown in Figure 35A. Groove 4001 can accommodate an O-ring. When positioned in place, with the chuck fixed to the PCB, the O-ring air-isolates the magnet cutout zone from the rest of the vacuum chuck, ensuring in particular that the vacuum is not impaired by the magnet cutout.

[0194] Figures 41A and 41B are similar to those shown in Figures 35A and 35B, and show respectively a plan view and a cross-sectional view of a chuck that includes a gap 4115 for thermally accessing a heating component, such as a heater (e.g., a resistive heater) 4105. The heater 4105 is shown positioned over cavity 4115 in the chuck so that it can be readily thermally adjusted (e.g., cooled). The resistive heater may be in the PCB (not shown in Figures 41A and 41B).

[0195] For example, Figure 41A shows an example of a heat dissipation system that may be included in any of the reading devices described herein. This heat dissipation system may be constructed such that any heat load generated by a heater 4105 in the reading device (e.g., in a PCB) can be dissipated correctly and effectively. A first heat dissipation configuration may be constructed to dissipate heat generated by a heater embedded in the PCB, hereinafter referred to as heat dissipation of the embedded heater. A second heat dissipation design may be constructed to dissipate heat generated by a thermoelectric cooler embedded in the vacuum chuck, hereinafter referred to as heat dissipation of the thermoelectric cooler. Both heat dissipation designs may utilize unique features of the vacuum chuck and accessory components for dissipating heat. Both designs can be used together, or as an assembly, or independently.

[0196] For example, the heat dissipation of a heater embedded in a vacuum chuck can be configured as a ventilation chamber. In FIG. 41A, a plan view of the chuck shows the heat dissipation aspect of the chuck. FIG. 41B shows a pair of air channels 4101 that supply a cooling chamber 4103 that can be part of or under (or otherwise connected to) the area where the heater is located. FIG. 41B shows the paths of a plurality of air elements (channels 4101, 4101') acting in this system. The air drawn into 4101 is warmed by heat including residual heat from a heater in a PCB (e.g., an installation area, not shown), may be covered or partially covered, and may flow over a through-cut 4115 area of the vacuum chuck that leads to a heater in the PCB (or one or more thermal vias in thermal communication with the heater). Cross-section A-A (shown in FIG. 41B) shows the air flow of two air elements, namely warm air 4105 and ambient air, when a fan fixed in the same plane as the chuck and centered on the through-cut 4115 is turned on. A 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 draw ambient air into the chuck and through-cut via two channels 4101, 4101' in the chuck. The system cyclically moves ambient air into the chuck and warm air out of the chuck continuously or intermittently to effectively dissipate any heat generated by the PCB heater.

[0197] Also described herein is a system for heat dissipation of an embedded heater. For example, the assembly shown in FIG. 42 can be configured to include both a chuck 4203 and a fan 4205. The airflow described above can be controlled by a fan 4205 fixed to the lower part of the chuck 42031. FIG. 42 shows a front view of the chuck 4203 and the fan 4205. The first arrow 4221 points to the vacuum chuck (upper structure), and the second set of arrows 4201, 4201' indicates the airflow path. FIG. 43 shows an example of the arrangement of a chuck 4303, a fan 4307, a PCB 4305 forming an installation surface (including, for example, an array of electrodes not shown), and a cartridge 4311. The cartridge can be held by vacuum through an opening (for example, in some of the electrodes).

[0198] FIG. 44 shows an example of a heat dissipation system for adjusting the temperature of a thermoelectric cooler via a vacuum chuck. In FIG. 45, an isometric view of a chuck (similar to that shown in FIG. 35A) is shown in FIG. 45B. The chuck shown includes a recess 4509 designed to allow a thermoelectric cooler (TEC) to fit therein.

[0199] FIGS. 45A - 45B show a plan view and a cross-sectional view, respectively, of a chuck similar to that shown in FIG. 35A. The cross-section shown in FIG. 45B (viewed from A - A) emphasizes the path of the heat generated by the thermoelectric cooling element 4525. The rectangle 4525 represents the TEC, and the arrows in the chuck indicate the heat spreading throughout the chuck. The device can be fixed under the lower part of the chuck and under the TEC, and then include one or more heat sinks of a desired size that absorb heat. Finally, two fans (shown in FIG. 46) fixed to each side of the heat sink cooperate to push hot air out of the entire system and draw ambient air into the system.

[0200] Figures 47A - 47C show an assembly of one or more devices configured for heat dissipation of a thermoelectric cooler. For example, FIG. 46 shows a front view of a chuck. The downward arrow 4613 in FIG. 46 indicates the path of heat in the chuck described in FIG. 45. Arrows 4611, 4611' indicate the channels of air pushed into the heat sink by the fan and the channels of air drawn out from the heat sink by the fan. The fans operate simultaneously in the same direction. FIGS. 47A - 47C show the assembly process and a plurality of components that may be included in this device and the method of using it. For example, FIG. 47A shows the chuck 4701, FIG. 47B shows the chuck 4701 and the heat sink 4703, and FIG. 47C shows the chuck 4701 and the heat sink 4703 as well as two fans 4709, 4709'. FIG. 48 shows an exploded view of a partial arrangement of a reading device assembly including the assembly of FIG. 47 (e.g., chuck 4801, heat sink 4803, fans 4809, 4809') and a PCB 4807 including drive electrodes and heaters (not visible). In addition, a cartridge 4811 is attached to the mounting surface of the PCB via a vacuum.

[0201] Operation zone Any of the devices described herein may include one or more operation zones that strategically arrange various possible operations to which droplets can be applied for the execution of a protocol. The goal of the multiplexing strategy is to adapt to various laboratory requirements in a more flexible and modular way. Strategically grouping the various stages of the protocol to be executed into operation zones may enable the protocol designer to define abstract targets on the board. The operation zone can be a fixed area below or above the electrode board used for reactions (i.e., mixing, confluence, heating, cooling, thermocycling, magnetic capture, discard, optical detection, etc.).

[0202] FIG. 55 shows an example of an electrode grid setup having a magnetic capture, a heater that can be an isothermal or thermal cycler, a Peltier that is an active cooling zone up to 4° C., a waste connection that reaches the top plate, passes through a channel, and enters a waste chamber, a mixing connection that reaches the top plate and passes through a channel, and an independent operating zone for optical detection. Thus, FIG. 55 shows an electrode grid having separate operating zones.

[0203] To better conform to various user needs and laboratory space, independent single modules each having its own power, environment, internal computer, and connection to a console unit for the user interface can be multiplexed. Additionally, the console unit for the user interface can be integrated to control functions such as scanning various modules as well as other laboratory required functions, such as sample ID and cartridge ID, and incorporating that information into a local laboratory or sample management system. The connection to the console unit can be either a wireless connection or a cable connection. FIG. 56 schematically shows four independently controlled 1-plex modules having a console unit.

[0204] Any of the methods (including the user interface) described herein can be embodied as software, hardware, or firmware and, when executed by a processor, cause the processor to execute any of a process including display, communication with the user, analysis, change of parameters (including timing, frequency, strength, etc.), decision, warning, etc., and can be described as a non-transitory computer-readable storage medium storing a set of instructions executable by a processor (such as a computer, tablet, smartphone, etc.).

[0205] As used herein, when a feature or element is referred to as being "above" another feature or element, it can be directly above that other feature or element, or intervening features and / or elements may be present. In contrast, when a feature or element is referred to as being "immediately above" another feature or element, no intervening features or elements are present. Also, when a feature or element is referred to as being "connected", "attached" or "coupled" to another feature or element, it can be directly connected, attached or coupled to that other feature or element, or it will be understood that intervening features and / or elements may be present. In contrast, when a feature or element is referred to as being "directly connected", "directly attached" or "directly coupled" to another feature or element, no intervening features or elements are present. Features and elements described or illustrated with respect to one aspect may apply to other aspects. Also, it will be understood by those skilled in the art that a reference to a structure or feature located "adjacent" to another feature may have portions that overlap above or below that adjacent feature.

[0206] The terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting of the invention. For example, the singular forms of articles used herein are intended to include the plural forms (unless the context clearly dictates otherwise). Further, "comprising" and / or "including", as used herein, specify the presence of the features, steps, acts, elements and / or components recited, but do not preclude the presence or addition of one or more other features, steps, acts, elements, components and / or groups thereof. As used in the present invention, "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ".

[0207] Terms indicating spatial relationships, such as "below", "beneath", "under", "above", "on", etc., may be used in this specification to facilitate the description of the relationship between one element or feature and another element or feature, as shown in the drawings. It will be understood that terms indicating spatial relationships are intended to encompass various orientations of the device during use or operation, in addition to the orientation shown in the drawings. For example, if the device in the figure is upside down, an element described as being "below" another element or feature will be located "above" the other element or feature. Thus, the exemplary term "below" can encompass both upper and lower arrangements. The device may be arranged in other ways (rotated 90° or in other orientations), and the descriptive terms indicating spatial relationships used herein will be interpreted accordingly. Similarly, terms such as "upward", "downward", "vertical", "horizontal", etc. are also used in this specification for illustrative purposes only (unless otherwise indicated).

[0208] The terms "first" and "second" may be used in this specification to describe various features / elements (including steps), but these features / elements should not be limited by these terms (unless the context otherwise indicates). These terms may be used to distinguish one feature / element from another. Thus, without departing from the teachings of the present invention, the first feature / element described below could also be referred to as the second feature / element, and similarly, the second feature / element described below could also be referred to as the first feature / element.

[0209] Throughout this specification and the following claims, unless the context otherwise requires, the term "comprising" and its variations, such as "comprises", mean that various components can be used together in methods and articles (e.g., compositions and devices including devices and methods). For example, it will be understood that the term "comprises" means the inclusion of any recited element or step, but does not mean the exclusion of any other element or step.

[0210] Generally, any of the devices and methods described herein should be understood to be inclusive, but all or subsets of components and / or steps may alternatively be exclusive and may be expressed as "consisting of" or "consisting essentially of" various configurations, steps, sub-components or sub-steps.

[0211] Unless otherwise expressly specified, all numerical values used in this specification and the claims, including those used in the examples, can be read as being preceded by the word "about" or "approximately" (even if such a word does not explicitly appear). The word "about" or "approximately" can be used to indicate that the value and / or position being described is within a reasonable expected range of values and / or positions when describing size and / or position. For example, a numerical value can have a numerical 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. Also, any numerical value set forth in this specification should be understood to include its value of about or approximately, unless the context otherwise indicates. For example, if the numerical value "10" is disclosed, then "about 10" is also disclosed. Any numerical range described in this specification is intended to include all sub-ranges subsumed therein. Also, when a numerical value is disclosed, it will be understood that "less than that numerical value", "greater than that numerical value" and the possible ranges between the numerical values are also disclosed, as appropriately understood by those skilled in the art. For example, if the numerical value "X" is disclosed, then "less than X" and "greater than X" (where X is a numerical value) are also disclosed. Also, throughout this application, data is provided in several different formats, and it will be understood that this data represents ranges of endpoints and starting points and any combination of data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it will be understood that data points greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, as well as data points between 10 and 15, are also considered to be disclosed. Also, it will be understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13 and 14 are also disclosed.

[0212] Although various illustrative aspects have been described above, numerous modifications may be made to various aspects without departing from the scope of the invention as described by the claims. For example, in many cases, the order in which the various method steps described are performed may be changed in alternative aspects, and in other alternative aspects, one or more method steps may be omitted altogether. Optional features of various apparatus and system aspects may be included in some aspects and not in others. Accordingly, the foregoing is provided primarily for illustrative purposes and should not be construed as limiting the scope of the invention to that set forth in the claims.

[0213] The examples and illustrations included herein are presented as examples of specific aspects in which the subject matter may be implemented and are not limiting. As noted above, other aspects may be utilized and derived therefrom in which structural and logical substitutions and changes may be made without departing from the scope of the present disclosure. Such aspects of the inventive subject matter may be referred to herein, merely for convenience, individually or collectively, as "the invention" (even if more than one is actually disclosed) without intending to voluntarily limit the scope of this application to any one invention or inventive concept. Accordingly, although specific aspects are illustrated and described herein, any structure that is presumed to achieve the same results may be used in place of the specific aspects shown. The present disclosure is intended to embrace any and all modifications or variations of the various aspects. Considering the foregoing, combinations of the above aspects with other aspects not specifically described herein will be apparent to those of ordinary skill in the art.

Prior Art Documents

Patent Documents

[0214]

Patent Document 1

Claims

1. A cartridge for a microfluidic device, having a lower part and an upper part, a flexible sheet of dielectric material having a first face and a second face, the first face forming the exposed lower face of the lower part of the cartridge, and at least the second face of the flexible sheet of dielectric material including a first hydrophobic face; a tension frame that holds the tension of the flexible sheet of dielectric material so that the flexible sheet of dielectric material is configured to bend under a vacuum of at least 50 kPa to fix the flexible sheet of dielectric material to the base of the reading device; a second sheet separate from the flexible sheet of dielectric material having a second hydrophobic face, the second sheet being disposed at the upper part of the cartridge; and a void separating the first hydrophobic face and the second hydrophobic face, the void including a separation distance exceeding 280 μm The cartridge includes.

2. The cartridge according to claim 1, further including a lip that extends around and protrudes from the flexible sheet of dielectric material.

3. The cartridge according to claim 1, wherein the tension frame includes an outer frame and an inner frame, and the flexible sheet of dielectric material is held between the outer frame and the inner frame.

4. The cartridge according to claim 1, further including a ground electrode at the upper part.

5. The cartridge according to claim 4, wherein the ground electrode is formed of a conductive ink.

6. The cartridge according to claim 4, wherein the ground electrode extends over more than 50% of the first face of the upper part.

7. The cartridge according to claim 1, wherein the upper part includes the top plate having a plurality of cavities within the thickness of the top plate, and the cavities are filled with a heat insulating material having a low heat mass and a low thermal conductivity.

8. The cartridge according to claim 7, wherein the heat insulating material includes air.

9. The cartridge according to claim 1, wherein the upper part includes polycarbonate and / or acrylic.

10. The cartridge according to claim 1, wherein the thickness of the flexible sheet of dielectric material is less than 30 microns.

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

12. The cartridge according to claim 1, wherein the gap includes a separation distance exceeding 400 μm.

13. A cartridge for a microfluidic device, having a lower part and an upper part, a flexible sheet of a thin elastomeric dielectric material having a first surface and a second surface, the first surface forming an exposed first surface, the flexible sheet; a tension frame configured to hold the flexible sheet of the dielectric material generally flat over the exposed first surface in a stretched state; an upper part; and a gap separating the flexible sheet and the upper part, the gap having a separation distance exceeding 280 μm comprising a cartridge.

14. The cartridge according to claim 13, wherein the second surface of the flexible sheet of the dielectric material includes a first hydrophobic coating.

15. The cartridge according to claim 13, further comprising a ground electrode having a second hydrophobic coating in the upper part.

16. The cartridge according to claim 13, wherein the flexible sheet of the elastomeric dielectric material is configured to bend under a vacuum of at least 50 kPa.

17. The cartridge according to claim 13, further comprising a ground electrode.

18. The cartridge according to claim 13, further comprising a ground electrode extending over a majority of the first surface of the upper part.

19. The cartridge according to claim 13, wherein the upper part includes a plurality of cavities therein configured to reduce the thermal mass of the top plate.

20. The cartridge according to claim 19, wherein the plurality of cavities are filled with air.

21. The cartridge according to claim 13, wherein the thickness of the flexible sheet of the dielectric material is less than 20 microns.

22. The cartridge according to claim 13, wherein the gap includes a separation distance exceeding 400 μm.

23. A cartridge for a microfluidic device, having a lower part and an upper part, a flexible sheet of a thin elastomeric material having a first surface and a second surface, the first surface forming a surface exposed on the outer surface of the cartridge, the flexible sheet; a tension frame configured to hold the flexible sheet of the elastomeric material generally flat over the exposed surface in a stretched state to fix the flexible sheet of the elastomeric material; an upper part having a first surface, a second surface, and a thickness therebetween; A void between the flexible sheet and the upper part, the void including a separation distance exceeding 280 μm; and An opening that communicates through the upper part into the void A cartridge including the above.

24. The cartridge according to claim 23, wherein the upper part includes a microfluidic channel configured to accommodate a fluid exceeding 1 ml.

25. The cartridge according to claim 23, further including a plurality of openings that communicate from the upper part of the cartridge into the void.

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