Moving magnet for magnetic bead-assisted separation

Abstract

Described herein are methods, systems, and devices that include providing a droplet on a surface adjacent to a magnet configured to provide a magnetic field in contact with the droplet, the droplet including one or more artifacts responsive to the magnetic field, and displacing the magnet proximate to the droplet, the magnetic field in contact with the droplet including a magnetic flux density of at least about 4 millitesla ("mT") to at least about 10 mT, thereby manipulating the one or more artifacts responsive to the magnetic field.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 353,474, filed June 17, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Many biological protocols rely on the use of magnetic beads to perform operations on DNA, proteins, and other biological assets. These magnetic beads typically need to be well suspended and mixed with various biological samples. The biological asset of interest can then precipitate from the solution and be captured on the magnetic beads. At this point, the beads need to be separated from the rest of the solution to extract and purify the biological asset of interest. To do so, a magnetic field can be introduced to pellet the magnetic beads into a smaller area.

[0003] Current methods do not adequately consider shear stress on the captured nucleic acid molecules. Summary of the Invention

[0004] An aspect of the present disclosure is a method for manipulating droplets, the method including: providing an electrowetting array adjacent to a magnet configured to apply a magnetic field to the electrowetting array in one or more of three directional axes; providing a droplet on the array including one or more artifacts responsive to the magnetic field; and actuating the magnet in one or more of three directional axes relative to the electrowetting array to separate the one or more artifacts from the droplet. In some embodiments, the magnet is moved parallel to the array in an x-direction. In some embodiments, the magnet is moved parallel to the array in a y-direction. In some embodiments, the magnet is moved orthogonal to the array. In some embodiments, the strength of the magnetic field is modulated. In some embodiments, the strength of the magnetic field is increased. In some embodiments, the strength of the magnetic field is decreased. In some embodiments, the magnet is a permanent magnet. In some embodiments, the magnet is an electromagnet or an electro-permanent magnet. In some embodiments, the electromagnet or electro-permanent magnet modulates the strength of the magnetic field in a time-dependent manner. In some embodiments, the magnet is positioned above the electrowetting array. In some embodiments, the magnet is moved orthogonally relative to the electrowetting array to separate the one or more artifacts from the droplet. In some embodiments, the magnet is positioned below the electrowetting array.

[0005] Another aspect of the present disclosure is a method of removing one or more artifacts from a droplet, the method comprising providing a magnet configured to apply a magnetic field to the droplet and actuating the magnet relative to the droplet to separate the one or more artifacts from the droplet, the droplet being less than 40 microliters. In some embodiments, the magnet is moved in an x-direction parallel to the array. In some embodiments, the magnet is moved in a y-direction parallel to the array. In some embodiments, the magnet is moved orthogonal to the array. In some embodiments, the strength of the magnetic field is modulated. In some embodiments, the strength of the magnetic field is increased. In some embodiments, the strength of the magnetic field is decreased. In some embodiments, the magnet is a permanent magnet. In some embodiments, the magnet is an electromagnet or an electro-permanent magnet. In some embodiments, the electromagnet or the electro-permanent magnet modulates the strength of the magnetic field in a time-dependent manner. In some embodiments, the magnet is disposed above the electrowetting array. In some embodiments, the magnet is moved orthogonally relative to the electrowetting array to separate the one or more artifacts from the droplet. In some embodiments, the magnet is positioned below the electrowetting array. In some embodiments, the droplet is less than 30 microliters. In some embodiments, the droplet is less than 20 microliters. In some embodiments, the droplet is less than 10 microliters.

[0006] Another aspect of the present disclosure is a method for manipulating droplets, the method comprising: providing the droplets on a surface adjacent to a magnet configured to provide a magnetic field in contact with the droplets, the droplets comprising one or more artifacts responsive to the magnetic field; and displacing the magnet proximate to the droplets, the magnetic field in contact with the droplets comprising a magnetic flux density of at least about 4 millitesla ("mT") to at least about 10 mT, thereby manipulating the one or more artifacts responsive to the magnetic field. In some embodiments, the magnet is displaced along an axis perpendicular to the surface. In some embodiments, the magnet is displaced at a distance of about 0 millimeters to about 15 millimeters from the surface. In some embodiments, the surface comprises an electrowetting array. In some embodiments, the one or more artifacts responsive to the magnetic field comprise nucleic acid molecules. In some embodiments, the one or more artifacts responsive to the magnetic field comprise a plurality of nucleic acid molecules. In some embodiments, the magnet is displaced along an axis parallel to the surface. In some embodiments, the method further comprises moving the magnet to maintain a magnetic flux density of at least about 4 millitesla ("mT") to at least about 10 mT in the magnetic field contacting the droplet. In some embodiments, the method further comprises moving the magnet to maintain a distance of about 0 millimeters to about 15 millimeters from the surface. In some embodiments, the droplets are less than 30 microliters. In some embodiments, the droplets are less than 20 microliters. In some embodiments, the droplets are less than 10 microliters. In some embodiments, the nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 20% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, the method further comprises moving the magnet to maintain the nucleic acid molecules at 100 kb or greater. In some embodiments, the method further comprises moving the magnet to maintain at least 20% of the plurality of nucleic acid molecules at 100 kb or greater. In some embodiments, the magnetic flux density is sufficient to immobilize one or more artifacts responsive to the magnetic field during one or more droplet operations. In some embodiments, the one or more droplet operations comprise agitating the surface.

[0007] Another aspect of the present disclosure is a method for manipulating droplets, the method comprising providing the droplets on a surface adjacent to a magnet configured to provide a magnetic field in contact with the droplets, the droplets comprising one or more artifacts responsive to the magnetic field; and displacing the magnet proximate to the droplets at a distance of about 0 millimeters to about 15 millimeters from the surface, thereby manipulating the one or more artifacts responsive to the magnetic field. In some embodiments, the magnet is displaced along an axis perpendicular to the surface. In some embodiments, the magnetic field in contact with the droplets comprises a magnetic flux density of at least about 4 millitesla ("mT") to at least about 10 mT. In some embodiments, the surface comprises an electrowetting array. In some embodiments, the one or more artifacts responsive to the magnetic field comprise nucleic acid molecules. In some embodiments, the one or more artifacts responsive to the magnetic field comprise a plurality of nucleic acid molecules. In some embodiments, the magnet is displaced along an axis parallel to the surface. In some embodiments, the method further comprises moving the magnet to maintain a magnetic flux density of at least about 4 millitesla ("mT") to at least about 10 mT in the magnetic field contacting the droplet. In some embodiments, the method further comprises moving the magnet to maintain a distance of about 0 millimeters to about 15 millimeters from the surface. In some embodiments, the droplet is less than 30 microliters. In some embodiments, the droplet is less than 20 microliters. In some embodiments, the droplet is less than 10 microliters. In some embodiments, the nucleic acid molecule comprises at least 100 kb. In some embodiments, at least 20% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, the method further comprises moving the magnet to maintain the nucleic acid molecule at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 20% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain the nucleic acid molecule at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 20% of the plurality of nucleic acid molecules greater than or equal to 100 kb.In some embodiments, the distance is sufficient to immobilize one or more artifacts responsive to the magnetic field during one or more droplet operations. In some embodiments, the one or more droplet operations include agitating the surface.

[0008] Another aspect of the present disclosure is a method for manipulating droplets, the method comprising: providing the droplets on a surface adjacent to a magnet configured to provide a magnetic field in contact with the droplets, the droplets comprising one or more artifacts responsive to the magnetic field, the one or more artifacts responsive to the magnetic field comprising nucleic acid molecules; and moving the magnet to maintain the nucleic acid molecules at 100 kb or greater, thereby manipulating the one or more artifacts responsive to the magnetic field. In some embodiments, the magnet is displaced along an axis perpendicular to the surface. In some embodiments, the magnetic field in contact with the droplets comprises a magnetic flux density of at least about 4 millitesla ("mT") to at least about 10 mT. In some embodiments, the surface comprises an electrowetting array. In some embodiments, the one or more artifacts responsive to the magnetic field comprise a plurality of nucleic acid molecules. In some embodiments, the magnet is displaced along an axis parallel to the surface. In some embodiments, the method further comprises moving the magnet to maintain a magnetic flux density of at least about 4 millitesla ("mT") to at least about 10 mT in the magnetic field in contact with the droplets. In some embodiments, the method further comprises moving the magnet and maintaining a distance of about 0 millimeters to about 15 millimeters from the surface. In some embodiments, the droplets are less than 30 microliters. In some embodiments, the droplets are less than 20 microliters. In some embodiments, the droplets are less than 10 microliters. In some embodiments, at least 20% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, the method further comprises maintaining at least 20% of the plurality of nucleic acid molecules at 100 kb or greater. In some embodiments, the method further comprises moving the magnet and maintaining the nucleic acid molecules at 100 kb or greater. In some embodiments, the method further comprises moving the magnet and maintaining at least 20% of the plurality of nucleic acid molecules at 100 kb or greater. In some embodiments, the distance is sufficient to immobilize one or more artifacts responsive to the magnetic field during one or more droplet operations. In some embodiments, the one or more droplet operations comprise agitating the surface.

[0009] Another aspect of the present disclosure is a system for manipulating droplets, the system including: a surface configured to support the droplets; a magnet adjacent to the surface, the magnet configured to provide a magnetic field in contact with the droplets, the droplets including one or more artifacts responsive to the magnetic field, the one or more artifacts responsive to the magnetic field including nucleic acid molecules; and a controller mechanically coupled to the magnet, the controller configured to displace the magnet in proximity to the droplets, maintaining the nucleic acid molecules at 100 kb or greater. In some embodiments, the magnet is displaced along an axis perpendicular to the surface. In some embodiments, the magnetic field in contact with the droplets includes a magnetic flux density of at least about 4 millitesla ("mT") to at least about 10 mT. In some embodiments, the surface includes an electrowetting array. In some embodiments, the one or more artifacts responsive to the magnetic field include a plurality of nucleic acid molecules. In some embodiments, the magnet is displaced along an axis parallel to the surface. In some embodiments, the controller is configured to maintain a magnetic flux density of the magnetic field in contact with the droplet of at least about 4 millitesla ("mT") to at least about 10 mT. In some embodiments, the controller is configured to maintain the magnet at a distance of about 0 millimeters to about 15 millimeters from the surface. In some embodiments, the droplets are less than 30 microliters. In some embodiments, the droplets are less than 20 microliters. In some embodiments, the droplets are less than 10 microliters. In some embodiments, at least 20% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, the system maintains at least 20% of the plurality of nucleic acid molecules at 100 kb or greater. In some embodiments, the system maintains the nucleic acid molecules at 100 kb or greater. In some embodiments, the system maintains at least 20% of the plurality of nucleic acid molecules at 100 kb or greater. In some embodiments, the distance is sufficient to immobilize one or more artifacts responsive to the magnetic field during one or more droplet operations. In some embodiments, one or more droplet operations include agitating the surface.

[0010] Another aspect of the present disclosure is a device for processing droplets, the device including: a surface configured to support the droplets; a magnet adjacent to the surface, the magnet configured to provide a magnetic field in contact with the droplets, the droplets including one or more artifacts responsive to the magnetic field, the one or more artifacts responsive to the magnetic field including nucleic acid molecules; and a controller mechanically coupled to the magnet, the controller configured to displace the magnet in proximity to the droplets, maintaining the nucleic acid molecules 100 kb or larger. In some embodiments, the magnet is displaced along an axis perpendicular to the surface. In some embodiments, the magnetic field in contact with the droplets includes a magnetic flux density of at least about 4 millitesla ("mT") to at least about 10 mT. In some embodiments, the surface includes an electrowetting array. In some embodiments, the one or more artifacts responsive to the magnetic field include a plurality of nucleic acid molecules. In some embodiments, the magnet is displaced along an axis parallel to the surface. In some embodiments, the controller is configured to maintain a magnetic flux density of at least about 4 millitesla ("mT") to at least about 10 mT in the magnetic field in contact with the droplet. In some embodiments, the controller is configured to maintain the magnet at a distance of about 0 millimeters to about 15 millimeters from the surface. In some embodiments, the droplets are less than 30 microliters. In some embodiments, the droplets are less than 20 microliters. In some embodiments, the droplets are less than 10 microliters. In some embodiments, at least 20% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, the distance is sufficient to immobilize one or more artifacts responsive to the magnetic field during one or more droplet operations. In some embodiments, the one or more droplet operations include agitating the surface.

[0011]

[0013] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0012] Incorporation by Reference All publications, patents, and patent applications mentioned herein are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the present disclosure contained herein, the present specification is intended to supersede and / or take precedence over any such conflicting material.

[0013] This specification incorporates by reference in its entirety International Application No. PCT / US2022 / 018549, filed March 2, 2022. [Brief explanation of the drawings]

[0014] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "Figure" and "FIG."). [Figure 1] We show that moving the magnetic field in one or more axes aligned with the plane of the EWOD surface allows for bead separation that is not possible with the EWOD alone. [Figure 2A]Adjusting the strength of the magnetic field can allow for a reduction in shear forces when drawing the magnetic beads through the supernatant droplet. [Figure 2B] Adjusting the strength of the magnetic field can allow for reduced shear forces when drawing the magnetic beads through the supernatant droplets. [Figure 3A] We show that introducing a magnet above the droplets can be useful for separating beads from small droplets or droplets with high surface tension. [Figure 3B] We show that introducing a magnet above the droplets can be useful for separating beads from small droplets or droplets with high surface tension. [Figure 3C] We show that introducing a magnet above the droplets can be useful for separating beads from small droplets or droplets with high surface tension. [Figure 4A] The magnet is shown moving to create clearance for other operations. Figure 4A shows the magnet in close proximity to the active surface. [Figure 4B] Figure 4B shows the magnet being moved orthogonally to provide clearance between the magnet and the surface. [Figure 4C] The magnet is shown moving to create clearance for other operations, and Figure 4C shows the active surface moving up and down to create agitation in the droplet surrounding the magnetic bead pellet. [Figure 5A] 1 shows a configuration for the synthesis and assembly of biopolymers (eg, DNA) using the systems and methods described herein. [Figure 5B] 1 shows a configuration for the synthesis and assembly of biopolymers (eg, DNA) using the systems and methods described herein. [Figure 6]1 shows an exemplary schematic Next-Generation Sequencing (NGS) workflow using the systems and methods described herein. The exemplary workflow includes manipulating a biological sample on an array (e.g., lysing cells, digesting proteins, and cleaning up DNA) as described herein. [Figure 7A] We demonstrate an application of vibration assisted electrowetting on a dielectric for DNA extraction using magnetic beads. [Figure 7B] We demonstrate an application of vibration-assisted electrowetting on a dielectric for DNA extraction using magnetic beads. [Figure 8A] It can be seen that droplets with high contact angles (FIG. 8A) tend to experience a greater response to vibration than droplets with lower contact angles (FIG. 8B). [Figure 8B] It can be seen that droplets with high contact angles (FIG. 8A) tend to experience a greater response to vibration than droplets with lower contact angles (FIG. 8B). [Figure 9A] 1 illustrates an embodiment of an electromechanical actuator. [Figure 9B] 1 illustrates an embodiment of an electromechanical actuator. [Figure 10A] 10 illustrates an additional embodiment of an electromechanical actuator. [Figure 10B] 10 illustrates an additional embodiment of an electromechanical actuator. [Figure 11] An embodiment is presented that efficiently couples the actuation force of an electromechanical actuator into droplet vibration and ultimately into effective mixing. [Figure 12] The magnet position and magnetic flux density (mT) measured at various distances from the EWOD surface are shown. [Figure 13]The magnetic flux density (mT) measured on the EWOD surface is shown as the magnet progresses from position (1) to position (50). Position (1) is when the magnet is 13.6 mm from the EWOD surface, and position (50) is when the magnet is 6.7 mm from the EWOD surface. [Figure 14] 1 depicts a cartoon of a computer system used in the arrays described herein. Detailed Description of the Invention

[0015] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed.

[0016] Many biological protocols rely on the use of magnetic beads to perform operations on DNA, proteins, and other biological assets. These magnetic beads typically need to be well suspended and mixed with various biological samples. The biological asset of interest can then precipitate from the solution and be captured on the magnetic beads. At this point, the beads need to be separated from the rest of the solution to extract and purify the biological asset of interest. To do so, a magnetic field can be introduced to pellet the magnetic beads into a smaller area. In the prior art, this separation of the supernatant from the bead pellet is accomplished by using electrowetting-based droplet manipulation to displace the droplet solution from the bead pellet.

[0017] However, this approach can pose challenges in certain scenarios. For example, if the droplet volume is relatively large and / or has low surface tension, a puddle may form rather than a spherical cap-shaped droplet. Furthermore, because the electrowetting force can only be applied to the periphery of the droplet / puddle, the puddle may be difficult to translate properly to separate it from the bead pellet using an EWOD (because the area in contact with the EWOD surface is large compared to the periphery).

[0018] An alternative approach, described herein and shown in FIG. 1, involves positioning a magnetic field rather than using an EWOD to position the supernatant. In this approach, friction / shear forces between the droplet and the surface help resist the force of the bead pellet as it is pulled from the droplet. Thus, by moving the magnetic field in at least one axis aligned with the plane of the EWOD surface, it is possible to achieve a separation between the bead pellet and the supernatant that is not possible with an EWOD alone.

[0019] Adding control over magnetic field strength can also be useful (Figures 2A-2B). For example, this can be used to reduce the rate at which the magnetic beads are pelleted, thereby reducing the shear forces experienced between the pelleting beads and the liquid supernatant (since shear forces are proportional to velocity). This modulation of magnetic field strength can be achieved in several different ways. In one embodiment, a permanent magnet can be moved orthogonally relative to the EWOD surface to increase or decrease the magnetic field strength in the vicinity of the magnetic beads. In an alternative embodiment, an electromagnet or electro-permanent magnet can be used instead of a permanent magnet to electronically modulate the magnetic field strength in a time-dependent manner.

[0020] Small (<40 μL) and / or high surface tension droplets can also pose problems for splitting the bead pellet from the supernatant. Separating droplets in which the magnetic beads occupy a relatively significant volume relative to the overall bead and droplet volume can also pose this problem. In these cases, it can be beneficial to use gravity and surface tension as additional forces to aid in the separation. This can be achieved, for example, by introducing a magnet above the droplet and bead pellet (Figures 3A-3C). This magnet can be a permanent magnet, an electromagnet, or an electro-permanent magnet. The magnet is positioned vertically above the droplet so that the bead pellet is pulled upward out of the droplet. The position and strength of the magnet can be adjusted to result in a clean separation between the bead pellet and the droplet supernatant.

[0021] When a magnet is used for magnetic bead manipulation on a surface, it can be beneficial to move the magnet away in a controlled manner for various reasons. In this method, the magnet is moved perpendicular to the active surface so that there is sufficient clearance between the magnet and the bottom surface of the fluid manipulation device. A similar effect can be achieved by moving the magnet parallel to the surface, but in a way that the magnet is completely outside the perimeter of the active surface and provides sufficient clearance below the surface.

[0022] One magnet position for this method is shown in FIG. 4A. The magnet is near the active surface of the fluid manipulation device. Another embodiment of the magnet position for this method is shown in FIG. 4B. The magnet moves orthogonally to provide clearance between the magnet and the surface. Another embodiment of the magnet position for this method is shown in FIG. 4C. The active surface moves up and down to create agitation in the droplet surrounding the magnetic bead pellet. In the embodiment of FIGS. 4B-4C, the magnetic field strength may be reduced, but is still strong enough to keep the magnetic bead pellet in place while the agitation process continues. Additionally, the agitation process can be replaced with another droplet operation. Droplet operations can include stationary droplets, droplets moving back and forth on beads using electrowetting, droplets being heated or cooled, small aliquots from droplets being removed, droplet splitting, droplet dispense, or any combination thereof.

[0023] Creating this clearance and introducing droplet manipulation while the beads are held in place acts as a supplement to the electrowetting forces if the forces are not sufficient to manipulate the droplet. Droplet manipulation while the magnet is still below the surface allows for improved droplet mobility and / or improved droplet manipulation.

[0024] definition Whenever the terms "at least," "greater than," or "greater than or equal to" precede the first number in a series of two or more numbers, the terms "at least," "greater than," or "greater than or equal to" apply to each and every number in the series. For example, 1, 2, or 3 or more is equivalent to 1 or more, 2 or more, or 3 or more.

[0025] Whenever the terms "no more than," "less than," or "less than or equal to" precede the first number in a series of two or more numbers, the "no more than," "less than," or "less than or equal to" applies to each and every number in the series. For example, 3, 2, or 1 or less is equivalent to 3 or less, 2 or less, or 1 or less.

[0026] As used in the specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, "a sample" includes multiple samples, including combinations thereof.

[0027] The terms "determining," "measuring," "evaluating," "assessing," "assaying," and "analyzing" are often used interchangeably herein to refer to forms of measurement. The terms include determining whether an element is present or not (e.g., detecting). These terms can include quantitative determination, qualitative determination, or quantitative and qualitative determination. Evaluation can be relative or absolute. "Detecting the presence of" can include determining the amount of something present in addition to determining presence or absence, depending on the context.

[0028] As used herein, the term "about" or "approximately," when referring to a measurable value such as an amount or concentration, is intended to encompass a variation of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount. For example, "about" may mean plus or minus 10%, according to practice in the art. Alternatively, "about" may mean a range of plus or minus 20%, plus or minus 10%, plus or minus 5%, or plus or minus 1% of a given value. Alternatively, particularly with respect to biological systems or biological processes, the term may mean within an order of magnitude, up to 5-fold, or up to 2-fold of a value. Where specific values may be described in the present application and claims, unless otherwise specified, the term "about" may be assumed to encompass an acceptable range of error for the particular value. Also, ranges, subranges, or both of values may be provided, and the ranges or subranges may include the endpoints of the ranges or subranges.

[0029] When values are described as ranges, such disclosure can be understood to include disclosure of all possible subranges within such ranges, as well as specific numerical values falling within such ranges, whether or not a specific numerical value or specific subrange is explicitly stated.

[0030] The terms "comprise," "have," and "include" are open-ended linking verbs. Any form or tense of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes," and "including," is also open-ended. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to having only those one or more steps, but also encompasses other unlisted steps.

[0031] As used herein, the term "droplet" generally refers to a discrete or finite volume of a fluid (e.g., a liquid). A droplet may be produced by one phase separated from another phase by an interface. A droplet may be a first phase that is phase-separated from another phase. A droplet may contain a single phase or multiple phases (e.g., an aqueous phase containing a polymer or an emulsion). A droplet may be a liquid phase disposed adjacent to a surface and in contact with a separate phase (e.g., a gas phase such as air).

[0032] The term "biological sample," as used herein, generally refers to biological material. Such biological material exhibits or can be biologically active. Such biological material can be or include deoxyribonucleic acid (DNA) molecules, ribonucleic acid (RNA) molecules, polypeptides (e.g., proteins), or any combination thereof. A biological sample (or sample) can be a tissue sample, such as a biopsy, core biopsy, needle aspirate, or fine needle aspirate. A sample can be a fluid sample, such as a blood sample, urine sample, fecal sample, or saliva sample. A sample can be a skin sample. A sample can be a buccal swab. A sample can be a plasma or serum sample. A sample can be a plant-derived sample, a water sample, or a soil sample. A sample can be extraterrestrial. An extraterrestrial sample can include biological material. A sample can be an acellular (or acellular) sample. An acellular sample can include extracellular polynucleotides. The extracellular polynucleotides may be isolated from a body sample that may be selected from the group consisting of blood, plasma, serum, urine, saliva, mucosal excretions, sputum, feces, and tears. The sample may comprise a eukaryotic cell or a plurality thereof. The sample may comprise a prokaryotic cell or a plurality thereof. The sample may comprise a virus. The sample may comprise a compound derived from an organism. The sample may be derived from a plant. The sample may be derived from an animal. The sample may be derived from an animal having or suspected of having a disease. The sample may be derived from a mammal.

[0033] As used herein, the term "electro-mechanical actuator" generally refers to a non-human structure that can be utilized to apply vibration and / or acoustic forces to the arrays described herein. By way of non-limiting example, electro-mechanical actuators include oscillating mechanisms or cantilevers, motor-driven linkages, and / or rotating masses. In some embodiments, the electro-mechanical actuators described herein are flexible structures with various flexible elements (e.g., linear flexures) or with conventional bearings.

[0034] As used herein, the term "electrowetting" generally refers to any liquid handling technique that uses a voltage applied to electrodes or other conductors to move a fluid on a surface. The surface tension and wetting properties of a fluid can be altered by an electric field using the electrowetting effect. The electrowetting effect can result from a change in the solid-liquid contact angle due to an applied potential difference between the solid and the liquid. When the fluid is provided as a droplet, the difference in wetting surface tension can vary across the width of the droplet, and the corresponding change in contact angle can provide the motive force to move the droplet without moving parts or physical contact.

[0035] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0036] Electrowetting devices and systems Electrowetting devices can be used to move individual droplets of water (or other aqueous, polar, or conducting solutions) from location to location. The surface tension and wetting properties of water can be changed by electric field strength using the electrowetting effect. The electrowetting effect can result from a change in the solid-liquid contact angle due to an applied potential difference between the solid and the liquid. The difference in wetting surface tension, which can vary across the width of the droplet, and the corresponding change in contact angle can provide the motive force to move the droplet without moving or physical contacting any parts. Electrowetting devices can include a grid of electrodes with a dielectric layer with appropriate electrical and surface priorities covering the electrodes, all of which is placed on a rigid insulating substrate. Further examples of electrowetting devices can be found in WO2021041709, which is incorporated herein by reference in its entirety.

[0037] The surface of the electrode grid can be prepared to have low adhesion to water. This can allow a water droplet to be moved along the surface by a small force generated by the electric field gradient and surface tension across the width of the droplet. A surface with low adhesion can reduce the trail left behind by the droplet. A smaller trail can reduce droplet cross-contamination and reduce sample loss during droplet movement. Low adhesion to the surface can also allow for low actuation voltages for droplet movement and repeatable behavior of droplet movement. There are several methods for measuring low adhesion between a surface and a droplet, including sliding angle and contact angle hysteresis, such as using a contact angle goniometer or a charge-coupled device (CCD) camera.

[0038] There can be several ways to achieve low surface adhesion, such as mechanically polishing, chemically etching, or a combination of these until smooth within a few nanometers, applying a coating to fill surface irregularities, applying a liquid to fill surface irregularities, chemically modifying the surface to create desired surface properties (hydrophobicity, hydrophilicity, resistance to biofouling, change with field strength, etc.).

[0039] Electrowetting on a dielectric (EWOD) for droplet manipulation In some embodiments, electrowetting on dielectrics (EWOD) is a phenomenon in which the wettability of an aqueous, polar, or conductive liquid (L) can be modulated through an electric field across a dielectric film between a droplet and a conductive electrode. Adding or subtracting charge from the electrode changes the wettability of the insulating dielectric layer, which is reflected in a change in the contact angle of the droplet. The change in contact angle can then cause the droplet to change shape, move, break up into smaller droplets, or coalesce with another droplet. Further examples of EWOD droplet actuation can be found in WO2021041709, which is incorporated herein by reference in its entirety.

[0040] Droplet movement, coalescence and breakup Droplets can be moved, coalesced, split, or any combination thereof on an open surface electrowetting device. The same principle applies to a two plate configuration (sandwiched droplets).

[0041] In some embodiments, applying a voltage to an electrode can make the surface thereon hydrophilic, and then the droplet can wet it. If a voltage is applied to two adjacent electrodes, the droplet can spread across both actuation electrodes. If a voltage is removed from an electrode and applied to another adjacent electrode, the surface returns to its original hydrophobic state and the droplet can be pushed out. By sequentially controlling the voltage applied to the electrode grid, the position of the droplet on the surface can be precisely controlled.

[0042] In some embodiments, when two droplets are pulled toward the same electrode, they may coalesce spontaneously due to surface tension. This principle can be applied to coalesce multiple droplets to create a larger volume droplet that spans multiple electrodes.

[0043] In some embodiments, a droplet can be split into two smaller ones through a series of voltages applied across multiple electrodes (at least three electrodes). In some embodiments, a single large droplet is consolidated onto a single electrode. In some embodiments, equal voltages are applied simultaneously to three adjacent electrodes, which can cause a single droplet to spread across the three adjacent electrodes. In some embodiments, turning off the center electrode can force the droplet to move to the two outer electrodes. Because both of the two outer electrodes have equal potentials, the droplet can then split into two smaller droplets.

[0044] EWOD-enabled magnetic bead wash Magnetic particles can be manipulated on the surface of a chip by a controllable local magnetic field. The magnetic particles can be made, for example, from microspheres. Control of the local magnetic field can be achieved, for example, by placing a solenoid, magnet, magnet pair, or any combination thereof near the particles, or by generating a magnetic field within the EWOD chip. Magnetic bead-based separation and washing can be performed on EWOD-enabled arrays. Droplets can be manipulated using actuation electrodes, which can also enable droplet positioning. Magnetic particles can be concentrated into small areas using magnetic fields. Liquids can be separated from magnetic particles by EWOD-based, dielectrophoresis-based, or other electromotive force-based actuation. Separation is possible in open-plate and two-plate systems. Because droplets can be positioned using EWOD actuation, fluids can also be aspirated from the chip using a liquid handling robot, leaving magnetic particles on the chip surface. Liquid removal can be achieved through a hole or holes in the array by using capillary forces, pneumatic forces, electromotive forces such as EWOD or dielectrowetting, or any combination thereof. This waste liquid can be collected in a reservoir located below the array. A computer-vision-based algorithm can be used to inform and provide feedback to the liquid handler and / or array about processes involving magnetic beads. The process can include, for example, aspiration of the supernatant, resuspension of the beads, preventing aspiration of the magnetic beads with the supernatant during supernatant removal, or any combination thereof.

[0045] vibration-assisted mixing Droplets can be mixed in a variety of ways. The present disclosure provides methods in which vibration of a digital microfluidic surface can be used to assist in mixing of liquids on the surface of a digital microfluidic device. Vibration can generate small-scale fluid motion within droplets on the surface of the digital microfluidic device. The motion can promote diffusion and rapidly accelerate the mixing process. An example of an advantage of vibration-assisted droplet mixing is efficient capture of DNA onto magnetic microparticles (e.g., beads), and ultimately higher yields of DNA extraction. In some embodiments, an electrowetting array with an open surface is provided.

[0046] A common problem with digital microfluidic platforms is achieving robust mixing with all types of reagents and droplets. For example, highly viscous droplets can be extremely difficult to mix effectively using purely electrowetting-based motion. These types of viscous droplets are important in a wide range of applications, including DNA extraction from highly concentrated sample materials, where the DNA needs to be efficiently bound to magnetic beads. Using purely electrowetting-based motion for mixing in these applications results in very poor mixing and, therefore, very poor DNA extraction from the sample droplets.

[0047] Described herein are device, system, and method implementations in which the application of vibration and / or acoustic forces to a digital microfluidic surface can be used to assist in the mixing of liquids on the surface. The vibrations, when tuned to the appropriate frequency and amplitude, generate small-scale fluid motion within the droplets that promotes diffusion and rapidly accelerates the mixing process.

[0048] Vibration also contributes to improved droplet mobility. This is especially true for droplets containing particles. In the absence of vibration, large particles may tend to settle at the interface between the droplet and the substrate. If these particles are present at the droplet's contact line, they may act to pin the droplet in place and limit its mobility. The introduction of vibration helps prevent particles from settling at the contact line and, in doing so, significantly improves the reliability of the electrowetting mobility of particle-carrying droplets.

[0049] Vibration-based mixing is synergistic with electrowetting-based mixing. While vibration mixing is effective at dispersing particles within portions of a droplet, it is often ineffective at macro-scale mixing throughout the droplet, especially for droplets with low contact angles with surfaces. Electrowetting-based droplet mixing helps address this issue, and both vibration and electrowetting can work together to rapidly and effectively achieve mixing of a wide variety of droplets of various compositions.

[0050] Aspects of the present disclosure include a method for processing droplets, the method comprising: providing an electrowetting array adjacent to a magnet configured to apply a magnetic field to the electrowetting array in one or more of three directional axes; providing a droplet on the array that includes one or more artifacts responsive to the magnetic field; and actuating the magnet in the one or more of the three directional axes relative to the array to separate the one or more artifacts from the droplet.

[0051] In some embodiments, the magnet is moved parallel to the array in an x-direction, in some embodiments, the magnet is moved parallel to the array in a y-direction, and in some embodiments, the magnet is moved orthogonal to the array.

[0052] In some embodiments, the strength of the magnetic field is modulated. In some embodiments, the strength of the magnetic field is increased. In some embodiments, the strength of the magnetic field is decreased. In some embodiments, the strength of the magnetic field is not modulated. In some embodiments, the strength of the magnetic field remains essentially the same. In some embodiments, the strength of the magnetic field remains the same.

[0053] In some embodiments, the magnet is a permanent magnet. In some embodiments, the magnet is an electromagnet or an electro-permanent magnet. In some embodiments, the electromagnet or the electro-permanent magnet modulates the strength of the magnetic field in a time-dependent manner.

[0054] In some embodiments, the magnet is positioned above the electrowetting array. In some embodiments, the magnet is moved orthogonally relative to the electrowetting array to separate the one or more artifacts from the droplet. In some embodiments, the magnet is positioned below the electrowetting array.

[0055] One aspect of the present disclosure includes a method of removing one or more artifacts from a droplet, the method including providing a magnet configured to apply a magnetic field to the droplet and actuating the magnet with respect to the droplet to separate the one or more artifacts from the droplet, wherein the droplet is less than 40 microliters.

[0056] In some embodiments, the magnet is moved parallel to the array in an x-direction, in some embodiments, the magnet is moved parallel to the array in a y-direction, and in some embodiments, the magnet is moved orthogonal to the array.

[0057] In some embodiments, the strength of the magnetic field is modulated. In some embodiments, the strength of the magnetic field is increased. In some embodiments, the strength of the magnetic field is decreased.

[0058] In some embodiments, the magnet is a permanent magnet. In some embodiments, the magnet is an electromagnet or an electro-permanent magnet. In some embodiments, the electromagnet or the electro-permanent magnet modulates the strength of the magnetic field in a time-dependent manner.

[0059] In some embodiments, the magnet is positioned above the electrowetting array. In some embodiments, the magnet is moved orthogonally relative to the electrowetting array to separate the one or more artifacts from the droplet. In some embodiments, the magnet is positioned below the electrowetting array.

[0060] In some embodiments, the droplets are less than 30 microliters. In some embodiments, the droplets are less than 20 microliters. In some embodiments, the droplets are less than 10 microliters.

[0061] Another aspect of the present disclosure is a method for manipulating droplets, the method comprising: providing the droplets on a surface adjacent to a magnet configured to provide a magnetic field in contact with the droplets, the droplets comprising one or more artifacts responsive to the magnetic field; and displacing the magnet proximate to the droplets, the magnetic field in contact with the droplets comprising a magnetic flux density of at least about 4 millitesla ("mT") to at least about 10 mT, thereby manipulating the one or more artifacts responsive to the magnetic field. In some embodiments, the magnet is displaced along an axis perpendicular to the surface. In some embodiments, the magnet is displaced at a distance of about 0 millimeters to about 15 millimeters from the surface. In some embodiments, the surface comprises an electrowetting array. In some embodiments, the one or more artifacts responsive to the magnetic field comprise nucleic acid molecules. In some embodiments, the one or more artifacts responsive to the magnetic field comprise a plurality of nucleic acid molecules. In some embodiments, the magnet is displaced along an axis parallel to the surface. In some embodiments, the method further comprises moving the magnet to maintain a magnetic flux density of at least about 4 millitesla ("mT") to at least about 10 mT in the magnetic field in contact with the droplet. In some embodiments, the method further comprises moving the magnet to maintain a distance of about 0 millimeters to about 15 millimeters from the surface. In some embodiments, the droplet is less than 30 microliters. In some embodiments, the droplet is less than 20 microliters. In some embodiments, the droplet is less than 10 microliters.

[0062] In some embodiments, the nucleic acid molecule comprises at least 100 kb. In some embodiments, the nucleic acid molecule comprises at least 10 kb to 200 kb. In some embodiments, the nucleic acid molecule comprises at least 10 kb to 20 kb, 10 kb to 40 kb, 10 kb to 50 kb, 10 kb to 70 kb, 10 kb to 90 kb, 10 kb to 110 kb, 10 kb to 130 kb, 10 kb to 150 kb, 10 kb to 170 kb, 10 kb to 190 kb, 10 kb to 200 kb, 20 kb to 40 kb, 20 kb to 50 kb, 20 kb to 70 kb, 20 kb to 90 kb, 20 kb to 110 kb. b, 20kb~130kb, 20kb~150kb, 20kb~170kb, 20kb~190kb, 20kb~200kb, 40kb~50kb, 40kb~70kb, 40kb~90kb, 40kb~11 0kb, 40kb~130kb, 40kb~150kb, 40kb~170kb, 40kb~190kb, 40kb~200kb, 50kb~70kb, 50kb~90kb, 50kb~110kb, 50kb~ 130kb, 50kb~150kb, 50kb~170kb, 50kb~190kb, 50kb~200kb, 70kb~90kb, 70kb~110kb, 70kb~130kb, 70kb~150kb, 7 0kb~170kb, 70kb~190kb, 70kb~200kb, 90kb~110kb, 90kb~130kb, 90kb~150kb, 90kb~170kb, 90kb~190kb, 90kb~200 kb, 110kb to 130kb, 110kb to 150kb, 110kb to 170kb, 110kb to 190kb, 110kb to 200kb, 130kb to 150kb, 130kb to 170kb, 130kb to 190kb, 130kb to 200kb, 150kb to 170kb, 150kb to 190kb, 150kb to 200kb, 170kb to 190kb, 170kb to 200kb, or 190kb to 200kb. In some embodiments, the nucleic acid molecule comprises at least 10 kb, 20 kb, 40 kb, 50 kb, 70 kb, 90 kb, 110 kb, 130 kb, 150 kb, 170 kb, 190 kb, or 200 kb.In some embodiments, the nucleic acid molecule comprises at least 10 kb, 20 kb, 40 kb, 50 kb, 70 kb, 90 kb, 110 kb, 130 kb, 150 kb, 170 kb, or 190 kb, hi some embodiments, the nucleic acid molecule comprises at least 200 kb to 1,000 kb. In some embodiments, the nucleic acid molecule is at least 200 kb to 300 kb, 200 kb to 400 kb, 200 kb to 500 kb, 200 kb to 600 kb, 200 kb to 700 kb, 200 kb to 800 kb, 200 kb to 900 kb, 200 kb to 1,000 kb, 300 kb to 400 kb, 300 kb to 500 kb, 300 kb to 600 kb, 300 kb to 700 kb, 300 kb to 800 kb, 300 kb to 900 kb, 300 kb to 1,000 kb, 400 kb to 500 kb, 400 kb to 600 kb, 400 kb to 700 kb, 00kb, 400kb to 800kb, 400kb to 900kb, 400kb to 1,000kb, 500kb to 600kb, 500kb to 700kb, 500kb to 800kb, 500kb to 900kb, 500kb to 1,000kb, 600kb to 700kb, 600kb to 800kb, 600kb to 900kb, 600kb to 1,000kb, 700kb to 800kb, 700kb to 900kb, 700kb to 1,000kb, 800kb to 900kb, 800kb to 1,000kb, or 900kb to 1,000kb. In some embodiments, the nucleic acid molecule comprises at least 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, or 1,000 kb. In some embodiments, the nucleic acid molecule comprises at least 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, or 900 kb. In some embodiments, the nucleic acid molecule comprises at least up to 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, or 1,000 kb.

[0063] In some embodiments, the nucleic acid molecule comprises at least 10 kb to 1,000 kb. In some embodiments, the nucleic acid molecule comprises at least 10 kb to 20 kb, 10 kb to 50 kb, 10 kb to 75 kb, 10 kb to 100 kb, 10 kb to 200 kb, 10 kb to 300 kb, 10 kb to 400 kb, 10 kb to 500 kb, 10 kb to 750 kb, 10 kb to 900 kb, 10 kb to 1,000 kb, 20 kb to 50 kb, 20 kb to 75 kb, 20 kb to 100 kb, 20 kb to 200 kb, 20 kb to 3 ...300 kb, 20 kb to 300 kb, 20 kb to 300 kb, 20 kb to 300 kb, 20 kb to 300 kb, 20 kb to 300 kb, 20 kb to 300 kb, 20 kb to 300 kb, 20 kb to 30 b~400kb, 20kb~500kb, 20kb~750kb, 20kb~900kb, 20kb~1,000kb, 50kb~75kb, 50kb~100kb, 50kb~200kb, 50kb~300kb, 50k b~400kb, 50kb~500kb, 50kb~750kb, 50kb~900kb, 50kb~1,000kb, 75kb~100kb, 75kb~200kb, 75kb~300kb, 75kb~400kb, 75 kb~500kb, 75kb~750kb, 75kb~900kb, 75kb~1,000kb, 100kb~200kb, 100kb~300kb, 100kb~400kb, 100kb~500kb, 100kb~75 0kb, 100kb~900kb, 100kb~1,000kb, 200kb~300kb, 200kb~400kb, 200kb~500kb, 200kb~750kb, 200kb~900kb, 200kb~1,00 Includes 0kb, 300kb to 400kb, 300kb to 500kb, 300kb to 750kb, 300kb to 900kb, 300kb to 1,000kb, 400kb to 500kb, 400kb to 750kb, 400kb to 900kb, 400kb to 1,000kb, 500kb to 750kb, 500kb to 900kb, 500kb to 1,000kb, 750kb to 900kb, 750kb to 1,000kb, or 900kb to 1,000kb. In some embodiments, the nucleic acid molecule comprises at least 10 kb, 20 kb, 50 kb, 75 kb, 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 750 kb, 900 kb, or 1,000 kb.In some embodiments, the nucleic acid molecule comprises at least 10 kb, 20 kb, 50 kb, 75 kb, 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 750 kb, or 900 kb.

[0064] In some embodiments, at least 20% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 30% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 40% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 50% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 60% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 70% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 80% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 90% of the plurality of nucleic acid molecules comprise at least 100 kb.

[0065] In some embodiments, the method further comprises moving the magnet to maintain the nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 20% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 30% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 40% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 50% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 60% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 70% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 80% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 90% of the plurality of nucleic acid molecules greater than or equal to 100 kb.

[0066] In some embodiments, the magnetic flux density is sufficient to immobilize one or more artifacts responsive to the magnetic field during the one or more droplet operations. In some embodiments, the one or more droplet operations include agitating the surface. In some embodiments, the one or more droplet operations include applying a voltage to at least one of the one or more electrodes to manipulate one or more reagent droplets, sample droplets, or both. In some embodiments, the one or more droplet operations include applying vibration to one or more reagent droplets, sample droplets, or both. In some embodiments, the one or more droplet operations include applying vibration to an electrowetting array. In some embodiments, the one or more droplet operations include the droplets being stationary. In some embodiments, the one or more droplet operations include moving the droplets back and forth on beads using electrowetting. In some embodiments, the one or more droplet operations include heating or cooling the droplets. In some embodiments, the one or more droplet operations include removing a small aliquot from the droplet. In some embodiments, the one or more droplet operations include droplet splitting. In some embodiments, the one or more droplet operations include dispensing a droplet.

[0067] Another aspect of the present disclosure is a method for manipulating droplets, the method comprising providing the droplets on a surface adjacent to a magnet configured to provide a magnetic field in contact with the droplets, the droplets comprising one or more artifacts responsive to the magnetic field; and displacing the magnet proximate to the droplets at a distance of about 0 millimeters to about 15 millimeters from the surface, thereby manipulating the one or more artifacts responsive to the magnetic field. In some embodiments, the magnet is displaced along an axis perpendicular to the surface. In some embodiments, the magnetic field in contact with the droplets comprises a magnetic flux density of at least about 4 millitesla ("mT") to at least about 10 mT. In some embodiments, the surface comprises an electrowetting array. In some embodiments, the one or more artifacts responsive to the magnetic field comprise nucleic acid molecules. In some embodiments, the one or more artifacts responsive to the magnetic field comprise a plurality of nucleic acid molecules. In some embodiments, the magnet is displaced along an axis parallel to the surface. In some embodiments, the method further comprises moving the magnet to maintain a magnetic flux density of at least about 4 millitesla ("mT") to at least about 10 mT in the magnetic field in contact with the droplet. In some embodiments, the method further comprises moving the magnet to maintain a distance of about 0 millimeters to about 15 millimeters from the surface. In some embodiments, the droplet is less than 30 microliters. In some embodiments, the droplet is less than 20 microliters. In some embodiments, the droplet is less than 10 microliters.

[0068] In some embodiments, the nucleic acid molecule comprises at least 100 kb. In some embodiments, the nucleic acid molecule comprises at least 100 kb to 1,000 kb. In some embodiments, the nucleic acid molecule comprises at least 100 kb to 200 kb, 100 kb to 300 kb, 100 kb to 400 kb, 100 kb to 500 kb, 100 kb to 600 kb, 100 kb to 700 kb, 100 kb to 800 kb, 100 kb to 900 kb, 100 kb to 1,000 kb, 200 kb to 300 kb, 200 kb to 300 kb, 200 kb to 400 kb, 200 kb to 500 kb, 200 kb to 600 kb, 200 kb to 700 kb, 200 kb to 800 kb, 200 kb to 900 kb, 200 kb to 1,000 kb, 200 kb to 3 ... 00kb~400kb, 200kb~500kb, 200kb~600kb, 200kb~700kb, 200kb~800kb, 200kb~900kb, 20 0kb~1,000kb, 300kb~400kb, 300kb~500kb, 300kb~600kb, 300kb~700kb, 300kb~800kb, 30 0kb~900kb, 300kb~1,000kb, 400kb~500kb, 400kb~600kb, 400kb~700kb, 400kb~800kb, 4 00kb~900kb, 400kb~1,000kb, 500kb~600kb, 500kb~700kb, 500kb~800kb, 500kb~900kb, 5 In some embodiments, the nucleic acid molecule comprises at least 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1000 kb, 1000 kb, 1000 kb, 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, or 1000 kb. In some embodiments, the nucleic acid molecule comprises at least 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 600 kb, 700 kb, 800 kb, or 900 kb.

[0069] In some embodiments, the nucleic acid molecule comprises at least 10 kb to 100 kb. In some embodiments, the nucleic acid molecule comprises at least 10 kb to 20 kb, 10 kb to 50 kb, 10 kb to 75 kb, 10 kb to 90 kb, 10 kb to 100 kb, 20 kb to 50 kb, 20 kb to 75 kb, 20 kb to 90 kb, 20 kb to 100 kb, 50 kb to 75 kb, 50 kb to 90 kb, 50 kb to 100 kb, 75 kb to 90 kb, 75 kb to 100 kb, or 90 kb to 100 kb. In some embodiments, the nucleic acid molecule comprises at least 10 kb, 20 kb, 50 kb, 75 kb, 90 kb, or 100 kb. In some embodiments, the nucleic acid molecule comprises at least 10 kb, 20 kb, 50 kb, 75 kb, or 90 kb.

[0070] In some embodiments, the nucleic acid molecule comprises at least 10 kb to 1,000 kb. In some embodiments, the nucleic acid molecule comprises at least 10 kb to 20 kb, 10 kb to 50 kb, 10 kb to 75 kb, 10 kb to 100 kb, 10 kb to 200 kb, 10 kb to 300 kb, 10 kb to 400 kb, 10 kb to 500 kb, 10 kb to 750 kb, 10 kb to 900 kb, 10 kb to 1,000 kb, 20 kb to 50 kb, 20 kb to 75 kb, 20 kb to 100 kb, 20 kb to 200 kb, 20 kb to 3 ...300 kb, 20 kb to 300 kb, 20 kb to 300 kb, 20 kb to 300 kb, 20 kb to 300 kb, 20 kb to 300 kb, 20 kb to 300 kb, 20 kb to 300 kb, 20 kb to 30 b~400kb, 20kb~500kb, 20kb~750kb, 20kb~900kb, 20kb~1,000kb, 50kb~75kb, 50kb~100kb, 50kb~200kb, 50kb~300kb, 50k b~400kb, 50kb~500kb, 50kb~750kb, 50kb~900kb, 50kb~1,000kb, 75kb~100kb, 75kb~200kb, 75kb~300kb, 75kb~400kb, 75 kb~500kb, 75kb~750kb, 75kb~900kb, 75kb~1,000kb, 100kb~200kb, 100kb~300kb, 100kb~400kb, 100kb~500kb, 100kb~75 0kb, 100kb~900kb, 100kb~1,000kb, 200kb~300kb, 200kb~400kb, 200kb~500kb, 200kb~750kb, 200kb~900kb, 200kb~1,00 Includes 0kb, 300kb to 400kb, 300kb to 500kb, 300kb to 750kb, 300kb to 900kb, 300kb to 1,000kb, 400kb to 500kb, 400kb to 750kb, 400kb to 900kb, 400kb to 1,000kb, 500kb to 750kb, 500kb to 900kb, 500kb to 1,000kb, 750kb to 900kb, 750kb to 1,000kb, or 900kb to 1,000kb. In some embodiments, the nucleic acid molecule comprises at least 10 kb, 20 kb, 50 kb, 75 kb, 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 750 kb, 900 kb, or 1,000 kb.In some embodiments, the nucleic acid molecule comprises at least 10 kb, 20 kb, 50 kb, 75 kb, 100 kb, 200 kb, 300 kb, 400 kb, 500 kb, 750 kb, or 900 kb.

[0071] In some embodiments, at least 20% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 30% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 40% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 50% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 60% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 70% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 80% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 90% of the plurality of nucleic acid molecules comprise at least 100 kb.

[0072] In some embodiments, the method further comprises moving the magnet to maintain the nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 20% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 30% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 40% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 50% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 60% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 70% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 80% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 90% of the plurality of nucleic acid molecules greater than or equal to 100 kb.

[0073] In some embodiments, the method further comprises moving the magnet to maintain the nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 20% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the distance is sufficient to immobilize one or more artifacts responsive to the magnetic field during the one or more droplet operations. In some embodiments, the one or more droplet operations comprise agitating the surface. In some embodiments, the one or more droplet operations comprise applying a voltage to at least one of the one or more electrodes to manipulate one or more reagent droplets, sample droplets, or both. In some embodiments, the one or more droplet operations comprise applying vibration to one or more reagent droplets, sample droplets, or both. In some embodiments, the one or more droplet operations comprise applying vibration to an electrowetting array. In some embodiments, the one or more droplet operations comprise the droplets being stationary. In some embodiments, the one or more droplet operations comprise moving the droplets back and forth on the beads using electrowetting. In some embodiments, the one or more droplet operations comprise heating or cooling the droplets. In some embodiments, the one or more droplet operations include removing a small aliquot from a droplet. In some embodiments, the one or more droplet operations include splitting the droplet. In some embodiments, the one or more droplet operations include dispensing the droplet.

[0074] Another aspect of the present disclosure is a method for manipulating droplets, the method comprising: providing the droplets on a surface adjacent to a magnet configured to provide a magnetic field in contact with the droplets, the droplets comprising one or more artifacts responsive to the magnetic field, the one or more artifacts responsive to the magnetic field comprising nucleic acid molecules; and moving the magnet to maintain the nucleic acid molecules 100 kb or larger, thereby manipulating the one or more artifacts responsive to the magnetic field. In some embodiments, the magnet is displaced along an axis perpendicular to the surface. In some embodiments, the magnetic field in contact with the droplets comprises a magnetic flux density of at least about 4 millitesla ("mT") to at least about 10 mT. In some embodiments, the surface comprises an electrowetting array. In some embodiments, the one or more artifacts responsive to the magnetic field comprise a plurality of nucleic acid molecules. In some embodiments, the magnet is displaced along an axis parallel to the surface. In some embodiments, the method further comprises moving the magnet to maintain a magnetic flux density of at least about 4 millitesla ("mT") to at least about 10 mT in the magnetic field in contact with the droplets. In some embodiments, the method further comprises moving the magnet to maintain a distance of about 0 millimeters to about 15 millimeters from the surface. In some embodiments, the droplet is less than 30 microliters. In some embodiments, the droplet is less than 20 microliters. In some embodiments, the droplet is less than 10 microliters.

[0075] In some embodiments, at least 20% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 30% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 40% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 50% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 60% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 70% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 80% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 90% of the plurality of nucleic acid molecules comprise at least 100 kb.

[0076] In some embodiments, the method further comprises maintaining at least 20% of the plurality of nucleic acid molecules at 100 kb or greater. In some embodiments, the method further comprises maintaining at least 30% of the plurality of nucleic acid molecules at 100 kb or greater. In some embodiments, the method further comprises maintaining at least 40% of the plurality of nucleic acid molecules at 100 kb or greater. In some embodiments, the method further comprises maintaining at least 50% of the plurality of nucleic acid molecules at 100 kb or greater. In some embodiments, the method further comprises maintaining at least 60% of the plurality of nucleic acid molecules at 100 kb or greater. In some embodiments, the method further comprises maintaining at least 70% of the plurality of nucleic acid molecules at 100 kb or greater. In some embodiments, the method further comprises maintaining at least 80% of the plurality of nucleic acid molecules at 100 kb or greater. In some embodiments, the method further comprises maintaining at least 90% of the plurality of nucleic acid molecules at 100 kb or greater.

[0077] In some embodiments, the method further comprises moving the magnet to maintain the nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 20% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 30% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 40% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 50% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 60% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 70% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 80% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the method further comprises moving the magnet to maintain at least 90% of the plurality of nucleic acid molecules greater than or equal to 100 kb.

[0078] In some embodiments, the distance is sufficient to immobilize one or more artifacts responsive to the magnetic field during the one or more droplet operations. In some embodiments, the one or more droplet operations include agitating the surface. In some embodiments, the one or more droplet operations include applying a voltage to at least one of the one or more electrodes to manipulate one or more reagent droplets, sample droplets, or both. In some embodiments, the one or more droplet operations include applying vibration to one or more reagent droplets, sample droplets, or both. In some embodiments, the one or more droplet operations include applying vibration to an electrowetting array. In some embodiments, the one or more droplet operations include the droplets being stationary. In some embodiments, the one or more droplet operations include moving the droplets back and forth on the beads using electrowetting. In some embodiments, the one or more droplet operations include heating or cooling the droplets. In some embodiments, the one or more droplet operations include removing a small aliquot from the droplets. In some embodiments, the one or more droplet operations include droplet splitting. In some embodiments, the one or more droplet operations include dispensing the droplets.

[0079] Another aspect of the present disclosure is a system for manipulating droplets, the system including: a surface configured to support the droplets; a magnet adjacent to the surface, the magnet configured to provide a magnetic field in contact with the droplets, the droplets including one or more artifacts responsive to the magnetic field, the one or more artifacts responsive to the magnetic field including nucleic acid molecules; and a controller mechanically coupled to the magnet, the controller configured to displace the magnet in proximity to the droplets, maintaining the nucleic acid molecules at 100 kb or greater. In some embodiments, the magnet is displaced along an axis perpendicular to the surface. In some embodiments, the magnetic field in contact with the droplets includes a magnetic flux density of at least about 4 millitesla ("mT") to at least about 10 mT. In some embodiments, the surface includes an electrowetting array. In some embodiments, the one or more artifacts responsive to the magnetic field include a plurality of nucleic acid molecules. In some embodiments, the magnet is displaced along an axis parallel to the surface. In some embodiments, the controller is configured to maintain a magnetic flux density of at least about 4 millitesla ("mT") to at least about 10 mT in the magnetic field in contact with the droplet. In some embodiments, the controller is configured to maintain the magnet at a distance of about 0 millimeters to about 15 millimeters from the surface. In some embodiments, the droplet is less than 30 microliters. In some embodiments, the droplet is less than 20 microliters. In some embodiments, the droplet is less than 10 microliters.

[0080] In some embodiments, at least 20% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 30% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 40% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 50% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 60% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 70% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 80% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 90% of the plurality of nucleic acid molecules comprise at least 100 kb.

[0081] In some embodiments, the system maintains at least 20% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the system maintains the nucleic acid molecules at or above 100 kb. In some embodiments, the system maintains at least 20% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the system maintains at least 30% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the system maintains at least 40% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the system maintains at least 50% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the system maintains at least 60% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the system maintains at least 70% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the system maintains at least 80% of the plurality of nucleic acid molecules at or above 100 kb. In some embodiments, the system maintains at least 90% of the plurality of nucleic acid molecules at or above 100 kb.

[0082] In some embodiments, the distance is sufficient to immobilize one or more artifacts responsive to the magnetic field during the one or more droplet operations. In some embodiments, the one or more droplet operations include agitating the surface. In some embodiments, the one or more droplet operations include applying a voltage to at least one of the one or more electrodes to manipulate one or more reagent droplets, sample droplets, or both. In some embodiments, the one or more droplet operations include applying vibration to one or more reagent droplets, sample droplets, or both. In some embodiments, the one or more droplet operations include applying vibration to an electrowetting array. In some embodiments, the one or more droplet operations include the droplets being stationary. In some embodiments, the one or more droplet operations include moving the droplets back and forth on the beads using electrowetting. In some embodiments, the one or more droplet operations include heating or cooling the droplets. In some embodiments, the one or more droplet operations include removing a small aliquot from the droplets. In some embodiments, the one or more droplet operations include droplet splitting. In some embodiments, the one or more droplet operations include dispensing the droplets.

[0083] Another aspect of the present disclosure is a device for processing droplets, the device including: a surface configured to support the droplets; a magnet adjacent to the surface, the magnet configured to provide a magnetic field in contact with the droplets, the droplets including one or more artifacts responsive to the magnetic field, the one or more artifacts responsive to the magnetic field including nucleic acid molecules; and a controller mechanically coupled to the magnet, the controller configured to displace the magnet in proximity to the droplets, maintaining the nucleic acid molecules 100 kb or larger. In some embodiments, the magnet is displaced along an axis perpendicular to the surface. In some embodiments, the magnetic field in contact with the droplets includes a magnetic flux density of at least about 4 millitesla ("mT") to at least about 10 mT. In some embodiments, the surface includes an electrowetting array. In some embodiments, the one or more artifacts responsive to the magnetic field include a plurality of nucleic acid molecules. In some embodiments, the magnet is displaced along an axis parallel to the surface. In some embodiments, the controller is configured to maintain a magnetic flux density of at least about 4 millitesla ("mT") to at least about 10 mT in the magnetic field in contact with the droplet. In some embodiments, the controller is configured to maintain the magnet at a distance of about 0 millimeters to about 15 millimeters from the surface. In some embodiments, the droplet is less than 30 microliters. In some embodiments, the droplet is less than 20 microliters. In some embodiments, the droplet is less than 10 microliters.

[0084] In some embodiments, at least 20% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 30% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 40% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 50% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 60% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 70% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 80% of the plurality of nucleic acid molecules comprise at least 100 kb. In some embodiments, at least 90% of the plurality of nucleic acid molecules comprise at least 100 kb.

[0085] In some embodiments, the distance is sufficient to immobilize one or more artifacts responsive to the magnetic field during the one or more droplet operations. In some embodiments, the one or more droplet operations include agitating the surface. In some embodiments, the one or more droplet operations include applying a voltage to at least one of the one or more electrodes to manipulate one or more reagent droplets, sample droplets, or both. In some embodiments, the one or more droplet operations include applying vibration to one or more reagent droplets, sample droplets, or both. In some embodiments, the one or more droplet operations include applying vibration to an electrowetting array. In some embodiments, the one or more droplet operations include the droplets being stationary. In some embodiments, the one or more droplet operations include moving the droplets back and forth on the beads using electrowetting. In some embodiments, the one or more droplet operations include heating or cooling the droplets. In some embodiments, the one or more droplet operations include removing a small aliquot from the droplets. In some embodiments, the one or more droplet operations include droplet splitting. In some embodiments, the one or more droplet operations include dispensing the droplets.

[0086] The present disclosure provides a computer system programmed to implement the disclosed methods. Figure 14 shows a computer system (1301) programmed or otherwise configured to manipulate a droplet or a plurality of droplets on the system described herein. The computer system (1301) can adjust various aspects of the disclosed sample manipulation, such as droplet size, droplet volume, droplet position, droplet velocity, droplet wetting, droplet temperature, droplet pH, beads in the droplet, number of cells in the droplet, droplet color, chemical concentration, biological substance concentration, or any combination thereof. The computer system (1101) can be a user's electronic device or a computer system located remotely from the electronic device. The electronic device can be a mobile electronic device.

[0087] The computer system (1301) includes a central processing unit (CPU, also referred to herein as "processor" and "computer processor") (1305), which may be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system (1301) also includes memory or memory locations (1310) (e.g., random access memory, read-only memory, flash memory), an electronic storage unit (1315) (e.g., a hard disk), a communication interface (1320) (e.g., a network adapter) for communicating with one or more other systems, and peripheral devices (1325), such as cache, other memory, data storage devices, electronic display adapters, or any combination thereof. The memory (1310), storage unit (1315), interface (1320), and peripheral devices (1325) communicate with the CPU (1305) via a communication bus (solid lines), such as a motherboard. The storage unit (1315) may be a data storage unit (or data repository) for storing data. The computer system (1301) may be operably coupled to a computer network ("network") (1330) using the communication interface (1320). The network (1330) may be the Internet, an extranet, or any combination thereof, or an intranet, extranet, or any combination thereof that communicates with the Internet. The network (1330) may, in some cases, be a telecommunications network, a data network, or any combination thereof. The network (1330) may include one or more computer servers, which may enable distributed computing, such as cloud computing.The network 1330 may implement a peer-to-peer network, which in some cases may allow devices coupled to the computer system 1301 to act as clients or servers, using the computer system 1301.

[0088] The CPU (1305) may execute a sequence of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as the memory (1310). The instructions may be directed to the CPU (1305), which may then program or configure the CPU (1305) to implement the methods of the present disclosure. Examples of operations performed by the CPU (1305) may include fetch, decode, execute, and writeback.

[0089] The CPU 1305 may be part of a circuit, such as an integrated circuit. One or more other components of the system 1101 may be included in the circuit. In some cases, the circuit is an application-specific integrated circuit (ASIC).

[0090] The storage unit (1315) may store files such as drivers, libraries, and saved programs. The storage unit (1315) may store user data, such as user preferences and user programs. The computer system (1301) may, in some cases, include one or more additional data storage units that are external to the computer system (1301), such as those located on remote servers in communication with the computer system (1301) through an intranet or the Internet.

[0091] The computer system 1301 may communicate with one or more remote computer systems via the network 1330. For example, the computer system 1301 may communicate with a user's remote computer system (e.g., a mobile electronic device). Examples of remote computer systems include a personal computer (e.g., a portable PC), a slate or tablet PC (e.g., an Apple® iPad, a Samsung® Galaxy Tab), a telephone, a smartphone (e.g., an Apple® iPhone, an Android-enabled device, a Blackberry®), or a personal digital assistant. A user may access the computer system 1301 via the network 1330.

[0092] The methods described herein may be implemented by machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system (1301), such as memory (1310) or electronic storage unit (1315). The machine-executable or machine-readable code may be provided in the form of software. During use, the code may be executed by the processor (1305). In some cases, the code may be retrieved from the storage unit (1315) and stored in memory (1310) for easy access by the processor (1305). In some situations, the electronic storage unit (1315) may be omitted, and the machine-executable instructions are stored in memory (1310).

[0093] The code may be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or may be compiled during run-time. The code may be supplied in a programming language that may be selected to enable the code to execute in a pre-compiled or as-compiled manner.

[0094] Aspects of the systems and methods provided herein, such as the computer system (1301), may be embodied in programming. Various aspects of the present technology may typically be thought of as "products" or "articles of manufacture" in the form of machine (or processor) executable code, associated data, or any combination thereof, carried on or embodied in some type of machine-readable medium. The machine-executable code may be stored in an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. A type of "storage" medium may include any or all of the tangible memory of a computer, processor, or its associated modules, such as various semiconductor memories, tape drives, disk drives, etc., which may provide non-transitory storage for software programming at any time. All or portions of the software may, from time to time, be communicated via the Internet or various other telecommunications networks. Such communication may, for example, enable the loading of software from one computer or processor to another, e.g., from a management server or host computer to the computer platform of an application server. Accordingly, another type of medium that may bear software elements includes optical, electrical, and electromagnetic waves, such as those used across physical interfaces between local devices, through wired and optical landline networks, and over various air links. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., may also be considered software-bearing media. As used herein, unless limited to non-transitory, tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[0095] Thus, a machine-readable medium such as a computer-executable code may take many forms, including, but not limited to, a tangible storage medium, a carrier wave medium, or a physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer, such as those that may be used to implement the databases, etc., shown in the figures. Volatile storage media may include dynamic memory, such as the main memory of a computer platform. Tangible transmission media include coaxial cables, copper wire, and optical fibers, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves, such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer-readable media include, for example, floppy disks, flexible disks, hard disks, magnetic tape, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punch cards paper tape, any other physical storage media with patterns of holes, RAM, ROM, PROMs and EPROMs, FLASH-EPROMs, any other memory chips or cartridges, carrier waves transmitting data or instructions, cables or links transmitting such carrier waves, or any other medium from which a computer may read programming code, data, or any combination thereof. Many of these forms of computer-readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0096] The computer system (1301) may comprise or be in communication with an electronic display (1335), which comprises a user interface (UI) (1340) for providing information related to, for example, droplet operations, sample operations, or a combination thereof. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0097] The methods and systems of the present disclosure may be implemented by one or more algorithms. The algorithms may be implemented by software when executed by the central processing unit (1105). The algorithms may, for example, provide additional liquid to the droplets, replace evaporated solvent in the droplets, map the droplets' paths, or any combination thereof.

[0098] System video, input, and control can be accessed through a web-based software application. User input through the software can include, for example, droplet movement, droplet size, and images of the array, and the user input can be recorded and stored in a cloud-based computing system. The stored user input can be accessed and retrieved, either in subsets or in its entirety, to provide machine learning-based algorithms. Droplet movement patterns can be recorded and analyzed for use in training navigation algorithms. The trained algorithms can be used to automate droplet movement. Spatial fluid properties can be recorded and analyzed for use in training protocol optimization and generation algorithms. The trained algorithms can be used to optimize biological protocols and droplet movement protocols or in the generation of new biological protocols and droplet movement protocols. Biological quality control techniques (e.g., amplification-based quantification methods, fluorescence-based, absorbance-based quantification, surface plasmon resonance methods, and capillary-electrophoretic methods for analyzing nucleic acid fragment sizes) can be used to analyze the effectiveness of workflows performed on arrays. Data from these techniques can then be used as input to machine learning algorithms to improve output. This process can be automated so that the system can iteratively improve output.

[0099] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided herein. While the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. Therefore, it is contemplated that the present invention also encompasses any such alternatives, modifications, variations, or equivalents. The following claims define the scope of the invention, and it is intended to cover methods and structures within the scope of these claims and their equivalents. [Example]

[0100] Example 1: Moving Magnet for Magnetic Bead-Assisted Separation An electrowetting-on-dielectric ("EWOD") device is provided as described in International Application No. PCT / US2019 / 019954, International Application No. PCT / US2020 / 048241, or International Application No. PCT / US2022 / 046102, each of which is incorporated by reference in its entirety. A magnet is coupled to the EWOD device by a member configured to displace the magnet along an axis perpendicular to a surface of the EWOD device.

[0101] The EWOD device is used for magnetic bead-assisted separation of DNA from cell lysates. The desired magnetic flux of a magnetic field exerted on magnetically responsive beads suspended in droplets placed on the EWOD device is proportional to the amount of DNA fragment shearing that can be tolerated for a given fragment size.

[0102] If little or no shear can be tolerated, the magnets are placed closer (e.g., 0 mm to 10 mm) to the surface of the EWOD device. Manipulating the magnetic field in this manner results in increased clearance close to the surface of the EWOD device, allowing for the presence of additional actuators for droplet manipulation (e.g., vibrators, heaters, coolers, electrodes, etc.).

[0103] In addition to moving perpendicular to the surface of the EWOD device, the member is configured to position the magnet parallel to the surface of the EWOD device. The member is configured to position the magnet parallel to the surface of the EWOD device along both the x-axis and y-axis parallel to the surface of the EWOD device. The ability of the described devices to have magnets that can be positioned both perpendicular and parallel to the surface of the EWOD device allows a user of the EWOD device to position magnets in parallel with the sequence of electrode activation, thereby varying the magnetic flux density of the magnetic field exerted on the magnetically responsive beads encapsulated within the droplets while simultaneously positioning the droplets themselves in sequence on the surface of the EWOD device.

[0104] Precise control of the flux density of the magnetic field exerted on magnetically responsive beads encapsulated in droplets during droplet manipulation enables a wide variety of assays and further droplet manipulations that can be utilized on EWOD devices.

Claims

[Claim 1] A method for processing droplets, a. A step of providing an electrowetting array, wherein the electrowetting array is adjacent to a magnet configured to apply a magnetic field to the electrowetting array in one or more of three directional axes, b. A step of providing droplets containing one or more artifacts that respond to the magnetic field onto the electrowetting array, c. The step of activating the magnet in one or more of the three directional axes relative to the electrowetting array in order to separate the one or more artifacts from the droplet. Methods that include...

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