Fluidic control techniques

Abstract

The present disclosure provides systems and methods for fluid control. In some embodiments, a method comprises a method of processing a droplet comprising a plurality of artifacts. The method may comprise providing a substrate comprising a plurality of electrodes disposed beneath a surface of the substrate. The method may further comprise providing the droplet comprising the plurality of artifacts on the substrate. The method may further comprise activating one or more electrodes of the plurality of electrodes to induce a pattern of motion of the droplet. The pattern of motion may be sufficient to reduce a radius of the shape of the plurality of artifacts.

Description

FLUIDIC CONTROL TECHNIQUESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 720069 filed on November 13, 2024, the entirety of which is incorporated herein by reference.BACKGROUND

[0002] Significant progress has been made in recent years in performing analysis on relatively clean, standardized input materials. This includes research in the biological and physical sciences, as well as in medical diagnostics. For example, commercially available DNA sequencing platforms are capable of sequencing a human genome for less than about $1,000 starting from a suitably prepared sample. This is, in part, attributable to automation of these platforms such that they require very little human intervention.

[0003] However, relatively less improvement has been achieved in transforming various nonstandardized and non-homogenous primary samples (e.g., blood, tissue, environmental samples) into the kinds of standardized input material suitable for use in the aforementioned analytical and diagnostic techniques. These "upstream" manipulations still require significant labor and the use of several devices (e.g., pipettes, incubators, centrifuges, etc.) such that they are the major source of expense (and variability and error of results). Fluid handling robots have been developed that somewhat automate these procedures, but they lack dynamic range in volume, lack critical functionality, and cannot typically perform a complete procedure from raw input to standardized output.SUMMARY

[0004] Various devices and applications exist for droplet-based processing of biological samples.Such devices and applications may entail the manipulation of the droplets or artifacts comprised in the droplets. The systems and methods described herein use electro wetting on a dielectric (EWOD) technology to perform various fluidic manipulations in a single device, thereby automating previously labor-intensive laboratory procedures. Described herein are certain improvements that result in more efficient and reliable mixing of fluidic droplets, aggregation ofPC17IB2025 / 000562artifacts such as cellular material or magnetic beads, and prevention of fouling on the EWOD surface.

[0005] In an aspect, provided herein is a method of processing a droplet comprising a plurality of artifacts. The method can comprise: providing a substrate comprising a plurality of electrodes disposed beneath a surface of the substrate; providing the droplet comprising the plurality of artifacts on the substrate; activating one or more electrodes of the plurality of electrodes to induce a pattern of motion of the droplet, wherein the pattern of motion is sufficient to change a geometry of the shape of the plurality of artifacts.

[0006] In some embodiments, the pattern of motion comprises: moving the droplet with respect to an x-axis; subsequently moving the droplet with respect to a y-axis at least two times; subsequently moving the droplet with respect to an x-axis.

[0007] In some embodiments, the pattern of motion is executed in proximity of a magnetic field.

[0008] In some embodiments, the pattern of motion comprises moving the droplet in a circular motion, a rectangular motion, an X-shape, or a linear motion.

[0009] In some embodiments, the magnetic field is generated by a magnet.

[0010] In some embodiments, the magnet is displaced below the substrate.

[0011] In some embodiments, the magnet is configured to translate with respect to the substrate.

[0012] In some embodiments, further comprising removing the plurality of artifacts from the droplet.

[0013] In some embodiments, the removing comprises: (i) actuating a movement of the droplet via EWOD; (ii) translating the magnet; or both.

[0014] In some embodiments, one or more surfactants are added to the droplet prior to the removing of the plurality of artifacts.

[0015] In another aspect, provided herein is a method of processing a droplet, the method comprising: providing a droplet on a surface; contacting the droplet with one or more surfactants; and removing one or more artifacts encapsulated within the droplet.

[0016] In some embodiments, (a) the providing the droplet on the surface occurs prior to (b) the contacting the droplet with the one or more surfactants.

[0017] In some embodiments, (b) the contacting the droplet with the one or more surfactants occurs prior to (c).

[0018] In some embodiments, the one or more artifacts comprise biofouling substances.PC17IB2025 / 000562

[0019] In some embodiments, in (b), the contacting the droplet with the one or more surfactants comprises pipetting, using a magnet, using vibration, or electrowetting.

[0020] In some embodiments, the one or more artifacts comprises one or more magnetic beads.

[0021] In some embodiments, further comprising providing a magnet in proximity to the substrate.

[0022] In some embodiments, the magnet is actuated.

[0023] In some embodiments, the pattern of motion causes the one or more magnetic beads to be in proximity to the magnet.

[0024] In some embodiments, the proximity to the magnet of the one or more magnetic beads reduces a radius of the one or more magnetic beads in the droplet.

[0025] In another aspect, provided herein is a method of selecting a frequency for vibrating a droplet on the surface of an electrowetting array comprising: determining a surface tension or viscosity of the droplet; and selecting a frequency of less than about 15 Hertz (Hz) for a droplet with lower surface tension or higher viscosity, and a frequency of greater than about 15 Hz for a droplet with higher surface tension or lower viscosity.

[0026] In some embodiments, a frequency of about 40 Hz to at least about 55 Hz is selected for a droplet comprising water.

[0027] In some embodiments, a frequency of about 2 Hz to at least about 15 Hz is selected for a droplet comprising ethanol or a surfactant.

[0028] In another aspect, provided herein is a method of vibrating a droplet comprising: providing a substrate comprising a plurality of electrodes disposed beneath a surface of the substrate; providing the droplet on the substrate; activating a plurality of electrodes, wherein the plurality of electrodes is in a pattern of motion.

[0029] In another aspect, provided herein is a method of processing a droplet, wherein the droplet comprises a plurality of artifacts, the method comprising: providing a substrate comprising a plurality of electrodes disposed beneath a surface of the substrate; providing the droplet comprising the plurality of artifacts on the substrate; resuspending the plurality of artifacts without mechanical agitation.

[0030] In some embodiments, the resuspending comprises activating an electrode of the plurality of electrodes.

[0031] In some embodiments, activating a plurality of electrodes comprises a temporal pattern.

[0032] In some embodiments, activating a plurality of electrodes excites a resonant mode of the droplet.PC17IB2025 / 000562

[0033] In some embodiments, activating a plurality of electrodes comprises a spatial pattern.

[0034] In some embodiments, the spatial pattern is a chessboard pattern.

[0035] In some embodiments, the spatial pattern is agnostic to droplet size.

[0036] In some embodiments, the spatial pattern is chosen to maximize perimeter between activated electrodes and inactivated electrodes.

[0037] In some embodiments, a portion of the droplet is localized.

[0038] In some embodiments, the droplet changes shape.

[0039] In some embodiments, the electrodes are activated in a pattern comprising activation of a plurality of electrodes adjacent horizontally

[0040] In some embodiments, the plurality of electrodes is activated in a pattern.

[0041] In some embodiments, the activation of the plurality of electrodes is sufficient to excite a resonance mode in the droplet but the activation of the electrodes is not sufficient to translate the droplet’s location. In some embodiments, the activation of the plurality of electrodes is sufficient to excite a near-resonance mode in the droplet but the activation of the electrodes is not sufficient to translate the droplet’s location.

[0042] In another aspect, provided herein is a method of immobilizing a droplet at a discrete location, the method comprising: providing a droplet on an electrowetting array, the array comprising a plurality of electrodes, wherein a subset of the plurality of electrodes comprises one or more adjacent electrodes forming a field on the surface comprising the discrete location; and activating or deactivating the one or more adjacent electrodes in a spatial and / or temporal pattern to localize the droplet at the discrete location.

[0043] In some embodiments, the spatial pattern is a cross shape.

[0044] In some embodiments, a horizontal and a vertical element of the plurality of electrodes are alternatively activated.

[0045] In some embodiments, a diameter of the discrete location is less than 2 millimeters (mm).

[0046] In some embodiments, the spatial pattern is a chessboard pattern.

[0047] In some embodiments, the pattern of motion is sufficient to reduce a radius of the shape of the plurality of artifacts.

[0048] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this 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 ofPC17IB2025 / 000562modifications in various obvious respects, all without departing from thedisclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.BRIEF DESCRIPTION OF THE DRAWINGS

[0049] 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 “Figure” and “FIG.” herein), of which:

[0050] FIG. 1 depicts positioning uncertainty for systems as described herein where the system comprises antagonistically driven electrodes with no reference voltage or grounding of the droplet, such that the equilibrium position will try to equalize as much projected area on activated electrodes as on deactivated electrodes.

[0051] FIG. 2 depicts an example of an electrode pattern of the present disclosure that can be used to move droplets of high and low contact-angles

[0052] FIG. 3 depicts exemplary electrode activation patterns for improving the localization of a droplet, as described herein.

[0053] FIG. 4 depicts examples of certain vibrational modes of a droplet of a high-contact-angle fluid (e.g., a solution of water) can increase the droplet’s apparent surface area above the electrodes and allow its movement on electrodes that would nominally be too large to resolve it.

[0054] FIG. 5 depicts a droplet with low surface tension drop (top) and higher surface tension drop (bottom) optimally agitated at 5 Hz and 47 Hz, respectively.

[0055] FIG. 6 depicts vigorous agitation accomplished for 150 pL, 100 pL, and 50 pL droplets with a checkerboard electrode activation pattern.

[0056] FIG. 7 depicts an exemplary application of DNA extraction using systems as disclosed including suspended beads before vibration-assisted aggregation of material and a pellet of beads and DNA has formed after vibration-assisted aggregation.

[0057] FIG. 8 depicts exemplary reactions of droplets in vibration where high contact angle droplets tend to experience greater response to vibration than droplets with lower contact angle.

[0058] FIG. 9 depicts electro-mechanical actuators as disclosed herein where the actuator may produce a gradient in the vibration energy across the length of the surface, or the whole surface may be translated vertically.

[0059] FIG. 10 depicts additional embodiments of electro-mechanical actuators.

[0060] FIG. 11 depicts an exemplary bead-molding embodiment where droplets are moved (i.e., relative to a bird's eye view) to force beads into tighter pellets.

[0061] FIG. 12 depicts an exemplary representation of a poloxamer triblock copolymer structure.

[0062] FIG. 13 depicts an exemplary representation of poloxamer-droplet interactions which stabilize at the droplet-substrate interface and stop the hydrophilic biomolecules from reaching the substrate.

[0063] FIG. 14 depicts results of a pacer test that measures droplet viscosity via successive testing of increasing rates of electro wetting movement (increasing force), where 1 cSt = 1 mm2 / s.

[0064] FIG. 15 shows an embodiment of efficiently coupling the actuation force of the electromechanical actuator into droplet vibration and, ultimately, to effective mixing.

[0065] FIG. 16 depicts a computer system that is programmed or otherwise configured to implement methods provided herein.DETAILED DESCRIPTION

[0066] While various embodiments of the 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. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0067] The present disclosure provides devices and methods for droplet operations on a surface. In some embodiments, the surface is overlying an electrowetting array. In some embodiments, the droplet comprises at least one biological sample. In some embodiments, the droplet is process for a biological application, e.g., high molecular weight extraction, hybridization, amplification, sequencing, or any other biological application. As used herein, an "array of electrodes" denotes a plurality of electrodes arranged on a substantially planar fashion and doesn't imply any particular geometry including a square, or regular arrangement. Array is used interchangeably with "grid" and can include triangular, hexagonal, or any shape or arrangement.PC17IB2025 / 000562Sample preparation

[0068] The systems and methods described herein can be used to transform biological samples (e.g., blood, tissue, environmental samples) into an enriched sample (e.g., DNA) suitably prepared for analysis (e.g., DNA sequencing). The methods can involve manipulation of aqueous samples, in the form of droplets, on an EWOD surface. The droplets can contain "artifacts", which as used herein, are solid materials that can be suspended in, but are not necessarily soluble in, the aqueous droplet. Examples of artifacts can include magnetic beads, cells, or cellular lysate. In some cases, the magnetic beads are used to bind to and aggregate certain DNA molecules (e.g., by hybridization to a probe on the magnetic beads).Apparatus

[0069] Systems as provided herein may comprise an electrowetting device. The electrowetting device may be used to move individual droplets of water (or other aqueous, polar, or conducting solution) from place to place. The surface tension and wetting properties of water may be altered by electric field strength using the electrowetting effect. The electrowetting effect arises from the change in solid-electrolyte contact angle due to an applied potential difference between the solid and the electrolyte. Differences in wetting surface tension that vary over the width of the droplet, and corresponding change in contact angle, may provide motive force to cause the droplets to move, without moving parts or physical contact. Electrowetting device may include a grid of electrodes with a dielectric layer (e.g. , a dielectric described elsewhere herein) with appropriate electrical and surface priorities overlaying electrodes, all laid on a substrate. The substrate may be insulating. The substate may be rigid. Additional description of apparatus that can be suitable for performing the methods described herein can be found in U.S. Patent Serial No. 11,123,729, U.S. Patent Application Serial No. 18 / 061,133, U.S. Patent Application Serial No. 18 / 462,365, U.S. Patent Application Serial No. 18 / 628,148, PCT Patent Application Serial No. US2023 / 068,630, and PCT Patent Application Serial No. US2023 / 068,253, each of which is incorporated by reference herein in its entirety. In some cases, these apparatus are modified (e.g., by inclusion of magnets) in order to perform the methods described herein.Electrowetting-based control of droplets

[0070] The methods described herein can use selective activation of electrodes on a substantially planar array to manipulate a fluidic drop on a surface. The activation can be in patterns thatPC17IB2025 / 000562change over time and space in order to localize a droplet to a location, prevent pinning of a droplet at a particular location, to move a droplet from one location to another, or to agitate a droplet (e.g., for purposes of mixing the droplet or facilitating aggregation of material within a droplet).

[0071] Under the paradigm for open-faced electrowetting on dielectric, droplets are situated atop electrodes that may be at a variety of potentials. The droplets minimize their energy state when situated halfway over a region of one charge and halfway over a region with another opposing charge. The droplet serves in this case as a dielectric in a “capacitive circuit”, easing the passage of electric field lines through the region. Thus, the droplets “prefer” to be situated with half of their area over electrodes that are “activated”, and they will generally move into a position that satisfies this condition if they are not in one.Localization of droplets

[0072] Spatial electrode activation patterns can be used to improve the localization of droplets on the EWOD grid. When driving the active electrodes with high frequency (>100 Hz) alternating electric field, drops are attracted to the boundary between inactive and active electrodes. This is because the droplets are driven towards being 50% on activated and 50% on deactivated electrodes and may spread to increase their surface area in approaching this objective. For simple contiguous electrode patterns this can result in droplets not having a well determined location. For example, with a 2x2 electrode active grid, a droplet will localize itself anywhere along the perimeter of the 2x2 grid (see, FIG. 1).

[0073] With reference to FIG. 1, depicted here is droplet positioning uncertainty for a 2x2 EWOD grid. The system comprises antagonistically driven electrodes with no reference voltage or grounding of the droplet. Activated electrodes 100 are shown in a darker shade than nonactivated electrodes 102. The equilibrium position of a droplet will try to equalize as much projected area on activated electrodes as on deactivated electrodes. Here, four droplet positions 104 are equally favorable, resulting in uncertainty of the actual droplet position that might be achieved.

[0074] An alternative is to use a spatial electrode pattern that is more determinative of the localization of the droplet with respect to the active electrodes. Following the design principles described herein leads to the design of a variety of useful patterns of activated electrodes thatcan be used to locate droplets. One such embodiment of this involves a cross shape with alternating activation of the vertical and horizontal element.

[0075] In another embodiment, shown in FIG. 2 is an electrode pattern that can be used to move droplets of high and low contact-angles. This “T” pattern is useful for its ability to hold droplets of varying volumes in a consistent manner. Here, the droplet 200 is on a grid of activated and deactivated 202 electrodes. Successive activation of a first 204 and second 206 electrode in a perpendicular direction to the first activated electrodes can hold successively larger droplets in place. This T-pattem can also be used as a portion (i.e., at one time point) of a time -variant pattern that moves a droplet in a direction of the first and second successively activated electrodes.

[0076] Other embodiments may involve diagonal crosses (see, FIG.3). In some instances, electrowetting is more efficient when pulling droplets, rather than pushing. An activated line of electrodes can pull a droplet in a motion roughly perpendicular to the line. If that line is temporally alternated with another perpendicular line of activated electrodes, the droplet will also have the same effect and be combined. Here, the droplet will be pulled towards the center of the two alternating lines, resulting in localization.

[0077] FIG. 3 depicts such electrode activation patterns for improving the localization of a droplet.In this figure, the left column of figures 300 shows a droplet located on a 2x2 grid 302, on a 3x3 grid 304, and on a 4x4 grid 306, where the activated electrodes are shown in gray 308.However, the electrodes are not uniformly activated. As shown in the second column 310 of FIG. 3, alternating activation of elements are used. For example, in the 2x2 configuration, separate diagonal elements 312 and 314 are activated, resulting in, overall, holding the droplet 302 on the 2x2 element. Similarly, for the 3x3 configuration a cross shape with alternating activation of vertical 316 and horizontal 318 elements is used. Finally, for the 4x4 array, a diagonal cross with alternating activation of the diagonal elements 310 and 322 are used.

[0078] The localization methods described herein may be used to localize droplets. The method may comprise providing a droplet on an electro wetting array comprising a plurality of electrodes. A subset of the plurality of electrodes may comprise one or more adjacent electrodes forming a field on the surface comprising a discrete location. The one or more adjacent electrodes may be activated or deactivated in a pattern to immobilize the droplet. The pattern may be a cross shape. The horizontal and vertical elements of the electrode may be alternatively activated. The diameter of the discrete location may be at least about 50 micrometers (pm) to atleast about 10 millimeters (mm). The diameter of the discrete location may at least about 50 gm, 60 gun, 70 gun, 80 gm, 90 gm, 100 gm, 200 gm, 300 gm, 400 gm, 500 gm, 600 gm, 700 gm, 800 gm, 900 gm, or more. The diameter of the discrete location may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mm.

[0079] Another aspect of the present disclosure provides a method of immobilizing a droplet at a discrete location, the method comprising: providing a droplet on an electrowetting array, the array comprising a plurality of electrodes, wherein a subset of the plurality of electrodes comprises one or more adjacent electrodes forming a field on the surface comprising the discrete location; and Activating or deactivating the one or more adjacent electrodes in a spatial and / or temporal pattern to immobilize the droplet at the discrete location. In some embodiments, the spatial pattern is a cross shape. In some embodiments, a horizontal and a vertical element of the plurality of electrodes are alternatively activated. In some embodiments, a diameter of the discrete location is less than 2 millimeters (mm). In some embodiments, a diameter of the discrete location comprises less than about 0.05 mm of error to about 2 mm of error. In some embodiments, a diameter of the discrete location comprises less than about 2 mm of error to about 1.75 mm of error, about 2 mm of error to about 1.5 mm of error, about 2 mm of error to about 1.25 mm of error, about 2 mm of error to about 1 mm of error, about 2 mm of error to about 0.75 mm of error, about 2 mm of error to about 0.5 mm of error, about 2 mm of error to about 0.25 mm of error, about 2 mm of error to about 0.1 mm of error, about 2 mm of error to about 0.05 mm of error, about 1.75 mm of error to about 1.5 mm of error, about 1.75 mm of error to about 1.25 mm of error, about 1.75 mm of error to about 1 mm of error, about 1.75 mm of error to about 0.75 mm of error, about 1.75 mm of error to about 0.5 mm of error, about 1.75 mm of error to about 0.25 mm of error, about 1.75 mm of error to about 0.1 mm of error, about 1.75 mm of error to about 0.05 mm of error, about 1.5 mm of error to about 1.25 mm of error, about 1.5 mm of error to about 1 mm of error, about 1.5 mm of error to about 0.75 mm of error, about 1.5 mm of error to about 0.5 mm of error, about 1.5 mm of error to about 0.25 mm of error, about 1.5 mm of error to about 0.1 mm of error, about 1.5 mm of error to about 0.05 mm of error, about 1.25 mm of error to about 1 mm of error, about 1.25 mm of error to about 0.75 mm of error, about 1.25 mm of error to about 0.5 mm of error, about 1.25 mm of error to about 0.25 mm of error, about 1.25 mm of error to about 0.1 mm of error, about 1.25 mm of error to about 0.05 mm of error, about 1 mm of error to about 0.75 mm of error, about 1 mm of error to about 0.5 mm of error, about 1 mm of error to about 0.25 mm of error, about 1 mm of error toPC17IB2025 / 000562about 0.1 mm of error, about 1 mm of error to about 0.05 mm of error, about 0.75 mm of error to about 0.5 mm of error, about 0.75 mm of error to about 0.25 mm of error, about 0.75 mm of error to about 0.1 mm of error, about 0.75 mm of error to about 0.05 mm of error, about 0.5 mm of error to about 0.25 mm of error, about 0.5 mm of error to about 0.1 mm of error, about 0.5 mm of error to about 0.05 mm of error, about 0.25 mm of error to about 0.1 mm of error, about 0.25 mm of error to about 0.05 mm of error, or about 0.1 mm of error to about 0.05 mm of error. In some embodiments, a diameter of the discrete location comprises less than about 2 mm of error, about 1.75 mm of error, about 1.5 mm of error, about 1.25 mm of error, about 1 mm of error, about 0.75 mm of error, about 0.5 mm of error, about 0.25 mm of error, about 0.1 mm of error, or about 0.05 mm of error. In some embodiments, a diameter of the discrete location comprises less than at least about 2 mm of error, about 1.75 mm of error, about 1.5 mm of error, about 1.25 mm of error, about 1 mm of error, about 0.75 mm of error, about 0.5 mm of error, about 0.25 mm of error, or about 0.1 mm of error. In some embodiments, a diameter of the discrete location comprises less than at most about 1.75 mm of error, about 1.5 mm of error, about 1.25 mm of error, about 1 mm of error, about 0.75 mm of error, about 0.5 mm of error, about 0.25 mm of error, about 0.1 mm of error, or about 0.05 mm of error. In some embodiments, the spatial pattern is a chessboard pattern. “Discrete location” as used herein may refer to the center of mass of the droplet.

[0080] Another aspect of the present disclosure provides a method of manipulating a droplet comprising: Providing an electrowetting array comprising a plurality of electrodes, wherein a subset of the plurality of electrodes is activated and another subset of the plurality of electrodes is deactivated, and wherein an activated electrode of the subset of the plurality of electrodes and a deactivated electrode of the another subset of the plurality of electrodes is adjacent; and providing a droplet on the electrowetting array, such that at least about 50% of the droplet is located on the activated electrode and at least about 50% of the droplet is located on the deactivated electrode. In some embodiments, the providing the droplet on the electrowetting array results in the droplet adjusting to cover a surface area above the activated electrode.Preventing droplet pinning

[0081] Counter-intuitively, the droplets can become not just localized but immobilized at a location.This can be undesirable for procedures that can involve subsequently moving the droplet.Without being held to any particular theory, it is believed that in the course of usingelectrowetting on dielectric to manipulate droplets, the buildup of static charges on and around the droplet, as well as direct contact between the droplet and the dielectric, can cause the droplet to become “pinned” and immobile. A method for mitigating this is to pulse the holding pattern underneath the droplet, activating the related electrodes at a low (<50%) duty cycle and thus reducing both the average downforce on the droplet and the rate of static buildup. In some embodiments, the duty cycle can be from about 50% to about 10%. In some embodiments, the duty cycle can be less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, or less than about 15%. In some embodiments, the duty cycle can be as low as about 10%.

[0082] Vibration can be useful for moving droplets. Certain vibrational modes of a droplet of a high-contact-angle fluid (e.g., a solution of water) can increase the droplet’s apparent surface area above the electrodes and allow its movement on electrodes that would nominally be too large to resolve it. As shown in FIG. 4, the droplet without vibration 400 becomes compressed 402 and spreads over a larger area of the surface 404 when vibrated 406. During the phase of vibration when the droplet is compressed against the tile its area is situated across multiple electrodes, allowing movement. This typically takes place at frequencies around 20-80Hz.Droplet mixing

[0083] The present disclosure provides systems and methods for electrowetting-based mixing of droplets. Mechanical agitation may also be used to mix one or more droplets. The mechanical agitation of droplets and puddles by, e.g., vibration or movement of a magnetic field may be capable of assisting with mixing of the droplets and puddles. However, electro wetting may also be employed in mixing droplets and puddles that are difficult to mix. Mixing can include resuspension of particles or artifacts (e.g., magnetic beads), within the drop optionally without the use of any mechanical agitation. This may be done by either exciting a resonant mode of the drop or by exciting a faraday wave within the puddle / droplet.

[0084] Electrowetting-based agitation and mixing may be accomplished by applying a temporally patterned and / or spatially patterned activation of electrodes in the electrode array. The temporal pattern of the electrode activation may include a single frequency of activation or multiple frequencies overlaid. The frequency that results in maximum agitation and mixing is dependent upon the fluid properties of the droplet, especially surface tension or viscosity. Droplets with low surface tension or high viscosity tend to respond more vigorously to lower frequency (<15Hz) stimulation, while drops with higher surface tension and low viscosity respond more vigorously at higher frequencies (>15 Hz). Droplets of greater size may response to lower frequencies while droplets of smaller size may respond to higher frequencies. At certain size ranges, for droplets of, e.g., water, frequencies in the range of 40 to 55 Hz may be most effective at producing a strong mixing response in the drop. At certain size ranges, for droplets of, e.g., 70% ethanol, frequencies in the range of 2 to 15 Hz may produce the strongest response (see, FIG. 5). As shown, FIG. 5 depicts a droplet with low surface tension drop 500 and higher surface tension drop 502 optimally agitated at 5 Hz and 47 Hz, respectively.

[0085] The electrode activation can also be spatially patterned in order to get a stronger mixing and agitation response. The spatial pattern of the active electrodes depends on the size of the drop being agitated. In general, the size of the active electrode should scale with the diameter of the drop. With reference to FIG. 6, to be able to effectively mix and agitate a range of different drop sizes with a single electrode activation pattern, a chessboard pattern 600 may be used. This pattern is relatively agnostic to drop size because it has a uniform number of activated and deactivated electrodes throughout the electrowetting array, thereby maximizing the perimeter between inactive and active electrodes, and allowing the droplet to spread easily no matter where it is positioned. As shown, FIG. 6 depicts vigorous agitation accomplished for 150 uL droplets 602, 100 uL droplets 604, and 50 pL 606 droplets with a checkerboard electrode activation pattern.

[0086] Another aspect of the present disclosure provides a method of selecting a frequency for vibrating a droplet on the surface of an electrowetting array comprising: a. determining a surface tension or viscosity of the droplet; and b. selecting a frequency of less than about 15 Hertz (Hz) for a droplet with lower surface tension or higher viscosity, and a frequency of greater than about 15 Hz for a droplet with higher surface tension or lower viscosity. In some embodiments, a frequency of about 40 Hz to at least about 55 Hz is selected for a droplet comprising water. In some embodiments, a frequency of about 2 Hz to at least about 15 Hz is selected for a droplet comprising ethanol or a surfactant.

[0087] Another aspect of the present disclosure provides a method of vibrating a droplet comprising: a. providing a substrate comprising a plurality of electrodes disposed beneath a surface of the substrate; b. providing the droplet on the substrate; c. activating a plurality of electrodes, wherein the plurality of electrodes is in a pattern of motion. In some embodiments, the resuspending comprises activating an electrode of the plurality of electrodes. In somePC17IB2025 / 000562embodiments, the activating a plurality of electrodes comprises a temporal pattern. In some embodiments, activating a plurality of electrodes excites a resonant mode of the droplet. In some embodiments, activating a plurality of electrodes comprises a spatial pattern. In some embodiments, the spatial pattern is a chessboard pattern. In some embodiments, the spatial pattern is agnostic to droplet size. In some embodiments, the spatial pattern is chosen to maximize perimeter between activated electrodes and inactivated electrodes. In some embodiments, a portion of the droplet is immobilized. In some embodiments, the droplet changes shape. In some embodiments, the electrodes are activated in a pattern comprising activation of a plurality of electrodes adjacent horizontally. In some embodiments, the plurality of electrodes is activated in a pattern. In some embodiments, the activation of the plurality of electrodes is sufficient to excite a resonance mode in the droplet but the activation of the electrodes is not sufficient to translate the droplet’s location. In some embodiments, the activation of the plurality of electrodes is sufficient to excite a near-resonance mode in the droplet but the activation of the electrodes is not sufficient to translate the droplet’s location.

[0088] Another aspect of the present disclosure provides a method of processing a droplet, wherein the droplet comprises a plurality of artifacts, the method comprising: a. providing a substrate comprising a plurality of electrodes disposed beneath a surface of the substrate; b. providing the droplet comprising the plurality of artifacts on the substrate; c. resuspending the plurality of artifacts without mechanical agitation. In some embodiments, the resuspending comprises activating an electrode of the plurality of electrodes. In some embodiments, the activating a plurality of electrodes comprises a temporal pattern. In some embodiments, activating a plurality of electrodes excites a resonant mode of the droplet. In some embodiments, activating a plurality of electrodes comprises a spatial pattern. In some embodiments, the spatial pattern is a chessboard pattern. 3 In some embodiments, the spatial pattern is agnostic to droplet size. In some embodiments, the spatial pattern is chosen to maximize perimeter between activated electrodes and inactivated electrodes. In some embodiments, a portion of the droplet is immobilized. In some embodiments, the droplet changes shape. In some embodiments, the electrodes are activated in a pattern comprising activation of a plurality of electrodes adjacent horizontally. In some embodiments, the plurality of electrodes is activated in a pattern. In some embodiments, the activation of the plurality of electrodes is sufficient to excite a resonance mode in the droplet but the activation of the electrodes is not sufficient to translate the droplet’s location. In some embodiments, the activation of the plurality of electrodes is sufficient to excitea near-resonance mode in the droplet but the activation of the electrodes is not sufficient to translate the droplet’s location.Magnets

[0089] Systems as disclosed herein may comprise a droplet on a surface, wherein the surface is configured to support the droplet. The droplet may comprise an artifact. The artifact may be at least one bead formed of a material configured to couple to a magnetic field. A magnet may be configured to supply a magnetic field. An actuator may be operatively coupled to the magnet, and the actuator may be configured to subject the magnetic field to translation along a plane parallel to the surface. A controller may be operatively coupled to the actuator. The controller may direct the actuator to subject the magnetic field to translation along the plane, such that while the magnetic field translates along the plane, the droplet undergoes motion along the surface. The actuator can be a switch. The actuator can comprise motor coupled to the magnet, wherein the motor is configured to translate the magnet along a direction parallel to the surface. In some embodiments, the system further comprises an electrode configured to supply an electric field to the surface, wherein the electric field and the magnetic field are sufficient to subject the droplet to the motion. In some embodiments, the actuator is configured to motion the magnet to translate along at least two axes parallel to the plane, and optionally in a third axis perpendicular to the first two axis (i.e., z-direction). In some embodiments, the magnetic comprises a permanent magnet. In some embodiments, the magnet comprises at least one electromagnet. In some embodiments, the actuator comprises a pivot, wherein the pivot is coupled to the surface. In some embodiments, the surface comprises a dielectric (e.g., a dielectric described elsewhere herein) disposed over one or more electrodes. In some embodiments, the one or more magnets are disposed below the surface. In some embodiments, the surface comprises a liquid layer. In some embodiments, the liquid layer comprises a liquid comprising an affinity for the surface.

[0090] Systems as disclosed herein may comprise a liquid layer adjacent to a dielectric layer (e.g., a dielectric described elsewhere herein) and a plurality of electrodes. The present disclosure provides a system for inducing motion in a droplet, comprising: (a) a surface configured to support the droplet comprising at least one bead formed of a material configured to couple to a magnetic field; (b) an actuator coupled a magnet, wherein the magnet is configured to supply the magnetic field, and wherein the actuator is configured to subject the magnetic field totranslation along a plane parallel to the surface; and (c) a controller operatively coupled to the actuator, wherein the controller is configured to direct the actuator to subject the magnetic field to translation along the plane, such that while the magnetic field translates along the plane, the droplet undergoes motion along the surface. In some embodiments, the actuator is a switch. In some embodiments, the actuator comprises a motor coupled to the magnet, wherein the motor is configured to translate the magnet along a direction parallel to the surface. In some embodiments, the system further comprises an electrode configured to supply an electric field to the surface, wherein the electric field and the magnetic field are sufficient to subject the droplet to the motion. In some embodiments, the actuator is configured to motion the magnet to translate along at least two axes parallel to the plane. In some embodiments, the magnetic comprises a permanent magnet. In some embodiments, the magnet comprises at least one electromagnet. In some embodiments, the actuator comprises a pivot, wherein the pivot is coupled to the surface. In some embodiments, the surface comprises a dielectric (e.g., a dielectric described elsewhere herein) disposed over one or more electrodes. In some embodiments, the one or more magnets are disposed below the surface. In some embodiments, the surface comprises a liquid layer. In some embodiments, the liquid layer comprises a liquid comprising an affinity for the surface. The system may include a magnetic bead-based separation unit for DNA size selection, DNA purification, protein purification, plasmid extraction and any other biological workflow that uses magnetic beads. The device may perform a number of simultaneous magnetic bead-based operations - from one to a million on a single chip.Dielectric

[0091] Systems as disclosed herein may comprise a droplet on a surface, wherein the surface is configured to support the droplet. In some embodiments, the surface comprises a dielectric (or dielectric layer) disposed over one or more electrodes. In some embodiments, the dielectric is a dielectric film. In some embodiments, the dielectric comprises a polymeric material. In some embodiments, the dielectric comprises a natural polymeric material, a synthetic polymeric material, a fluorinated material, a surface modification, or any combination thereof. In some embodiments, the natural polymeric material comprises shellac, amber, wool, silk, natural rubber, cellulose, wax, chiton, or any combination thereof. In some embodiments, the synthetic polymeric material comprises polyethylene,PC17IB2025 / 000562polypropylene, polystyrene, poly etheretherketone (PEEK), polyimide, polyacetal, polysilfone, polyphenulene ether, polyphenylene Sulfide (PPS), polyvinyl chloride, synthetic rubber, neoprene, nylon, polyacrylonitrile, polyvinyl butyral, silicone, parafilm, polyethylene terephthalate, polybutylene terephthalate, polyamides, polyoxymethlyene, polycarbonate, polymethylpentene, polyphenylene oxide (Polyphenyl ether), polyphthalamide (PPA), polylactic acid, synthetic cellulose ethers (e.g., methyl cellulose, ethyl cellulose, propyl cellulose, hydroxyethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose (HPC), hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose (HPMC), ethyl hydroxyethyl cellulose), paraffins, microcrystalline wax, epoxy, or any combination thereof. In some embodiments, the fluorinated material comprises polytetrafluoroethylene (PTFE), tetrafluoroethylene (TFE), fluorinated ethylenepropylene copolymer (FEP), polyvinylidene fluoride (PVDF), perfluoroalkoxytetrafluoroethylene copolymer (PF A), perfluoromethyl vinylether copolymer (MFA), ethylenechlorotrifluoroethylene copolymer (ECTFE), ethylene -tetrafluoroethylene copolymer (ETFE), perfluoropolyether (PFPE), polychlorotetrafluoroethylene (PCTFE), or any combination thereof. In some embodiments, the surface modification comprises silicone, silane, fluoro-polymer treatment, parylene coating, any other suitable surface chemistry modification process, ceramic, clay minerals, bentonite, kaolinite, vermiculite, graphite, molybdenum disulfide, mica, boron nitride, sodium formate, sodium oleate, sodium palmitate, sodium sulfate, sodium alginate, or any combination thereof.

[0092] In some embodiments, the surface comprises a liquid layer adjacent to the dielectric and a plurality of electrodes. In some embodiments, the liquid layer comprises silicone oils, fluorinated oils, ionic liquids, mineral oils, ferrofluids, polyphenyl ether, vegetable oil, esters of saturated fatty and dibasic acids, grease, fatty acids, triglycerides, polyalphaolefin, polyglycol hydrocarbons, other Non-hydrocarbon synthetic oils, or any combination thereof. In some embodiments, the liquid layer comprises surfactants, electrolytes, rheology modifier, wax, graphite, graphene, molybdenum disulfide, PTFE particles, or any combination thereof.Vibration

[0093] Liquid droplets can be mixed in a variety of methods. The present disclosure provides methods by which vibration of a digital micro fluidic surface can be used to assist in the mixingPC17IB2025 / 000562of liquids on the surface of the digital microfluidic device. The vibration may produce small- scale fluidic motion within a droplet on the surface of the digital micro fluidic device. The motion may encourage diffusion and rapidly speed up the mixing process. The result is, e.g., efficient capture of the DNA onto the magnetic microparticles and ultimately to a higher yield DNA extraction. In some embodiments, an electrowetting array comprising an open surface is provided.

[0094] Vibration based mixing is synergistic with electrowetting based mixing. While vibration mixing is effective at dispersing particles within portions of a liquid droplet, it is often less effective at macro-scale mixing across the entire droplet, especially for droplets with low contact angle with the surface. Electro wetting -based droplet mixing helps address this problem and with both vibration and electrowetting acting together, mixing of a wide variety of droplets of various compositions can be accomplished rapidly and effectively.

[0095] A common problem with digital micro fluidics platforms is achieving robust mixing with all varieties of reagents and droplets. Highly viscous liquid droplets, for example, can be extremely difficult to mix effectively using a purely electrowetting based motion. These kinds of viscous droplets are important in a wide range of applications including DNA extraction from highly concentrated sample material where DNA needs to be efficiently bound to magnetic beads. Using purely electrowetting based motion to mix in these applications results in very poor mixing and therefore very poor DNA extraction from the sample droplet.

[0096] Implementation of devices, systems, and methods by which vibration and / or application of acoustic forces to the digital micro fluidic surface can be used to assist in the mixing of liquids on the surface is described herein. The vibration, when tuned to the appropriate frequency and amplitude, produces small-scale fluidic motion within the droplet that encourages diffusion and rapidly speeds up the mixing process. FIG. 7 illustrates one such application of this technique for the extraction of DNA using magnetic beads. In FIG. 7, the viscosity of the DNA sample inhibits efficient mixing unless vibration is used. While using mixing, efficient capture of the DNA onto the magnetic microparticles is achieved, ultimately resulting in a higher yield DNA extraction. The upper image 700 shows suspended beads 702 and DNA 704 in the droplet 706 before vibration-assisted mixing (i.e., aggregation of material). While the lower figure 708 shows a pellet 710 of beads and DNA has formed after vibration-assisted mixing on an EWOD surface 712. The mixing can be performed by moving 714 the substrate 716. As used herein, "mixing" does not necessarily imply making a sample more homogenous. In fact, the samplecan become less homogenous when mixing, e.g., if a magnetic force is used to aggregate magnetic beads.

[0097] Vibration also contributes to enhanced mobility of droplets. This is especially true for droplets that contain particulates. Without vibration, large particles can tend to settle at the interface between the droplet and the substrate. When these particles are present at the droplet’s contact line they can act to pin the droplet in place, restricting its mobility. The introduction of vibration can help keep particles from settling at the contact line and, in doing so, greatly improves the reliability of electro wetting mobility of particulate-carrying droplets.

[0098] Vibration also contributes to controlled immobility of droplets. The present disclosure provides systems and methods for immobilizing droplets. The method may comprise providing a droplet on an electro wetting array comprising a plurality of electrodes. A subset of the plurality of electrodes may comprise one or more adjacent electrodes forming a field on the surface comprising a discrete location. The one or more adjacent electrodes may be activated or deactivated in a pattern to immobilize the droplet. The pattern may be a cross shape. The horizontal and vertical elements of the electrode may be alternatively activated. The diameter of the discrete location may be at least about 50 micrometers (pm) to at least about 10 millimeters (mm). The diameter of the discrete location may at least about 50 pm, 60 pm, 70 pm, 80 pm, 90 pm, 100 pm, 200 pm, 300 pm, 400 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, or more. The diameter of the discrete location may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mm.

[0099] The vibration frequency and amplitude may need to be tuned to the droplet and the system dynamics. The resonant dynamics of the droplet depends on a number of factors including the volume, surface tension, density, and viscosity of the droplet. Droplets with higher contact angles tend to exhibit a greater response to the vibration while droplets that spread more readily on the surface (and have a lower contact angle) require greater amplitude to achieve comparable mixing. With reference to FIG. 8, high contact angle droplets 800 tend to experience greater response to vibration than droplets with lower contact angle 802. In order to achieve sufficient mixing of droplets using vibration, the entire digital micro fluidics device or parts of it can be displaced anywhere from a few micrometers to few millimeters. A displacement between 0.1 mm and 10mm is a suitable range for this purpose. In the vibration assisted mixing schemes described above, typical frequencies of vibrations range from 1 Hz to 20 kHz. In some cases, the droplets shown in FIG. 8 have different volumes and resonant modes. Droplets with higherPC17IB2025 / 000562contact angles may exhibit a greater response to the vibration while droplets that spread more readily on the surface (and have a lower contact angle) may require greater amplitude to achieve comparable mixing. With continued reference to FIG. 8, the substrate 804 has an electrode grid 806 disposed thereon. The electrode surface 808 can be made slippery for the movement of fluid thereon, as described herein. The substate can be vibrated 810 to produce a resonant mode 812 in the droplet.

[0100] Beyond vibration, in some embodiments, other methods to mix difficult-to-mix droplets may be used. If assistance is needed with resuspending magnetic beads, an alternating magnetic field may be used to resuspend and mix magnetic beads within a droplet. This may be accomplished with the use of rotating permanent magnets or with electromagnetic coils oriented in multiple axes around the droplet. Using the electrode grid itself, it is possible to mix the droplet by exciting a resonance using an alternating current circuit that oscillates the voltage on electrodes beneath the droplet. This actuation may benefit from having a low impedance path to the droplet itself in order to increase the magnitude of the response.

[0101] The present disclosure provides methods of electrowetting-based droplet mixing. In some embodiments, electrowetting-based droplet mixing comprises both vibration and electro wetting. In some embodiments, vibration and electrowetting act together and mix a wide variety of droplets. In some embodiments, vibration and electrowetting of various compositions may be accomplished rapidly and effectively. The frequency for electro wetting-based droplet mixing may be modulated. The amplitude for electrowetting-based droplet mixing may be modulated.

[0102] The vibration of the digital microfluidic surface may be accomplished through a number of means. In one embodiment, the surface itself may be used as a spring element whereby one end of the surface is fixed while the other is attached to an electro-mechanical actuator. With reference to FIG. 9, in some embodiments, the electro-mechanical actuator is an oscillating mechanism or cantilever 900. This produces a gradient in the vibration energy across the length of the surface. Droplets positioned closer to the vibrating edge 902 of the cantilever will experience much greater amplitude than those closer to the fixed end 904. For example, electromagnetic actuators, voice coil actuators, piezoelectric actuators, ultrasonic transducers, rotating eccentric masses, motor driven linkage, and brushed / brushless / stepper motors with oscillating linkage mechanisms may be used.

[0103] In another embodiment 906, the whole surface is translated vertically 908. This may be accomplished with an electro-mechanical actuator comprising various flexible elements 910PC17IB2025 / 000562(e.g. a linear flexure) or with bearings. This can produce uniform vibration amplitude across the entire surface 912 having the EWOD electrodes 914 disposed thereon (assuming a sufficiently rigid substrate is used). In some embodiments, one or more springs and / or other shock absorbers can be positioned beneath the surfaces described herein to facilitate vibration application to the surface via whole surface vertical translation.

[0104] The vibration mechanisms of constraining the motion of the surface described herein may be actuated through a number of different methods including electromagnetic actuators, piezoelectric actuators, ultrasonic transducers, rotating eccentric masses, as well as brushed / brushless / stepper motors with oscillating linkage mechanisms (Figure 13A).

[0105] Electromagnetic voice coil actuators can be actuated at a wide range of frequencies and amplitudes and these can be controlled independently. Additionally, a variety of waveforms can be used to excite the actuator to achieve different effects. A sine wave can be used, for example, to produce quiet oscillation while a square wave may be used to excite the surface much more aggressively.

[0106] The embodiments depicted in FIG. 10 result in a dynamic system that can be characterized in order to efficiently couple the actuation force into droplet vibration and ultimately to effective mixing (as shown in FIG. 15). The resulting dynamic system may be modified or augmented through the use of external elements such as passive springs, dampers, or masses. These elements can be used, for example, to shift the resonant frequency of the system to one that is aligned with a resonant frequency of the droplets on the surface. This can be done passively or actively (with actuated spring elements). In some embodiments a disposable widget may be attached to the system between the surface and the system to modify the system for greater stability and reliable performance. This widget may be a sponge clip and may facilitate the vibration of the surface within the system.

[0107] In some embodiments the system may be leveled by the user through the use of a digital leveling interface. This digital leveling may increase the functionality of the vibrational mixing and decrease the occurrence of vibrational induced droplet splitting. This leveling interface may instruct the user on how to properly level the system and may ensure that it has been properly leveled through a leveling module configured to detect an angle of the surface.

[0108] Turning to FIG. 10, a voice coil actuator 1000 may be used to generate vibration 1002. The voice coil actuator may comprise a permanent magnetic field assembly 1004 and a coil assembly 1006. The voice coil actuator may be actuated at a wide range of frequencies andamplitudes. Such an embodiment can have an air gap 1008 and a flux path 1010. The voice coil actuator may be excited via a variety of waveforms. A sine wave can be used, for example, to produce quiet oscillation while a square wave may be used to excite the surface much more aggressively.

[0109] In some embodiments, a motor driven linkage 1012 may be used to generate vibration. A conventional brushless, brushed, or stepper motor 1014 may used to turn a shaft 1016. The shaft may be connected to a rigid-body or flexural linkage, which when turned, results in oscillatory motion 1018 of the output. The shaft can turn an eccentric shaft 1020 which turns around a bearing 1022. The motor driven linkage may be augmented with, for example, passive spring, mass, and damping elements, to improve efficiency and allow for larger amplitude oscillations with less input power. For example, spring elements may be placed between the output of the linkage and the oscillating substrate. This embodiment is much less dependent on the system dynamics of the surface and motion constraint mechanisms but, as a result, may require more power input in order to achieve equivalent vibration output to a well-tuned dynamic system.

[0110] In some embodiments, a rotating eccentric mass may be used to generate vibration. The rotating eccentric mass may be off-center from the point of rotation. A motor may be mounted to an oscillating substrate (either directly or through coupling springs). The motor may spin an offset mass to create an oscillating acceleration. The operation of the rotating eccentric mass may cause an uneven centripetal force, which may in turn cause the motor to move backwards and forwards. The acceleration amplitude and frequency may be directly linked.[OHl] While the resonant frequency of the system may be excited at or close to the resonant peak in order to achieve best efficiency between input and output power. It may also be advantageous in some embodiments to excite the system far from the resonant frequency. This may be beneficial, for example, if it is desirable to excite the droplet with a roughly equivalent amplitude across a wide range of frequencies. This particular case can be readily achieved by tuning the system’s natural frequency to be low relative to the desired frequency range and results in a near constant acceleration amplitude across a broad frequency range.

[0112] In another embodiment, a closed loop control can be utilized for fine control of amplitude independently from frequency. This may be accomplished, for example, with the use of an accelerometer mounted to the vibrating platform. A microcontroller may communicate with the sensor and calculate the acceleration amplitude in real time. Given some desired accelerationamplitude, the microcontroller can adjust the amplifier gain and modulate the drive waveform of the vibration actuator in order to precisely control the vibration amplitude.Bead molding

[0113] The present disclosure provides devices and methods for manipulating one or more droplets.The one or more droplets may comprise one or more artifacts. The one or more artifacts may be beads. In bead-based biological assays, electrowetting forces can be used to separate droplets from beads. The droplets may be provided on a device. The droplets may be provided on the surface of a dielectric substrate (e.g., a dielectric described elsewhere herein). During separations, beads may be suspended in a droplet to be pelleted together. The beads may be pelleted together using a magnet as described herein. The beads may be pelleted using a magnet coupled to an actuator through techniques described herein. The magnet may be adjacent to the droplet. The magnet may be adjacent to a dielectric substrate. The magnet may be beneath the dielectric substrate. The magnet may be above the surface of the dielectric substrate. The geometry of the pellet may affect the electrowetting force required to successfully remove the droplet from the bead pellet.

[0114] The smaller the radius of the bead pellet, the easier it can be to separate the droplet from the pellet. As described herein, one can use the contact line of the droplet on the surface to force bead pellets into a tighter pellet with smaller radii. One such embodiment, referred to as beadmolding is shown in FIG. 11. Here, the droplet 1100 containing a bead 1102 may be moved in any suitable pattern to push the beads into a tighter pellet using the periphery (contact line) of the droplet. The pattern in FIG. 11, includes moving to the right (denoted 1) and up and down repeatedly (denoted 2, 3, 4), however the exact pattern can be varied.

[0115] In some cases, the droplet is moved in an “H” pattern such that droplets containing a bead pellet will be stretched to the right, moved up and down repeatedly, stretched to the left, and then again moved up and down repeatedly. This stretch and subsequent movement tensions the contact line of the droplet against the bead pellet, molding it into a tighter circle with a smaller radius thus making the subsequent droplet separation achievable with less electro wetting force. In addition to “H”-molding, other droplet movement patterns may be used to produce similar outcomes such as “©’’-molding or “X”-molding, where the droplet is moved in a wide circle or a cross shape, respectively.PC17IB2025 / 000562

[0116] Aspects of the present disclosure provides a method of processing a droplet comprising a plurality of artifacts, the method comprising: a. providing a substrate comprising a plurality of electrodes disposed beneath a surface of the substrate; b. providing the droplet comprising the plurality of artifacts on the substrate; c. activating one or more electrodes of the plurality of electrodes to induce a pattern of motion of the droplet, wherein the pattern of motion is sufficient to reduce a radius of the shape of the plurality of artifacts. In some embodiments, the pattern of motion comprises: moving the droplet with respect to an x-axis; subsequently moving the droplet with respect to a y-axis at least two times; subsequently moving the droplet with respect to an x-axis. In some embodiments, the pattern of motion is executed in proximity of a magnetic field. In some embodiments, the pattern of motion is in a proximity of the magnetic field such that the proximity is sufficient to exert the magnetic field onto at least a subset of artifacts of the plurality of artifacts. In some embodiments, the subset of artifacts comprises at least about 30 % of the plurality of artifacts to about 99 % of the plurality of artifacts. In some embodiments, the subset of artifacts comprises at least about 30 % of the plurality of artifacts to about 40 % of the plurality of artifacts, about 30 % of the plurality of artifacts to about 50 % of the plurality of artifacts, about 30 % of the plurality of artifacts to about 60 % of the plurality of artifacts, about 30 % of the plurality of artifacts to about 70 % of the plurality of artifacts, about 30 % of the plurality of artifacts to about 75 % of the plurality of artifacts, about 30 % of the plurality of artifacts to about 80 % of the plurality of artifacts, about 30 % of the plurality of artifacts to about 85 % of the plurality of artifacts, about 30 % of the plurality of artifacts to about 90 % of the plurality of artifacts, about 30 % of the plurality of artifacts to about 95 % of the plurality of artifacts, about 30 % of the plurality of artifacts to about 97 % of the plurality of artifacts, about 30 % of the plurality of artifacts to about 99 % of the plurality of artifacts, about 40 % of the plurality of artifacts to about 50 % of the plurality of artifacts, about 40 % of the plurality of artifacts to about 60 % of the plurality of artifacts, about 40 % of the plurality of artifacts to about 70 % of the plurality of artifacts, about 40 % of the plurality of artifacts to about 75 % of the plurality of artifacts, about 40 % of the plurality of artifacts to about 80 % of the plurality of artifacts, about 40 % of the plurality of artifacts to about 85 % of the plurality of artifacts, about 40 % of the plurality of artifacts to about 90 % of the plurality of artifacts, about 40 % of the plurality of artifacts to about 95 % of the plurality of artifacts, about 40 % of the plurality of artifacts to about 97 % of the plurality of artifacts, about 40 % of the plurality of artifacts to about 99 % of the plurality of artifacts, about 50 % of the plurality of artifacts toPC17IB2025 / 000562about 60 % of the plurality of artifacts, about 50 % of the plurality of artifacts to about 70 % of the plurality of artifacts, about 50 % of the plurality of artifacts to about 75 % of the plurality of artifacts, about 50 % of the plurality of artifacts to about 80 % of the plurality of artifacts, about 50 % of the plurality of artifacts to about 85 % of the plurality of artifacts, about 50 % of the plurality of artifacts to about 90 % of the plurality of artifacts, about 50 % of the plurality of artifacts to about 95 % of the plurality of artifacts, about 50 % of the plurality of artifacts to about 97 % of the plurality of artifacts, about 50 % of the plurality of artifacts to about 99 % of the plurality of artifacts, about 60 % of the plurality of artifacts to about 70 % of the plurality of artifacts, about 60 % of the plurality of artifacts to about 75 % of the plurality of artifacts, about 60 % of the plurality of artifacts to about 80 % of the plurality of artifacts, about 60 % of the plurality of artifacts to about 85 % of the plurality of artifacts, about 60 % of the plurality of artifacts to about 90 % of the plurality of artifacts, about 60 % of the plurality of artifacts to about 95 % of the plurality of artifacts, about 60 % of the plurality of artifacts to about 97 % of the plurality of artifacts, about 60 % of the plurality of artifacts to about 99 % of the plurality of artifacts, about 70 % of the plurality of artifacts to about 75 % of the plurality of artifacts, about 70 % of the plurality of artifacts to about 80 % of the plurality of artifacts, about 70 % of the plurality of artifacts to about 85 % of the plurality of artifacts, about 70 % of the plurality of artifacts to about 90 % of the plurality of artifacts, about 70 % of the plurality of artifacts to about 95 % of the plurality of artifacts, about 70 % of the plurality of artifacts to about 97 % of the plurality of artifacts, about 70 % of the plurality of artifacts to about 99 % of the plurality of artifacts, about 75 % of the plurality of artifacts to about 80 % of the plurality of artifacts, about 75 % of the plurality of artifacts to about 85 % of the plurality of artifacts, about 75 % of the plurality of artifacts to about 90 % of the plurality of artifacts, about 75 % of the plurality of artifacts to about 95 % of the plurality of artifacts, about 75 % of the plurality of artifacts to about 97 % of the plurality of artifacts, about 75 % of the plurality of artifacts to about 99 % of the plurality of artifacts, about 80 % of the plurality of artifacts to about 85 % of the plurality of artifacts, about 80 % of the plurality of artifacts to about 90 % of the plurality of artifacts, about 80 % of the plurality of artifacts to about 95 % of the plurality of artifacts, about 80 % of the plurality of artifacts to about 97 % of the plurality of artifacts, about 80 % of the plurality of artifacts to about 99 % of the plurality of artifacts, about 85 % of the plurality of artifacts to about 90 % of the plurality of artifacts, about 85 % of the plurality of artifacts to about 95 % of the plurality of artifacts, about 85 % of the plurality of artifacts to about 97 % of the plurality ofPC17IB2025 / 000562artifacts, about 85 % of the plurality of artifacts to about 99 % of the plurality of artifacts, about 90 % of the plurality of artifacts to about 95 % of the plurality of artifacts, about 90 % of the plurality of artifacts to about 97 % of the plurality of artifacts, about 90 % of the plurality of artifacts to about 99 % of the plurality of artifacts, about 95 % of the plurality of artifacts to about 97 % of the plurality of artifacts, about 95 % of the plurality of artifacts to about 99 % of the plurality of artifacts, or about 97 % of the plurality of artifacts to about 99 % of the plurality of artifacts. In some embodiments, the subset of artifacts comprises at least about 30 % of the plurality of artifacts, about 40 % of the plurality of artifacts, about 50 % of the plurality of artifacts, about 60 % of the plurality of artifacts, about 70 % of the plurality of artifacts, about 75 % of the plurality of artifacts, about 80 % of the plurality of artifacts, about 85 % of the plurality of artifacts, about 90 % of the plurality of artifacts, about 95 % of the plurality of artifacts, about 97 % of the plurality of artifacts, or about 99 % of the plurality of artifacts. In some embodiments, the subset of artifacts comprises at least at least about 30 % of the plurality of artifacts, about 40 % of the plurality of artifacts, about 50 % of the plurality of artifacts, about 60 % of the plurality of artifacts, about 70 % of the plurality of artifacts, about 75 % of the plurality of artifacts, about 80 % of the plurality of artifacts, about 85 % of the plurality of artifacts, about 90 % of the plurality of artifacts, about 95 % of the plurality of artifacts, or about 97 % of the plurality of artifacts. In some embodiments, the subset of artifacts comprises at least at most about 40 % of the plurality of artifacts, about 50 % of the plurality of artifacts, about 60 % of the plurality of artifacts, about 70 % of the plurality of artifacts, about 75 % of the plurality of artifacts, about 80 % of the plurality of artifacts, about 85 % of the plurality of artifacts, about 90 % of the plurality of artifacts, about 95 % of the plurality of artifacts, about 97 % of the plurality of artifacts, or about 99 % of the plurality of artifacts. In some embodiments, the pattern of motion comprises moving the droplet in a circular motion, a rectangular motion, an X-shape, or a linear motion. In some embodiments, the magnetic field is generated by a magnet. In some embodiments, the magnet is displaced below the substrate. In some embodiments, the magnet is configured to translate with respect to the substrate. In some embodiments, the method further comprises removing the plurality of artifacts from the droplet. In some embodiments, the removing comprises: (i) actuating a movement of the droplet via EWOD; (ii) translating the magnet; or both. In some embodiments, one or more surfactants are added to the droplet prior to the removing of the plurality of artifacts.Biofouling

[0117] The present disclosure provides systems and methods for preventing biofouling. Droplets comprising biomolecules (proteins, DNA, RNA, etc.) being transported across a substrate through electro wetting may lose mobility over time due to the phenomenon of biofouling.Biomolecules could adsorb on the dielectric substrate of the electrowetting system and change the surface properties through biofouling. For example, proteins can adsorb to a hydrophobic substrate via hydrophobic interaction. When the substrate is fouled with biomolecules, the original properties of the virgin material may be lost and thus droplet mobility will cease.

[0118] The present disclosure provides systems and methods for using poloxamers or pluronics in droplets to prevent biofouling. Poloxamers (often referred to by the trade name Pluronic, see FIG. 12) can be added to a droplet to prevent biofouling. Poloxamers have a hydrophobic portion 1200 which allows the molecule to stabilize at the droplet-substrate interface and two or more hydrophilic groups 1202 which stop the hydrophilic biomolecules from reaching the substrate through molecular interactions.

[0119] With reference to FIG. 13, depicted here is an exemplary representation of poloxamer- droplet interactions. The pluronic layer 1300 stabilize at the interface between the droplet 1302 and the substrate 1304, thereby stopping the hydrophilic biomolecules from reaching and biofouling the substrate.

[0120] The present disclosure provides systems and methods for adding one or more droplets comprising one or more surfactants to a sample droplet (as described herein) to prevent biofouling. Surfactants may be characterized by their ability to manipulate a fluid. Surfactants may “break down” a fluid into one or more phases according to one or more properties. In the disclosed systems and methods, a liquid dispenser may dispense one or more droplets comprising one or more surfactants onto a sample droplet on an electrowetting array. The sample droplet may comprise one or more artifacts. The one or more artifacts may be one or more beads. The one or more beads may be configured to be responsive to a magnetic field as described herein. The one or more droplets comprising surfactant may be dispensed at a distance from the sample droplet, and these droplets can be contacted with one another using, e.g., magnetic field or electrowetting-on-dielectric (EWOD). The droplets may be mixed using, e.g., vibration as described herein. Upon mixing of the droplets using a vibration, a magnetic field, and / or EWOD, the same vibration, magnetic field, and / or EWOD may be used to separate the one or more artifacts of the sample droplet from the droplet. In some embodiments, the one orPC17IB2025 / 000562more surfactants comprise Poloxamers. A surfactant may be configured to prevent biofouling or to remove biofouling substances.

[0121] In some embodiments, one or more surfactants are contacted with a sample droplet. In some embodiments, the surfactants are contacted with the sample droplet prior to an actuation of separating one or more artifacts from the sample droplet in order to facilitate removal of the artifacts. In some embodiments, the addition of the surfactant to the sample droplet prior to removal of the artifacts within the sample droplet leads to reduced contamination of the artifacts from the sample droplet. In some embodiments, the contamination of the one or more artifacts upon removal of the sample droplet treated with a surfactant in contrast to a sample droplet containing the same artifacts but is not treated with the surfactant is reduced by at least about 5 % to at least about 90 %. In some embodiments, the contamination of the one or more artifacts upon removal of the sample droplet treated with a surfactant in contrast to a sample droplet containing the same artifacts but is not treated with the surfactant is reduced by at least about 1 % to at least about 90 %. In some embodiments, the contamination of the one or more artifacts upon removal of the sample droplet treated with a surfactant in contrast to a sample droplet containing the same artifacts but is not treated with the surfactant is reduced by at least about 1 % to at least about 5 %, at least about 1 % to at least about 10 %, at least about 1 % to at least about 15 %, at least about 1 % to at least about 20 %, at least about 1 % to at least about 30 %, at least about 1 % to at least about 40 %, at least about 1 % to at least about 50 %, at least about 1 % to at least about 60 %, at least about 1 % to at least about 70 %, at least about 1 % to at least about 80 %, at least about 1 % to at least about 90 %, at least about 5 % to at least about 10 %, at least about 5 % to at least about 15 %, at least about 5 % to at least about 20 %, at least about 5 % to at least about 30 %, at least about 5 % to at least about 40 %, at least about 5 % to at least about 50 %, at least about 5 % to at least about 60 %, at least about 5 % to at least about 70 %, at least about 5 % to at least about 80 %, at least about 5 % to at least about 90 %, at least about 10 % to at least about 15 %, at least about 10 % to at least about 20 %, at least about 10 % to at least about 30 %, at least about 10 % to at least about 40 %, at least about 10 % to at least about 50 %, at least about 10 % to at least about 60 %, at least about 10 % to at least about 70 %, at least about 10 % to at least about 80 %, at least about 10 % to at least about 90 %, at least about 15 % to at least about 20 %, at least about 15 % to at least about 30 %, at least about 15 % to at least about 40 %, at least about 15 % to at least about 50 %, at least about 15 % to at least about 60 %, at least about 15 % to at least about 70 %, at least about 15 % to at least about 80 %, atPC17IB2025 / 000562least about 15 % to at least about 90 %, at least about 20 % to at least about 30 %, at least about 20 % to at least about 40 %, at least about 20 % to at least about 50 %, at least about 20 % to at least about 60 %, at least about 20 % to at least about 70 %, at least about 20 % to at least about 80 %, at least about 20 % to at least about 90 %, at least about 30 % to at least about 40 %, at least about 30 % to at least about 50 %, at least about 30 % to at least about 60 %, at least about 30 % to at least about 70 %, at least about 30 % to at least about 80 %, at least about 30 % to at least about 90 %, at least about 40 % to at least about 50 %, at least about 40 % to at least about 60 %, at least about 40 % to at least about 70 %, at least about 40 % to at least about 80 %, at least about 40 % to at least about 90 %, at least about 50 % to at least about 60 %, at least about 50 % to at least about 70 %, at least about 50 % to at least about 80 %, at least about 50 % to at least about 90 %, at least about 60 % to at least about 70 %, at least about 60 % to at least about 80 %, at least about 60 % to at least about 90 %, at least about 70 % to at least about 80 %, at least about 70 % to at least about 90 %, or about 80 % to at least about 90 %. In some embodiments, the contamination of the one or more artifacts upon removal of the sample droplet treated with a surfactant in contrast to a sample droplet containing the same artifacts but is not treated with the surfactant is reduced by at least about 1 %, at least about 5 %, at least about 10 %, at least about 15 %, at least about 20 %, at least about 30 %, at least about 40 %, at least about 50 %, at least about 60 %, at least about 70 %, at least about 80 %, or about 90 %. In some embodiments, the contamination of the one or more artifacts upon removal of the sample droplet treated with a surfactant in contrast to a sample droplet containing the same artifacts but is not treated with the surfactant is reduced by at least about at least about 1 %, at least about 5 %, at least about 10 %, at least about 15 %, at least about 20 %, at least about 30 %, at least about 40 %, at least about 50 %, at least about 60 %, at least about 70 %, or about 80 %. In some embodiments, the contamination of the one or more artifacts upon removal of the sample droplet treated with a surfactant in contrast to a sample droplet containing the same artifacts but is not treated with the surfactant is reduced by at least about at most about 5 %, at least about 10 %, at least about 15 %, at least about 20 %, at least about 30 %, at least about 40 %, at least about 50 %, at least about 60 %, at least about 70 %, at least about 80 %, or about 90 %.

[0122] In some embodiments, the contamination of the one or more artifacts upon removal of the sample droplet treated with a surfactant in contrast to a sample droplet containing the same artifacts but is not treated with the surfactant is reduced by at least about 5 %, at least about 10 %, at least about 15 %, at least about 20 %, at least about 25 %, at least about 30 %, at leastPC17IB2025 / 000562about 35 %, at least about 40 %, at least about 45 %, at least about 50 %, at least about 55 %, at least about 60 %, or more. In some embodiments, one or more surfactants are contacted with a sample droplet. In some embodiments, the surfactants are contacted with the sample droplet prior to an actuation of separating one or more artifacts from the sample droplet in order to facilitate removal of the artifacts. In some embodiments, when one or more artifacts are removed from a sample droplet, the one or more artifacts may comprise residual moisture and / or residual sample droplet content. In some embodiments, the addition of the surfactant to the sample droplet prior to removal of the artifacts by a vibration, a magnetic field, and / or EWOD leads to a reduced moisture content of the artifacts. In some embodiments, the addition of the surfactant to the sample droplet prior to removal of the artifacts by a vibration, a magnetic field, and / or EWOD leads to a reduced moisture content of the artifacts that is more reduced than a sample droplet comprising of the same physical composition and comprising artifacts of the same composition but are not treated with the same surfactant (or any surfactant). In some embodiments, the moisture content of the one or more artifacts upon removal of the sample droplet treated with a surfactant in contrast to a sample droplet containing the same artifacts but is not treated with the surfactant is reduced by at least about at least about 1 % to at least about 90 %. In some embodiments, the moisture content of the one or more artifacts upon removal of the sample droplet treated with a surfactant in contrast to a sample droplet containing the same artifacts but is not treated with the surfactant is reduced by at least about 1 % to at least about 5 %, at least about 1 % to at least about 10 %, at least about 1 % to at least about 15 %, at least about 1 % to at least about 20 %, at least about 1 % to at least about 30 %, at least about 1 % to at least about 40 %, at least about 1 % to at least about 50 %, at least about 1 % to at least about 60 %, at least about 1 % to at least about 70 %, at least about 1 % to at least about 80 %, at least about 1 % to at least about 90 %, at least about 5 % to at least about 10 %, at least about 5 % to at least about 15 %, at least about 5 % to at least about 20 %, at least about 5 % to at least about 30 %, at least about 5 % to at least about 40 %, at least about 5 % to at least about 50 %, at least about 5 % to at least about 60 %, at least about 5 % to at least about 70 %, at least about 5 % to at least about 80 %, at least about 5 % to at least about 90 %, at least about 10 % to at least about 15 %, at least about 10 % to at least about 20 %, at least about 10 % to at least about 30 %, at least about 10 % to at least about 40 %, at least about 10 % to at least about 50 %, at least about 10 % to at least about 60 %, at least about 10 % to at least about 70 %, at least about 10 % to at least about 80 %, at least about 10 % to at least about 90 %, at least about 15 % to at leastPC17IB2025 / 000562about 20 %, at least about 15 % to at least about 30 %, at least about 15 % to at least about 40 %, at least about 15 % to at least about 50 %, at least about 15 % to at least about 60 %, at least about 15 % to at least about 70 %, at least about 15 % to at least about 80 %, at least about 15 % to at least about 90 %, at least about 20 % to at least about 30 %, at least about 20 % to at least about 40 %, at least about 20 % to at least about 50 %, at least about 20 % to at least about 60 %, at least about 20 % to at least about 70 %, at least about 20 % to at least about 80 %, at least about 20 % to at least about 90 %, at least about 30 % to at least about 40 %, at least about 30 % to at least about 50 %, at least about 30 % to at least about 60 %, at least about 30 % to at least about 70 %, at least about 30 % to at least about 80 %, at least about 30 % to at least about 90 %, at least about 40 % to at least about 50 %, at least about 40 % to at least about 60 %, at least about 40 % to at least about 70 %, at least about 40 % to at least about 80 %, at least about 40 % to at least about 90 %, at least about 50 % to at least about 60 %, at least about 50 % to at least about 70 %, at least about 50 % to at least about 80 %, at least about 50 % to at least about 90 %, at least about 60 % to at least about 70 %, at least about 60 % to at least about 80 %, at least about 60 % to at least about 90 %, at least about 70 % to at least about 80 %, at least about 70 % to at least about 90 %, or about 80 % to at least about 90 %.. In some embodiments, the moisture content of the one or more artifacts upon removal of the sample droplet treated with a surfactant in contrast to a sample droplet containing the same artifacts but is not treated with the surfactant is reduced by at least about 1 %, at least about 5 %, at least about 10 %, at least about 15 %, at least about 20 %, at least about 30 %, at least about 40 %, at least about 50 %, at least about 60 %, at least about 70 %, at least about 80 %, or about 90 %. In some embodiments, the moisture content of the one or more artifacts upon removal of the sample droplet treated with a surfactant in contrast to a sample droplet containing the same artifacts but is not treated with the surfactant is reduced by at least about 1 %, at least about 5 %, at least about 10 %, at least about 15 %, at least about 20 %, at least about 30 %, at least about 40 %, at least about 50 %, at least about 60 %, at least about 70 %, or about 80 %. In some embodiments, the moisture content of the one or more artifacts upon removal of the sample droplet treated with a surfactant in contrast to a sample droplet containing the same artifacts but is not treated with the surfactant is reduced by at least about at most about 1%, at most about 5 %, at most about 10 %, at most about 15 %, at most about 20 %, at most about 30 %, at most about 40 %, at most about 50 %, at most about 60 %, at most about 70 %, at most about 80 %, at most 90 %.Measuring viscosity

[0123] The present disclosure provides systems and methods for measuring the viscosity of one or more droplets. The one or more droplets may be sample droplets. The one or more droplets may comprise a biological sample and may be used for biological applications. The one or more droplets may be provided on the surface of an electrowetting array. The mobility of drops manipulated by electrowetting forces and vibration depends on their viscosity, surface tension, density, and other properties. This means the drop viscosity can be measured using output responses of droplet mobility.

[0124] A method of moving a droplet across a surface by electrowetting can be used to measure or index the droplet viscosity. For example, mobility may be tested using a protocol to move a droplet by electrowetting at one rate across a row of electrodes for one lap, and then increasing the rate for the next lap, and so on, until the droplet failed to track the moving electrode pattern. This maximum velocity is related to the viscous dissipation inside the droplet, and so to its viscosity, thus this velocity measurement is related to droplet viscosity. The accuracy of this viscosity measurement depends on the relative viscosities of the mobile droplet and of the oil layer and is more accurate for a lower- viscosity oil underlayer (1 cSt rather than 5 cSt).Referring to FIG. 14, using the low-viscosity oil, an experiment was conducted to differentiate and rank water-glycerol droplet mixtures successfully with a precision of 10 cSt (i.e., one could differentiate 10 and 20 cSt droplets, but not 10 and 12 cSt droplets). For the same droplets with a range of viscosities tested, the electrowetting force was found to be relatively constant, supporting the idea that changing velocity was due to changing viscosity alone, and not to other factors like density or surface tension changing in the series of droplets. FIG. 14 depicts results of a pacer test that measures droplet viscosity via successive testing of increasing rates of electro wetting movement (increasing force), where 1 cSt = 1 mm2 / s.Example 1 - Droplet vibration

[0125] FIG. 15 shows an embodiment of efficiently coupling the actuation force of the electromechanical actuator into droplet vibration and, ultimately, to effective mixing.PC17IB2025 / 000562

[0126] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 16 shows a computer system 1601 that is programmed or otherwise configured to manipulate droplet operations. The computer system 1601 can regulate various aspects of the present disclosure, such as, for example, controlling vibrations and / or vibration-assisted mixing, controlling a magnetic field with respect to a droplet provided on the surface of an array, controlling electrode activation, or controlling the power source to the electrowetting device. The computer system 1601 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0127] The computer system 1601 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 1605, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 1601 also includes memory or memory location 1610 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 1615 (e.g., hard disk), communication interface 1620 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1625, such as cache, other memory, data storage and / or electronic display adapters. The memory 1610, storage unit 1615, interface 1620 and peripheral devices 1625 are in communication with the CPU 1605 through a communication bus (solid lines), such as a motherboard. The storage unit 1615 can be a data storage unit (or data repository) for storing data. The computer system 1601 can be operatively coupled to a computer network (“network”) 1630 with the aid of the communication interface 1620. The network 1630 can be the Internet, an internet and / or extranet, or an intranet and / or extranet that is in communication with the Internet. The network 1630 in some cases is a telecommunication and / or data network. The network 1630 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 1630, in some cases with the aid of the computer system 1601, can implement a peer-to-peer network, which may enable devices coupled to the computer system 1601 to behave as a client or a server.

[0128] The CPU 1605 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 1610. The instructions can be directed to the CPU 1605, which can subsequently program or otherwise configure the CPU 1605 to implement methods of the presentPC17IB2025 / 000562disclosure. Examples of operations performed by the CPU 1605 can include fetch, decode, execute, and writeback.

[0129] The CPU 1605 can be part of a circuit, such as an integrated circuit. One or more other components of the system 1601 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0130] The storage unit 1615 can store files, such as drivers, libraries and saved programs. The storage unit 1615 can store user data, e.g., user preferences and user programs. The computer system 1601 in some cases can include one or more additional data storage units that are external to the computer system 1601, such as located on a remote server that is in communication with the computer system 1601 through an intranet or the Internet.

[0131] The computer system 1601 can communicate with one or more remote computer systems through the network 1630. For instance, the computer system 1601 can communicate with a remote computer system of a user. Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 1601 via the network 1630.

[0132] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 1601, such as, for example, on the memory 1610 or electronic storage unit 1615. The machine executable or machine-readable code can be provided in the form of software. During use, the code can be executed by the processor 1605. In some cases, the code can be retrieved from the storage unit 1615 and stored on the memory 1610 for ready access by the processor 1605. In some situations, the electronic storage unit 1615 can be precluded, and machine-executable instructions are stored on memory 1610.

[0133] The code can be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.

[0134] Aspects of the systems and methods provided herein, such as the computer system 1601, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor)executable code and / or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a harddisk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted 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.

[0135] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, 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. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and / or data. 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.

[0136] The computer system 1601 can include or be in communication with an electronic display 1635 that comprises a user interface (UI) 1640 for providing, for example, the patterns of droplet movement and / or electrode actuation described herein. Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0137] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 1605. The algorithm can, for example, optimize and execute the patterns of droplet movement and / or electrode actuation described herein.

[0138] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0139] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0140] 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. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the 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 thePC17IB2025 / 000562invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

WHAT IS CLAIMED IS:

1. A method of processing a droplet comprising a plurality of artifacts, the method comprising:a. providing a substrate comprising a plurality of electrodes disposed beneath a surface of the substrate;b. providing the droplet comprising the plurality of artifacts on the substrate;c. activating one or more electrodes of the plurality of electrodes to induce a pattern of motion of the droplet, wherein the pattern of motion is sufficient to change a geometry of the shape of the plurality of artifacts.

2. The method of claim 1 , wherein the pattern of motion comprises:a. moving the droplet with respect to an x-axis;b. subsequently moving the droplet with respect to a y-axis at least two times; c. subsequently moving the droplet with respect to an x-axis.

3. The method of claim 1 or 2, wherein the pattern of motion is executed in proximity of a magnetic field.

4. The method of any one of claims 1-3, wherein the pattern of motion comprises moving the droplet in a circular motion, a rectangular motion, an X-shape, or a linear motion.

5. The method of any one of the preceding claims, wherein the magnetic field is generated by a magnet.

6. The method of claim 5, wherein the magnet is displaced below the substrate.

7. The method of claim 5, wherein the magnet is configured to translate with respect to the substrate.

8. The method of any one of the preceding claims, further comprising removing the plurality of artifacts from the droplet.

9. The method of claim 8, wherein the removing comprises:(i) actuating a movement of the droplet via EWOD;(ii) translating the magnet; or both.

10. The method of any one of the preceding claims, wherein one or more surfactants are added to the droplet prior to the removing of the plurality of artifacts.

11. A method of processing a droplet, the method comprising:a. providing a droplet on a surface;b. contacting the droplet with one or more surfactants; andc. removing one or more artifacts encapsulated within the droplet.

12. The method of claim 11, wherein (a) the providing the droplet on the surface occurs prior to (b) the contacting the droplet with the one or more surfactants.

13. The method of claim 11 , wherein (b) the contacting the droplet with the one or more surfactants occurs prior to (c).

14. The method of claim 11, wherein the one or more artifacts comprise biofouling substances.

15. The method of claim 11, wherein in (b), the contacting the droplet with the one or more surfactants comprises pipetting, using a magnet, using vibration, or electro wetting.

16. The method of any one of claims 1-5, wherein the one or more artifacts comprises one or more magnetic beads.

17. The method of claim 16, further comprising providing a magnet in proximity to the substrate.

18. The method of claim 17, wherein the magnet is actuated.

19. The method of claim 18, wherein the pattern of motion causes the one or more magnetic beads to be in proximity to the magnet.

20. The method of claim 19, wherein the proximity to the magnet of the one or more magnetic beads reduces a radius of the one or more magnetic beads in the droplet.

21. A method of selecting a frequency for vibrating a droplet on the surface of an electro wetting array comprising:a. determining a surface tension or viscosity of the droplet; andb. selecting a frequency of less than about 15 Hertz (Hz) for a droplet with lower surface tension or higher viscosity, and a frequency of greater than about 15 Hz for a droplet with higher surface tension or lower viscosity.

22. The method of claim 21, wherein a frequency of about 40 Hz to at least about 55 Hz is selected for a droplet comprising water.

23. The method of claim 21, wherein a frequency of about 2 Hz to at least about 15 Hz is selected for a droplet comprising ethanol or a surfactant.

24. A method of vibrating a droplet comprising:a. providing a substrate comprising a plurality of electrodes disposed beneath a surface of the substrate;b. providing the droplet on the substrate;c. activating a plurality of electrodes, wherein the plurality of electrodes is in a pattern of motion.

25. A method of processing a droplet, wherein the droplet comprises a plurality of artifacts, the method comprising:a. providing a substrate comprising a plurality of electrodes disposed beneath a surface of the substrate;b. providing the droplet comprising the plurality of artifacts on the substrate;c. resuspending the plurality of artifacts without mechanical agitation.

26. The method of claim 25, wherein the resuspending comprises activating an electrode of the plurality of electrodes.

27. The method of claim 24 or 25, wherein activating a plurality of electrodes comprises a temporal pattern.

28. The method of claim 24 or 25, wherein activating a plurality of electrodes excites a resonant mode of the droplet.

29. The method of claim 24 or 25, wherein activating a plurality of electrodes comprises a spatial pattern.

30. The method of claim 29, wherein the spatial pattern is a chessboard pattern.

31. The method of claim 29, wherein the spatial pattern is agnostic to droplet size.

32. The method of claim 29, wherein the spatial pattern is chosen to maximize perimeter between activated electrodes and inactivated electrodes.

33. The method of any one of the preceding claims, wherein a portion of the droplet is localized.

34. The method of any one of the preceding claims, wherein the droplet changes shape.

35. The method of claim 24 or 25, wherein the electrodes are activated in a pattern comprising activation of a plurality of electrodes adjacent horizontally36. The method of claim 24 or 25, wherein the plurality of electrodes is activated in a pattern.

37. The method of claim 24 or 25, wherein the activation of the plurality of electrodes is sufficient to excite a resonance mode in the droplet but the activation of the electrodes is not sufficient to translate the droplet’s location.

38. The method of claim 24 or 25, wherein the activation of the plurality of electrodes is sufficient to excite a near-resonance mode in the droplet but the activation of the electrodes is not sufficient to translate the droplet’s location.

39. A method of immobilizing a droplet at a discrete location, the method comprising:a. providing a droplet on an electrowetting array, the array comprising a plurality of electrodes, wherein a subset of the plurality of electrodes comprises one or more adjacent electrodes forming a field on the surface comprising the discrete location; andb. activating or deactivating the one or more adjacent electrodes in a spatial and / or temporal pattern to localize the droplet at the discrete location.

40. The method of claim 39, wherein the spatial pattern is a cross shape.

41. The method of claim 39, wherein a horizontal and a vertical element of the plurality of electrodes are alternatively activated.

42. The method of any one of claims 39-41, wherein a diameter of the discrete location is less than 2 millimeters (mm).

43. The method of claim 39, wherein the spatial pattern is a chessboard pattern.

44. The method of claim 1 , wherein the pattern of motion is sufficient to reduce a radius of the shape of the plurality of artifacts.

45. The method of claim 1 , wherein the one or more electrodes are activated at a low duty cycle.

Images