Directional control of droplet movement using differential wetting

Abstract

To provide a device for controlling the movement of liquid droplets. [Solution] A set of electrode pads is arranged to define one or more trajectories on which a droplet can move across a series of electrode pads. The surface on the electrode pads is dielectric, smooth, and slippery over the droplet. In some cases, the smooth surface is formed as a thin layer of a second liquid that does not mix with the droplet. The surface has a wetting affinity for a liquid that can be individually varied in a manner controlled by the application of a voltage to each electrode pad. The control device is designed to move the droplet on the surface by modifying the wetting properties of the varying wetting portion on each electrode pad. The device is designed to have the smooth hydrophobic surface open so that no electrodes or plates overlap or face the droplet.

Description

[Technical Field]

[0001] This invention relates to directing the movement of droplets using differential wetting. [Background technology]

[0002] This application claims priority under U.S. Provisional Application 62 / 811,018, filed February 27, 2019, entitled "Directing Motion of Droplets Using Differential Wetting," and U.S. Provisional Application 62 / 636,268, filed February 28, 2018, entitled "Manufacturing Methods for Electrowetting Microfluidic Chip." These provisional applications are cited by reference.

[0003] This application relates to the control and manipulation of liquids or gases in small devices, typically ranging from milliliters to submicroliters in scale. [Overview of the project]

[0004] Overall, in a first embodiment, the present invention features a device for controlling the movement of a droplet. The device comprises a set of electrode pads configured in an array or path that defines one or more trajectories on which a droplet moves along a series of electrodes. The device comprises a surface provided on the electrode pads that is dielectric, smooth to within 2 μm, has a sliding angle of 5 degrees or less for a 5 μl droplet, and has a wetting affinity for a liquid that can be modified by applying a voltage to the electrode pad. The device further comprises a control device designed to move a droplet along the trajectory by modifying the wetting properties of a portion of the surface on each electrode pad, the wetting properties being modified by controlling the charging and discharging of the electrode pads in a desired arrangement.

[0005] Overall, in a second embodiment, the present invention features a device for controlling the movement of a liquid droplet. The device comprises a smooth hydrophobic surface having portions that have a controllably variable wetting affinity for a liquid. These variable wetting portions are configured as an array or path defining one or more trajectories on which the liquid droplet can move over a series of the variable wetting portions. The device further comprises a control device designed to move the liquid droplet along the trajectory by varying the wetting properties of the variable wetting portions of the surface. The device is configured with the smooth hydrophobic surface open, and is designed so that no electrodes or plates overlap or face the liquid droplet.

[0006] Overall, in a third embodiment, the present invention features a device for controlling the movement of a droplet. The device includes a solid surface that is textured to hold a thin layer of a second liquid that does not mix with the droplet and an upper surface of the second liquid that forms a slippery liquid-liquid surface for the droplet, has a sliding angle of 5 degrees or less for a 5 μl droplet, and has a controllably variable wetting affinity for the droplet. The variable wetting portion is configured as an array or path that defines one or more trajectories on which the droplet can move over a continuous series of the variable wetting portions. The device further includes a control device designed to move the droplet along the trajectory by varying the wetting affinity of the variable wetting portion of the liquid-liquid surface.

[0007] Overall, in a fourth embodiment, the present invention features a device for controlling the movement of a liquid droplet. The device comprises a set of electrode pads configured in an array or path defining one or more trajectories on which a liquid droplet moves over a series of electrodes. The device comprises a dielectric surface provided on the electrode pads, which is smooth to within 1 μm, and which is formed as a thin layer of a second liquid that does not mix with the droplet, and as an upper surface of the second liquid that forms a slippery liquid-liquid surface for the droplet, having a sliding angle of 5 degrees or less for a 5 μl droplet, and having a portion that has a wetting affinity for the liquid, which can be individually varied by applying a voltage to control each electrode pad, and the variable wetting portions are configured in an array or path defining one or more trajectories on which the droplet can move over a series of the variable wetting portions. The second liquid is on the surface of a substrate solid substrate that is textured to hold the second liquid against gravity. The apparatus further includes a control device designed to move droplets along the trajectory by modifying the wetting properties of the wetting portion of the surface on each electrode pad, the wetting properties being modified by controlling the charging and discharging of the electrode pads in a desired arrangement. The apparatus is designed with the smooth hydrophobic surface open so that no electrodes or plates overlap or face the droplets.

[0008] Overall, in a fifth embodiment, the present invention features a method, in which a droplet is introduced to a surface on a set of electrode pads configured in an array or path defining one or more trajectories on which the droplet moves along a series of electrodes. The surface is dielectric, smooth to within 2 μm, has a sliding angle of 5 degrees or less for a 5 μl droplet, and has a wetting affinity for a liquid that can be modified by applying a voltage to the electrode pads. The variable wetting portion is configured in an array or path defining one or more trajectories on which the droplet can move along a series of the variable wetting portions. The wetting properties of a portion of the surface on each electrode pad are controlled to move the droplet along the trajectory, and the wetting properties are modified by controlling the charging and discharging of the electrode pads in a desired alignment. The surface is designed to have the smooth hydrophobic surface open so that no electrodes or plates overlap or face the droplet.

[0009] Embodiments of the present invention may include one or more of the following features: The electromotive force may be less than 100V, less than 80V, less than 50V, less than 40V, less than 30V, or less than 20V. The electrodes may be printed on a substrate using printed circuit board technology or manufactured using thin-film transistor (TFT), active matrix, or passive matrix backplane technology. Various smoothing levels are preferred, ranging from 5 μm, 2 μm, 1 μm, 500 nm, 200 nm, or 100 nm. The surface can be made smooth to within 1 μm by polishing. The surface can be made smooth to within 1 μm by coating, which is applied by at least one of spin coating, spray coating, dipping coating, or vapor deposition. The surface coating may be made of a dielectric and hydrophobic material. The surface can be made smooth to within 1 μm by stretching a sheet polymer and removing wrinkles. The sliding angle can be imparted to the surface by creating hydrophobicity through patterning or texture processing. The sliding angle of a 5 μl droplet may be 5 degrees, 3 degrees, 2 degrees, or 1 degree or less. A set of electrode pads can be configured as an array or path defining one or more trajectories on which a droplet can move over a series of electrode pads, and the variable wetting portion is a dielectric surface on the electrode pads. The wetting of the variable wetting portion of the surface can be varied by exposure to light. The variable wetting portion of the surface can be actuated by optoelectrowetting. The variable wetting portion of the surface can be actuated by photoelectrowetting. The smooth surface may have one or more holes, for example, for introducing droplets or reactants or allowing light to pass through. The apparatus may include stations for one or more purposes from the group consisting of distribution, mixing, heating, cooling, application of magnetic fields, application of electric fields, addition of reagents, optical inspection or assay, and isolation or purification of proteins, peptides, or other biomacromolecules. The apparatus may include an acoustic transducer configured to introduce droplets into the apparatus.The apparatus may include a microdiaphragm pump configured to introduce droplets into the apparatus. Other methods for introducing or injecting droplets include inkjet nozzles of an inkjet printer, syringe pumps, capillary tubes, or pipettes. The second liquid may be an oil that has a wetting affinity for a solid and is retained on the textured surface of the solid.

[0010] The above advantages and features represent only representative embodiments and are presented solely to aid in understanding the invention. It should be understood that these are not intended to be interpreted as limiting the invention as defined in the claims. Further features and advantages of embodiments of the invention will become apparent by referring to the following description, drawings, and claims. [Brief explanation of the drawing]

[0011] [Figure 1A] This is a plan view showing droplets on an electrowetting surface. [Figure 1B] This is a side cross-sectional view showing droplets on an electrowetting surface. [Figure 2A] This is a side cross-sectional view showing droplets on an electrowetting surface. [Figure 2B] This is a side cross-sectional view showing droplets on an electrowetting surface. [Figure 3A] This is a side cross-sectional view showing droplets on an electrowetting surface. [Figure 3B] This is a side cross-sectional view showing droplets on an electrowetting surface. [Figure 3C] This is a side cross-sectional view showing droplets on an electrowetting surface. [Figure 4A] This is a side cross-sectional view showing droplets on an electrowetting surface. [Figure 4B] This is a side cross-sectional view showing droplets on an electrowetting surface. [Figure 4C] This is a side cross-sectional view showing droplets on an electrowetting surface. [Figure 5A] It is a side cross-sectional view showing a droplet on an electro-wetting surface. [Figure 5B] It is a side cross-sectional view showing a droplet on an electro-wetting surface. [Figure 6A] It is a side cross-sectional view showing a droplet on an electro-wetting surface. [Figure 6B] It is a side cross-sectional view showing a droplet on an electro-wetting surface. [Figure 6C] It is a side cross-sectional view showing a droplet on an electro-wetting surface. [Figure 7A] It is a side cross-sectional view of a printed circuit board. [Figure 7B] It is a side cross-sectional view of a printed circuit board. [Figure 8A] It is a side cross-sectional view of a printed circuit board. [Figure 8B] It is a side cross-sectional view of a printed circuit board. [Figure 8C] It is a side cross-sectional view of a printed circuit board. [Figure 8D] It is a side cross-sectional view of a printed circuit board. [Figure 8E] It is a side cross-sectional view of a printed circuit board. [Figure 8F] It is a side cross-sectional view of a printed circuit board. [Figure 9A] It is a side cross-sectional view of a printed circuit board. [Figure 9B] It is a diagram showing a manufacturing process. [Figure 9C] It is a diagram showing a manufacturing process. [Figure 9D] It is a diagram showing a manufacturing process. [Figure 9E] It is a side cross-sectional view of a printed circuit board. [Figure 10A] It is a side cross-sectional view showing a droplet on an electro-wetting surface. [Figure 10B] It is a side cross-sectional view showing a droplet on an electro-wetting surface. [Figure 10C] It is a side cross-sectional view showing a droplet on an electro-wetting surface. [Figure 10D] This is a side cross-sectional view showing droplets on an electrowetting surface. [Figure 10E] This is a side cross-sectional view showing droplets on an electrowetting surface. [Figure 10F] This is a side cross-sectional view showing droplets on an electrowetting surface. [Figure 10G] This is a side cross-sectional view showing droplets on an electrowetting surface. [Figure 10H] This is a side cross-sectional view showing droplets on an electrowetting surface. [Figure 10I] This is a side cross-sectional view showing droplets on an electrowetting surface. [Figure 11A] This is a perspective view of the inspection device. [Figure 11B] This is a plan view of the processing station for electrowetting devices. [Figure 11C] This is a plan view of the processing station for electrowetting devices. [Figure 11D] This is a plan view of the processing station for electrowetting devices. [Figure 11E] This is a plan view of the processing station for electrowetting devices. [Figure 11F] This is a perspective view of an electrowetting device processing station. [Figure 11G] This is a side cross-sectional view of an electrowetting device processing station. [Figure 11H] This is a perspective view of an electrowetting device processing station. [Figure 11I] This is a side cross-sectional view of an electrowetting device processing station. [Figure 11J] This is a side cross-sectional view of an electrowetting device processing station. [Figure 12A] This is an exploded view of two components of an electrowetting device. [Figure 12B] This is a side cross-sectional view of a microfluidic device. [Figure 12C] This is a side cross-sectional view of a microfluidic device. [Modes for carrying out the invention]

[0012] The description of the present invention is organized as follows: I. Introduction Liquid electrowetting (LLEW) for IA electrowetting. Electrowetting (EWOD) onto dielectrics for IB droplet operation. II. Method for Manufacturing an Electrowetting Array II.A. Circuit boards for electrowetting II.B. Creation of a smooth dielectric surface on an electrode array II.B.1. Smoothing with photoresist / epoxy / embedding compounds (a) Fabrication of dielectrics on smooth photoresists / epoxy resins / embedding compounds (i) Deposit a thin film coating as a dielectric. (ii) Bonding polymer films to form a dielectric on top II.B.2. Formation of a smooth dielectric surface by polishing with an excess amount of photoresist II.B.3. Polymer Films as Smooth Dielectric Surfaces II.C. Create a slippery surface as the final product. II.C.1. Modifying Solid Dielectrics to Achieve Hydrophobicity (a) Surface chemical modification (functionalization) (b) Surface topography modification (i) Creation of micropillars (ii) Microsphere II.D. Slippery liquid coatings and electrowetting of liquids onto liquid surfaces (LLEW) II.D.1. Droplets on a Thin Film Liquid Layer of LLEW Create a textured solid for II.D.2.LLEW. II.D.3. Apply lubricant to the textured solid. III. Intrinsic properties of liquid electrowetting (LLEW) on top of other liquids III.A. Low Operating Voltage III.B. Cleaning of the device surface by washing. IV. Application of electrowetting IV.A. Any large open face IV.B. Droplet movement, integration, and division IV.C. Lab in a Box (Desktop Digital Wet Lover) IVD Processing Station IV.D.1. Mixing Station IV.D.2. Incubation Station IV.D.3. Magnetic Bead Station IV.D.4. Nucleic Acid Delivery Station IV.D.5. Optical Inspection Station IV.D.6. Loading / unloading via acoustic liquid handler or microdiaphragm-based pump dispenser V. Alternative Embodiments Droplets located on a VA open surface (single-plate configuration) and sandwiched between two plates (double-plate configuration) VB Optoelectrowetting and Photoelectrowetting VC Optoelectrowetting VD Photoelectrowetting VE software and hardware VF and other alternatives

[0013] I. Introduction Referring to Figure 1, an electrowetting device is used to move individual water droplets (or other aqueous solutions, polar solutions, conductive solutions) to different locations. The surface tension and wetting properties of water can be modified by the electric field strength using the electrowetting effect. The electrowetting effect arises from the change in the contact angle between the solid and the electrolyte due to the applied voltage difference between the solid and the electrolyte. The difference in wet surface tension, which varies across the width of the droplet, and the corresponding change in the contact angle, can generate a propulsive force to move the droplet without requiring any moving parts or physical contact. The electrowetting device (100) comprises a grid of electrodes (120) in which dielectric layers (130) with appropriate electrical and surface priority overlap the electrodes (120), all located on a fixed insulating substrate (140).

[0014] In some cases, it is desirable to prepare the electrode grid surface (130) to reduce the adhesion force to water. This allows the droplet (110) to move along the surface due to a small force created by the gradient in the electric field and surface tension across the entire width of the droplet. Reduced surface adhesion reduces the trail left behind after the droplet moves. Fewer trails reduce droplet cross-contamination and minimize sample loss during droplet movement. Furthermore, reduced surface adhesion enables droplet movement at low operating voltages and allows for repeatable droplet movement behavior. Various methods exist for measuring the low adhesion force between a surface and a droplet.

[0015] • Sliding angle: When a surface is raised from a horizontal position, what angle does a given droplet size begin to move under gravity? For example, if a 5 μl droplet is held at 4 degrees, but the surface allows the droplet to slide at 5 degrees, the sliding angle for a 5 μl droplet is sometimes said to be 5 degrees. In various applications, sliding angles of 10 degrees, 5 degrees, 3 degrees, 2 degrees, and 1 degree for a 5 μl droplet may be desirable. It is said that as the sliding angle decreases, the surface becomes more slippery, and the voltage required for droplet movement across the entire surface decreases overall.

[0016] • Contact angle hysteresis: On surfaces with low surface adhesion, when a droplet moves across the entire surface, the contact angle between the leading edge and the surface becomes approximately the same as the contact angle between the trailing edge and the surface, and this is largely determined by the surface tension of the liquid. When a droplet moves across the entire surface with high adhesion, the contact angles at the leading and trailing edges will separate. If the liquid surface hydrophobicity is high and the surface energy is low, the difference in angles becomes small. In some cases, gradually increasing contact angle hysteresis (i.e., the difference between the leading and trailing edge contact angles) of 15, 10, 7, 5, 3, and 2 degrees may be desirable.

[0017] Various methods exist to achieve low surface adhesion. For example, these include mechanical polishing until the surface is smooth to within a few nanometers, applying a coating to fill in irregular areas on the surface, and chemically modifying the surface to bring about desired surface properties (such as hydrophobicity, hydrophilicity, and variation with electric field strength).

[0018] Liquid electrowetting (LLEW) for IA electrowetting. Referring to Figures 2A and 2B, the electrowetting mechanism, known as "electrowetting of liquid onto liquid" (LLEW), utilizes the electrowetting phenomenon that occurs at the liquid-liquid-gas interface (200). When a water droplet (110) rests on the surface of a layer of low-surface-energy liquid (210) (such as oil) and is substantially surrounded by air (or gas such as vapor), a liquid-liquid-gas interface is created at the contact line (200). The oil (210) is stabilized in place on the solid substrate by the textured surface (220) of the solid substrate, and the conductive layer of the metal electrode (120) can be embedded in the solid body. Referring to Figure 2B, when a potential is applied along the entire length of the droplet (110), the liquid-liquid-gas interface (200) causes the droplet (110) to wet the oil (210) and spread across the entire surface, while still remaining on top of the oil (210).

[0019] Referring to Figures 3A, 3B, and 3C, the electrowetting technique of liquid onto liquid can be used to manipulate droplets (110) containing biological and chemical samples. In Figure 3A, the droplet (110) moves from left to right, and is attracted to the leftmost electrode by applying a positive voltage to the leftmost electrode (120a) of the three electrodes, followed by applying an electric field to the liquid-liquid interface to increase wetting. In Figure 3B, the voltage is recovered from the leftmost electrode (120a) and applied to the center electrode (120b). As wetting increases on the center electrode (120b), the droplet is attracted to the center in Figure 3B. In Figure 3C, the voltage is recovered from the leftmost electrode (120a) and the center electrode (120b) and applied to the rightmost electrode (120c). As wetting increases on the rightmost electrode (120c), the droplet is attracted to the right.

[0020] Referring to Figures 4A, 4B, and 4C, differential wetting is used to merge two droplets (110a) and (110b) on the LLEW surface (200) on electrode arrays (120d), (120e), and (120f). In Figure 4A, the two droplets are attracted to the leftmost electrode (120d) and the rightmost electrode (120f). In Figure 4B, the voltage is removed from the leftmost electrode (120d) and the rightmost electrode (120f) and applied to the center electrode (120e). Merging begins as the two droplets are attracted to the center (120e) from the left and right. In Figure 4C, the two droplets are fully merged.

[0021] Referring to Figures 3A, 3B, 3C, 4A, 4B, and 4C, such microfluidic selective wetting devices can perform microfluidic droplet operations such as droplet transport, droplet integration, droplet mixing, droplet splitting, droplet distribution, and droplet shape modification. When applied to microfluidic devices, this LLEW droplet operation enables automated biological experiments, such as liquid assays, in medical diagnostic devices and many love-on-a-chip applications.

[0022] Electrowetting (EWOD) onto dielectrics for IB droplet operation. Referring to FIGS. 5A and 5B, electro-wetting on dielectric (EWOD) is a phenomenon where the wettability of an aqueous solution, polar solution, or conductive solution may be adjusted through an electric field on a dielectric film (530) between a droplet and a conductive electrode (120). When charge is added to or removed from the electrode (120), the wettability of the insulating dielectric layer (530) changes, and this change in wettability is reflected in the change in the contact angle (540) of the droplet (110). When the contact angle changes, the droplet (110) may change shape, move, split into smaller droplets, or integrate with another droplet. As represented by Equation 2, the contact angle (540) varies according to the applied voltage.

[0023] The wetting behavior (wetting or wettability) of a liquid on a solid surface indicates how well the liquid spreads on the solid surface. The wettability of a droplet on a solid surface surrounded by air is regulated by the interfacial tension between the solid medium, liquid medium, and the expected medium. For a fixed droplet, wettability is measured from the perspective of the contact angle (540) with the solid surface. Wettability is regulated by Young's equation: γ SL =γ SG +γ LG cos(θ e )(Equation 1) where γ SL is the solid-liquid surface tension, γ LG is the liquid-air surface tension, γ SG is the solid-gas surface tension, and θ e is the contact angle under equilibrium conditions.

[0024] Gabriel Lippmann observed that the capillary level of mercury in an electrolyte changes when a voltage is applied. Subsequently, this phenomenon (electrocapillarity) is described by the Lippmann-Young equation: cos(θ u )=cos(θ0)+1 / γ LG * 1 / 2 * C * U 2 (Equation 2) In the equation, θ0 is the contact angle when the electric field is zero (i.e., no voltage is applied), and θ u c is the contact angle when a voltage U is applied, and c is the capacitance per unit area between the electrode and the droplet.

[0025] II. Method for Manufacturing an Electrowetting Array Electrowetting devices used for the transfer and mixing of biological fluids may consist of an array of electrodes (120) on an insulating substrate, a thin layer of dielectric (130), and, if necessary, a final slippery coating. Sometimes, the dielectric layer may provide sufficiently hydrophobic and slippery behavior by itself, by additional chemicals or modifications of the surface topography, or independently thereof.

[0026] The electrode grid (120) on the insulating substrate may be manufactured using one or more of the following methods—printed circuit board manufacturing, CMOS or HV CMOS, or other semiconductor assembly methods—using thin-film transistors (TFTs), active matrix, or passive matrix backplane technology, or any other method by which conductive circuits can be laid on an insulating substrate. To keep the biological fluids separated while moving and mixing, the surface of the electrode array may be coated with a dielectric by one of the many methods described below.

[0027] PCBs and surface electrodes may be manufactured using thin-film transistors (TFTs), active-matrix, or passive-matrix backplane technology.

[0028] The chemical properties and texture of the dielectric surface interacting with the droplet determine the voltage required for good and repeated droplet movement. Due to the chemical composition and physical texture, droplets on electrowetting devices may experience two phenomena when moving: droplet fixation and contact angle hysteresis. Droplet fixation occurs when a droplet becomes stuck on any localized surface defect while in motion. Contact angle hysteresis is the difference between the contact angle that advances a moving droplet and the contact angle that recedes it. Due to droplet fixation and high contact angle hysteresis, droplets on electrowetting surfaces may require significantly higher voltages. The chemical composition of the surface, its texture and slipperiness, and its smoothness can also cause droplets to leave trails when in motion. These trails can be as simple as a single molecule.

[0029] To reduce fixation, contact angle hysteresis, and trails left behind by droplets, the dielectric covering the electrode array is typically smoothed and then chemically modified to create a low surface energy surface. Surface energy is the energy related to intermolecular forces at the interface between two media. Droplets interacting with a low surface energy surface are repelled by the surface and are considered hydrophobic. Sometimes, the dielectric layer itself provides a sufficiently slippery surface for droplet movement.

[0030] The following sections describe various materials used to manufacture electrowetting devices: substrates for laying conductive materials, conductive materials for electrodes and interconnections, dielectric materials, methods for depositing dielectric materials, dielectric and hydrophobic coating materials that provide a smooth surface for droplet movement and a slippery surface.

[0031] II.A. Circuit boards for electrowetting Electrowetting microfluidic devices may be formed by creating a slippery surface (in the sense of low surface energy) directly on an electrode array (120). The electrode array consists of conductive plates (120) that are electrically charged to actuate droplets. The electrodes in the array may be arranged in any layout, e.g., a rectangular grid or a set of discrete paths. The electrodes themselves can be made of any combination of conductive metals (e.g., gold, silver, copper, nickel, aluminum, platinum, titanium), conductive oxides (indium tin oxide, aluminum-doped zinc oxide), and semiconductors (e.g., silicon dioxide). The substrate for laying out the electrode array may be any insulating material of any thickness and rigidity.

[0032] Electrode arrays may be manufactured on standard rigid and flexible printed circuit board substrates. Substrates for PCBs can be FR4 (glass epoxy), FR2 (glass epoxy), or insulated metal substrates (IMS), polyimide films (examples of commercial brands include Kapton and Pyralux), polyethylene terephthalate (PET), ceramic, or other commercially available substrates with thicknesses ranging from 1 μm to 3000 μm. Thicknesses of 500 μm to 2000 μm may be preferred in certain applications.

[0033] Electrode arrays may also be fabricated from conductive and semiconductor elements manufactured using active-matrix technologies such as thin-film transistor (TFT) technology, as well as passive-matrix technologies. Electrode arrays can also be made from arrays of pixels manufactured using conventional CMOS or HV-CMOS manufacturing techniques.

[0034] Electrode arrays can also be manufactured on transparent conductive materials such as indium tin oxide (ITO), aluminum-doped zinc oxide (AZO), fluorine-doped tin oxide (FTO), polyethylene terephthalate (PET), and any other insulating substrates deposited on a sheet of glass.

[0035] Electrode arrays can also be manufactured from metals deposited on glass, polyethylene terephthalate (PET), and other insulating substrates.

[0036] Referring to Figure 6A, in some cases, the electrowetting microfluidic device (100) consists of coplanar electrodes (electrodes on the same layer) without a second plate, and the droplet (110) may rest on an open surface above the plane of the electrodes. In this configuration, the reference electrode (120g) (usually a ground signal) and the driving electrode (120h) are on the same plane, placed on a substrate for a printed circuit board together with a thin insulator on the electrodes. The droplet rests on this insulating layer and is not sandwiched between the two plates. In these cases, sometimes the reference electrode (120g) has a different geometric arrangement compared to the driving electrode. In most cases, the dielectric elements or layers are arranged so that the droplet (110) does not come into contact with an electrode (120) of a different polarity, and as a result, the droplet is exposed only to an electric field and not to an electric current.

[0037] Referring to Figure 6B, in some cases, an electrowetting microfluidic device may consist of two layers of electrodes (one for the reference electrode (120g) and one for the driving electrode (120h)), with one layer on top of the other within the substrate (140) (as opposed to an electrode sandwich with the droplet between plates). Here, the droplet (110) may rest on an open surface and be located on both layers of electrodes. The two layers of electrodes, (120g) and (120h), are typically separated by an extremely thin insulator layer (602) (10 nm to 30 μm). Usually, the layer with the reference electrode (120g) is close to the droplet. Sometimes, the uppermost reference electrode (120g) is in direct contact with the droplet. The reference electrode layer may be less than 500 nm thick and may be coated with a hydrophobic material. The second layer with the reference electrode may be a single continuous trace of any arbitrary shape.

[0038] Referring to Figure 6C, in other configurations, the layers may be arranged from top to bottom as follows: a hydrophobic / insulating layer (130), a layer with electrodes (typically reference or ground) (120g), a dielectric layer (602), a driving electrode layer (120h), and an insulating substrate (140). The droplet (110) rests on the hydrophobic / insulating layer (130) on the uppermost open surface. Since the electrodes (120) are usually metal, it may be desirable that they all be covered with an insulator or dielectric (130) to prevent chemical reactions between the droplet (110) and the electrodes.

[0039] In constructing an electrowetting microfluidic device (100), multiple layers (1-50 layers) of lamination may be used to separate multiple layers (2-50 layers) of electrical interconnect routing. One of the outermost layers of lamination may contain an electrode pad (120) to actuate a droplet and may also contain a reference electrode. The interconnect may connect the electrical pad to a high voltage for driving and capacitance sensing. The operating voltage may be between 5V and 350V. This operating voltage may be an AC signal or a DC signal.

[0040] II.B. Creation of a smooth dielectric surface on an electrode array To electrically isolate droplets from the electrode array, a dielectric layer (130) may be applied to the upper surface of the electrode array (120). Preferably, the upper surface of this dielectric layer (130) may be formed to have little or no resistance to the movement of droplets, and as a result, droplets may be moved by a weak operating voltage (less than 100V, 80V, 50V, 40V, 30V, 20V, 15V, 10V, or 8V DC, depending on the degree of smoothness, slipperiness, and hydrophobicity). To obtain a slippery, low-resistance surface, the dielectric surface may have a smooth surface topography and be hydrophobic, or otherwise reduce droplet adhesion.

[0041] Smooth topographic surfaces are typically characterized by their roughness value. Experiments have shown that as the surface becomes smoother, the voltage required to produce droplet movement changes. Smoothnesses of 2 μm, 1 μm, and 500 nm may be desirable.

[0042] A smooth dielectric surface on an electrode array may be formed by some combination of the following techniques, for example: 1. A two-step process to obtain a relatively smooth surface involves patching surface defects and then covering them with a dielectric material. The defect patching step is typically done with a photoresist, epoxy, or filling compound. The second dielectric layer may be the same material or a polymer film. 2. The second method involves depositing excess photoresist or epoxy onto the electrode array, followed by polishing the excess material to the desired thickness and surface roughness. 3. The third method involves spreading and bonding a thin polymer film onto the surface.

[0043] To prevent droplets from adhering to the smoothed dielectric surface (130), the surface may be further modified to make it slippery by one or more of the following methods: 1. Process to modify the interface chemistry. 2. Process for modifying surface topography 3. Process of applying a slippery liquid coating. Here, we also introduce a novel electrowetting mechanism called liquid-on-liquid electrowetting (LLEW).

[0044] The following sections detail various methods for modifying rough, non-slippery electrode array surfaces into smooth, slippery surfaces.

[0045] II.B.1. Smoothing with photoresist / epoxy / embedding compounds Referring to Figures 7A and 7B, a printed circuit board (PCB) manufactured by a typical process has a rough surface due to defects such as canyons (gaps) between electrodes, holes (also known as vias) for establishing connections between multilayers, holes for joining through-hole components, and other manufacturing errors. Typical dimensions of surface defects range from 30 μm to 300 μm, as small as about 1 μm, and may vary depending on the manufacturing process.

[0046] Various methods may be used alone or in combination to reduce these surface defects and to obtain planes with roughness values ​​of more or less than 1 μm, and they may result in desirable wetting properties and behavior at low voltages.

[0047] Smooth surfaces can be obtained by flowing photoresist, epoxy, embedding compound, or liquid polymer between grooves. Some photoresists in mind may flow between grooves smaller than 10 μm in size in all dimensions and have a kinematic viscosity of less than 8500 centipoise. Commercially available SU-8 photoresist is a good example. A suitable liquid polymer for this application is liquid polyimide.

[0048] Referring to Figure 8A, to fill the valleys between electrodes (120), a nearly flattened surface (802) of the electrode array can be obtained by coating (804) with a photoresist, epoxy, filling compound, liquid polymer, or other dielectric material. The material should have gap-filling properties that allow it to flow into small gaps (e.g., 100 μm (width) × 35 μm (height)) and fill larger gaps. The coating may then be cured to obtain a surface with a desired roughness value of around 1 μm. The metal electrode surfaces may be exposed or covered with the coating.

[0049] (a) Fabrication of dielectrics on smooth photoresists / epoxy resins / embedding compounds Once surface defects are patched by flowing a photoresist or filling compound or epoxy (804), the top surface of the electrode array is more or less planarized. The nearly planar surface may have metal electrodes (120) that require an additional dielectric coating (810) to separate droplets from the charged electrodes while the electric field allows the droplets to spread in areas where they can still be affected by the electric field. The thickness of this coating (810) may be any value between 10 nm and 30 μm. The dielectric layer (810) is formed as a thin film by bonding a polymer film, by various deposition techniques of thin films via various coating methods, as described below, or by any other thin film deposition technique.

[0050] (i) Deposit a thin film coating as a dielectric. Referring to Figure 8B, the top flattened surface (802) (exposed metal electrode (120) and photoresist (804) from the first application in Figure 8A) may be coated with an additional layer of the same photoresist (or epoxy, or embedding compound) material, or with a different material having different dielectric, bonding, and smoothing properties, to form a dielectric layer (810) that electrically isolates the droplet from the electrode. The photoresist may be applied by spin coating, spray coating, or dipping coating.

[0051] The planarized surface (802) may also be coated with a thin film (810) of dielectric material by some form of chemical vapor deposition. Often, this type of deposition results in a film following topography of the coated surface. A class of commercially available materials for vapor deposition is called conformal coating materials and is well-suited for mass production. Conformal coating materials include parylene conformal coatings, epoxy conformal coatings, polyurethane conformal coatings, acrylic conformal coatings, and fluorocarbon conformal coatings. Other coating materials that may be used with vapor deposition include silicon dioxide, silicon nitride, hafnium oxide, tantalum pentoxide, and titanium dioxide.

[0052] (ii) Bond the polymer film to form a dielectric on the top layer. Referring to Figure 8C, to separate droplets from the electrodes, the top flattened surface (802) (metal electrodes (120) and photoresist (804)) may be coated with an additional layer (816) of polymer film. The film (816) may be stretched to remove wrinkles and make it even smoother. The polymer film may be held on the electrode array by thermal bonding, vacuum suction, electrostatically drawing it down, or simply by mechanical holding.

[0053] II.B.2. Formation of a smooth dielectric surface by polishing with an excess amount of photoresist Referring to Figure 8D, a smooth dielectric surface may be obtained by coating the electrode array with a photoresist or other curable dielectric material (820), and then polishing the top surface (822) to obtain a smooth surface (824). The photoresist / dielectric material may be coated using techniques such as spin coating, spray coating, vapor deposition, or immersion coating.

[0054] A first step in this process may be to coat the electrode array (120) with a curable dielectric to a thickness (820) significantly greater than the height of the electrodes. For example, if the electrodes have a height of 35 μm, the thickness of the dielectric coating on the top surface of the electrodes may be at least 70 μm. The dielectric can then be polished (822) with fine abrasives and chemical slurries, typically using a polishing pad larger than the electrode grid array. The buffing process may be continued until the dielectric on the electrodes reaches a desired thickness (15 μm to 500 nm) above the electrodes. Typically, the polishing process also smooths the surface to a surface roughness value of less than 1 μm, and more preferably smoother than 500 nm, 200 nm, or 100 nm. After polishing, it may be desirable to add a hydrophobic coating. A thin, smooth surface with or without a hydrophobic coating may provide sufficient electrowetting force to move droplets at lower voltages.

[0055] II.B.3. Polymer Films as Smooth Dielectric Surfaces Referring to Figure 8E, in some cases a thin polymer film (830) (1 μm to 20 μm) may be used to form a smooth dielectric surface directly on the electrode array. In this case, pretreatment does not require patching some of the valleys with photoresist, epoxy, or filling compounds, and these depressions (832) may be left filled with air. Instead, the film may be applied directly to the electrode surface before modification. In these cases, the film is first stretched to remove any wrinkles (834), and then adhered to the electrode surface. Polymer films with low surface free energy may be used for such applications. Many fluorinated polymers such as PTFE (polytetrafluoroethylene), ETFE (ethylene tetrafluoroethylene), FEP (fluorinated ethylene propylene), and PFA (perfluoroalkoxyalkane) are other low surface energy fluoropolymers that may be suitable for electrowetting. Polydimethylsiloxane (PDMS) is another low surface energy material that may be used as a dielectric for electrowetting. To further reduce surface energy for weak adhesion and excellent electrowetting droplet movement, these low-surface-energy polymer films may sometimes require an additional layer of hydrophobic material. Films made from polymers with slightly higher surface free energy, such as polypropylene, polyimide, Mylar, and polyvinylidene fluoride (PVDF), are also suitable for electrowetting; however, they may require an additional hydrophobic coating or surface modification to facilitate droplet movement.

[0056] II.C. Create a slippery surface as the final product. The surface of electrowetting microfluidic devices may be further treated to reduce or eliminate adhesion to the upper surface of droplets. This additional treatment may allow droplets to be repeatedly moved from one location to another by a low operating voltage. To transform a smooth dielectric surface into a surface that is slippery and less adhesive to droplets, the surface of the dielectric material may be converted to a hydrophobic surface via chemical modification or surface topography modification. Alternatively, this slippery surface may be created by creating a thin layer of lubricant directly on the smooth dielectric or on the electrode array. A hydrophobic coating material may be such that a 1 μl droplet on a surface tilted at an angle of 3 degrees or more will slide off. These methods are described in detail in the following sections.

[0057] II.C.1. Modifying Solid Dielectrics to Achieve Hydrophobicity In some cases, a smooth dielectric surface may not have a surface energy low enough to allow the movement of droplets induced by electrowetting. To further reduce the surface energy, the dielectric surface may be chemically or topographically modified.

[0058] (a) Surface chemical modification (functionalization) Referring to Figure 8F, the surface energy may be reduced by chemical modification, for example, by coating the electrodes (120) and / or dielectrics (130) with hydrophobic or low surface energy materials (840) such as fluorocarbon-based polymers (fluoropolymers) or other hydrophobic surface coatings. The hydrophobic coating may be applied by spin coating, dipping coating, spray coating, chemical vapor deposition, or other methods.

[0059] In some cases, it is preferable to choose a fluorocarbon conformal coating, which can act as both a dielectric (insulating droplets from the charge of the electrical pad while allowing the electric field to propagate) and a hydrophobic (reducing adhesion and allowing smooth droplet movement) coating.

[0060] (b) Surface topography modification To induce hydrophobicity on the surface of a dielectric, its topography may be modified at a microscopic level. Such modification may include patterning the surface to create the deposition of micropillars, or microspheres.

[0061] (i) Creation of micropillars Referring to Figure 9A, the micropillar structure (910) may be fabricated on a film of dielectric layer (130). This top layer above the electrode array acts as a hydrophobic surface.

[0062] Referring to Figures 9B, 9C, and 9D, micropillar structures may be created as dielectrics on electrodes by a first heat-bonded polymer film (920) made of polypropylene, polytetrafluoroethylene (PTFE), Mylar, ethylenetetrafluoroethylene (ETFE), fluorinated ethylene propylene (FEP), perfluoroalkoxyalkane (PFA), other fluorocarbon-based polymers, or other low-surface-energy polymers. The polymer surface may be pressed against a micropillar template (922), such as a polycarbonate film (or other porous film as a template) having pores of 1 μm to 5 μm in size. Referring to Figure 9C, the polycarbonate micropillar template may imprint itself onto the dielectric film by heat and pressure (924). Referring to Figure 9D, when the polycarbonate film is peeled off, it leaves behind microscopic pillar-like structures (910).

[0063] In an alternative, the micropillar structure (910) may be fabricated with polydimethylsiloxane (PDMS) elastomer on the planarized electrode array (after the electrode array has been planarized). In this method, the PDMS elastomer may be cast as a thin film by spin coating. Subsequently, the polycarbonate film may be pressed against the PDMS surface. The PDMS cell membrane may be cured to strengthen it. Subsequently, the polycarbonate film may be dissolved.

[0064] In other options, polymers (ETFE, PTFE, FEP, PFA, PP, Mylar, PVDC) or elastomers (PDMS, silicone) may be bonded to the electrode array and then laser-etched to create micropillars.

[0065] Alternatively, the photoresist material may be deposited onto an electrode array and then etched with a laser to create micropillars. The photoresist may also be patterned and etched using photolithography techniques.

[0066] (ii) Microsphere Referring to Figure 9E, an alternative method to topographic modification for obtaining a slippery or low-adhesion surface is by depositing microspheres (930) with particle sizes ranging from 200 nm to 2 μm. The microspheres may be densely packed to make the surface hydrophobic. A suitable candidate for such microsphere particles is silica beads. To make the surface slippery, these microspheres may be coated with organically functional alkoxysilane molecules. Alternatively, fluorocarbon-based microspheres (PTFE, ETFE) may be deposited, eliminating the need for additional coating.

[0067] II.D. Slippery liquid coatings and electrowetting of liquids onto liquid surfaces (LLEW) II.D.1. Droplets on a Thin Film Liquid Layer of LLEW In LLEW, droplets may rest on a thin film of low-surface-energy oil that provides lubrication. This oil film may form on a low-surface-energy textured solid surface. The textured solid and lubricating oil may be selected so that the lubricating oil is suitable for completely wetting the solid and preferentially avoids interacting with the droplets. Once the textured solid body is filled with oil, a thin layer of oil forms directly on top of the oil-filled object. The self-leveling properties of the upper oil layer can mask any non-uniformity in the topography of the underlying surface. Thus, the surface of an electrode array with significant roughness (tens of micrometers) can be transformed into an almost molecularly smooth surface by the thin film of lubricating oil.

[0068] These molecularly smooth surfaces impart only minimal friction to droplet movement, and droplets may experience little to no fixation. Droplets on such smooth surfaces may have very low contact angle hysteresis (as low as 2 degrees). As a result, low contact angle hysteresis and no droplet fixation can lead to very low operating voltages (1V to 100V) even under vigorous droplet manipulation.

[0069] In a solid body, oil may be trapped within the irregularities or pores that make up the solid's texture. In contrast to a layer of oil on a smooth, untextured surface, oil in a textured solid may have reduced effects of gravity due to its sufficient affinity to the solid surface and molecular interactions. The trapping of oil by the texture may allow the surface to retain its oil layer and its properties when tilted or inverted. Because the oil does not leave the solid surface, a moving droplet rides on the lubricant, and it interacts only with the surface of the lubricant, and not with the underlying textured solid. As a result, the droplet may leave little to no trail on the underlying solid. If the oil does not mix with the droplet, the droplet may move over a liquid film layer without turbidity between two consecutive droplets traversing the path.

[0070] Textured solids can have regular or irregular micro-textures. Examples include: A solid having a microscopic pillar structure on a micron scale, with regularly spaced pillars. A solid having regularly spaced voids; the voids may be of any arbitrary shape. • A random matrix of fibers. A solid having a microscopic pillar structure on a micron scale, with irregularly spaced pillars. A solid having regularly spaced voids; the voids may be of any arbitrary shape. Porous materials such as porous Teflon, porous polycarbonate, porous polypropylene, porous paper, and porous textiles can be used as solids processed with irregular or regular microtextures.

[0071] The lubricant may be any low-energy oil, such as silicone oil, DuPont Krytox oil, Fluorinert FC-70, or other oils. The lubricant may be selected to be non-miscible with the droplet. A lubricant that does not mix with the droplet's solvent may improve the performance of the lubricant or droplet resting on the oil, as it reduces the diffusion of contents from the droplet to the oil and vice versa. The viscosity of the lubricant affects the mobility of the droplet during electrowetting; lower viscosity results in greater mobility. A suitable lubricant is generally non-volatile and does not mix with the target droplet resting on it. If the droplet contains biological components, a biocompatible oil may be desirable. In LLEW devices with on-chip heating elements for incubation and thermal cycling (e.g., for polymerase chain reactions), the oil may be selected to withstand heating and high temperatures. An oil with a sufficiently high dielectric constant may reduce the operating voltage that causes droplet movement.

[0072] Create a textured solid for II.D.2.LLEW. In LLEW, an oil-filled, textured solid can act as an electrical barrier between the electrode array and the droplets, and may also provide a slippery surface for the movement of the droplets. There are many different ways in which a textured dielectric surface can be created on an electrode array.

[0073] Textured solid surfaces may be formed on electrode arrays by bonding polymers or other dielectric materials as a film. The film itself may be textured before being bonded to the electrode array. Alternatively, an untextured film may be bonded to the electrode array and then textured by laser etching, chemical etching, or photolithography.

[0074] Alternatively, a layer of photosensitive material, such as photoresist (SU-8), may be coated onto the electrode array. The photoresist may be patterned by chemical etching, laser etching, or other photolithography techniques.

[0075] Alternatively, textured solids may be created by coating an ultrathin layer of elastomer material, such as PDMS, onto an electrode array, and then using soft lithography techniques to selectively create holes. Following the creation of the thin elastomer layer, the surface of the PDMS may also be laser-etched to create a texture.

[0076] Alternatively, textured solids may be created as follows: Processes that involve conformal coating, liquid-developable (LPI) solder masks, or dry-film-developable solder masks. • The process of etching the surface of this coating, either by laser or by physical stamping. • A process of directly growing a polymer material mesh on an electrode array. • A process of multiplying one molecule at a time to obtain the desired structure.

[0077] II.D.3. Apply lubricant to the textured solid. The textured solid layer may be filled with lubricant by spin coating, spraying, dipping, brushing, or by dispensing from a reservoir.

[0078] Lubricants can sometimes be prevented from leaking out of the LLEW chip by creating a physical or chemical barrier around the device.

[0079] III. Intrinsic properties of liquid electrowetting (LLEW) on top of other liquids LLEW arrays possess two desirable inherent properties for handling biological samples. Because of the smooth surface of the LLEW array, the electrowetting operating voltage can be significantly reduced. Furthermore, the surface structure of the LLEW reduces cross-contamination between specimens by minimizing the trail left behind by droplets and improving the cleaning mechanism.

[0080] III.A. Low Operating Voltage The near-molecular-level smoothness of the oil surface on the LLEW electrode array may reduce or eliminate droplet fixation. A droplet, being an aqueous solution resting on the oil surface, experiences little to no resistance from the surface, resulting in a small difference between its advance and receding angles. Eliminating these two phenomena may result in a lower operating voltage. The droplet may start at a low voltage of around 1V.

[0081] In LLEW devices, droplets resting on a thin layer of oil never physically come into contact with the solid dielectric substrate below the oil. This can reduce or eliminate the amount of material left behind and, therefore, cross-contamination between samples passing through the same spot.

[0082] III.B. Cleaning of the device surface by washing. When an LLEW device becomes contaminated with solid particles such as dust, droplets may be manipulated over the contaminants as part of a cleaning routine to remove them from the liquid film surface. This cleaning routine can be further extended to clean the entire surface of the electrowetting device. For example, the cleaning routine may be used between two biological experiments on an LLEW microfluidic chip to reduce cross-contamination. In some cases, when a droplet remains in one place for an extended period, a small number of molecules may diffuse from the droplet into the oil below. Any residue left behind by the droplet through diffusion can be cleaned up with a similar cleaning routine.

[0083] As droplets move across the LLEW device, they may carry and deplete the oil film from the surface. The oil on the surface can be replenished by injecting oil from an external reservoir, such as an inkjet cartridge, syringe pump, or other distribution mechanism.

[0084] Between two consecutive experiments, the lubricating oil surface may be completely washed and replaced with a layer of fresh oil to prevent cross-contamination.

[0085] IV. Application of electrowetting IV.A. Any large open face The droplets may be operated on an open surface without being sandwiched between the electrode array and the cover plate (neutral glass, or upper electrode array, or simply a large outer electrode). Sometimes, the cover plate above the droplets may be used so as not to be in physical contact with the droplets.

[0086] Electrode arrays and electrowetting on open surfaces and arbitrarily large areas enable the driving of droplets in volumes between 1 nanoliter and 1 milliliter (a six-order-of-magnitude difference). This implementation digitally demonstrates multiscale fluid manipulation on a single device.

[0087] Arbitrarily large two-dimensional arrays (grids) of electrodes can be prepared for electrowetting droplet driving. Compared to a fixed one-dimensional trajectory, a two-dimensional array provides multiple paths for the droplet. These grids can be utilized to avoid cross-contamination between droplets of two different compositions. For example, a two-dimensional grid may allow multiple droplets to be started in parallel. Droplets with different solutes may be run on separate parallel trajectories to reduce contamination. Multiple separate biological experiments may be run in parallel.

[0088] IV.B. Droplet movement, integration, and division The droplets may be moved, merged, and / or split on the open surface of the electrowetting device. The same principle applies to a two-plate configuration (with the droplet sandwiched between them).

[0089] Figures 10A, 10B, and 10C show the movement of a droplet (110) on an array electrode (120). In Figure 10A, applying a voltage to electrode (120i) makes the overlapping surface hydrophilic, allowing the droplet to then penetrate it. When the voltage is removed from electrode (120i) and applied to another adjacent electrode (120j), the surface returns to its original hydrophobic state, as shown in Figure 10C, and the droplet is pushed out. By continuously controlling the voltage applied to the electrode grid, the position of the droplet on the surface can be precisely controlled.

[0090] Referring to Figures 10D, 10E, and 10F, two droplets can merge. When two droplets are drawn toward the same electrode (120k), they naturally merge due to surface tension. This principle can be applied to merging many droplets to create a larger volume droplet that spreads across multiple electrodes.

[0091] Referring to Figures 10G, 10H, and 10I, a droplet may split into two smaller droplets due to a sequence of voltages applied across multiple electrodes (at least three). In Figure 10G, a single large droplet merges onto a single electrode (120l). In Figure 10H, equal voltages are applied simultaneously to three adjacent electrodes, causing the single droplet to spread across the three adjacent electrodes. In Figure 10I, cutting the central electrode (120l) forces the droplet to move out onto the two outer electrodes (120m) and (120n). Due to the equal potentials on both of the two outer neighboring electrodes, the droplet then splits into two smaller droplets.

[0092] IV.C. Lab in a Box (Desktop Digital Wet Lover) Any combination of the manufacturing methods described so far may be used in the applications discussed in this section.

[0093] Figure 11A shows a “desktop digital wet lab” (1100) based on digital microfluidics. This device may provide a versatile machine capable of automating a wide variety of biological protocols / assays / tests. The box may have a lid that can be opened and closed. The lid may have a transparent window (1102) for observing the movement of droplets on an electrode array, which may be formed as a digital microfluidic chip. The box may house a digital microfluidic chip (100) that can move, integrate, and divide droplets, where the droplets can carry biological reagents. The microfluidic chip may also have one or more heaters or coolers (1128) that can heat droplets to a high temperature of about 150°C or cool them to a low temperature of about -20°C.

[0094] The droplets may be distributed onto the tip through one or more “liquid dispenser” droppers. Each liquid dispenser could be an electrofluidic pump, syringe pump, simple tubing, robotic pipette, inkjet nozzle, acoustic dispensing device, or other pressure-driven or non-pressure-driven device. The droplets may be fed into the liquid dispensers from a reservoir called a “cartridge.” A “love-in-box” may have hundreds of cartridges that connect directly to the microfluidic tip.

[0095] Droplets may be moved from a digital microfluidic tip onto a microplate. A microplate is a plate with wells that can hold a sample. A microplate can have any number of wells, from 1 to 1 million, on a single plate. Multiple microplates may be connected to tips in a box. Electrowetting tips with various geometric shapes may be used to distribute droplets from the microfluidic tip to the microplate. In some cases, the dispensing tip may be in the form of a cone, similar to a pipette tip. In another form, the dispensing aperture may simply be a cylinder. In yet another form, the dispensing device may be two parallel plates with a gap between them. In yet another form, the dispensing device may be a single open surface with droplets moving on an open surface. The dispensing mechanism may also use many other mechanisms, such as electrofluidic pumps, syringe pumps, tubing, capillaries, paper, wicks, or just a simple hole in the tip.

[0096] "Love in a Box" may have an atmosphere-controlled environment to adjust the internal temperature, humidity, and oxygen concentration. The inside of the box may be under vacuum.

[0097] The digital microfluidic chip 130 in the center of the box may be removed, washed, and replaced.

[0098] Digital microfluidic devices may include sensors such as optical spectrometers and acoustic transducers to perform various assays.

[0099] Digital microfluidic devices may include magnetic bead-based separation units for DNA size selection, DNA purification, protein purification, plasmid collection, and any other biological workflow using magnetic beads. The devices may perform many—from 1 to 100,000—magnetic bead-based operations simultaneously on a single chip.

[0100] The box may be equipped with multiple cameras that view the chip from the top, sides, and bottom. The cameras may be used on the chip to position the droplet, measure its volume, measure the degree of mixing, and analyze the reaction in progress. Information from these sensors may be provided as feedback to a computer that controls the electrical flow to the electrodes, so that the droplet can be precisely controlled to achieve a high throughput rate through accurate droplet positioning, mixing, etc.

[0101] Love-in-boxes may be used to perform microplate operations such as plate stamping, serial dilution, plate replication, and plate rearrangement.

[0102] A Love-in-Box may include instruments for PCR amplification and DNA assembly (Gibson assembly, Golden Gate assembly), molecular cloning, DNA library construction, RNA library construction, DNA sequencing, single-cell sorting, cell incubation, cell culture, cell assay, cell lysis, DNA extraction, protein extraction, RNA extraction, and RNA and cell-free protein expression.

[0103] IVD Processing Station The electrowetting chip (with or without a love-in-box enclosure) may contain one or more stations for various functions.

[0104] IV.D.1. Mixing Station Referring to Figure 11B, an electrowetting device may incorporate one or more mixing stations (1120). On the left, there is a 2x2 assembly of electrowetting-based mixing stations, which may operate in parallel. A single mixer (1120) has a 3x3 grid of driving electrodes. Each mixing station (1120) may be used to mix biological samples, chemical reagents, and liquids. For example, droplets of two reagents may be brought together to a mixing station and then mixed by moving the combined droplet around eight electrodes outside the 3x3 grid, or by moving it through another pattern designed to mix the two original droplets. The center-to-center distance between each mixer may be 9 mm, equivalent to the spacing of a standard 96-well plate.

[0105] The parallel mixing station (1120) may be extended to have many different configurations. Each individual mixer may consist of any number of drive electrodes in an A×B pattern (1122). Furthermore, the spacing between mixers is arbitrary and may be modified to suit the application (such as other SDS plates). The parallel mixing station may also have any number of individual mixers in an M×N pattern (1122). The parallel mixing station may have any configuration of top plate, including, but not limited to, open-face plates, closed plates, or closed plates with liquid inlets.

[0106] IV.D.2. Incubation Station Referring to Figure 11C, an electrowetting chip may include one or more incubation stations (1128). Each individual incubator (1128) may integrate one or more functions applied to the liquid sample, such as mixing, heating (to a temperature up to, for example, 150°C), or cooling (to, for example, -20°C), to compensate for fluidity loss due to evaporation and to homogenize the sample temperature. Heating or cooling may be performed by thermocouples or evaporative heat exchangers on the substrate. In some cases, individualized heating elements may allow each station to be controlled to a separate temperature (e.g., -20°C, 25°C, 37°C, 95°C) depending on the heat transfer output of each element and the level of heat conduction between stations.

[0107] A parallel incubation station may be configured to have the same configuration as any of the parallel mixed stations.

[0108] IV.D.3. Magnetic Bead Station Referring to Figure 11D, a magnetic bead washing station (1134) may contain a sample on an electrode grid comprising nucleic acids, proteins, cells, buffers, magnetic beads, washing buffers, elution buffers, and other liquids (1136). The station may be configured to mix the sample and reagents and apply heating or other treatments in a sequential order to perform nucleic acid isolation, cell isolation, protein isolation, peptide purification, biopolymer isolation or purification, immunoprecipitation, in vitro diagnostics, exosome isolation, cell activation, cell elongation, and / or isolation of specific biomolecules. In addition to mixing and heating liquids, each magnetic bead station may have the ability to locally generate and stop strong and varying magnetic fields, thereby, for example, moving the magnetic beads to the bottom of the electrowetting tip. Each magnetic bead station may also have the ability to remove excess supernatant and washings by the force of electrowetting or other forces.

[0109] In some cases, the sample may be on an open surface with a single-plate electrowetting device. In other cases, the sample may be sandwiched between two plates. Multiple magnetic bead stations may be configured to operate in parallel, as described above for parallel mixing stations.

[0110] IV.D.4. Nucleic Acid Delivery Station Referring to Figure 11E, an electrowetting chip may contain one or more nucleic acid delivery stations (1140). Each individual parallel nucleic acid delivery station may be designed to deliver genetic material (1142), other nucleic acids, and biologics into cells by various insertion methods. This delivery may be performed by applying a strong magnetic field, a strong electric field, ultrasound, laser beams, or other techniques. One or more nucleic acid delivery stations may be configured as singletons on the electrowetting device, or multiple nucleic acid delivery stations may be supplied to operate in parallel.

[0111] IV.D.5. Optical Inspection Station Referring to Figures 11F and 11G, one or more optical inspection stations (1150) using optical detection and assay methods may be provided on an electrowetting device (100). A light source (1152) (broad-spectrum, single-frequency, or other illumination) may pass through an optical system (1154) (filters, diffraction gratings, mirrors, etc.) to adjust the light, and then illuminate a sample (1156) located on the electrowetting device. An optical detector on the opposite side of the electrowetting device is configured to detect the spectrum of light passing through the sample for analysis. Optical inspection may be used for measuring nucleic acid concentration, nucleic acid quality, cell density, degree of mixing between two liquids, sample volume, fluorescence of a sample, absorption of a sample, protein quantification, colorimetric quantification assays, and other biological assays.

[0112] As shown in Figure 11F, the sample (1156) may be on an open surface with a single-plate electrowetting device (100). As shown in Figure 11G, the sample (1156) may be sandwiched between two plates (100) and (1160). In some cases, the electrowetting tip and electrode may be transparent. In some cases, the electrode on which the sample is placed may have holes to allow light from a light source to pass through the sample and reach an optical detector, or to introduce the sample, reagent, or reactant.

[0113] Referring to Figure 11H, optical detection (1150) is performed on samples arranged in a 2×2 sample format, a 96-well plate format for optical detection, or an M×N format, which can reach up to one million samples. Samples and corresponding units of measurement may be arranged in any regular or irregular format.

[0114] IV.D.6. Loading / unloading via acoustic liquid handler or microdiaphragm-based pump dispenser Referring to Figures 11I and 11J, an electrowetting device may include one or more stations (1160) for loading biological samples, chemical reagents, and liquids from a source well, plate, or reservoir onto an electrowetting tip (100).

[0115] In Figure 11I, droplets may be placed on the electrowetting surface by acoustic droplet ejection. The source plate may hold liquid in the well (1164) and may be coupled to the piezoelectric transducer (1162) via an acoustically coupled fluid (1166). Acoustic energy from the piezoelectric acoustic transducer (1162) may be concentrated on the sample in the well (1164). Note that in Figure 11I, the electrowetting tip (100) is placed on top and inverted. Note that droplets (110) adhere to the electrowetting tip (100) because an additional wetting force is induced by the voltage, which contributes to the droplet sorting function of the apparatus (1160). Droplets (1168) ejected from the well (1164) by acoustic energy may adhere to the upper electrowetting device (100) or be incorporated into droplets moved to the acoustic injection station.

[0116] Referring to Figure 11J, the electrowetting device may include one or more stations (1180) designed to place biological samples, chemical reagents, and liquids (1182) onto the electrowetting tip by a microdiaphragm pump (1184) dispenser.

[0117] Either the acoustic droplet dispensing technique shown in Figure 11I or the microdiaphragm pump (1184) can be used to distribute fluid droplets of picoliters, nanoliters, or microliters in volume. An electrowetting device (100), positioned above the source plate (Figure 11I), captures and holds droplets (1168) dispensed from the well plate by the force of electrowetting. In this manner, samples containing nucleic acids, proteins, cells, salts, buffers, enzymes, and any other biological and chemical reagents can be distributed onto the electrowetting chip. In one alternative version (Figure 11J), the electrowetting plate (100) is at the bottom, and the acoustic droplet dispensing transducer ((1162) in Figure 11I), or the microdiaphragm pump (1184), is at the top. A valve (1186) and a larger microdiaphragm pump (1188) may be used to measure the flow rate to the microdiaphragm pump (1184). In this method, a dispenser can be used to place the sample anywhere on the electrowetting tip.

[0118] In some cases, the electrowetting tip has an open-plate configuration (without a second plate), and droplets may be placed directly on the tip. In some cases, the electrowetting tip may have a second plate between the electrode array and the ground electrode, sandwiching the droplets. In some cases, the second plate (a cover plate with or without ground) may have holes to allow the droplets to pass through. In some cases, droplets may be placed first on the open plate, and then the second plate is added. In some cases, the liquid placed on the electrowetting tip is prepared to perform the workflow when the tip is inside an acoustic liquid handler. In some cases, the liquid placed on the electrowetting tip is prepared to perform the workflow when the tip is positioned outside an acoustic liquid handler or a microdiaphragm pump. In some cases, the liquid is placed on the electrowetting tip while the workflow is being performed. In some cases, an acoustic droplet injector or microdiaphragm pump may be mounted on a positionable carriage (somewhat like a 3D printer nozzle) that can move on the electrowetting device, resulting in droplets being injected at specific locations on the electrowetting device.

[0119] Other options for introducing or injecting droplets may include inkjet nozzles in inkjet printers, syringe pumps, capillary tubes, or pipettes.

[0120] In some cases, both the source and destination can be an electrowetting chip. In this scenario, the chip may be configured such that the electrode arrays face each other. In some cases, droplets may be moved between upper and lower electrowetting chips, or between front and rear electrowetting chips, using an acoustic field or electric field and differential wet affinity. Here, there are acoustic transducers and coupling fluid on both sides of the chip. In some cases, the sample on the electrowetting chip may be the source, and the well plate may be the destination. Here, the sample is moved from the electrowetting chip onto the well plate using acoustic droplet ejection.

[0121] The spacing between wells in the well plate, and therefore the format in which the liquid is placed on (and moved away from) the electrowetting tip, may be a standard well plate configuration, another SDS well plate format, or any arbitrary format. The number of wells in the plate may be any number ranging from 1 to 1 million.

[0122] Electrowetting tips, loaded with samples from acoustic droplet dispensing devices or microdiaphragm pump devices, may be combined with one or more functions such as a mixing station, incubation station, magnetic bead station, nucleic acid delivery station, or optical inspection station.

[0123] V. Alternative Embodiments Droplets located on a VA open surface (single-plate configuration) and sandwiched between two plates (double-plate configuration) Referring to Figure 12A, for electrowetting droplet operation, droplets may be placed on an open surface (single plate) (1200), (100) or sandwiched between two plates (double plate) (100), (1202), (1210). In the double plate configuration (1202), droplets can be sandwiched between two plates (100), (1210) typically divided into 100 μm to 500 μm sections. The double plate configuration has an electrode (120) on one side for supplying the operating voltage, and simultaneously supplying a reference electrode (typically a common ground signal) on the opposite side (1210). Constant contact of the droplet with the reference electrode in the double plate configuration increases the force from the electric field on the droplet, and thus provides robust control over the droplet. Droplets in the double plate configuration (1210) can be separated at a lower operating voltage. In a single-plate configuration (1200), the drive electrode and the reference electrode are on the same side.

[0124] Two-plate electrowetting systems may be improved by the surface treatments described above. In a two-plate system, droplets are sandwiched between plates separated by a small distance. The space between the plates may be filled with another fluid or simply air. By smoothing the liquid-facing surfaces of the two plates to 2 μm, 1 μm, or 500 nm using the techniques described above, the two-plate system may operate at low voltage, reduce droplet fixation, reduce residual orbits, reduce cross-contamination, and reduce sample loss.

[0125] VB Optoelectrowetting and Photoelectrowetting Referring to Figures 12B and 12C, directly applying a potential to an array electrode is one method of acting on droplets using electrowetting; however, there are alternative electrowetting mechanisms that differ from this conventional electrowetting mechanism. Two notable mechanisms, both of which use light to actuate droplets, are described below—opto-electrowetting and photo-electrowetting. The general principles for fabricating electrowetting arrays and creating smooth and slippery surfaces, as described above, are applicable not only to the conventional electrowetting described earlier, but also to opto-electrowetting and photo-electrowetting, and other forms of electrowetting.

[0126] A liquid film may be placed on a grid of photoconductors to result in "optoelectrowetting of the liquid onto the liquid." Instead of having a grid of electrodes in a lubricating liquid layer, the grid may be formed from the photoactive photoconductor to the grid of pads, or as a single line of photoconductors. Light illuminating the photoconductor may form a pattern and result in an electrowetting effect. Textured solids and oils may be selected to be sufficiently transparent to light so that the substrate surface is exposed to light to produce differential wetting.

[0127] VC Optoelectrowetting Referring to Figure 12B, the optoelectrowetting mechanism (1230) may use a photoconductor (1232) located directly beneath a conventional electrowetting circuit (on the left, (100)) to which an AC power supply (1234) is attached. Under normal (dark) conditions, the majority of the system's impedance is in the photoconductive region (1232) (since it is nonconductive), and therefore the majority of the voltage drop occurs here. However, when light (1236) is shone on the system, carrier generation and recombination cause spikes in the conductivity of the photoconductor (1232), and the voltage drop across the photoconductor (1232) is reduced. As a result, a voltage-dependent voltage drop occurs across the insulating layer (130), changing the contact angle of (540) relative to (1238).

[0128] VD Photoelectrowetting Conventional electrowetting is observed in droplets located on a dielectric-coated conductor (a stack of liquid (110) / insulator (130) / conductor (120)), but photoelectrowetting can be observed by replacing the conductor (120) with a semiconductor (1252) (a stack of liquid / insulator / semiconductor).

[0129] Incident light (1254) above the bandgap of semiconductor (1252) creates light-induced carriers in the depletion region of the semiconductor (1252) below through the creation of electron-hole pairs. This leads to a modification of the capacitance of the insulator / semiconductor stack (130) / (1252), resulting in a modification of the contact angle of droplets remaining on the surface of the stack. The figure illustrates the principle of the photoelectrowetting effect. At zero bias (0V), if the insulator is hydrophobic, the conducting droplet (1258) has a large contact angle (left image). As the bias is increased (positive for p-type semiconductors, negative for n-type semiconductors), the droplet (1260) spreads out—i.e., the contact angle decreases (center image). In the presence of light (1254) (which has energy exceeding the band gap of the semiconductor (1252)), the droplet (1262) expands further at the insulator / semiconductor interface (130) / (1252) due to the cross-sectional reduction rate of the thickness of the space charge region (right image).

[0130] VE software and hardware The various processes described herein may be performed by appropriately programmed general-purpose computers, specialized computers, and computing devices. Generally, a processor (e.g., one or more microprocessors, one or more microcontrollers, one or more digital signal processors) receives instructions (e.g., from memory or similar devices), executes those instructions, and thereby performs one or more processes defined by those instructions. Instructions may be concretely expressed in one or more computer programs, one or more scripts, or other forms. Processes may be performed in one or more microprocessors, central processing units (CPUs), computing devices, microcontrollers, digital signal processors, or similar devices, or in any combination thereof. Programs that perform processes, and operational data, may be stored and transmitted using various media. In some cases, hardwired circuits or custom hardware may be used instead of, or in combination with, some or all of the software instructions that can perform the processes. Algorithms other than those described may be used.

[0131] Programs and data may be stored in various media appropriate for the purpose, or in combinations of heterogeneous media that can be read and / or written by a computer, processor, or similar device. Media may include non-volatile media, volatile media, optical or magnetic media, dynamic RAM (DRAM), static RAM, floppy disks, flexible disks, hard disks, magnetic tape, any other magnetic media, CD-ROMs, DVDs, any other optical media, punch cards, paper tape, any other physical media with a pattern of holes, RAM, PROMs, EPROMs, FLASH-EEPROMs, other memory chips, or cartridges, or other memory technologies. Transmission media may include coaxial cables, copper wires, and fiber optics, including wires encompassing a system bus coupled to a processor.

[0132] Databases may run using a database management system or an ad-hoc memory configuration scheme. Alternative database structures to those described are readily available. Databases may be stored locally or remotely from devices accessing the data within such databases.

[0133] In some cases, processing may be performed in a network environment that includes a computer communicating with one or more devices (e.g., via a communication network). The computer may communicate directly or indirectly with the devices via any wired or wireless medium (e.g., the Internet, LAN, WAN or Ethernet, Token Ring, telephone lines, cable lines, wireless communication channels, optical communication lines, commercial online service providers, bulletin board systems, satellite communication links, or any combination of the above). Each of the devices may itself consist of a computer or other computing device based on an Intel®, Pentium®, or Centrino® processor, which is suitable for communicating with the computer. Any number and type of devices may communicate with the computer.

[0134] A server computer or centrally located authority may be necessary, unnecessary, or desirable. In various cases, the network may or may not include a central authority device. Various processing functions may run on a central authority server, one of several distributed servers, or other distributed devices.

[0135] VF and other alternatives For the reader's convenience, the above description focuses on representative samples of all possible embodiments that teach the principles of the invention and convey the best form intended for its implementation. Throughout this application and its associated file history, when the term “invention” is used, it refers to the entire set of ideas and principles described; in contrast, the formal definition of the protected exclusive intellectual property rights is explicitly stated in the claims, which have exclusive control. The description does not attempt to thoroughly enumerate all possible variations. Other undescribed variations or improvements are possible. Where multiple alternative embodiments are described, in many cases it is possible to combine elements of different embodiments or elements of the embodiments described herein with other improvements or variations that are not explicitly described. Unless otherwise clearly stated, the listing of items does not imply that any or all of the items are mutually exclusive, or that any or all of the items are comprehensive in any category. In many cases, one feature or group of features may be used separately from the entire apparatus or method described. Many such undescribed variations and improvements are within the exact scope of the following claims, and others are as well.

Claims

1. A method for moving a plurality of droplets on an open surface, A step of providing the plurality of droplets onto an electrode array adjacent to an open surface, The process of heating the plurality of droplets, A step of moving the plurality of droplets on the open surface by modifying the wetting characteristics of the open surface, Methods that include...

2. The method according to claim 1, wherein the open surface comprises a dielectric material disposed adjacent to the electrode array.

3. The method according to claim 1, wherein the polymer is spread and bonded onto the electrode array.

4. The method according to claim 1, wherein the open surface comprises an uppermost layer containing a liquid layer.

5. The method according to claim 4, wherein the liquid layer does not mix with the plurality of droplets.

6. The method according to claim 4, wherein the liquid layer is dielectric or hydrophobic.

7. The method according to claim 1, wherein the open surface comprises one or more orbitals of the electrode array for moving the plurality of droplets.

8. The method according to claim 1, further comprising the step of distributing the plurality of droplets from a dispenser of a positionable carriage.

9. The method according to claim 8, wherein the dispenser comprises a microdiaphragm pump, an acoustic transducer, an inkjet nozzle, a syringe pump, a capillary tube, or a pipette device.

10. The method according to claim 8, further comprising the step of moving the positionable carriage to a specific location on the electrode array.

11. The method according to claim 1, wherein the open surface does not have an electrode or plate that overlaps or faces it.

12. A system for moving a plurality of droplets on an open surface, An electrode array adjacent to an open surface, wherein the plurality of droplets are provided on the open surface, One or more heating elements connected to the open surface, wherein the heating elements heat the plurality of droplets on the open surface, One or more processors connected to the electrode array, wherein the one or more processors modify the wetting properties of the open surface, and the wetting properties cause the plurality of droplets to move on the open surface, A system that includes these features.

13. The system according to claim 12, wherein the open surface comprises a dielectric material disposed adjacent to the electrode array.

14. The system according to claim 12, wherein the polymer is spread and bonded onto the electrode array.

15. The system according to claim 12, wherein the open surface comprises an uppermost layer including a liquid layer.

16. The system according to claim 15, wherein the liquid layer does not mix with the plurality of liquid droplets.

17. The system according to claim 15, wherein the liquid layer is dielectric or hydrophobic.

18. The system according to claim 12, wherein the open surface comprises one or more orbitals of the electrode array for moving the plurality of droplets.

19. The system according to claim 12, further comprising a dispenser configured to distribute the plurality of liquid droplets.

20. The system according to claim 19, wherein the dispenser comprises a microdiaphragm pump, an acoustic transducer, an inkjet nozzle, a syringe pump, a capillary tube, or a pipette device.

21. The system according to claim 19, wherein the dispenser is configured to move to a specific location on the electrode array.

22. The system according to claim 12, wherein the open surface does not have any overlapping or facing electrodes or plates.

Images