Patent Publication Number: US-2015075985-A1

Title: Droplet Dispensing Device and Methods

Description:
RELATED PATENT APPLICATIONS 
     This application claims priority to U.S. Patent Application No. 60/910,897, filed on Apr. 10, 2007, entitled “Droplet dispensing methods for droplet microactuators”; and U.S. Patent Application No. 60/980,202, filed on Oct. 17, 2007, entitled “Droplet dispensing designs and methods for droplet actuators”; the entire disclosures of which are incorporated herein by reference. 
    
    
     GOVERNMENT INTEREST 
     This invention was made with government support under DK066956-02 awarded by the National Institutes of Health of the United States. The United States Government has certain rights in the invention. 
    
    
     BACKGROUND 
     Droplet actuators are used to conduct a wide variety of droplet operations. A droplet actuator typically includes a substrate associated with electrodes configured for conducting droplet operations on a droplet operations surface thereof and may also include a second substrate arranged in a generally parallel fashion in relation to the droplet operations surface to form a gap in which droplet operations are effected. The gap is typically filled with a filler fluid that is immiscible with the fluid that is to be subjected to droplet operations on the droplet actuator. Among the droplet operations which may be effected on a droplet actuator is the dispensing of a droplet from a fluid source. There is need in the art for improved approaches to dispensing droplets on a droplet actuator. 
     BRIEF DESCRIPTION OF THE INVENTION 
     The invention provides a method of forming multiple droplets on a droplet actuator. The method may, for example, involve providing a droplet actuator. Various basic droplet actuator structures are described herein and/or are known in the art. These may be modified as described herein to perform the unique methods of the invention. In one embodiment, the modified droplet of the invention includes a base substrate having: (i) droplet operation electrodes configured for conducting one or more droplet operations; (ii) a perimeter barrier surrounding the electrodes comprising multiple openings, each opening approximately adjacent to one or more electrodes of the droplet operation electrodes; and (iii) a flow path exterior to the perimeter barrier and arranged to flow fluid through the multiple openings into proximity with the one or more electrodes. Droplets may be dispensed by flowing fluid through the flow path, through the openings in the perimeter barrier and into proximity with the one or more electrodes and conducting one or more droplet operations to form droplets on the droplet operation electrodes. 
     In another embodiment, the method of forming multiple droplets on a droplet actuator, includes providing fluid on one or more activated electrodes and draining fluid from around the activated electrodes, leaving droplets on the activated droplet operation electrodes. Fluid may, for example, be provided on activated electrodes by (i) flowing fluid onto at least a portion of the droplet operation electrodes; and (ii) activating one or more of the droplet operation electrodes. 
     Another embodiment relates to a method of dispensing one or more sub-droplets from a droplet on a droplet actuator, the method including: (i) providing a path of electrodes in proximity to a droplet; (ii) activating electrodes in the path of electrodes to form the droplet into a slug arranged along the path of electrodes and transport the slug along the path of electrodes; and (iii) selectively deactivating electrodes in the path of electrodes at a trailing end of the slug to pinch off one or more sub-droplets from the trailing end of the slug. 
     Yet another embodiment relates to a method of dispensing one or more sub-droplets from a droplet on a droplet actuator, the method: (i) providing a path of electrodes in proximity to a droplet; (b) activating electrodes in the path of electrodes to form the droplet into a slug arranged along the path of electrodes and transport the slug along the path of electrodes; and (c) selectively deactivating electrodes in the path of electrodes at a trailing end of the slug to pinch off one or more sub-droplets from the trailing end of the slug. 
     In another aspect, the method of dispensing one or more sub-droplets from a droplet on a droplet actuator makes use of a droplet actuator comprising: (i) a base substrate comprising electrodes configured for conducting droplet operations; and (ii) a top substrate separated from the base substrate to form a gap, the top plate comprising: (1) a reservoir; and (2) an opening forming a fluid path from the reservoir into the gap. The reservoir opening may be arranged such that when a fluid is provided in the reservoir, the fluid is brought into proximity to a first electrode, which first electrode is adjacent to a second electrode. The method may include (a) causing the first and second electrodes to be activated, thereby causing fluid to flow from the reservoir onto the first and second electrodes; and (b) deactivating the first electrode, causing a droplet to form on the second electrode and causing the remaining fluid to return substantially to the reservoir. 
     The invention also provides method of dispensing one or more sub-droplets from a droplet on a droplet actuator including a base substrate with a droplet operation electrodes configured for conducting droplet operations and a recessed reservoir region configured for holding a droplet in proximity to one or more of the electrodes. The droplet actuator may also include a top substrate separated from the base substrate to form a gap. The method may include (a) causing a first electrode adjacent to the recessed reservoir region and a second electrode adjacent to the first electrode to be activated, thereby causing fluid to flow from the reservoir onto the first and second electrodes; and (b) deactivating the first electrode, causing a droplet to form on the second electrode and causing the remaining fluid to return substantially to the recessed reservoir region. 
     In another aspect, the invention provides a method of dispensing one or more sub-droplets from a droplet on a droplet actuator having a set of electrodes with a set of successively smaller substantially crescent shaped planar electrodes, arranged concentrically substantially in a common plane along a common axis positioned midway between vertices of the substantially crescent-shaped electrodes, wherein each successively smaller electrode is positioned adjacent to the next larger electrode. The droplet actuator may also include a set of planar dispensing electrodes substantially in a common plane with the crescent shaped electrodes, arranged substantially along the common axis of the crescent. In some cases, the droplet actuator includes a top substrate separated from the base substrate to form a gap. The method generally involves (a) ausing a first electrode adjacent to the recessed reservoir region and a second electrode adjacent to the first electrode to be activated, thereby causing fluid to flow from the reservoir onto the first and second electrodes; and (c) deactivating the first electrode (or an electrode intermediate to the crescent shaped electrodes and the terminal activated electrode or electrodes), causing a droplet to form on the second electrode and causing the remaining fluid to return substantially to the recessed reservoir region. 
     A further aspect of the invention is a droplet actuator having a base substrate with (a) droplet operation electrodes configured for conducting one or more droplet operations; (b) a perimeter barrier surrounding the electrodes comprising multiple openings, each opening approximately adjacent to one or more electrodes of the droplet operation electrodes; and (c) a flow path formed in the perimeter barrier and arranged to flow fluid through the multiple openings into proximity with the one or more electrodes. 
     Another droplet actuator of the invention includes (a) a base substrate having electrodes configured for conducting droplet operations; and (b) a top substrate separated from the base substrate to form a gap, the top plate comprising: (i) a reservoir; and (ii) an opening forming a fluid path from the reservoir into the gap; wherein the reservoir opening is arranged such that when a fluid is provided in the reservoir, the fluid is brought into proximity to a first one of the electrodes. 
     Still another aspect relates to a droplet actuator with (a) a base substrate comprising: (i) droplet operation electrodes configured for conducting droplet operations; and (ii) a recessed reservoir region configured for holding a droplet in proximity to one or more of the droplet operation electrodes; and (b) a top substrate separated from the base substrate to form a gap. 
     A further droplet actuator embodiment includes a set of electrodes comprising a set of successively smaller substantially crescent shaped planar electrodes, arranged: concentrically; or substantially in a common plane along a common axis positioned midway between vertices of the substantially crescent-shaped electrodes, wherein each successively smaller electrode is positioned adjacent to the next larger electrode. 
     In another method aspect, the invention provides a method of manipulating a droplet on a droplet actuator, the method comprising: (a) providing a droplet actuator comprising: (i) a reservoir electrode comprising an array of multiple, independently controllable electrodes; (ii) a structure proximate the reservoir electrode comprising an opening; (iii) a transfer electrode positioned in fluid communication with both the reservoir electrode and the opening; and (iv) a flow path through the opening, transfer electrode and the reservoir electrode; and (b) flowing fluid through the flow path. 
     Another method of the invention relates to forming a droplet on a droplet actuator, the method comprising: (a) providing a droplet actuator comprising: (i) a reservoir electrode; (ii) a structure proximate the reservoir electrode comprising an opening; (iii) a transfer electrode positioned in fluid communication with both the reservoir electrode and the opening, wherein the transfer electrode at least partially overlaps with the opening; and (iv) a flow path through the opening and transfer electrode and the reservoir electrode; and (b) flowing fluid through the flow path. 
     Yet another method of manipulating a droplet on a droplet actuator according to the invention includes (a) providing a droplet actuator comprising: (i) a droplet operation electrode configured for conducting one or more droplet operations; (ii) a structure comprising an opening; and (iii) a reservoir electrode proximate both the droplet operation electrode and the opening; and (b) providing a flow path through the opening, reservoir electrode and droplet operation electrode. 
     The invention also provides a method of manipulating a droplet on a droplet actuator, the method including the following steps: (a) supplying a droplet to a reservoir electrode; (b) embedding an electrode within the reservoir electrode; (c) selectively activating electrodes in a path of electrodes that includes the embedded electrode to form the droplet into a slug arranged along the path of electrodes and to transport the slug along the path of electrodes; and (d) selectively deactivating electrodes in the path of electrodes at a trailing end of the slug to pinch off one or more sub-droplets from the trailing end of the slug. 
     In still another aspect, the method of manipulating droplets on a droplet actuator includes: (a) providing a droplet actuator comprising: (i) a reservoir electrode; (ii) a structure proximate the reservoir electrode comprising an opening; (iii) a plurality of electrode arrays respectively in fluid communication with the reservoir electrode; and (iv) a plurality of flow paths through the opening, reservoir electrode and each respective electrode array; and (b) flowing fluid through at least one of the flow paths. 
     The invention also provides a method of manipulating droplets on a droplet actuator, the method comprising: (a) providing a droplet actuator comprising a structure comprising an opening in fluid connection with a plurality of flow paths; and (b) flowing fluid through the plurality of flow paths. 
     In another aspect, the invention provides method of manipulating droplets on a droplet actuator, the method comprising: (a) providing a droplet actuator comprising: (i) a structure comprising an opening in fluid connection with a plurality of other openings; (ii) a plurality of fluid reservoirs respectively in fluid communication with each of the other openings; (iii) a plurality of electrodes in respective fluid communication with the fluid reservoirs; and (iv) a plurality of flow paths through the opening, the other openings, the reservoirs and the electrodes; and (b) flowing fluid through the plurality of flow paths. 
     The invention provides a method of manipulating a droplet on a droplet actuator, the method comprising: (a) supplying a droplet to a reservoir electrode; (b) embedding an electrode within the reservoir electrode; (c) selectively activating the embedded electrode so as to retain a portion of the droplet proximate the embedded electrode; and (d) evacuating another portion of the droplet from the reservoir electrode. 
     Another method of dispersing magnetic beads within a droplet in a droplet actuator includes: (a) providing a droplet actuator, comprising: (i) a plurality of transport electrodes configured to transport the droplet; and (ii) a magnet field present at a portion of the plurality of transport electrodes; (b) transporting the droplet along the plurality of transport electrodes away from the magnetic field; and (c) transporting the droplet along the plurality of transport electrodes towards the magnetic field. 
     The invention provides a method of manipulating a droplet comprising magnetic beads within a droplet actuator, the method comprising: (a) providing a droplet actuator, comprising: (i) a plurality of transport electrodes configured to transport the droplet; and (ii) a magnetic field present at a portion of the plurality of transport electrodes; and (b) positioning a magnetic shielding material in the droplet actuator to selectively minimize the magnetic field. 
     The invention also provides a method of re-suspending particulate within a droplet in a droplet actuator, the method comprising: (a) providing a droplet actuator, comprising: (i) a plurality of independently controllable reservoir electrodes configured to manipulate a droplet; and (ii) a plurality of transport electrodes in fluid communication with the plurality of reservoir electrodes; and (b) independently operating the plurality of reservoir electrodes to cause the particulate to re-suspend within the droplet. 
     The invention provides a method of re-suspending particulate within a droplet in a droplet actuator, the method comprising: (a) providing a droplet actuator, comprising: (i) a reservoir electrode configured to manipulate a droplet; and (ii) a plurality of transport electrodes in fluid communication with the reservoir electrode; (b) separating a slug of the droplet from the droplet on the reservoir electrode; and (c) recombining the slug with the droplet at the reservoir electrode. 
     Moreover, the invention provides a method of re-suspending particulate within a droplet in a droplet actuator, the method comprising: (a) providing a droplet actuator, comprising: (i) a reservoir electrode configured to manipulate a droplet; and (ii) a plurality of transport electrodes in fluid communication with the reservoir electrode; and (b) selectively applying across the reservoir electrode a voltage from an alternating current source to agitate the droplet. 
     In another aspect, the invention provides a method of manipulating a droplet comprising magnetic beads within a droplet actuator, the method comprising: (a) providing a droplet actuator, comprising: (i) a plurality of transport electrodes configured to transport the droplet; and (ii) a magnetic field present at a portion of the plurality of transport electrodes; and (b) positioning a plurality of magnets so as to selectively minimize the magnetic field. 
     In yet another aspect, the invention provides a method of dispensing magnetic beads within a droplet on a droplet actuator, the method comprising: (a) providing a droplet actuator, comprising: (i) top and bottom plates; (ii) a plurality of magnetic fields respectively present proximate the top and bottom plates, wherein at least one of the magnet fields is selectively alterable; and (iii) a plurality of transport electrodes positioned along at least one of the top and bottom surfaces; (b) positioning the droplet between the top and bottom surfaces; and (c) selectively altering at least one of the magnetic fields. 
     The invention also provides a method of splitting a droplet comprising a magnetic bead in a droplet actuator, the method comprising: (a) providing a droplet actuator comprising: (i) a plurality of transport electrodes configured to transport the droplet; and (ii) a magnetic field present at the plurality of transport electrodes; (b) immobilizing the magnetic bead using the magnetic field; and (c) using the plurality of transport electrodes to split the droplet into first and second droplets, wherein the magnetic bead remains substantially immobilized. 
     Further, the invention provides a method of splitting a droplet comprising a magnetic bead in a droplet actuator, the method comprising: (a) providing a droplet actuator comprising: (i) a plurality of transport electrodes configured to transport the droplet, the plurality including an elongated electrode having a length at least twice that of a transport electrode of the plurality; and (b) splitting the droplet using the elongated electrode. 
     The invention also provides a method of splitting a droplet comprising a magnetic bead in a droplet actuator, the method comprising: (a) providing a droplet actuator comprising: (i) a plurality of transport electrodes configured to transport the droplet, the plurality including a segmented electrode having at least one of a column and row of segments; and (b) splitting the droplet using the segmented electrode. 
     Further, the invention provides a method of detecting a component of supernatant, the method comprising: (a) removing excess unbound antibody from a plurality of beads; (b) adding a chemiluminescent substrate to the beads; and (c) detecting the component of the supernatant. 
     These and other aspects of the invention will be apparent from the ensuing description and claims. 
     DEFINITIONS 
     As used herein, the following terms have the meanings indicated. 
     “Activate” with reference to one or more electrodes means effecting a change in the electrical state of the one or more electrodes which results in a droplet operation. 
     “Bead,” with respect to beads on a droplet actuator, means any bead or particle that is capable of interacting with a droplet on or in proximity with a droplet actuator. Beads may be any of a wide variety of shapes, such as spherical, generally spherical, egg shaped, disc shaped, cubical and other three dimensional shapes. The bead may, for example, be capable of being transported in a droplet on a droplet actuator or otherwise configured with respect to a droplet actuator in a manner which permits a droplet on the droplet actuator to be brought into contact with the bead, on the droplet actuator and/or off the droplet actuator. Beads may be manufactured using a wide variety of materials, including for example, resins, and polymers. The beads may be any suitable size, including for example, microbeads, microparticles, nanobeads and nanoparticles. In some cases, beads are magnetically responsive; in other cases beads are not significantly magnetically responsive. For magnetically responsive beads, the magnetically responsive material may constitute substantially all of a bead or one component only of a bead. The remainder of the bead may include, among other things, polymeric material, coatings, and moieties which permit attachment of an assay reagent. Examples of suitable magnetically responsive beads are described in U.S. Patent Publication No. 2005-0260686, entitled, “Multiplex flow assays preferably with magnetic particles as solid phase,” published on Nov. 24, 2005, the entire disclosure of which is incorporated herein by reference for its teaching concerning magnetically responsive materials and beads. The beads may include one or more populations of biological cells adhered thereto. In some cases, the biological cells are a substantially pure population. In other cases, the biological cells include different cell populations, e.g., cell populations which interact with one another. 
     “Dispense,” “dispensing” and the like means a droplet operation in which a droplet is formed from a larger volume of fluid. In some embodiments, the droplet is formed atop an electrode on a droplet operations substrate. The larger volume of fluid may, for example, be a continuous fliud source, a relatively large volume of fluid extending into a fluid path and/or reservoir associated with a droplet actuator, or a source droplet associated with a droplet actuator surface. The larger volume of fluid may me loaded on a droplet actuator, partially loaded on a droplet actuator, or otherwise associated with a droplet actuator in sufficient proximity with an electrode to effect a dispensing operation. 
     “Droplet” means a volume of liquid on a droplet actuator that is at least partially bounded by filler fluid. For example, a droplet may be completely surrounded by filler fluid or may be bounded by filler fluid and one or more surfaces of the droplet actuator. Droplets may take a wide variety of shapes; nonlimiting examples include generally disc shaped, slug shaped, truncated sphere, ellipsoid, spherical, partially compressed sphere, hemispherical, ovoid, cylindrical, and various shapes formed during droplet operations, such as merging or splitting or formed as a result of contact of such shapes with one or more surfaces of a droplet actuator. 
     “Droplet operation” means any manipulation of a droplet on a droplet actuator. A droplet operation may, for example, include: loading a droplet into the droplet actuator; dispensing one or more droplets from a source droplet; splitting, separating or dividing a droplet into two or more droplets; transporting a droplet from one location to another in any direction; merging or combining two or more droplets into a single droplet; diluting a droplet; mixing a droplet; agitating a droplet; deforming a droplet; retaining a droplet in position; incubating a droplet; heating a droplet; vaporizing a droplet; cooling a droplet; disposing of a droplet; transporting a droplet out of a droplet actuator; other droplet operations described herein; and/or any combination of the foregoing. The terms “merge,” “merging,” “combine,” “combining” and the like are used to describe the creation of one droplet from two or more droplets. It should be understood that when such a term is used in reference to two or more droplets, any combination of droplet operations sufficient to result in the combination of the two or more droplets into one droplet may be used. For example, “merging droplet A with droplet B,” can be achieved by transporting droplet A into contact with a stationary droplet B, transporting droplet B into contact with a stationary droplet A, or transporting droplets A and B into contact with each other. The terms “splitting,” “separating” and “dividing” are not intended to imply any particular outcome with respect to size of the resulting droplets (i.e., the size of the resulting droplets can be the same or different) or number of resulting droplets (the number of resulting droplets may be 2, 3, 4, 5 or more). The term “mixing” refers to droplet operations which result in more homogenous distribution of one or more components within a droplet. Examples of “loading” droplet operations include microdialysis loading, pressure assisted loading, robotic loading, passive loading, and pipette loading. 
     “Immobilize” with respect to magnetically responsive beads, means that the beads are substantially restrained in position in a droplet or in filler fluid on a droplet actuator. For example, in one embodiment, immobilized beads are sufficiently restrained in position to permit execution of a splitting operation on a droplet, yielding one droplet with substantially all of the beads and one droplet substantially lacking in the beads. 
     “Magnetically responsive” means responsive to a magnetic field. “Magnetically responsive beads” include or are composed of magnetically responsive materials. Examples of magnetically responsive materials include paramagnetic materials, ferromagnetic materials, ferrimagnetic materials, and metamagnetic materials. Examples of suitable paramagnetic materials include iron, nickel, and cobalt, as well as metal oxides, such as Fe 3 O 4 , BaFe 12 O 19 , CoO, NiO, Mn 2 O 3 , Cr 2 O 3 , and CoMnP. 
     “Washing” with respect to washing a magnetically responsive bead means reducing the amount and/or concentration of one or more substances in contact with the magnetically responsive bead or exposed to the magnetically responsive bead from a droplet in contact with the magnetically responsive bead. The reduction in the amount and/or concentration of the substance may be partial, substantially complete, or even complete. The substance may be any of a wide variety of substances; examples include target substances for further analysis, and unwanted substances, such as components of a sample, contaminants, and/or excess reagent. In some embodiments, a washing operation begins with a starting droplet in contact with a magnetically responsive bead, where the droplet includes an initial amount and initial concentration of a substance. The washing operation may proceed using a variety of droplet operations. The washing operation may yield a droplet including the magnetically responsive bead, where the droplet has a total amount and/or concentration of the substance which is less than the initial amount and/or concentration of the substance. Other embodiments are described elsewhere herein, and still others will be immediately apparent in view of the present disclosure. 
     The terms “top” and “bottom” are used throughout the description with reference to the top and bottom substrates of the droplet actuator for convenience only, since the droplet actuator is functional regardless of its position in space. 
     When a given component, such as a layer, region or substrate, is referred to herein as being disposed or formed “on” another component, that given component can be directly on the other component or, alternatively, intervening components (for example, one or more coatings, layers, interlayers, electrodes or contacts) can also be present. It will be further understood that the terms “disposed on” and “formed on” are used interchangeably to describe how a given component is positioned or situated in relation to another component. Hence, the terms “disposed on” and “formed on” are not intended to introduce any limitations relating to particular methods of material transport, deposition, or fabrication. 
     When a liquid in any form (e.g., a droplet or a continuous body, whether moving or stationary) is described as being “on”, “at”, or “over” an electrode, array, matrix or surface, such liquid could be either in direct contact with the electrode/array/matrix/surface, or could be in contact with one or more layers or films that are interposed between the liquid and the electrode/array/matrix/surface. 
     When a droplet is described as being “on” or “loaded on” a droplet actuator, it should be understood that the droplet is arranged on the droplet actuator in a manner which facilitates using the droplet actuator to conduct one or more droplet operations on the droplet, the droplet is arranged on the droplet actuator in a manner which facilitates sensing of a property of or a signal from the droplet, and/or the droplet has been subjected to a droplet operation on the droplet actuator. 
     Further, the terms “top” and “bottom” or “horizontal” and “vertical” are sometimes used with reference to portions of the figures. These terms are used with reference to regions of the figures and are not intended to limit the orientation in space of the actual elements of the invention. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1A ,  1 B, and  1 C show a top view of a droplet dispensing portion of a droplet actuator in which fluid is flowed through multiple openings into proximity with droplet operations electrodes; 
         FIGS. 2A ,  2 B and  2 C show a top view of a droplet dispensing portion of a droplet actuator in which fluid is flowed across and/or retracted from activated electrodes to form droplets; 
         FIG. 3  shows a top view of a droplet dispensing portion of another embodiment of a droplet actuator in which fluid is flowed across and/or retracted from activated electrodes to form droplets; 
         FIGS. 4A ,  4 B,  4 C, and  4 D illustrate a top view of a droplet dispensing configuration of a portion of a droplet actuator in which droplets are transported across electrodes using droplet operations to form droplets; 
         FIG. 5  illustrates a top view of another droplet dispensing configuration of a portion of a droplet actuator in which droplets are transported across electrodes using droplet operations to form droplets; 
         FIGS. 6A ,  6 B, and  6 C show a side view of a segment of a droplet actuator and illustrate a droplet dispensing process that forms small droplets from a large droplet by use of electrowetting, gravity forces, and capillary forces; 
         FIGS. 7A ,  7 B, and  7 C show a side view of a portion of a droplet actuator in which a reduced gap height is used to facilitate dispensing of droplets; 
         FIG. 8  illustrates a top view of a droplet dispensing configuration of a portion of a droplet actuator for efficiently handling varying volumes of liquid in the fluid reservoir; 
         FIGS. 9A ,  9 B, and  9 C illustrates a top view of another droplet dispensing configuration of a portion of a droplet actuator for efficiently handling varying volumes of liquid in the fluid reservoir; 
         FIG. 10  illustrates a top view of yet another droplet dispensing configuration of a portion of a droplet actuator for efficiently handling varying volumes of liquid in the fluid reservoir; 
         FIGS. 11A ,  11 B, and  11 C illustrate a top view of another droplet dispensing configuration of a portion of a droplet actuator for efficiently handling varying volumes of liquid in the fluid reservoir; 
         FIGS. 12A ,  12 B, and  12 C illustrate a top view of yet another droplet dispensing configuration of a portion of a droplet actuator for efficiently handling varying volumes of liquid in the fluid reservoir; 
         FIGS. 13A ,  13 B, and  13 C illustrate an electrode array of a droplet actuator and shows a droplet dispensing process in which droplets are dispensed diagonally in multiple directions; 
         FIG. 14  illustrates a top view of a reservoir droplet dispensing configuration of a droplet actuator in relation to an opening for loading\unloading fluid; 
         FIGS. 15A ,  15 B,  15 C,  15 D,  15 E, and  15 F illustrate multiple top views, respectively, of multiple example reservoir droplet dispensing configurations of a droplet actuator, shown in relation to an opening for loading and/or unloading fluid; 
         FIGS. 16A ,  16 B, and  16 C illustrate multiple top views of certain example openings in relation to a fluid reservoir of a droplet actuator; 
         FIG. 17  illustrates a top view of a droplet dispensing configuration of a portion of a droplet actuator and illustrates a process of dispensing droplets; 
         FIG. 18  illustrates another view of the droplet dispensing configuration and process of dispensing droplets of  FIG. 17 ; 
         FIG. 19  illustrates a top view of another droplet dispensing configuration of a portion of a droplet actuator and illustrates another process of dispensing droplets; 
         FIG. 20A  illustrates another top view of the droplet dispensing configuration of  FIG. 17  and illustrates a process of agitating droplets and/or priming the fluid reservoir in a droplet actuator; 
         FIG. 20B  illustrates yet another top view of the droplet dispensing configuration of  FIG. 17  and illustrates a process of agitating fluid in a droplet actuator; 
         FIG. 21A  illustrates a top view of a droplet dispensing configuration of a portion of a droplet actuator and illustrates a process of disposing of a 1× size droplet in a droplet actuator; 
         FIG. 21B  illustrates another top view of the droplet dispensing configuration of  FIG. 21A  and illustrates a process of disposing of a 2× size droplet in a droplet actuator; 
         FIG. 22A  illustrates a top view of a dual-purpose droplet dispensing configuration of a portion of a droplet actuator and illustrates a process of dispensing droplets in a droplet actuator; 
         FIG. 22B  illustrates another top view of the dual-purpose droplet dispensing configuration of  FIG. 22A  and illustrates a process of disposing of droplets in a droplet actuator; 
         FIG. 23A  illustrates a top view of an example droplet dispensing configuration for dispensing droplets in multiple directions from a single reservoir in a droplet actuator; 
         FIG. 23B  illustrates a top view of another example droplet dispensing configuration for dispensing droplets in multiple directions from a single reservoir in a droplet actuator; 
         FIG. 23C  illustrates a top view of yet another example droplet dispensing configuration for dispensing droplets in multiple directions from a single reservoir in a droplet actuator; 
         FIG. 24A  illustrates a top view of a portion of a droplet actuator for parallel distribution of fluid to multiple fluid reservoirs using a single opening; 
         FIG. 24B  illustrates a cross-sectional view of the droplet actuator taken along line AA of  FIG. 24A ; 
         FIG. 25A  illustrates a top view of a portion of a droplet actuator for serial distribution of fluid to multiple fluid reservoirs using a single opening; 
         FIG. 25B  illustrates a cross-sectional view of the droplet actuator taken along line BB of  FIG. 25A ; 
         FIGS. 26A and 26B  illustrate top views of an example droplet dispensing configuration of a droplet actuator that includes a droplet forming electrode that is embedded in a larger reservoir electrode; and 
         FIG. 26C  illustrates a top view of an example droplet dispensing configuration of a droplet actuator that includes multiple droplet forming electrodes that are embedded in a larger reservoir electrode. 
     
    
    
     DESCRIPTION 
     The invention provides an improved droplet actuator and methods of making and using the droplet actuator. Various aspects of the invention provide enhanced droplet dispensing relative to existing droplet actuators. Enhanced droplet dispensing may, for example, include aspects which provide enhanced efficiency, throughput, scalability, and/or droplet uniformity. Other aspects provide improved unloading of droplets from a droplet actuator relative to existing droplet actuators. The various aspects of the invention described in the ensuing sections may be provided on a droplet actuator individually or in any combination with other aspects. 
     Droplet Dispensing Structures and Methods 
       FIGS. 1A ,  1 B and  1 C show top views of various embodiments of a region of a droplet operations surface  129  of a droplet actuator showing a droplet dispensing configuration  100 . The illustrated embodiment is useful, among other things, for dispensing multiple droplets in a substantially simultaneous manner. Configuration  100  includes a fluid reservoir  128 . Fluid reservoir  128  is defined by wall  110 , by the substrate that forms the droplet operations surface  129  and optionally by a top substrate (not shown). It will be appreciated that any of a wide variety of configurations is possible, so long as the configuration provides a fluid path that permits liquid  126  to flow under appropriate conditions from the reservoir  128  onto the droplet operations surface  129 . 
     Wall  110  of fluid reservoir  128  may include multiple openings  114 . Each opening  114  provides a fluid path from the reservoir  128  to the droplet operations surface  129 . In some embodiments, surfaces of the wall  110 , the top substrate (not shown), and/or the bottom substrate  129 , associated with openings  114  may be sufficiently hydrophobic in character to inhibit the flow of liquid  126  through openings  114 . A hydrophobic coating, such as a Teflon® coating can be used to achieve this purpose. In other embodiments, flow may be inhibited by keeping the openings sufficiently small and/or by including physical flow barriers in proximity to the openings. The inhibition of flow may be overcome by forcing fluid into reservoir  128 , e.g., using a pressure source and/or a vacuum source. 
     As illustrated in  FIG. 1A , droplet dispensing operations may take place on three sides of fluid reservoir  128 . Fluid reservoir  128  essentially projects onto a droplet operations surface  129  so that droplets may be dispensed on three sides thereof. In a dispensing operation, liquid  126  is forced through openings  114  into proximity with electrodes  118 . When liquid  126  is in proximity with electrodes  118 , electrodes  118  may be used to conduct droplet dispensing operations.  FIG. 1B  illustrates an alternative arrangement in which droplets are dispensed in multiple directions from a centrally located reservoir  128 .  FIG. 1C  shows another embodiment in which droplets are dispensed in parallel in a single direction from a reservoir  128 . 
     One or more electrodes  118  may be provided in association with the droplet operations surface and/or the top substrate (when present). The electrodes  118  are configured for conducting one or more droplet operations on the droplet operations surface  129 , e.g., dispensing of droplets on the droplet operations surface  129 . 
     In operation, at a certain pressure level, liquid  126  fills fluid reservoir  128  without passing through openings  114 . At a certain higher pressure level, liquid  126  flows through openings  114  into sufficient proximity with electrodes  118  to permit electrodes  118  to facilitate one or more droplet operations. 
     In one embodiment, when one or more of electrodes  118  is activated, liquid  126  in reservoir  128  may be retracted to leave droplets of fluid on electrodes  118 . In this embodiment, pressure source  130  provides the force needed to push out and pulling back the volume of liquid  126  within fluid reservoir  128 . For example, the supply of liquid  126  may be held under pressure via pressure source  130 , which is a variable pressure source. 
     In another embodiment, additional electrodes adjacent to electrodes  118  may be activated, further extending liquid  126  onto the droplet operations surface. Intermediate electrodes, such as electrodes  118 , may be deactivated to cause the formation of droplets on the additional electrodes. As illustrated by this embodiment, a change in pressure from the pressure source may not be required to facilitate droplet formation, though in some cases droplet formation may be enhanced by a change in pressure from the pressure source. 
       FIGS. 1B and 1C  illustrate embodiments which are similar to the embodiments illustrated in  FIG. 1A . As illustrated in  FIG. 1B , fluid reservoir  128  may be provided within droplet operations surface so that fluid may be dispensed in multiple directions on the surface. In the specifically illustrated embodiment, droplets may be dispensed radially in four directions from a central fluid source. Another embodiment, droplets may be dispensed radially in 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more directions from a central fluid source. Other embodiments permit dispensing from a central fluid source, but the dispensing path is not necessarily radially oriented relative to the central fluid source. Further, as illustrated in  FIG. 1C , the fluid reservoir  128  may extend alongside droplet operations surface  129  so that droplets are dispensed on one side thereof. 
     It will be appreciated that the embodiment of  FIGS. 25A and 25B  (discussed below) is an alternative aspect of the embodiment illustrated in  FIG. 1 . In  FIG. 1 , the reservoir  128  is oriented on generally the same plane as the droplet operations surface  129 . In contrast, in  FIGS. 25A and 25B , the fluid source bringing is located in a substantially different plane relative to the droplet operations surface. It should also be noted that the fluid source in  FIGS. 25A and 25B  may in other embodiments be located in substantially the same plane as the droplet operations surface  129 . 
       FIGS. 2A ,  2 B and  2 C show top view of droplet dispensing configurations  200  of a portion of a droplet actuator. The illustrated embodiment is useful, among other things, for dispensing multiple droplets from a source fluid  226 . The droplets may, for example, be dispensed onto a droplet operations surface  229 . 
     As illustrated in  FIG. 2A , configuration  200  includes a fluid reservoir  228 , though it will be appreciated that in some cases the fluid reservoir could represent substantially the entire droplet operations surface  229 . As illustrated in  FIG. 2A , fluid reservoir  228  is defined by walls  210 , by the substrate that forms the droplet operations surface  229  and optionally by a top substrate (not shown). A path or as illustrated here, an array  214  of electrodes  218  is associated with the droplet operations surface  229  and/or associated with the top substrate (not shown) within area of fluid reservoir  228  defined by walls  210 . Other electrodes  222  may be provided outside the fluid reservoir or in some cases the fluid reservoir may take up substantially the entire droplet operations surface. Electrode array  214  is illustrated as an array of N×M electrodes, within which there may be individual control of each electrodes or of specific sets of electrodes. Of course, in alternative embodiments, paths or other patterns of electrodes will suffice, for example, see  FIGS. 2B and 2C . 
     An arrangement of droplet operations electrodes  222  may be included, fed by electrode array  214 , for conducting subsequent droplet operations using dispensed droplets  234 . Droplet operations electrodes  222  may also be provided in various paths or arrays. 
     Fluid reservoir  228  may be filled or partially filled with a volume of liquid  226  from which droplets may be dispensed. Droplets are dispensed by providing activated electrodes within the filled region of fluid reservoir  228 . When the liquid  226  is retracted, droplets remain on the activated electrodes. In the specific example illustrated, a pressure source  230  provides the force for pushing out and pulling back the volume of liquid  226  within fluid reservoir  228 . For example, the pressure source  230  may be a variable pressure source. One of more pressure sources may be used as needed. 
     In operation, liquid  226  may be flowed into fluid reservoir  228  so that liquid  226  covers a portion of, or substantially all of, electrode array  214 . Liquid  226  may then be retracted or otherwise removed from transport electrodes  222 . Selected electrodes  218  may be activated prior to retracting liquid  226 , so that droplets  234  are retained on the activated electrodes  218 . In one embodiment, an array of electrodes, including every other electrode  218  is activated, resulting in formation of an array of droplets. The droplets are left behind on the activated electrodes  218  in the wake of the retracting or otherwise removing liquid  226 . Upon formation, droplets  234  may be subjected to droplet operations using electrodes  218  and or other electrodes  222  exterior to the reservoir  228 . 
       FIGS. 2B and 2C  illustrate examples of alternative arrangements to the arrangement shown in  FIG. 2A .  FIG. 2B  illustrates an arrangement in which electrodes  218  are provided in paths rather than in an array.  FIG. 2C  illustrates an arrangement in which multiple walls  218  separate individual paths of electrodes  218 . 
       FIG. 3  illustrates a top view of a droplet dispensing configuration  300  of a portion of a droplet actuator. Droplet dispensing configuration  300  is substantially the same as droplet dispensing configuration  200  of  FIG. 2 , except that a pressure mechanism (e.g., pressure source  230 ) is replaced or supplemented with an electrowetting mechanism as the energy source for moving the volume of liquid  226  across the droplet forming electrodes  218 . In the example illustrated, a series of flow electrodes  310 , such as flow electrodes  310   a ,  310   b ,  310   c ,  310   d ,  310   e , and  310   f , are arranged at the outer edges of electrode array  214 , as shown in  FIG. 3 . Flow electrodes  310  provide an electrowetting mechanism for moving the volume of liquid  222  across the droplet forming electrodes  218  in the process of forming droplets  234 . Each electrode  310  may, for example, be several times larger, e.g., 2×, 3×, 4×, 5×, 6×, or larger, as compared to the area of a droplet operations electrode  218 . 
     In operation, flow electrodes  310  are activated to draw liquid  226  across droplet forming electrodes  218 . Certain of the droplet forming electrodes  218  are activated. Flow electrodes  310  are then deactivated, causing the liquid  226  to retract and leaving droplets  234  on the activated droplet forming electrodes. 
       FIGS. 4A ,  4 B,  4 C, and  4 D illustrate a top view of a droplet dispensing configuration  400  of a portion of a droplet actuator and illustrate a droplet dispensing process that dispenses droplets as liquid flows in one direction (as compared to the flow in and retract schemes illustrated in  FIGS. 2 and 3 ). Droplet dispensing configuration  400  may include a reservoir electrode  410 , which may, in one embodiment, be an electrode of a source fluid reservoir. Droplet dispensing configuration  400  may also include a reservoir electrode  414 , which may, in one embodiment, be an electrode of a destination fluid reservoir. Droplet dispensing configuration  400  further includes a set of transport electrodes  418  that are arranged between reservoir electrode  410  and reservoir electrode  414 . In another embodiment, one or both of the reservoir electrode and the destination electrode may be replaced with one or more droplet operations electrodes, such as transport electrodes  418 . 
       FIG. 4A  shows an example of a first step of a droplet dispensing process in which reservoir electrode  410  only is activated and, thus, substantially all of the volume of a liquid  422  is present at reservoir electrode  410 . Liquid  422  is the liquid from which droplets to be subjected to droplet operations may be dispensed. 
       FIG. 4B  shows an example of a second step of the droplet dispensing process in which reservoir electrode  410  remains activated and transport electrodes  418  and reservoir electrode  414  are activated. As a result, the volume of liquid  422  extends from reservoir electrode  410 , across all transport electrodes  418 , and to reservoir electrode  414 . In doing so, the volume of fluid that originated at reservoir electrode  410  is substantially distributed across reservoir electrode  410 , transport electrodes  418 , and reservoir electrode  414 . Additional fluid may also be drawn into the gap from an external fluid source (not shown) associated with reservoir  422 . A substantially continuous “slug” of liquid  422  is thus formed from reservoir electrode  410  to reservoir electrode  414 . 
       FIG. 4C  shows an example of a third step of the droplet dispensing process in which reservoir electrode  410  is deactivated, every other of transport electrode  418  only is activated, and reservoir electrode  414  is activated. As the slug of liquid  422  changes its footprint and moves across transport electrodes  418  and toward reservoir electrode  414 , a droplet, such as a droplet  426 , is left behind on each transport electrode  418  that is activated. Ideally, reservoir electrode  410  is deactivated followed sequentially by deactivation of a series of one or more of the intermediate transport electrode  418 , sequentially forming droplets  426  from the trailing liquid at each of the activated electrodes. 
       FIG. 4D  shows an example of a fourth step of the droplet dispensing process in which, after forming a certain number of droplets  426 , reservoir electrode  414  remains activated and the remaining volume of liquid  422  (excluding droplets  426   a  and  426   b ) is collected at reservoir electrode  414 .  FIG. 4D  shows, for example, a droplet  426   a  and a droplet  426   b  that are formed on certain transport electrodes  418  that are activated. Of course, a wide variety of droplet arrangements is possible, depending on which of the electrodes  418  remain activated and which are deactivated. 
       FIG. 5  illustrates a top view of another example of a droplet dispensing configuration  500  of a portion of a droplet actuator. Like the embodiment illustrated in  FIG. 4 , this embodiment dispenses droplets from a trailing end of a moving slug of liquid. Droplet dispensing configuration  500  may include a path of electrodes  510 . As illustrated, the path is arranged in a loop, but any arrangement that forms a path along which a slug of liquid can be transported is suitable. A “slug” of liquid  518  is provided from which droplets to be subjected to droplet operations may be formed. Electrodes are activated to cause the slug of liquid  518  to be transported around the loop of electrodes  510 . In the wake of the moving slug of liquid  518 , certain electrodes  510 , e.g., every other electrode  510 , may remain activated, thereby forming droplets  522  on these certain electrodes  510 , as the slug continues to be transported away from the trailing activated electrodes. In the loop embodiment, transport electrodes  514  may be used for transporting liquid  518  and droplets  522  in and out of the loop for further droplet operations. 
       FIGS. 6A ,  6 B, and  6 C illustrate a side view (cross-section) of a segment of a droplet actuator  600  and show a droplet dispensing process that forms small droplets from a large droplet. Droplet actuator  600  may include a bottom substrate  614  that is separated from a top substrate  618  by a gap. An electrode  622  and one or more transport electrodes  626  may be associated with bottom substrate  614 . A fluid reservoir  630  or other fluid source may be associated with top substrate  618 . Fluid reservoir  630  may, for example, be a well that opens to, or otherwise includes a fluid path extending to, the gap between bottom substrate  614  and top substrate  618 . A droplet  634  may be contained within fluid reservoir  630 , from which droplets may be dispensed. 
       FIG. 6A  shows an example of a first step of a droplet dispensing process. Droplet  634  is substantially contained within fluid reservoir  630 . Without the use electrowetting and when all electrodes are deactivated, liquid supply droplet  634  stays substantially within the well of fluid reservoir  630 . 
       FIG. 6B  shows an example of a second step of the droplet dispensing process in which electrode  622  and the adjacent transport electrode  626  are both activated in order to generate sufficient pressure difference in the gap of droplet actuator  600  to cause liquid supply droplet  634  to flow out of fluid reservoir  630  and onto electrode  622  and transport electrode  626 . 
       FIG. 6C  shows an example of a third step of the droplet dispensing process in which electrode  622  is deactivated and the adjacent transport electrode  626  remains activated. Capillary forces cause liquid supply droplet  634  to return to fluid reservoir  630 , leaving a droplet  638  behind that is formed on transport electrode  626 . 
       FIGS. 7A ,  7 B, and  7 C illustrate a side view of a portion of a droplet actuator  700  and a droplet dispensing process. The droplet dispensing process forms a sub-droplet from a source droplet by making use of electrowetting in combination with other forces, such as surface tension and/or capillary forces. Droplet actuator  700  may include a bottom substrate  714  that is separated from a top substrate  718  by a gap  732 . Top substrate  718  and bottom substrate  714  establish droplet operations surfaces  716 , facing gap  732 . An electrode  722  and one or more droplet operations electrodes, such as transport electrodes  726  may be associated with bottom substrate  714 . 
     A fluid reservoir  730  may be formed by providing a region between top substrate  718  and bottom substrate  714  of increased gap height relative to the height of the gap  732  in the droplet operations region of the droplet actuator. In the illustrated embodiment, the gap  730  forming the fluid reservoir may be formed by features within bottom substrate  714  only, top substrate  718  only, or within the combination of bottom substrate  714  and top substrate  718 . Alternatively, the fluid reservoir  730  may be formed by a separate structure that abuts the top substrate  718  and bottom substrate  714 , such that the height of gap  730  is established by substrates or structures other than the top substrate  718  and bottom substrate  714 . For example a reservoir or other fluid source may abut top substrate  718  and bottom substrate  714  and provide a fluid source and fluid path for supplying liquid to the droplet operations surface of the droplet actuator. A liquid supply droplet  734  may be contained within gap  730 , from which droplets to be subjected to droplet operations may be dispensed. The reservoir formed by gap  730  or its alternatives may itself be coupled in fluid communication with an external liquid supply source. 
       FIG. 7A  shows a first step of a droplet dispensing process. Liquid supply droplet  734  is provided and substantially contained within fluid reservoir  730  in proximity with electrode  722 . When electrode  722  is deactivated, liquid supply droplet  734  remains substantially within fluid reservoir  730 . 
       FIG. 7B  shows an example of a second step of the droplet dispensing process. Electrode  722  and the adjacent electrode  726  are both activated in order to cause liquid supply droplet  734  to flow into gap  732  onto electrode  722  and transport electrode  726 . 
       FIG. 7C  shows an example of a third step of the droplet dispensing process. Electrode  722  is deactivated and the adjacent transport electrode  726  remains activated. A portion of liquid supply droplet  734  returns to fluid reservoir  730 , leaving a droplet  738  on transport electrode  726 . 
       FIG. 8  illustrates a top view of a droplet dispensing configuration  800  of a portion of a droplet actuator. Droplet dispensing configuration  800  includes a fluid reservoir  810  that may be formed in association with a single droplet operations substrate or between two substrates of a droplet actuator that are separated by a gap. Disposed within fluid reservoir  810  may be one or more electrodes for efficiently performing operations on the volume of liquid therein. The volume of liquid is variable. In one example, fluid reservoir  810  may include an electrode  814 , an electrode  818 , and an electrode  822  within the area of fluid reservoir  810 . A barrier  824  may be provided to serve as a boundary of fluid reservoir  810 , separating the reservoir from the remainder of the droplet operations surface. The barrier  824  includes an opening  850  through which liquid may flow into proximity with adjacent electrode  826  that feeds a set of droplet operations electrodes  830 . 
     Electrode  814 , electrode  818 , and electrode  822  may be, for example, individually-controlled concentric crescent moon-shaped electrodes that are widest at the opening of fluid reservoir  810  and narrowest opposite the opening of fluid reservoir  810 , as shown in  FIG. 8 . As illustrated, the reservoir electrodes are formed from substantially perfect circles; however, it will be appreciated that angles may be introduced, and a variety of shapes may be employed in which the electrode is thickest in proximity to electrode  826  and narrowest at a point which is generally distal to electrode  826 . As the volume of liquid (not shown) within fluid reservoir  810  varies, e.g., due to the process of dispensing droplets via electrode  826  and transport electrodes  830 , certain of one or more electrodes  814 ,  818 , and  822  are activated for most efficient operations on the liquid. All three electrodes may be activated to cause larger volumes of liquid to flow into proximity with electrode  826 . Reservoir electrodes  814  and  818  may be activated together for smaller volumes. Reservoir  814  may be activated alone for still smaller volumes. As a result, the volume of liquid may be moved efficiently into proximity with electrode  826 . Once in proximity with electrode  826 , droplet operations for dispensing subdroplets may be executed using electrode  826  and electrodes  830 , e.g., by activating a row of electrodes to cause liquid to flow onto the droplet operations surface and deactivating an intermediate one or more of the electrodes to produce a subdroplet on one or more of the electrodes on the droplet operations surface. 
       FIGS. 9A ,  9 B, and  9 C illustrates a top view of another droplet dispensing configuration  900 , which is similar to the configuration  800  illustrated in  FIG. 8 . Droplet dispensing configuration  900  includes a fluid reservoir  910  that may be formed on a single substrate or between two substrates of a droplet actuator that are separated by a gap. One or more reservoir electrodes  922  and/or  914  are disposed within fluid reservoir  910 . 
     In one example, fluid reservoir  910  may includes a central H-shaped reservoir electrode  922 , which is also illustrated in  FIG. 9B . The H-shaped electrode includes two generally parallel segments  922   a / 922   b  joined (at a point other than the end-point) by a connecting segment  922   c . As illustrated, the two generally parallel segments  922   a / 922   b  are positioned generally at right angles relative to the connecting segment  922   c ; however, it will be appreciated that obtuse or acute angles may be employed as alternatives. The connecting segment  922   c  connects the two generally parallel segments  922   a / 922   b  at a point other than the end-point, two gaps A and B (see  FIG. 9B ) are formed, one gap A at the top and one gap B at the bottom portion of the H-shaped electrode. One or more droplet operations electrodes, such as droplet dispensing electrodes  926  may be inset into either of these gaps. In an alternative embodiment, the connecting segment  922   c  connects the two generally parallel segments  922   a / 922   b  at an endpoint proximal to the droplet dispensing electrodes, thereby forming a U-shaped reservoir electrode rather than an H-shaped reservoir electrode. In one embodiment, an H-shaped electrode is provided having first and second gaps (A and B) and a droplet operations electrode  924  positioned in one of the gaps. The droplet dispensing electrodes  926  may be associated with additional droplet operations electrodes  930  configured for conducting droplet operations using dispensed droplets. 
     Fluid reservoir  910  may also include two L-shaped electrodes  914  and  918 , which is also illustrated in  FIG. 9C . One of the L-shaped electrodes  918  may be reflected along a vertical axis, i.e., it may be a mirror image of an “L.” Each of the L-shaped electrodes  914  and  918  includes an elongated segment  914   a / 918   a  and a shorter segment  914   a / 914   b . The elongated segments  914   a / 918   a  may in some embodiments be placed at a right angle relative to the corresponding shorter segments  914   a / 914   b . The two L-shaped electrodes may be electrically coupled to one another such that they function as a single electrode. An L-shaped electrode  914  and a mirror image L-shaped electrode  918  may be aligned with the horizontal segments  914   b / 918   b  facing each other and a gap D formed therebetween. This arrangement also provides a gap C between the horizontal vertical members of the L-shaped electrodes  914 / 918 . In one embodiment, an L-shaped electrode is provided along with a mirror image of an L-shaped electrode, where the horizontal portions of the two L-shaped electrodes are aligned with each other and separated to form a gap therebetween, and a droplet operations electrode is positioned in the gap. The droplet dispensing electrodes  926  may be associated with additional droplet operations electrodes  930  configured for conducting droplet operations using dispensed droplets. 
     In another embodiment, an L-shaped electrode is provided along with a mirror image of an L-shaped electrode, where the horizontal portions of the two L-shaped electrodes are aligned with each other and separated to form a gap therebetween. An H-shaped electrode is provided in the gap between the vertical members of the L-shaped electrodes, such that a gap in the H-shaped electrode is generally aligned with the gap between the horizontal members of the L-shaped electrodes. A first droplet operations electrode is provided at least partially in the gap of the H-shaped electrode that is aligned with the gap between the horizontal members of the L-shaped electrodes. A second droplet operations electrode is provided at least partially in the gap formed by the horizontal members of the L-shaped electrodes. 
     Electrode  914 , electrode  918 , and electrode  922  may be, for example, individually-controlled electrodes of differing size, location, and shape, as shown in  FIG. 9 . In this way, as the volume of liquid (not shown) within fluid reservoir  910  varies over time, due to the process of dispensing droplets via electrode  926  and transport electrodes  930 , certain of one or more electrodes  914 ,  918 , and  922  are activated for most efficient operation on the liquid. 
     In operation, the H-shaped electrode  922  and L-shaped electrodes  914 / 918  may be activated together to cause larger volumes of liquid to flow into proximity with droplet dispensing electrodes. Further, the H-shaped electrode  922  and L-shaped electrodes  914 / 918  may be activated together with droplet dispensing electrode  926   a  to cause larger volumes of liquid to flow into proximity with droplet dispensing electrode  926   b . Electrodes  926   b  and  930  may then be used to dispense a droplet. For smaller volumes, the H-shaped electrode  922  or L-shaped electrodes  914 / 918  may be activated individually to cause liquid to flow into proximity with electrode  926   a  or  926   b , as the case may be. Once in proximity with the appropriate droplet dispensing electrodes  926   a  or  926   b , droplet operations for dispensing subdroplets may be executed using droplet dispensing electrode  926   a  and/or  926   b  and droplet operations electrodes  930 , e.g., by activating a row of electrodes to cause liquid to flow onto the droplet operations surface and deactivating an intermediate one or more of the electrodes to produce a subdroplet on one or more of the electrodes on the droplet operations surface. 
       FIG. 10  illustrates a top view of yet another droplet dispensing configuration  1000  of a portion of a droplet actuator for efficiently handling varying volumes of liquid in the fluid reservoir. Droplet dispensing configuration  1000  includes a fluid reservoir  1010  that may be formed on a droplet actuator substrate or between two substrates of a droplet actuator that are separated by a gap. Disposed within fluid reservoir  1010  may be one or more electrodes for efficiently performing operations on the volume, which is variable, of liquid therein. Additionally, an opening in a barrier  1016  that serves as the boundary of fluid reservoir  1010  is adjacent to an electrode  1018  that feeds a set of transport electrodes  1022 . 
     In one example, fluid reservoir  1010  may include electrode array  1014 , which may be multiple individually-controlled electrodes that are arranged in an array, such as checkerboard pattern, within the area of fluid reservoir  1010 , as shown in  FIG. 10 . As the volume of liquid (not shown) within fluid reservoir  1010  varies over time, due to the process of dispensing droplets via electrode  1018  and transport electrodes  1022 , certain electrodes of electrode array  1014  are activated as necessary to bring the fluid into proximity with electrode  1018  so that electrodes  1018  and  1022  may be employed to dispense droplets from the fluid. 
       FIGS. 11A ,  11 B, and  11 C illustrates a top view of yet another droplet dispensing configuration  1100  of a portion of a droplet actuator for efficiently handling varying volumes of liquid in the fluid reservoir. Droplet dispensing configuration  1100  includes a fluid reservoir  1110  that may be formed on a droplet actuator substrate or between two substrates of a droplet actuator that are separated by a gap. Disposed within fluid reservoir  1110  may be one or more electrodes  1114  for performing droplet dispensing operations on the various volumes of liquid therein. Additionally, an opening in a barrier  1116  that serves as the boundary of fluid reservoir  1110  is adjacent to a droplet dispensing electrode  1118  that feeds a set of transport electrodes  1122 . 
     Electrodes  1114  may be, for example, individually-controlled elongated (e.g., finger-shaped) electrodes that are widest at the opening of fluid reservoir  1110  and narrowest opposite the opening of fluid reservoir  1110 . When an electrode is activated, liquid will tend to become oriented at the widest end of the electrode in proximity with the droplet operations electrode  1118 . Opposite sets of electrodes can be electrically coupled so that they can operate as single electrodes. For example, electrodes A can be electrically coupled so that they are activated and deactivated together. Similarly, electrodes A can be electrically coupled so that they are activated and deactivated together. More electrodes  1114  can be activated to handle greater volumes of fluid, and less electrodes  1114  can be activated to handle smaller volumes of fluid. As illustrated, electrodes  1114  include three electrodes, including matching pair A, matching pair B and single electrode C. Of course, any number of electrodes  114  can be used, limited only by the expediency of efficient design. In various embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more electrodes  114  are provided. 
     In one mode of operation, electrodes  1114 A, B and C are activated alone for dispensing droplets from larger volumes of liquid, electrodes  1114 B and C or  1114 A and B are activated alone for dispensing droplets from intermediate volumes of liquid, and electrode  1114 C is activated alone for dispensing droplets from a still smaller volume of liquid. 
       FIG. 11B  illustrates a related embodiment in which the reservoir electrodes  114  are generally elongated teardrop shapes. Having wider end proximal to the droplet operations electrode  1118  and tapering towards the tip, which is distal to the droplet operations electrode. Further, the electrodes are generally arrayed in a fan-type layout layout. 
       FIG. 11C  illustrates another embodiment in which the droplet operations electrode  118  is divided into sub-electrodes. These sub-electrodes may be used to dispense smaller droplets from the reservoir electrodes. 
       FIGS. 12A ,  12 B and  12 C illustrates a top view of yet another droplet dispensing configuration  1200  of a portion of a droplet actuator. Droplet dispensing configuration  1200  includes a fluid reservoir  1210  that may be formed on a droplet actuator substrate or between two substrates of a droplet actuator that are separated by a gap. An electrode  1214  may be disposed within fluid reservoir  1210 . An opening  1230  in a barrier  1216  serves as a fluid path from reservoir  1210  onto electrode  1218  that feeds a set of transport electrodes  1222  on a droplet operations surface. 
     Electrode  1214  may be, for example, an electrode that is elongated in a manner which provides pull back on the droplet during the droplet dispensing operation, where the pull back is at a right angle or acute angle to the direction in which the droplet is being dispensed. In this example, when electrode  1214  is activated during the pull-back phase of the droplet dispensing operation, the volume of liquid within fluid reservoir  1210  the liquid tends to conform to the shape of electrode  1214 , resulting in a pull away from electrode  1218  and transport electrodes  1222 . 
       FIG. 12B  illustrates a similar configuration in which the reservoir electrode  1214  is thickest at a point which is proximal to electrode  1218  and tapers in a proximal direction relative to electrode  1218 .  FIG. 12B  illustrates another similar configuration in which electrode  1218  is inset in a gap in reservoir electrode  1214 . 
     Referring to  FIG. 12C , an example of a droplet dispensing process involves activation of reservoir electrode  1214 , electrode  1218  and electrode  1222 , followed by deactivation of electrode  1218  to leave a droplet on electrode  1222 . Similar processes are envisaged in which multiple electrodes  1222  are used to pull a longer droplet slug onto the droplet operations surface, followed by deactivation of one or more intermediate electrodes to form droplets on the droplet operations surface. 
       FIGS. 13A ,  13 B, and  13 C illustrate an electrode array  1300  of a droplet actuator and illustrate a droplet dispensing process in which droplets are dispensed diagonally. For example, electrode array  1300  may be formed of an array of electrodes  1310 , e.g., electrowetting electrodes.  FIG. 13A  shows that a droplet  1314  from which droplets are to be dispensed is held upon certain electrodes  1310  which have been activated.  FIG. 13B  shows that certain electrodes  1310  that are diagonal to droplet  1314  may be activated, thereby extending fingers of fluid from droplet  1314  and causing the formation of diagonally located sub-droplets  1318 , as shown in  FIG. 13C . The dispensing may be on a single diagonal, forming two droplets, and/or on two diagonals, forming multiple droplets. In other embodiments in which the electrode array may be formed using electrodes having more than four sides, more than four droplets may be formed. 
     Fluid Loading and Unloading Structures and Methods 
     In the following embodiments of the invention, which are described in  FIGS. 14 through 26C , the “opening” may, for example, be an opening in a substrate of a droplet actuator through which fluid, such as sample fluid, may be loaded into the droplet actuator and/or unloaded from the droplet actuator. Furthermore, the opening may be any shape. 
       FIG. 14  illustrates a top view of a reservoir droplet dispensing configuration  1400  of a droplet actuator in relation to an opening for loading/unloading fluid. Reservoir droplet dispensing configuration  1400  is associated with a fluid reservoir that may be formed between two substrates of a droplet actuator that are separated by a gap. Reservoir droplet dispensing configuration  1400  includes an electrode array  1410  that is formed of multiple electrodes. In one example, electrode array  1410  may be formed of individually controlled electrodes  1414   a  through  1414   i  that are arranged in a 3×3 array.  FIG. 14  also shows an opening  1418  in a substrate of the droplet actuator. The interaction of opening  1418  with electrode array  1410  may be facilitated via a transfer electrode  1422 . Transfer electrode  1422  is used to assist in the transfer of fluid that is supplied through opening  1418  onto electrode array  1410 . In this example, opening  1418  is positioned to at least partially overlap with transfer electrode  1422 , as shown in  FIG. 14 . Additionally, electrode array  1410  feeds an arrangement of electrodes  1426 , e.g., electrowetting electrodes, onto which droplets (not shown) may be dispensed and by which the droplets may be subjected to droplet operations. 
     In the example reservoir droplet dispensing configuration  1400  of  FIG. 14 , electrode array  1410  provides a fluid reservoir that may be several times the area of a single electrode  1426 . In the example shown in  FIG. 14 , electrode array  1410  provides a fluid reservoir that may be about 9 times the area of a single electrode  1426 . Additionally, electrode array  1410  of reservoir configuration  1400  provides improved control for dispensing droplets onto electrodes  1426  via the individually controlled electrodes  1414 , as compared with one large reservoir electrode. Other example reservoir configurations for providing improved control and interaction with the opening of a droplet actuator are described with reference to  FIGS. 15A through 26C . 
       FIGS. 15A ,  15 B,  15 C,  15 D,  15 E, and  15 F illustrate multiple top views, respectively, of various example reservoir droplet dispensing configurations of a droplet actuator, shown in relation to an opening for loading and/or unloading fluid. 
       FIG. 15A  shows a reservoir droplet dispensing configuration  1500  that is positioned in relation to an opening  1510 . In particular, opening  1510  is positioned to at least partially overlap with a transfer electrode  1512  of reservoir configuration  1500 . Transfer electrode  1512  is used to assist in the transfer of fluid that is supplied through opening  1510  onto a ring-shaped reservoir electrode  1514 , e.g., circular or oval shape of any designer-defined width. Additionally, on a side of ring-shaped reservoir electrode  1514  that may be opposite to transfer electrode  1512  is an arrangement of electrodes  1516 , e.g., electrowetting electrodes, onto which droplets (not shown) may be dispensed from ring-shaped reservoir electrode  1514  and subjected to droplet operations. 
       FIG. 15B  shows a reservoir droplet dispensing configuration  1520  that is substantially the same as reservoir droplet dispensing configuration  1500  of  FIG. 15A  except that ring-shaped reservoir electrode  1514  of  FIG. 15A  is replaced with a segmented ring-shaped reservoir electrode  1524 . The segment may be individually controlled or electrically coupled together to operate as a single electrode. 
       FIG. 15C  shows a reservoir droplet dispensing configuration  1530  that is substantially the same as reservoir droplet dispensing configuration  1500  of  FIG. 15A  except that ring-shaped reservoir electrode  1514  of  FIG. 15A  is replaced with a polygon-shaped reservoir electrode  1534 , e.g., square, rectangular, hexagonal, pentagonal, hexagonal, etc., shape of any designer-defined width. 
       FIG. 15D  shows a reservoir droplet dispensing configuration  1540  that is substantially the same as reservoir droplet dispensing configuration  1500  of  FIG. 15A  except that ring-shaped reservoir electrode  1514  of  FIG. 15A  is replaced with a segmented band-shaped reservoir electrode  1544 . Each segment may be individually controlled for providing further control as compared with the continuous ring-shaped reservoir electrode  1514  of  FIG. 15A  and/or the continuous band-shaped reservoir electrode  1534  of  FIG. 15C . 
       FIG. 15E  shows a reservoir droplet dispensing configuration  1550  that is substantially the same as reservoir droplet dispensing configuration  1500  of  FIG. 15A  except that ring-shaped reservoir electrode  1514  of  FIG. 15A  is replaced with a set of elongated electrodes  1554  that are arranged as, for example, spokes in a wheel between transfer electrode  1512  and electrodes  1516 . In this example, each elongated electrode  1554  is rectangle-shaped and may be individually controlled for providing improved control. 
       FIG. 15F  shows a reservoir droplet dispensing configuration  1560  that is substantially the same as reservoir droplet dispensing configuration  1550  of  FIG. 15E  except that elongated electrodes  1554  of  FIG. 15E , which are rectangle-shaped, are replaced with a set of elongated electrodes  1564  that are triangle-shaped. Again, elongated electrodes  1564  are arranged as, for example, spokes in a wheel between transfer electrode  1512  and electrodes  1516 , with the points of the triangles pointing inward. Each elongated electrode  1564  may be individually controlled for providing improved control. 
       FIGS. 16A ,  16 B, and  16 C illustrate multiple top views of certain example openings in relation to a fluid reservoir  1600  of a droplet actuator. Fluid reservoir  1600  may include a reservoir electrode  1610  feeding, for example, a line of electrodes  1614 , e.g., electrowetting electrodes, onto which droplets (not shown) are dispensed from reservoir electrode  1610  and by which droplets may be subjected to droplet operations. The interaction of the reservoir electrode, such as reservoir electrode  1610 , with the opening through which, for example, sample fluid may be loaded into a droplet actuator may be effected by the relative position of the opening to the reservoir electrode. 
       FIG. 16A  shows an opening  1618  that has a diameter that may be, for example, about one third to about one half the width of reservoir electrode  1610 . Additionally,  FIG. 16A  shows three example positions of opening  1618  relative to reservoir electrode  1610 . In a first example, about half of the area of opening  1618  overlaps reservoir electrode  1610 . In a second example, about less than half of the area of opening  1618  overlaps reservoir electrode  1610 . In a third example, substantially none of the area of opening  1618  overlaps reservoir electrode  1610 . 
       FIG. 16B  shows an opening  1622  that has a diameter that may be, for example, about two times the diameter of opening  1618  of  FIG. 16A . Additionally,  FIG. 16B  shows three example positions of opening  1622  relative to reservoir electrode  1610 . In a first example, about half of the area of opening  1622  overlaps reservoir electrode  1610 . In a second example, about less than half of the area of opening  1622  overlaps reservoir electrode  1610 . In a third example, substantially none of the area of opening  1622  overlaps reservoir electrode  1610 . 
       FIG. 16C  shows an opening  1626  that has a diameter that may be, for example, about three times the diameter of opening  1618  of  FIG. 16A . Additionally,  FIG. 16C  shows three example positions of opening  1626  relative to reservoir electrode  1610 . In a first example, about half of the area of opening  1626  overlaps reservoir electrode  1610 . In a second example, about less than half of the area of opening  1626  overlaps reservoir electrode  1610 . In a third example, substantially none of the area of opening  1626  overlaps reservoir electrode  1610 . 
       FIG. 17  illustrates a top view of a droplet dispensing configuration  1700  of a portion of a droplet actuator and illustrates a process of dispensing droplets. Droplet dispensing configuration  1700  may include a reservoir electrode  1710  that feeds, for example, a line of electrodes  1714 , e.g., electrowetting electrodes  1714   a ,  1714   b , and  1714   c . Droplets (not shown) from reservoir electrode  1710  may be dispensed from reservoir electrode  1710  onto electrodes  1714  and subjected to droplet operations. 
       FIG. 18  illustrates another view of the droplet dispensing configuration  1700  and the process of dispensing droplets of  FIG. 17 . 
     Additionally,  FIGS. 17 and 18  show electrodes  1714   a ,  1714   b , and  1714   c , where electrode  1714   a  is embedded within reservoir electrode  1710  and an opening  1718  near reservoir electrode  1710 . Referring to  FIGS. 17 and 18 , the process of dispensing droplets via droplet dispensing configuration  1700  may include, but is not limited to, the following steps. 
     At step 1, reservoir electrode  1710 =ON, electrode  1714   a =OFF, electrode  1714   b =OFF, and electrode  1714   c =OFF. At this step, a quantity of fluid is distributed substantially across the area of reservoir electrode  1710  only and substantially no fluid and/or droplets are present atop electrodes  1714   a ,  1714   b , and  1714   c.    
     At step 2, reservoir electrode  1710 =ON, electrode  1714   a =ON, electrode  1714   b =OFF, and electrode  1714   c =OFF. At this step, fluid from reservoir electrode  1710  is pulled atop electrode  1714   a  due to the activation of electrode  1714   a.    
     At step 3, reservoir electrode  1710 =ON, electrode  1714   a =ON, electrode  1714   b =ON, and electrode  1714   c =OFF. At this step, a finger of fluid from reservoir electrode  1710  is pulled along both electrode  1714   a  and electrode  1714   b  due to the activation of both electrode  1714   a  and electrode  1714   b.    
     At step 4, reservoir electrode  1710 =ON, electrode  1714   a =ON, electrode  1714   b =ON, and electrode  1714   c =ON. At this step, the finger of fluid from reservoir electrode  1710  is pulled further along electrodes  1714  to span electrode  1714   a , electrode  1714   b , and electrode  1714   c  due to the activation of electrode  1714   a , electrode  1714   b , and electrode  1714   c.    
     At step 5, reservoir electrode  1710 =OFF, electrode  1714   a =ON, electrode  1714   b =ON, and electrode  1714   c =ON. At this step, reservoir electrode  1710  is deactivated, which releases the fluid at reservoir electrode  1710  to take a shape that is suitable for dispensing a droplet. In particular, fluid atop reservoir electrode  1710  is allowed to reach equilibrium toward the slug of fluid that spans across electrode  1714   a , electrode  1714   b , and electrode  1714   c . This step may be conducted at higher frequency relative to the other steps. 
     At step 6, reservoir electrode  1710 =ON, electrode  1714   a =ON, electrode  1714   b =OFF, and electrode  1714   c =ON. At this step, electrode  1714   b  is deactivated and reservoir electrode  1710  is reactivated, which pulls a portion of the slug back toward reservoir electrode  1710  and causes the slug of liquid to split at electrode  1714   b , which is serving as the electrode, leaving behind a droplet at electrode  1714   c.    
       FIG. 19  illustrates a top view of another droplet dispensing configuration  1900  of a portion of a droplet actuator and illustrates another process of dispensing droplets. Droplet dispensing configuration  1900  may include a central reservoir electrode  1910 , a first side reservoir electrode  1912 , and a second side reservoir electrode  1914 . Central reservoir electrode  1910  may have a tapered geometry, as shown in  FIG. 19 . First side reservoir electrode  1912  and second side reservoir electrode  1914  may be triangular in shape and fitted to central reservoir electrode  1910 , as shown in  FIG. 19 . The combination of central reservoir electrode  1910 , first side reservoir electrode  1912 , and second side reservoir electrode  1914  forms a substantially rectangular or square reservoir electrode that is segmented for improved control. In particular, the segments are shaped in a manner to assist in the droplet dispensing process. 
     The narrow end of central reservoir electrode  1910  feeds, for example, a line of electrodes  1918 , e.g., electrowetting electrodes  1918   a ,  1918   b , and  1918   c , onto which droplets are dispensed from central reservoir electrode  1910  and by which droplets may be subjected to droplet operations. More specifically,  FIG. 19  shows electrodes  1918   a ,  1918   b , and  1918   c , where electrode  1918   a  is embedded within the narrow end of central reservoir electrode  1910  and an opening  1922  near central reservoir electrode  1910 . Referring to  FIG. 19 , the process of dispensing droplets via droplet dispensing configuration  1900  may include, but is not limited to, the following steps. 
     At step 1, central reservoir electrode  1910 =ON, first side reservoir electrode  1912 =ON, second side reservoir electrode  1914 =ON, electrode  1918   a =OFF, electrode  1918   b =OFF, and electrode  1918   c =OFF. At this step, a quantity of fluid is distributed substantially across the combined area of central reservoir electrode  1910 , first side reservoir electrode  1912 , and second side reservoir electrode  1914  and substantially no fluid and/or droplets are present atop electrodes  1918   a ,  1918   b , and  1918   c.    
     At step 2, central reservoir electrode  1910 =ON, first side reservoir electrode  1912 =ON, second side reservoir electrode  1914 =ON, electrode  1918   a =ON, electrode  1918   b =OFF, and electrode  1918   c =OFF. At this step, fluid from central reservoir electrode  1910  is pulled atop electrode  1918   a  due to the activation of electrode  1918   a.    
     At step 3, central reservoir electrode  1910 =ON, first side reservoir electrode  1912 =OFF, second side reservoir electrode  1914 =OFF, electrode  1918   a =ON, electrode  1918   b =ON, and electrode  1918   c =OFF. At this step, a finger of fluid from central reservoir electrode  1910  is pulled along both electrode  1918   a  and electrode  1918   b  due to the activation of both electrode  1918   a  and electrode  1918   b . Additionally, because first side reservoir electrode  1912  and second side reservoir electrode  1914  are deactivated, the fluid at central reservoir electrode  1910  takes on a shape that is suitable to assist in the droplet dispensing process, as shown in  FIG. 19 . 
     At step 4, central reservoir electrode  1910 =ON, first side reservoir electrode  1912 =OFF, second side reservoir electrode  1914 =OFF, electrode  1918   a =ON, electrode  1918   b =ON, and electrode  1918   c =ON. At this step, the finger of fluid from central reservoir electrode  1910  is pulled further along electrodes  1918  to span electrode  1918   a , electrode  1918   b , and electrode  1714   c  due to the activation of electrode  1918   a , electrode  1918   b , and electrode  1918   c  and the deactivation of first side reservoir electrode  1912  and second side reservoir electrode  1914 . 
     At step 5, central reservoir electrode  1910 =ON, first side reservoir electrode  1912 =ON, second side reservoir electrode  1914 =ON, electrode  1918   a =ON, electrode  1918   b =OFF, and electrode  1918   c =ON. At this step, electrode  1918   b  is deactivated and the pull of central reservoir electrode  1910 , which is now activated, draws a portion of the slug back toward central reservoir electrode  1910  and causes the slug of liquid to split at electrode  1918   b , which is serving as the electrode, leaving a droplet at electrode  1918   c.    
     At step 6, central reservoir electrode  1910 =ON, first side reservoir electrode  1912 =ON, second side reservoir electrode  1914 =ON, electrode  1918   a =OFF, electrode  1918   b =OFF, and electrode  1918   c =ON. At this step, the volume of fluid is pulled back across the combined area of central reservoir electrode  1910 , first side reservoir electrode  1912 , and second side reservoir electrode  1914  and no fluid is present atop electrodes  1918   a  and  1918   b . A droplet remains at electrode  1918   c.    
     Referring to steps 1 through 6 of the process of dispensing droplets via droplet dispensing configuration  1900 , the necessity to entirely deactivate the reservoir electrode is avoided. More specifically, central reservoir electrode  1910  remains activated throughout all steps of electrode activation sequence  1900  and first side reservoir electrode  1912  and second side reservoir electrode  1914  only are sequenced on and off. 
       FIG. 20A  illustrates another top view of droplet dispensing configuration  1700  of  FIG. 17  and illustrates a process of agitating droplets and/or priming the fluid reservoir in a droplet actuator. Referring to  FIG. 20A , the process of agitating droplets via droplet dispensing configuration  1700  may include, but is not limited to, the following steps. 
     At step 1, reservoir electrode  1710 =ON, electrode  1714   a =ON, and electrode  1714   b =OFF. In this step, a quantity of fluid is distributed substantially across the combined area of reservoir electrode  1710  and electrodes  1714   a  and no fluid is present atop  1714   b.    
     At step 2, reservoir electrode  1710 =ON, electrode  1714   a =OFF, and electrode  1714   b =OFF. In this step, electrode  1714   a  is deactivated which causes fluid at electrode  1714   a  to be drawn back to reservoir electrode  1714   a  and substantially no fluid is present atop  1714   b.    
     The process of agitating droplets via droplet dispensing configuration  1700  alternates between steps 1 and 2 in order to achieve a droplet agitation operation. Alternatively, alternating between steps 1 and 2 may be used in order to prime the liquid that is supplied via opening  1718  onto reservoir electrode  1710 . This priming operation may be carried out at the same time that other droplet operations are being performed. 
       FIG. 20B  illustrates yet another top view of droplet dispensing configuration  1700  of  FIG. 17  and illustrates a process of agitating fluid in a droplet actuator. The process of agitating fluid via droplet dispensing configuration  1700  may include, but is not limited to, the following steps. 
     At step 1, reservoir electrode  1710 =ON, electrode  1714   a =ON, and electrode  1714   b =OFF. In this step, a quantity of fluid is distributed substantially across the combined area of reservoir electrode  1710  and electrodes  1714   a  and substantially no fluid is present atop electrode  1714   b.    
     At step 2, reservoir electrode  1710 =ON, electrode  1714   a =OFF, and electrode  1714   b =OFF. In this step, electrode  1714   a  is deactivated which causes fluid at electrode  1714   a  to be drawn back to reservoir electrode  1714   a  and substantially no fluid is present atop electrode  1714   b.    
     At step 3, reservoir electrode  1710 =OFF, electrode  1714   a =OFF, and electrode  1714   b =OFF. In this step, by deactivating reservoir electrode  1710 , the fluid thereon is allowed to be substantially evacuated through opening  1718 , which provides a mechanism for disaggregating beads (not shown) in a fluid reservoir. 
     The process of agitating fluid via droplet dispensing configuration  1700  may repeatedly loop through steps 1, 2, and 3 in order to achieve a droplet agitation operation. For example, once beads (not shown) are loaded into the fluid reservoir, such as reservoir electrode  1710 , the beads tend to settle onto the surface of the fluid reservoir due to gravity. However, in order to resuspend them for use in an assay, the beads can be resuspended by loading fluid into the droplet actuator via opening  1718  and then returning the fluid back through opening  1718  (e.g., by switching off reservoir electrode  1710  in step 3). This action causes recirculation and resuspends the beads. 
       FIG. 21A  illustrates a top view of a droplet dispensing configuration  2100  of a portion of a droplet actuator and illustrates a process of disposing of a 1× size droplet in a droplet actuator. Droplet dispensing configuration  2100  includes a line of electrodes  2110  (e.g., electrowetting electrodes  2110   a ,  2110   b ,  2110   c , and  2110   d  for disposing of a 1× size droplet  2114  through an opening  2118  of a droplet actuator. In this example, opening  2118  is located in close proximity to electrode  2110   d . The 1× size refers to the approximate footprint of the droplet in relation to the approximate area of a single electrode  2110 . The process of disposing of a 1× size droplet via droplet dispensing configuration  2100  may include, but is not limited to, the following steps. 
     At step 1, electrode  2110   a =ON, electrode  2110   b =OFF, electrode  2110   c =OFF, and electrode  2110   d =OFF. In this step, 1× size droplet  2114  is held at electrode  2110   a  due to the activation of electrode  2110   a  only. 
     At step 2, electrode  2110   a =OFF, electrode  2110   b =ON, electrode  2110   c =OFF, and electrode  2110   d =OFF. In this step, electrode  2110   a  is deactivated and its neighbor, electrode  2110   b , is activated. This causes 1× size droplet  2114  to move from electrode  2110   a  to electrode  2110   b , which is in a direction that is toward opening  2118 . 
     At step 3, electrode  2110   a =OFF, electrode  2110   b =OFF, electrode  2110   c =ON, and electrode  2110   d =OFF. In this step, electrode  2110   b  is deactivated and its neighbor, electrode  2110   c , is activated. This causes 1× size droplet  2114  to move from electrode  2110   b  to electrode  2110   c , which is in a direction that is toward opening  2118 . 
     At step 4, electrode  2110   a =OFF, electrode  2110   b =OFF, electrode  2110   c =OFF, and electrode  2110   d =ON. In this step, electrode  2110   c  is deactivated and its neighbor, electrode  2110   d , is activated. This causes 1× size droplet  2114  to move from electrode  2110   c  to electrode  2110   d , which is located in close proximity to opening  2118 . 
     At step 5, electrode  2110   a =OFF, electrode  2110   b =OFF, electrode  2110   c =OFF, and electrode  2110   d =OFF. In this step, electrode  2110   d  is deactivated, which allows 1× size droplet  2114  to be evacuated from the droplet actuator (i.e., disposed of) through opening  2118 . 
       FIG. 21B  illustrates another top view of the droplet dispensing configuration  2100  of  FIG. 21A  and illustrates a process of disposing of a 2× size droplet in a droplet actuator. For example,  FIG. 21B  shows a 2× size droplet  2116  atop droplet dispensing configuration  2100 . The 2× size refers to the approximate footprint of the droplet in relation to the approximate area of a single electrode  2110 . The process of disposing of a 2× size droplet via droplet dispensing configuration  2100  may include, but is not limited to, the following steps. 
     At step 1, electrode  2110   a =ON, electrode  2110   b =OFF, electrode  2110   c =OFF, and electrode  2110   d =OFF. In this step, 2× size droplet  2116  is held at electrode  2110   a  due to the activation of electrode  2110   a  only. 
     At step 2, electrode  2110   a =OFF, electrode  2110   b =ON, electrode  2110   c =OFF, and electrode  2110   d =OFF. In this step, electrode  2110   a  is deactivated and its neighbor, electrode  2110   b , is activated. This causes 2× size droplet  2116  to move from electrode  2110   a  to electrode  2110   b , which is in a direction that is toward opening  2118 . 
     At step 3, electrode  2110   a =OFF, electrode  2110   b =OFF, electrode  2110   c =ON, and electrode  2110   d =OFF. In this step, electrode  2110   b  is deactivated and its neighbor, electrode  2110   c , is activated. This causes 2× size droplet  2116  to move from electrode  2110   b  to electrode  2110   c , which is in a direction that is toward opening  2118 . 
     At step 4, electrode  2110   a =OFF, electrode  2110   b =OFF, electrode  2110   c =ON, and electrode  2110   d =ON. In this step, both electrode  2110   c  and its neighbor, electrode  2110   d , are activated. This causes 2× size droplet  2116  to change shape and spread across both electrode  2110   c  and electrode  2110   d , which creates a slug of fluid that is located in close proximity to opening  2118 . 
     At step 5, electrode  2110   a =OFF, electrode  2110   b =OFF, electrode  2110   c =OFF, and electrode  2110   d =ON. In this step, electrode  2110   c  is deactivated, which leaves electrode  2110   d  only activated. This releases a portion of the volume of 2× size droplet  2116  to be evacuated from the droplet actuator (i.e., disposed of) through opening  2118 , which leaves the balance of the volume of 2× size droplet  2116  at electrode  2110   d.    
     At step 6, electrode  2110   a =OFF, electrode  2110   b =OFF, electrode  2110   c =OFF, and electrode  2110   d =OFF. In this step, electrode  2110   d  is deactivated, which allows the balance of the volume of 2× size droplet  2116  from step 5 to be evacuated from the droplet actuator (i.e., disposed of) through opening  2118 . 
       FIG. 22A  illustrates a top view of a dual-purpose droplet dispensing configuration  2200  of a portion of a droplet actuator and illustrates a process of dispensing droplets in a droplet actuator. Dual-purpose droplet dispensing configuration  2200  includes an array of multiple electrodes  2210  that serve as the fluid reservoir of a droplet actuator (not shown). In one example, electrodes  2210   a  through  2210   i  are arranged in a 3×3 array, as shown in  FIG. 22A . Arranged on one side of the array of electrodes  2210  may be a line of electrodes  2214 , such as electrodes  2214   a  and  2214   b , which may be, for example, electrowetting electrodes. Electrodes  2210  and electrodes  2214  may be individually controlled. Located, for example, near the side of the array of electrodes  2210  that is opposite electrodes  2214  may be an opening  2218 . Additionally,  FIG. 22A  shows all electrodes  2210  and electrodes  2214  in an activated state and a quantity of fluid  2222  that is distributed atop the combined area of electrodes  2210  and electrodes  2214 . 
       FIG. 22A  shows dual-purpose droplet dispensing configuration  2200  in one step of a droplet dispensing operation in a droplet actuator. In one example, the droplet dispensing process may be substantially the same as the droplet dispensing process that is described with reference to  FIGS. 17 and 18 . 
       FIG. 22B  illustrates another top view of dual-purpose droplet dispensing configuration  2200  of  FIG. 22A  and illustrates a process of disposing of droplets in a droplet actuator.  FIG. 22B  shows a droplet  2224  that is located atop electrode  2214   a . In this example, droplet  2224  is to be transported from electrode  2214   a  to electrode  2214   a , then to electrode  2210   b , then to electrode  2210   e , then to electrode  2210   h , and evacuated from the droplet actuator (i.e., disposed of) through opening  2218 . The droplet disposal process may be substantially the same as the droplet disposal process that is described with reference to  FIG. 21A . 
     An aspect of the dual-purpose droplet dispensing configuration  2200  of  FIGS. 22A and 22B  is that the same droplet dispensing configuration may be suited for both a droplet dispensing operation and a droplet disposal operation. 
       FIG. 23A  illustrates a top view of an example droplet dispensing configuration  2300  for dispensing droplets in multiple directions from a single reservoir in a droplet actuator. Droplet dispensing configuration  2300  may include a central reservoir electrode  2310 , which may be, for example, square or rectangular in shape, and multiple lines of electrodes  2312 . For example, a first line of electrodes  2312  may be arranged at a first side of central reservoir electrode  2310 , a second line of electrodes  2312  may be arranged at a second side of central reservoir electrode  2310 , a third line of electrodes  2312  may be arranged at a third side of central reservoir electrode  2310 , and a fourth line of electrodes  2312  may be arranged at a fourth side of central reservoir electrode  2310 , as shown in  FIG. 23A . In this example, the first electrode  2312  of each line of electrodes  2312  may be embedded in central reservoir electrode  2310 . 
     Additionally, an opening  2314  is substantially centrally located in relation to central reservoir electrode  2310 . The diameter of opening  2314  may be suitably sized such that a portion of opening  2314  may overlap the first electrode  2312  of each line of electrodes  2312 . In this way, the presence or absence of central reservoir electrode  2310  may be optional. 
     An aspect of droplet dispensing configuration  2300  of  FIG. 23A  is that it provides a single reservoir from which droplets may be dispensed in multiple directions, such as, but not limited to, four directions. Another aspect of droplet dispensing configuration  2300  is that the presence or absence of the central electrode, such as central reservoir electrode  2310 , may be optional. 
       FIG. 23B  illustrates a top view of another example droplet dispensing configuration  2320  for dispensing droplets in multiple directions from a single reservoir in a droplet actuator. Droplet dispensing configuration  2320  may include a central reservoir electrode  2322 , which may be, for example, square or rectangular in shape, and multiple side electrodes  2324  for feeding multiple lines of electrodes  2312 , which are described in  FIG. 23A . For example, a side electrode  2324   a  that feeds a first line of electrodes  2312  may be arranged at a first side of central reservoir electrode  2322 , a side electrode  2324   b  that feeds a second line of electrodes  2312  may be arranged at a second side of central reservoir electrode  2322 , a side electrode  2324   c  that feeds a third line of electrodes  2312  may be arranged at a third side of central reservoir electrode  2322 , a side electrode  2324   d  that feeds a fourth line of electrodes  2312  may be arranged at a fourth side of central reservoir electrode  2322 , as shown in  FIG. 23B . In this example, the first electrode  2312  of each line of electrodes  2312  may be embedded in each of the respective side electrodes  2324 . 
     Additionally, opening  2314  is substantially centrally located in relation to central reservoir electrode  2322 . The diameter of opening  2314  may be suitably sized such that a portion of opening  2314  may overlap each of the side electrodes  2324 . In this way, the presence or absence of central reservoir electrode  2322  may be optional. 
     An aspect of droplet dispensing configuration  2320  of  FIG. 23B  is that it provides a single reservoir from which droplets may be dispensed in multiple directions, such as, but not limited to, four directions. Another aspect of droplet dispensing configuration  2320  is that the presence or absence of the central electrode, such as central reservoir electrode  2322 , may be optional. 
       FIG. 23C  illustrates a top view of yet another example droplet dispensing configuration  2340  for dispensing droplets in multiple directions from a single reservoir in a droplet actuator. Droplet dispensing configuration  2340  may include a central reservoir electrode  2342 , which may be, for example, square, rectangular, circular, hexagonal, or octagonal in shape, and a distribution electrode  2344  that substantially surrounds central reservoir electrode  2342 . Furthermore, the geometry of distribution electrode  2344  has multiple platforms  2346  (see  FIG. 23C ) for feeding multiple lines of electrodes  2312 , which are described in  FIG. 23A . 
     For example, a first platform  2346  of distribution electrode  2344  feeds a first line of electrodes  2312 , a second platform  2346  of distribution electrode  2344  feeds a second line of electrodes  2312 , a third platform  2346  of distribution electrode  2344  feeds a third line of electrodes  2312 , a fourth platform  2346  of distribution electrode  2344  feeds a fourth line of electrodes  2312 , a fifth platform  2346  of distribution electrode  2344  feeds a fifth line of electrodes  2312 , a sixth platform  2346  of distribution electrode  2344  feeds a sixth line of electrodes  2312 , a seventh platform  2346  of distribution electrode  2344  feeds a seventh line of electrodes  2312 , an eighth platform  2346  of distribution electrode  2344  feeds an eighth line of electrodes  2312 , as shown in  FIG. 23C . In this example, the first electrode  2312  of each line of electrodes  2312  may be embedded in each of the respective platforms  2346 . 
     Additionally, opening  2314  is substantially centrally located in relation to central reservoir electrode  2342 . The diameter of opening  2314  may be suitably sized such that a portion of opening  2314  may overlap a portion of distribution electrode  2344 . In this way, the presence or absence of central reservoir electrode  2342  may be optional. 
     An aspect of droplet dispensing configuration  2340  of  FIG. 23C  is that it provides a single reservoir from which droplets may be dispensed in multiple directions, such as, but not limited to, eight directions. Another aspect of droplet dispensing configuration  2340  is that the presence or absence of the central electrode, such as central reservoir electrode  2342 , may be optional. 
     Referring to  FIGS. 23A ,  23 B, and  23 C, the geometries of the reservoir configurations are not limited to those shown in  FIGS. 23A ,  23 B, and  23 C only. In other embodiments, the geometries of the reservoir configurations may be modified to any shape that is suitable for dispensing droplets in any number of directions. Additionally, opening  2314  is not limited to circular. Alternatively, opening  2314  may be any geometry that is suited to correspond with the geometries of the reservoir configurations. 
       FIG. 24A  illustrates a top view of a portion of a droplet actuator  2400  for parallel distribution of fluid to multiple fluid reservoirs using a single opening. Additionally,  FIG. 24B  illustrates a cross-sectional view of droplet actuator  2400  taken along line AA of  FIG. 24A . Referring to  FIGS. 24A and 24B , droplet actuator  2400  may include a bottom substrate  2410  that is separated from a top substrate  2412  by a gap. A set of multiple droplet dispensing configurations  2414  may be associated with bottom substrate  2410 . In one example, droplet actuator  2400  may include droplet dispensing configurations  2414   a  through  2414   h , as shown in  FIG. 24A . Furthermore, each droplet dispensing configuration  2414  may be formed of a reservoir electrode  2416  that feeds a line of electrodes  2418 , e.g., electrowetting electrodes. 
     Droplet actuator  2400  further includes a central opening  2420  that is fluidly connected to multiple openings  2424 , which correspond to the respective droplet dispensing configurations  2414 , via respective fluid channels  2426 . For example, central opening  2420  is fluidly connected to openings  2424   a  through  2424   h  via fluid channels  2426   a  through  2426   h , respectively. Additionally, openings  2424   a  through  2424   h  correspond to droplet dispensing configurations  2414   a  through  2414   h , respectively. Furthermore, at least a portion of openings  2424   a  through  2424   h  may overlap each respective reservoir electrode  2416  of droplet dispensing configurations  2414   a  through  2414   h , as shown in  FIGS. 24A and 24B . 
     In operation, a quantity of fluid, such as a quantity of sample fluid  2428 , may be loaded into droplet actuator  2400  via central opening  2420 . Fluid  2428  then flows in a substantially simultaneous manner through fluid channels  2426  and fills openings  2424   a  through  2424   h , thereby supplying fluid  2428  in a substantially simultaneous manner to each respective reservoir electrode  2416  of the corresponding droplet dispensing configurations  2414   a  through  2414   h.    
     Optionally, a quantity of fluid  2428  may be loaded into droplet actuator  2400  via any one of the openings  2424   a  through  2424   h . However, in this instance, droplet dispensing configurations  2414   a  through  2414   h  may not be supplied with fluid  2428  in a substantially simultaneous manner, as fluid  2428  may reach the respective droplet dispensing configurations  2414  at slightly different times. Optionally, a quantity of fluid  2428  may be loaded into a certain droplet dispensing configuration  2414  only via its associated opening  2424 . For example, droplet dispensing configuration  2414   c  only may be loaded via opening  2424   c.    
     In another embodiment, openings  2424  are absent from droplet actuator  2400 . Instead, fluid may be supplied from central opening  2420  only, then flow through fluid channels  2426  to droplet dispensing configurations  2414 . 
     In yet another embodiment, the fluid paths, such as fluid channels  2426 , may lead to any type of electrode, as the invention is not limited to the fluid paths leading to reservoir electrodes only. 
       FIG. 25A  illustrates a top view of a portion of a droplet actuator  2500  for serial distribution of fluid to multiple fluid reservoirs using a single opening. Additionally,  FIG. 25B  illustrates a cross-sectional view of droplet actuator  2500  taken along line BB of  FIG. 25A . 
     Referring to  FIGS. 25A and 25B , droplet actuator  2500  may include a bottom substrate  2510  that is separated from a top substrate  2512  by a gap. A set of multiple droplet dispensing configurations  2514  may be associated with bottom substrate  2510 . In one example, droplet actuator  2500  may include droplet dispensing configurations  2514   a  through  2514   c , as shown in  FIG. 25A . Furthermore, each droplet dispensing configuration  2514  may be formed of a reservoir electrode  2516  that feeds a line of electrodes  2518 , e.g., electrowetting electrodes. 
     Droplet actuator  2500  further includes a fluid channel  2520  that is fluidly connected to multiple openings  2522 , which correspond respectively to the multiple droplet dispensing configurations  2514 . For example, fluid channel  2520  is fluidly connected to openings  2522   a  through  2522   c , which correspond to droplet dispensing configurations  2514   a  through  2514   c , respectively. 
     Furthermore, at least a portion of openings  2522   a  through  2522   c  may overlap each respective reservoir electrode  2516  of droplet dispensing configurations  2514   a  through  2514   c , as shown in  FIGS. 25A and 25B . 
     In operation, a quantity of fluid, such as a quantity of sample fluid  2528 , may be loaded into droplet actuator  2400  via fluid channel  2520 . Fluid  2428  then flows through fluid channel  2520  and reaches openings  2522   a  through  2522   c  in a substantially serial manner, thereby supplying fluid  2528  in a substantially sequential manner to each respective reservoir electrode  2516  of the corresponding droplet dispensing configurations  2514   a  through  2514   c . In one example, via fluid channel  2520 , fluid  2428  may first reach droplet dispensing configuration  2514   a , then droplet dispensing configuration  2514   b , and then droplet dispensing configuration  2514   c.    
     In another embodiment, the fluid path, such as fluid channel  2520 , may lead to any type of electrode, as the invention is not limited to the fluid path leading to reservoir electrodes only. 
       FIGS. 26A and 26B  illustrate top views of an example droplet dispensing configuration  2600  of a droplet actuator that includes a droplet forming electrode that is embedded in a larger reservoir electrode. Droplet dispensing configuration  2600  may include a reservoir electrode  2610  having a droplet forming electrode  2614  embedded therein, as shown in  FIGS. 26A and 26B . Reservoir electrode  2610  may be, for example, several times larger in area than droplet forming electrode  2614 . Additionally,  FIGS. 26A and 26B  show an opening  2618  that is associated with reservoir electrode  2610 . 
     In  FIG. 26A , both reservoir electrode  2610  and droplet forming electrode  2614  are activated. Consequently, a quantity of fluid, such as sample fluid  2622 , that is supplied via opening  2618  is atop the combined area of reservoir electrode  2610  and droplet forming electrode  2614 . 
     In  FIG. 26B , reservoir electrode  2610  is deactivated and droplet forming electrode  2614  only is activated. Consequently, the quantity of fluid  2622  that is atop reservoir electrode  2610  (see  FIG. 26A ) may be evacuated through opening  2618 , leaving a droplet  2626  atop droplet forming electrode  2614  only. 
       FIG. 26C  illustrates a top view of an example droplet dispensing configuration  2630  of a droplet actuator that includes multiple droplet forming electrodes that are embedded in a larger reservoir electrode. Droplet dispensing configuration  2630  may include a reservoir electrode  2632  having multiple droplet forming electrodes  2634  (e.g., droplet forming electrodes  2634   a ,  2634   b ,  2634   c , and  2634   d ) embedded therein, as shown in  FIG. 26C . Reservoir electrode  2632  may be, for example, several times larger in area than each droplet forming electrode  2634 . Additionally,  FIG. 26C  shows opening  2618  that is positioned substantially in a central area of reservoir electrode  2632 . 
     In  FIG. 26C , reservoir electrode  2632  is deactivated and droplet forming electrodes  2634   a ,  2634   b ,  2634   c , and  2634   d  are activated. Consequently, any quantity of fluid that may have been atop reservoir electrode  2632  may be evacuated through opening  2618 , leaving a droplet  2626  atop droplet forming electrodes  2634   a ,  2634   b ,  2634   c , and  2634   d  only. 
     The invention is not limited to the example embodiments shown in  FIGS. 1 through 26A ,  26 B, and  26 C. The scope of the invention may include any combinations of the example embodiments shown in  FIGS. 1 through 26A ,  26 B, and  26 C. Additionally, variations of the example embodiments shown in  FIGS. 1 through 26A ,  26 B, and  26 C may utilize, for example, pressure, electrowetting, gravity effect, capillary force, and any combinations thereof as the energy source for moving a volume of liquid in a droplet actuator. Furthermore, variations of the example embodiments shown in  FIGS. 1 through 26A ,  26 B, and  26 C may include fluid reservoirs, electrodes, and openings of any size, shape, and/or geometry, such as but not limited to, rectangular, square, circular, oval, hexagonal, and octagonal. 
     Droplet Actuator 
     For examples of droplet actuator architectures that are suitable for use with the present invention, see U.S. Pat. No. 6,911,132, entitled, “Apparatus for Manipulating Droplets by Electrowetting-Based Techniques,” issued on Jun. 28, 2005 to Pamula et al.; U.S. patent application Ser. No. 11/343,284, entitled, “Apparatuses and Methods for Manipulating Droplets on a Printed Circuit Board,” filed on filed on Jan. 30, 2006; U.S. Pat. No. 6,773,566, entitled, “Electrostatic Actuators for Microfluidics and Methods for Using Same,” issued on Aug. 10, 2004 and U.S. Pat. No. 6,565,727, entitled, “Actuators for Microfluidics Without Moving Parts,” issued on Jan. 24, 2000, both to Shenderov et al.; Pollack et al., International Patent Application No. PCT/US 06/47486, entitled, “Droplet-Based Biochemistry,” filed on Dec. 11, 2006, the disclosures of which are incorporated herein by reference. As described above, the droplet actuators include a droplet operations surface on which droplet operations are conducted. The droplet actuators also include electrodes configured for conducting droplet operations. 
     The droplet operations electrodes are often described here as being associated with the droplet operations surfaces, but it should be appreciated that they may be associated with any substrate of the droplet actuator, including the top and/or bottom substrates, as well as substrates which are intermediate to the top and bottom substrates, such as side walls or sealants coupling the top and bottom substrates. Further, in the various embodiments described, the top substrate may or may not be present. Various embodiments are described as using capillary forces, surface tension forces pressure sources to cause fluid to flow. It will be appreciated that in each of these embodiments any combination of capillary forces, surface tension forces, pressure sources (positive or negative) and/or other forces may be employed. Further, throughout the disclosure, the droplet actuator is typically described as having top and bottom substrates, but it will be appreciated that in embodiments that don&#39;t specifically require the droplet to be constrained between two substrates for operability, a single substrate will suffice. In embodiments that include a reservoir separated from the droplet operations surface by a reservoir wall, liquid may be introduced into the reservoir by a fluid path established in the top plate, the bottom plate and/or a side of the droplet actuator between the top and bottom plates. In addition to the various droplet dispensing protocols described herein, it should be noted that in each embodiment, a droplet may be dispensed by activating one or more of the reservoir electrodes and two or more droplet operations electrodes followed by deactivating a droplet operations electrode that is intermediate between the terminal activated droplet operations electrode and the one or more reservoir electrodes. With reference to the examples described herein, in various embodiments, 2, 3, 4, 5 or more droplet operations electrodes may be activated, followed by deactivation of an intermediate one of these droplet operations electrode to form a droplet on the terminal activated electrode or electrodes. Further, in the various embodiments described herein, a first droplet operations electrode may be adjacent to, partially embedded in or completely embedded in a reservoir electrode. 
     Fluids 
     For examples of fluids that may be subjected to droplet operations using the approach of the invention, see the patents listed in section 7.3, especially International Patent Application No. PCT/US 06/47486, entitled, “Droplet-Based Biochemistry,” filed on Dec. 11, 2006. In some embodiments, the fluid includes a biological sample, such as whole blood, lymphatic fluid, serum, plasma, sweat, tear, saliva, sputum, cerebrospinal fluid, amniotic fluid, seminal fluid, vaginal excretion, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudates, exudates, cystic fluid, bile, urine, gastric fluid, intestinal fluid, fecal samples, fluidized tissues, fluidized organisms, biological swabs and biological washes. In some embodiment, the fluid includes a reagent, such as water, deionized water, saline solutions, acidic solutions, basic solutions, detergent solutions and/or buffers. In some embodiments, the fluid includes a reagent, such as a reagent for a biochemical protocol, such as a nucleic acid amplification protocol, an affinity-based assay protocol, a sequencing protocol, and/or a protocol for analyses of biological fluids. 
     Filler Fluids 
     The gap is typically filled with a filler fluid. The filler fluid may, for example, be a low-viscosity oil, such as silicone oil. Other examples of filler fluids are provided in International Patent Application No. PCT/US 06/47486, entitled, “Droplet-Based Biochemistry,” filed on Dec. 11, 2006. 
     Example Method of High-Throughput Droplet Dispensing 
     One example approach for providing a high-throughput droplet dispensing operation in a droplet actuator may include, but is not limited to, the steps of (1) providing an array of individually-controlled electrodes in the path of a liquid from which droplets to be subjected to droplet operations may be formed, such as shown in  FIGS. 2 and 3 ; (2) providing, under a certain pressure, a volume of liquid that substantially covers the array of individually-controlled electrodes, such as shown in  FIGS. 2 and 3 ; (3) activating certain individually-controlled electrodes, such as every other individually-controlled electrode; (4) reducing the pressure in order to cause the volume of liquid to retract starting from one end of the array of individually-controlled electrodes; and (5) forming a droplet on certain activated electrodes, such as every other electrode, in the wake of the retracting fluid, such as shown in  FIGS. 2 and 3 . 
     CONCLUDING REMARKS 
     The foregoing detailed description of embodiments refers to the accompanying drawings, which illustrate specific embodiments of the invention. Other embodiments having different structures and operations do not depart from the scope of the present invention. 
     This specification is divided into sections for the convenience of the reader only. Headings should not be construed as limiting of the scope of the invention. 
     It will be understood that various details of the present invention may be changed without departing from the scope of the present invention. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation, as the present invention is defined by the claims as set forth hereinafter.