Abstract:
The objective of this research is to model and design a microfluidic system that uses electrostatic fields to induce movement of discrete droplets of solution. Of particular interest is movement of droplets of H 2 O for use in biological testing with lab-on a-chip and μTAS systems. Using computer modeling, the electric-fields for planar electrode configurations positioned on an insulating substrate are calculated for a hemispherical drop of H 2 O on the substrate at various positions. From these electric-fields the force on the drop is calculated. These models show that electrostatic actuation of droplets of H 2 O is possible. However, as the complexity of the model increases the properties of the system become less desirable and actuation may not be possible. Using microfabrication techniques, the modeled microfluidic systems have been built for testing using a Kapton substrate with copper electrodes. Hexadecenyltrichlorosilane (HTS), a self-assembled monolayer, and its oxidant have been studied and found capable of providing hydrophobic and hydrophilic surface coatings for the systems.

Description:
RELATED APPLICATIONS 
       [0001]    In addition to the patent applications cited herein, each of which is incorporated herein by reference, this patent application is related to and claims priority to U.S. Provisional Patent Application No. 60/892,285, filed on Mar. 1, 2007, entitled “Droplet actuator architectures;” U.S. Provisional Patent Application No. 60/895,784, filed on Mar. 20, 2007, entitled “Single metal layer microactuator structures;” and U.S. Provisional Patent Application No. 60/980,463, filed on Oct. 17, 2007, entitled “Droplet actuator architectures;” the entire disclosures of which are incorporated herein by reference. 
     
    
     GRANT INFORMATION 
       [0002]    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. 
     
    
     FIELD OF THE INVENTION 
       [0003]    The present invention generally relates to the field of conducting droplet operations in a droplet actuator. In particular, the present invention is directed to droplet actuator structures. 
       BACKGROUND OF THE INVENTION 
       [0004]    Droplet actuators are used to conduct a wide variety of droplet operations. A droplet actuator typically includes a substrate associated with electrodes 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. Surfaces exposed to the gap are typically hydrophobic. Electrodes that are associated with one or both substrates are arranged for conducting a variety of droplet operations, such as droplet transport and droplet dispensing. There is a need for alternative approaches to configuring and wiring electrodes in a droplet actuator. 
       BRIEF DESCRIPTION OF THE INVENTION 
       [0005]    The invention provides example approaches to configuring and wiring electrodes in a droplet actuator. Droplet actuators employing the designs of the invention are useful for conducting a variety of droplet operations. 
         [0006]    In one set of embodiments, the droplet actuator of the invention includes various single-layer wiring configurations for mitigating the constraints and drawbacks that are associated with single-layer designs, such as wireability constraints, limited mechanisms for performing droplet operations, electrostatic interference from wires, and any combinations thereof. A plurality of transport electrodes, reservoir electrodes, fluid reservoirs, and wires can be provided on a single-layer of a droplet actuator in varying arrangements. Transport electrodes may be configured to impart a gradient force to a droplet of sufficient force to manipulate the droplet. Electrostatic interference reducing structures may also be provided. 
         [0007]    In another set of embodiments, the droplet actuator of the invention can include a reference electrode that is situated on one substrate that is separated by a gap from a second substrate and one or more control electrodes that are situated on the second substrate. The control electrodes may be placed such that the second substrate is interposed between the control electrodes and the first substrate. A substantially planar substrate may be provided comprising an anisotropic conductive element. Recessed regions may be provided wherein electrodes are arranged in the recessed regions. A dispensing electrode configuration may be provided comprising a reservoir electrode and one or more droplet dispensing electrodes. 
       DEFINITIONS 
       [0008]    As used herein, the following terms have the meanings indicated. 
         [0009]    “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. 
         [0010]    “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. 
         [0011]    “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. 
         [0012]    “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. 
         [0013]    “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. 
         [0014]    “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. 
         [0015]    “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. 
         [0016]    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. 
         [0017]    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. 
         [0018]    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. 
         [0019]    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. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0020]      FIG. 1  illustrates a top view of a wiring structure of a portion of a droplet actuator, which is one embodiment of a single-layer wiring structure; 
           [0021]      FIG. 2  illustrates a top view of a wiring structure of a portion of a droplet actuator, which is another embodiment of a single-layer wiring structure; 
           [0022]      FIG. 3  illustrates a top view of a wiring structure of a portion of a droplet actuator, which is yet another embodiment of a single-layer wiring structure; 
           [0023]      FIG. 4  illustrates a top view of a wiring structure of a portion of a droplet actuator, which is yet another embodiment of a single-layer wiring structure; 
           [0024]      FIG. 5  illustrates a top view of a wiring structure of a portion of a droplet actuator, which is yet another embodiment of a single-layer wiring structure; 
           [0025]      FIG. 6  illustrates a top view of a prior art transport electrode of a droplet actuator and illustrates how the electrode wiring may influence a droplet footprint; 
           [0026]      FIG. 7  illustrates a top view of a single transport electrode of a droplet actuator, which is one embodiment of an electrode structure for reducing the negative effects of electrostatic interference; 
           [0027]      FIG. 8  illustrates a top view of a single transport electrode of a droplet actuator, which is another embodiment of an electrode structure for reducing the negative effects of electrostatic interference; 
           [0028]      FIG. 9  illustrates a side view of a segment of a droplet actuator, which is yet another embodiment of an electrode structure for reducing the negative effects of electrostatic interference; 
           [0029]      FIG. 10  illustrates a side view of a segment of a droplet actuator, which is yet another embodiment of an electrode structure for reducing the negative effects of electrostatic interference; 
           [0030]      FIG. 11  illustrates a side view of a segment of a droplet actuator, which is one embodiment of an electrode structure for improving droplet operations and/or ease of manufacture; 
           [0031]      FIG. 12  illustrates a side view of a segment of a droplet actuator, which is another embodiment of an electrode structure for improving droplet operations and/or ease of manufacture; 
           [0032]      FIG. 13  illustrates a side view of a segment of a droplet actuator, which is yet another embodiment of an electrode structure for improving droplet operations and/or ease of manufacture and/or assembly; 
           [0033]      FIG. 14  illustrates a side view of a segment of a droplet actuator, which is yet another embodiment of an electrode structure for improving droplet operations and/or ease of manufacture and/or assembly; 
           [0034]      FIG. 15  illustrates a side view of a segment of a droplet actuator, which is yet another embodiment of an electrode structure for improving droplet operations and/or ease of manufacture; and 
           [0035]      FIG. 16  illustrates a side view of a segment of a droplet actuator, which is yet another embodiment of an electrode structure for improving droplet operations. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0036]    The invention provides a droplet actuator that has improved wiring and/or electrode structures and methods of making and/or using the droplet actuator. The droplet actuator of the invention exhibits numerous advantages over droplet actuators of the prior art. In various embodiments, the droplet actuator of the invention includes various single-layer wiring configurations for mitigating the constraints and drawbacks that are associated with single-layer designs, such as wireability constraints, limited mechanisms for performing droplet operations, electrostatic interference from wires, and any combinations thereof. 
         [0037]    In other embodiments, the droplet actuator of the invention includes a reference electrode that is situated on one substrate that is separated by a gap from a second substrate and one or more control electrodes that are situated on the second substrate. The control electrodes may be placed such that the second substrate is interposed between the control electrodes and the first substrate. Droplet actuators employing the designs of the invention are useful for conducting a variety of droplet operations. 
       Example Single-Layer Wire/Electrode Configurations 
       [0038]      FIG. 1  illustrates a top view of a wiring structure  100  of a portion of a droplet actuator. Wiring structure  100  is provided on a substrate (not shown), which may, for example, be made from any suitably electrically resistant substance, such as a semiconductor chip or a printed circuit board. Wiring structure  100  is one embodiment of a single-layer wiring structure that may, among other things, provide improved wireability. Wiring structure  100  may include a droplet operations region  110 . A U-shaped transport bus  114  is disposed within droplet operations region  110 . U-shaped transport bus  114  is connected to one or more fluid reservoir electrodes  118  via one or more dispensing electrodes  120  for dispensing droplets (not shown). U-shaped transport bus  114  is formed of multiple transport electrodes  122  for transporting droplets that are dispensed from fluid reservoir electrodes  118 , which are arranged around the outer perimeter of U-shaped transport bus  114 . In one example, U-shaped transport bus  114  is connected to six fluid reservoir electrodes  118 , as shown in  FIG. 1 . 
         [0039]    Wiring structure  100  may further include a contact pad region  126 . Multiple control signal contact pads  130  are disposed within contact pad region  126 . The multiple control signal contact pads  130  are electrically connected to fluid reservoir electrodes  118  and transport electrodes  122 . More specifically,  FIG. 1  shows a layout of wire segments  134  that are connected at one end to contact pads  130  and are oriented toward droplet operations region  110  at the opposite end. The layout of wire segments  134  has a certain wiring density. Wire segments  134  have a certain trace width, w 1 . 
         [0040]    Additionally, wiring structure  100  may include a wire region  138  that may translate, in some embodiments, the wiring density of wire segments  134  of contact pad region  126  to a certain greater wiring density of droplet operations region  110 . For example,  FIG. 1  shows a layout of wire segments  142  that have a certain trace width, w 2 , and that are a continuation of wire segments  134  of contact pad region  126 . More specifically,  FIG. 1  shows that one end of wire segments  142  are connected to wire segments  134 . In the illustrated embodiment, the opposite end of wire segments  142  are oriented in a tight group toward the center of droplet operations region  110 . In particular, a layout of wire segments  146  is disposed within a central area of droplet operations region  110  for connecting to fluid reservoir electrodes  118  and transport electrodes  122 . Wire segments  146  have a certain trace width, w 3 , and are a continuation of wire segments  142  of wire region  138 . 
         [0041]    The combination of wire segments  134  of contact pad region  126 , wire segments  142  of wire region  138 , and wire segments  146  of droplet operations region  110  provide a complete electrical connection between contact pads  130  and fluid reservoir electrodes  118  and between contact pads  130  and transport electrodes  122 . In order to minimize the electrostatic interference from the wires to the electrodes, the width, w 3 , of wire segments  146  may be substantially minimized, while the width of the wires may increase as they approach contact pads  130 . In one example, the width, w 3 , of wire segments  146  may be about 10 microns, the width, w 2 , of wire segments  142  may be about 25 microns, and the width, w 1 , of wire segments  134  may be about 75 microns. 
         [0042]    In the nonlimiting example of  FIG. 1 , the outermost contact pads  130  are connected to fluid reservoir electrodes  118 , which may be bused together, and to dispensing electrodes  120 , which may be bused together, while the innermost contact pads  130  are independently connected to transport electrodes  122 . The centermost area of U-shaped transport bus  114  provides a clearance region and, therefore, each wire connection for transport electrodes  122  is inside U-shaped transport bus  114 . As a result, wiring structure  100  is an example of a single-layer structure that allows easy wiring access to multiple transport electrodes  122  for providing independent control thereof. 
         [0043]      FIG. 2  illustrates a top view of a wiring structure  200  of a portion of a droplet actuator. Wiring structure  200  is another embodiment of a single-layer wiring structure that may, among other things, provide improved wireability. Wiring structure  200  may include multiple transport electrodes  210  for transporting droplets (not shown) that are dispensed from multiple fluid reservoir electrodes  214  (e.g., fluid reservoir electrodes  214   a ,  214   b ,  214   c , and  214   d ). In one example, transport electrodes  210  in combination with fluid reservoir electrodes  214  are arranged in a cross pattern, as shown in  FIG. 2 . By busing multiple electrodes together, wiring structure  200  provides a single-layer design that uses a concentric approach to wiring radial paths of transport electrodes  210  and fluid reservoir electrodes  214 . In one example, a set of wires  218  approach transport electrodes  210  and fluid reservoir electrodes  214  from a single entry point and are distributed in a substantially concentric fashion such that certain transport electrodes  210  and fluid reservoir electrodes  214  are bused together, as shown in  FIG. 2 . 
         [0044]      FIG. 3  illustrates a top view of a wiring structure  300  of a portion of a droplet actuator. Wiring structure  300  is yet another embodiment of a single-layer wiring structure that may, among other things, provide improved wireability. Wiring structure  300  may include multiple transport electrodes  310  for transporting droplets (not shown) that are dispensed from multiple fluid reservoir electrodes  314  (e.g., fluid reservoir electrodes  314   a  and  314   b ). In one example, transport electrodes  310  are arranged in a line between fluid reservoir electrodes  314   a  and  314   b . Additionally, wiring structure  300  may include a droplet storage array  318 . In one example, droplet storage array  318  may include a line of transport electrodes  322   a  that feeds a fluid reservoir electrode  326   a , a line of transport electrodes  322   b  that feeds a fluid reservoir electrode  326   b , and a line of transport electrodes  322   c  that feeds a fluid reservoir electrode  326   c , as shown in  FIG. 3 . 
         [0045]    The single-layer design of wiring structure  300  provides multiple types of electrodes, such as transport electrodes  310 , fluid reservoir electrodes  314 , and fluid reservoir electrodes  326 , that are wired for independent control. For example, a set of wires  338  are provided from contact pad region  330  to individual fluid reservoir electrodes  326   a ,  326   b , and  326   c . Additionally, a set of wires  342  is provided from contact pad region  330  to individual transport electrodes  310  and fluid reservoir electrode  314   a  and  314   b , as shown in  FIG. 3 . 
         [0046]    The single-layer design of wiring structure  300  also provides electrodes, such as transport electrodes  322 , that are, in the illustrated embodiment, bused together for common control thereof. For example, a contact pad region  330  is shown from which a set of bus wires  334  is provided to transport electrodes  322   a ,  322   b , and  322   c , as shown in  FIG. 3 . 
         [0047]    The single-layer design of wiring structure  300  allows the capacity of storage arrays, such as droplet storage array  318 , to be maximized based on the number of control signals, such as N×M control signals. In one example, the capacity of the storage array may be N number of wires  334  times M number of wires  338 . 
       Example Single-Layer Electrostatic Energy Gradient Configurations 
       [0048]      FIG. 4  illustrates a top view of a wiring structure  400  of a portion of a droplet actuator. Wiring structure  400  is one embodiment of a single-layer wiring structure that uses an area gradient to control electrostatic energy for conducting droplet operations. Wiring structure  400  may include a fluid reservoir electrode  410 , a transport electrode  414 , a fluid reservoir electrode  418 , and a transport electrode  422 . Arranged between transport electrode  414  and transport electrode  422  is an electrode pair  426  that is formed of a first tapered elongated transport electrode  430  and a second tapered elongated transport electrode  434 . More specifically, elongated transport electrode  430  and  434  are each narrow at one end and wide at the other end. The narrow end of elongated transport electrode  430  is oriented adjacent to the wide end of elongated transport electrode  434 , as shown in  FIG. 4 . A set of control wires  438  is provided to all electrodes of wiring structure  400 . In particular, electrode pair  426  requires two control wires  438  only, rather than multiple control wires that would be required when using multiple individual transport electrodes to span the same distance as electrode pair  426 . As a result, wiring structure  400  provides a single-layer design that minimizes the number of control lines needed to perform droplet operations, while maintaining suitable control of droplet operations. 
         [0049]    The area gradient of electrode pair  426  may be used to conduct droplet operations between fluid reservoir electrode  410  and fluid reservoir electrode  418  as follows. In a first example, a droplet (not shown) is transported from fluid reservoir electrode  410  to fluid reservoir electrode  418 . Transport electrode  414  is activated and the droplet is dispensed from fluid reservoir electrode  410  to transport electrode  414 . In doing so, the droplet at transport electrode  414  overlaps slightly the narrow end of elongated transport electrode  434 . Transport electrode  414  is then deactivated and elongated transport electrode  434  is activated. Due to the area gradient along the length of elongated transport electrode  434 , the droplet moves from its narrow end to its wide end. Once the droplet is at the wide end of elongated transport electrode  434  and overlapping slightly transport electrode  422 , elongated transport electrode  434  is deactivated and transport electrode  422  is activated in order to move the droplet onto transport electrode  422 . Transport electrode  422  may then be deactivated and fluid reservoir electrode  418  activated in order to transport the droplet to fluid reservoir electrode  418 . 
         [0050]    In a second example, the droplet is transported from fluid reservoir electrode  418  to fluid reservoir electrode  410 . Transport electrode  422  is activated and the droplet is dispensed from fluid reservoir electrode  418  to transport electrode  422 . In doing so, the droplet at transport electrode  422  overlaps slightly the narrow end of elongated transport electrode  430 . Transport electrode  422  is then deactivated and elongated transport electrode  430  is activated. Due to the area gradient along the length of elongated transport electrode  430 , the droplet moves from its narrow end to its wide end. Once the droplet is at the wide end of elongated transport electrode  430  and overlapping slightly transport electrode  414 , elongated transport electrode  430  is deactivated and transport electrode  414  is activated in order to move the droplet onto transport electrode  414 . Transport electrode  414  may then be deactivated and fluid reservoir electrode  410  activated in order to transport the droplet to fluid reservoir electrode  410 . 
         [0051]    Wiring structure  400  is not limited to the geometry of electrode pair  426  for providing an area gradient to control electrostatic energy. Any geometry that provides a continuous area gradient in a certain direction is suitable. For example, other geometries that provide an area gradient may include, but are not limited to, electrodes containing interior voids, such as patterns of circular or square voids that form a density gradient. This density gradient may create an effective electrode area gradient along a certain direction. 
         [0052]      FIG. 5  illustrates a top view of a wiring structure  500  of a portion of a droplet actuator. Wiring structure  500  is one embodiment of a single-layer wiring structure that uses a voltage gradient to control electrostatic energy for conducting droplet operations. Wiring structure  500  is substantially the same as wiring structure  400  of  FIG. 4 , except that electrode pair  426  of wiring structure  400  is replaced with an elongated transport electrode  510 . 
         [0053]    Elongated transport electrode  510  has a first voltage control V 1  that is connected to one end and a second voltage control V 2  that is connected to its opposite end. In this way, a voltage gradient may be developed from one end to the other of elongated transport electrode  510 . This voltage gradient is a function of the voltage difference between V 1  and V 2  and the resistance per unit length R of electrode  510 . As a result, wiring structure  500  may reduce the number of control lines that are needed to transport a droplet over a certain distance, while maintaining suitable control of droplet transport operations. 
         [0054]    In one example, a droplet (not shown) may be dispensed from fluid reservoir electrode  410  to transport electrode  414 . A certain voltage is applied at voltage control V 1  and a certain higher voltage is applied at voltage control V 2 , thereby creating a voltage gradient along elongated transport electrode  510 . In one example, the voltage gradient between voltage control V 1  and V 2  may range from about 0 volts to about 300 volts. Due to the voltage gradient along the length of elongated transport electrode  510 , a proportional gradient of electrostatic energy develops along the length of elongated transport electrode  510 , which results in the movement of the droplet from the end that is connected to V 1  (the lower voltage) to the end that is connected to V 2  (the higher voltage). In this way, the droplet may be moved from transport electrode  414  to transport electrode  422 , and ultimately to fluid reservoir electrode  418 . 
         [0055]    Alternatively, a droplet actuator may include a combination of both the electrode area gradient of  FIG. 4  and the electrode voltage gradient of  FIG. 5  in order to create an electrostatic energy gradient for use as the mechanism for performing droplet operations. 
       Example Single-Layer Wire Interference Reducing Configurations 
       [0056]      FIG. 6  illustrates a top view of a prior art transport electrode  600  of a droplet actuator.  FIG. 6  illustrates how the electrode wiring may influence a droplet footprint. A droplet  618  is disposed upon a transport electrode  610 . A control wire  614  provides the control voltage to transport electrode  610 . When transport electrode  610  is activated, electrostatic interference from control wire  614  may influence the geometry of droplet  618 . Droplet  618  may extend along the path of control wire  614 , which distorts its geometry, and may adversely effect droplet operations.  FIGS. 7 ,  8 ,  9 , and  10  illustrate exemplary techniques for reducing, preferably substantially eliminating, the effects of electrostatic interference from wires in a droplet actuator. 
         [0057]      FIG. 7  illustrates a top view of a single transport electrode  700  of a droplet actuator. Transport electrode  700  may be substantially the same as transport electrode  600  of  FIG. 6 , except that transport electrode  700  provides a second control wire  714  that is opposite first control wire  614 . Control wire  714 , in addition to control wire  614 , provides the control voltage to transport electrode  610 . As a result, when transport electrode  610  is activated, the electrostatic interference from control wire  714  creates a substantially equal and opposite pull to the electrostatic interference from control wire  614 . Consequently, droplet  618  is maintained at substantially the center of transport electrode  610 , as shown in  FIG. 7 , instead of shifting toward control wire  614  in the manner that is illustrated in  FIG. 6 . Although some droplet distortion may occur, droplet  618  in  FIG. 7  remains substantially centered and its symmetry is substantially maintained. The first and second control wires may be independently connected to the same signal contact pad. Alternatively, only one of the two control wires may be connected to the signal contact pad and the remaining control wire may be a wire shaped stub that is connected to the electrode. 
         [0058]      FIG. 8  illustrates a top view of a single transport electrode  800  of a droplet actuator. Transport may include a transport electrode  810  and its associated control wire  814 . Transport electrode  800  provides an interface region  818  between transport electrode  810  and wire  814 . The metal that forms interface region  818  is tapered from the width of transport electrode  810  to the width of wire  814 , as shown in  FIG. 8 . The height and width of the taper within interface region  818  may vary. 
         [0059]      FIG. 9  illustrates a side view of a segment of a droplet actuator  900 . Droplet actuator  900  includes yet another embodiment of an electrode structure that may, among other things, reduce the effects of electrostatic interference from wires. Droplet actuator  900  may include a first substrate, such as a top substrate  910 , and a second substrate, such as a bottom substrate  914 . Top substrate  910  may be formed of substrate  918  and a ground electrode  922 . Bottom substrate  914  may be formed of substrate  926  and a transport electrode  930  that has an associated control wire  934 . A dielectric layer  938  is typically deposited atop transport electrode  930  and control wire  934 . Additionally, an electrically conductive shield  942  is deposited atop dielectric layer  938 , as shown in  FIG. 9 . Shield  942  is substantially aligned with control wire  934 . Shield  942  may be formed of any material, such as copper or aluminum, that is suitable for providing electrostatic shielding. Top substrate  910  and bottom substrate  914  are arranged in order to provide a gap therebetween that provides a fluid flow path. In one example, a droplet  950  may be transported along the gap. 
         [0060]    The position of shield  942  is such that it provides electrostatic shielding between droplet  950  and control wire  934 . The presence of shield  942  reduces, preferably substantially eliminates, the electrostatic attraction between droplet  950  and control wire  934  as compared with the electrostatic attraction between droplet  950  and transport electrode  930 . Optionally, shield  942  may overlap transport electrode  930  in order to reduce, preferably substantially eliminate, any fringing fields at the boundary therebetween. The amount of overlap may, in some embodiments, be optimized in order to minimize the reduction in the effective size of transport electrode  930 . The embodiment of  FIG. 9  uses two layers of metal, but this extra metal layer does not require vias or connections and, thus, the design remains simple. In some embodiments, shield  942  may serve as an electrical connection for controlling the reference potential of the droplet. 
         [0061]      FIG. 10  illustrates a side view of a segment of a droplet actuator  1000 . Droplet actuator  1000  includes yet another embodiment of an electrode structure that may, among other things, reduce the effects of electrostatic interference from wires. Droplet actuator  1000  is substantially the same as droplet actuator  900  of  FIG. 9 , except that the electrostatic shielding (e.g., shield  942 ) is replaced with another dielectric layer  1010 . Again, dielectric layer  1010  is substantially aligned with control wire  934  and is in addition to dielectric layer  938 , as shown in  FIG. 10 . The presence of the additional dielectric layer  1010  reduces, preferably substantially eliminates, the electrostatic attraction between droplet  950  and control wire  934  as compared with the electrostatic attraction between droplet  950  and transport electrode  930 . 
       Example Electrode Structures for Droplet Actuators 
       [0062]      FIG. 11  illustrates a side view of a segment of a droplet actuator  1100 . Droplet actuator  1100  may, among other things, provide improved droplet operations and/or ease of manufacture in a droplet actuator. Droplet actuator  1100  may include a first substrate  1110  and a second substrate  1112  that are arranged with a gap  1114  therebetween. A hydrophobic coating  1116  is disposed on an inner surface of first substrate  1110  (i.e., facing gap  1114 ). One or more control electrodes  1118  are disposed on an outer surface of first substrate  1110  (i.e., facing away from gap  1114 ). A reference electrode  1120  is disposed on an inner surface of second substrate  1112  (i.e., facing gap  1114 ). A hydrophobic coating  1116  is disposed on an inner surface of reference electrode  1120  (i.e., facing gap  1114 ). 
         [0063]    First substrate  1110  may, for example, be formed of a thin film of any nonconductive material, such as, but not limited to, Teflon® and Kapton® polyimide film. In one example, the thickness of the thin film material may be from about 1 mil to a few mils. Alternatively, first substrate  1110  may be formed of a thick film of any nonconductive material, such as, but not limited to, glass. In one example, the thickness of the thick film material may be from about 100 microns to about 1 millimeter. In either case, first substrate  1110  must be suitably thin to allow the electric fields of control electrodes  1118  to influence a droplet, such as a droplet  1122 , that is to be subjected to droplet operations. Furthermore, the presence of an insulator layer (e.g., first substrate  1110 ) between control electrodes  1118  and droplet  1122  may require an increase in electrode voltage relative to droplet actuators of the prior art, in order to ensure a suitable electric field at droplet  1122 . 
         [0064]    Second substrate  1112  may be, for example, a glass substrate. Control electrodes  1118  and reference electrode  1120  may be formed of a conductive material, such as, but not limited to, copper. Alternatively, reference electrode  1120  may be formed of indium tin oxide (ITO). Typically the portion of the substrate on which droplet operations are to take place are made from a hydrophobic material and/or include a hydrophobic coating. The insulating support and hydrophobic coating may be the same material and/or different materials, e.g., an insulating layer with a non-wetting surface. The non-wetting surface may be provided by, for example, but not limited to, a film coating, a chemical surface treatment, physical structures, wettability patterns, a liquid oil layer, and any combinations thereof. 
         [0065]    Optionally, an additional support structure may be provided in combination with first substrate  1110 , particularly when first substrate  1110  is formed of a thin film material. In one example, a rigid support structure  1124  supports the perimeter of first substrate  1110 . For example, rigid support structure  1124  may have an opening in order to accommodate control electrodes  1118  that are on the outer surface of first substrate  1110 , as shown in  FIG. 11 . In one example, support structure  1124  is formed of glass. Optionally, a spacer element  1126  may be provided at the perimeter of droplet actuator  1100  in order to establish the height of gap  1114 , as shown in  FIG. 11 . The spacer element  1126  may serve as a rigid support structure alone or in combination with support structure  1124 . 
         [0066]      FIG. 12  illustrates a side view of a segment of a droplet actuator  1200 . Droplet actuator  1200  may, among other things, provide improved droplet operations and/or ease of manufacture in a droplet actuator. Droplet actuator  1200  is substantially the same as droplet actuator  1100  of  FIG. 11 , except that first substrate  1110 , which is a nonconductive substrate, is replaced with a first substrate  1210 , which is a conductive substrate that has anisotropic conductivity. In one example, first substrate  1210  is formed of Z-axis electrically conductive tape, such as 3M™ Anisotropic Conductive Film from 3M Corporation (St. Paul, Minn.). Z-axis tape is formed of an insulator layer within which is embedded multiple parallel wires that are oriented across the thickness of the insulator layer and placed on a certain pitch according to a desired wire density. Z-axis tape is used, for example, in interconnect systems wherein alignment to metal pads, such as control electrodes  1118 , is not critical. Conductive substrate  1210  may be used alone or in combination with rigid support structure  1124 . 
         [0067]      FIG. 13  illustrates a side view of a segment of a droplet actuator  1300 . Droplet actuator  1300  may, among other things, provide improved droplet operations and/or ease of manufacture and/or assembly in a droplet actuator. Droplet actuator  1300  is substantially the same as droplet actuator  1100  of  FIG. 11  except that droplet actuator  1300  may include further structural support. For example,  FIG. 13  shows the inclusion of a bed-of-nails system  1310  upon which control electrodes  1118  may rest in order to provide electrical contact thereto. The arrangement of first substrate  1110  and second substrate  1112  may be in the form of a cartridge  1312  that is separable from bed-of-nails system  1310 . Additionally, bed-of-nails system  1310  provides rigid support to cartridge  1312 . Cartridge  1312  may include control electrodes  1118  that are permanently disposed upon first substrate  1110 . Alternatively, cartridge  1312  may include first substrate  1110  without control electrodes  1118  disposed thereon. More specifically, control electrodes  1118  can be instead incorporated permanently into bed-of-nails system  1310 . In this example, a cost savings is realized because control electrodes  1118  are not lost upon disposal of cartridge  1312  and because control electrodes  1118  are not processed in the manufacture of each cartridge  1312 . Additionally, in this example, first substrate  1110  may be formed of plastic, which is inexpensive. 
         [0068]      FIG. 14  illustrates a side view of a segment of a droplet actuator  1400 . Droplet actuator  1400  may, among other things, provide improved droplet operations and/or ease of manufacture and/or assembly in a droplet actuator. Droplet actuator  1400  is substantially the same as droplet actuator  1100  of  FIG. 11  except that first substrate  1110 , which is a nonconductive substrate of uniform thickness, is replaced with a first substrate  1410 . First substrate  1410  is designed to accommodate control electrodes  1118  on its outer surface and also to provide a structural support mechanism. More specifically, first substrate  1410  may include one or more protrusions  1412  that are located between control electrodes  1118 , as shown in  FIG. 14 . The one or more protrusions  1412  provide additional structural support over and above a substrate of a thin uniform thickness only. First substrate  1410  that has protrusions  1412  may be formed of, for example, a semiconductor material via, for example, a mask and etch process. Protrusions  1412  may be formed using standard semiconductor processes. Protrusions  1412  may rest upon a planar support structure  1416 , such as a glass substrate. Protrusions  1412  thus form a waffle-like structure that have arrays or patterns of indentations in which electrodes may be configured. 
         [0069]      FIG. 15  illustrates a side view of a segment of a droplet actuator  1500 . Droplet actuator  1500  may, among other things, provide improved droplet operations and/or ease of manufacture in a droplet actuator. In particular, droplet actuator  1500  may be used for dispensing or metering droplets and for conducting other droplet operations. Droplet actuator  1500  may include a pull-back electrode  1510  that is disposed on the outer surface of first substrate  1110  or otherwise associated with first substrate  1110 . Pull-back electrode  1510  may be situated substantially, or in some cases entirely, aligned with a fluid reservoir (not shown). A certain quantity of fluid  1512  may be provided at pull-back electrode  1510 . A pinch-off electrode  1514  and a droplet-forming electrode  1516  are disposed on the inner surface of second substrate  1112 . Pinch-off electrode  1514  and droplet-forming electrode  1516  are used in metering a droplet to be subjected to droplet operations along one or more transport electrodes  1518 , which are disposed on the outer surface of first substrate  1110 . A gap in reference electrode  1120 , which may be formed by, for example, etching, is formed to accommodate pinch-off electrode  1514  and droplet-forming electrode  1516 . Droplet actuator  1500  may include the hydrophobic coating  1116  atop reference electrode  1120 , pinch-off electrode  1514 , and droplet-forming electrode  1516 . 
         [0070]    In operation, pinch-off electrode  1514  and droplet-forming electrode  1516  are activated in order to pull a finger of fluid from fluid  1512  at pull-back electrode  1510  onto droplet-forming electrode  1516 . Fluid  1512  is grounded via reference electrode  1120  that is opposite pull-back electrode  1510 . Once the finger of fluid is formed across pinch-off electrode  1514  and droplet-forming electrode  1516 , which are not in the same plane as pull-back electrode  1510 , pinch-off electrode  1514  is deactivated and a droplet (not shown) remains on droplet-forming electrode  1516 , which is activated. The continued droplet operations of the resulting droplet may be effected using the one or more transport electrodes  1518 , which are not in the same plane as pinch-off electrode  1514  and droplet-forming electrode  1516 . 
         [0071]    Alternatively, a ground electrode may be provided on first substrate  1110 , opposite pinch-off electrode  1514  and droplet-forming electrode  1516 . Alternatively, pull-back electrode  1510 , pinch-off electrode  1514 , droplet-forming electrode  1516 , and transport electrodes  1518  may be arranged in any combination on any plane. 
         [0072]      FIG. 16  illustrates a side view of a segment of a droplet actuator  1600 . Droplet actuator  1600  may, among other things, provide improved droplet operations in a droplet actuator. In particular, droplet actuator  1600  may be used for conducting droplet operations. Droplet actuator  1600  is substantially the same as droplet actuator  1100  of  FIG. 11  except that transport electrodes  1118  are disposed upon the inner surface of first substrate  1110  (i.e., facing gap  1114 ) and are coated with a hydrophobic dielectric layer  1610 . Additionally, second substrate  1112  of  FIG. 11 , which is substantially nonconductive, is replaced with a second substrate  1612 , which is a conductive material, such as, but not limited to, a copper or aluminum foil or plate. Additionally, second substrate  1612  is coated with a hydrophobic dielectric layer  1610 . Alternatively, transport electrodes  1118  may be disposed on the outer surface of first substrate  1110 , as shown in  FIG. 11 . One or more observation openings may be provided in the foil in order to allow observation of a droplet on the droplet actuator and/or sensing of a property of a droplet on a droplet actuator. 
       Droplet Actuator 
       [0073]    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. Nos. 6,773,566, entitled, “Electrostatic Actuators for Microfluidics and Methods for Using Same,” issued on Aug. 10, 2004 and 6,565,727, entitled, “Actuators for Microfluidics Without Moving Parts,” issued on Jan. 24, 2000, both to Shenderov et al.; and 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. 
       Fluids 
       [0074]    For examples of fluids that may be subjected to droplet operations using the approach of the invention, see the patents listed in section 8.5, 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 
       [0075]    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. 
       Method of Providing Improved Single-Layer Microactuator Structures 
       [0076]    Referring to  FIGS. 1 through 10 , one approach for providing improved single metal layer designs for droplet microactuators may include, but is not limited to, the steps of (1) providing mechanisms for improved wireability, such as providing certain electrode configurations with improved wiring accessibility, radial wiring, and bus wiring; (2) creating electrostatic energy gradients as the droplet manipulation mechanism, such as providing an electrode area gradient and/or an electrode voltage gradient; and (3) reducing electrostatic interference from the electrode wires to the droplet, such as by providing electrostatic shielding. 
       Method of Providing a Bi-Planar Droplet Actuator Structure 
       [0077]    Referring to  FIGS. 11 through 16 , one approach for providing a structure for a droplet actuator may include, but is not limited to, the steps of (1) providing a first multilayer plate that is formed, for example, of a first nonconductive substrate having a hydrophobic coating on one surface and an arrangement of conductive transport electrodes on its opposite surface; (2) providing a second multilayer plate that is formed, for example, of a second nonconductive substrate, where a conductive reference electrode is disposed atop the second nonconductive substrate and where a hydrophobic coating is disposed atop the ground electrode; (3) arranging the first and second multilayer plates with a gap therebetween such that the hydrophobic coating and the transport electrodes of the first plate are facing toward and away from the gap, respectively, and such that the hydrophobic coating of the second plate is facing toward the gap; and (4) optionally providing additional structural support mechanisms. 
       Concluding Remarks 
       [0078]    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. 
         [0079]    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. 
         [0080]    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.