Abstract:
The invention provides a method of circulating magnetically responsive beads within a droplet in a droplet actuator. The invention also provides methods for splitting droplets. The invention, in one embodiment, makes use of a droplet actuator with top and bottom substrates, a plurality of magnetic fields respectively present proximate the top and bottom substrates, wherein at least one of the magnet fields is selectively alterable, and a plurality of droplet operations electrodes positioned along at least one of the top and bottom surfaces. A droplet is positioned between the top and bottom surfaces and at least one of the magnetic fields is selectively altered.

Description:
RELATED APPLICATIONS 
       [0001]    This application is a continuation of and claims priority to U.S. patent application Ser. No. 14/746,276, entitled “Manipulation of Beads in Droplets and Methods for Manipulating Droplets,” filed Jun. 22, 2015, the application of which is a continuation of and claims priority to U.S. patent application Ser. No. 14/308,110, entitled “Bead Incubation and Washing on a Droplet Actuator” filed Jun. 18, 2014, now U.S. Pat. No. 9,086,345, issued Jul. 21, 2015, the application of which is a divisional of and claims priority to U.S. patent application Ser. No. 12/761,066, entitled “Manipulation of Beads in Droplets and Methods for Manipulating Droplets,” filed Apr. 15, 2010, now U.S. Pat. No. 8,809,068, issued Aug. 19, 2014, the application of which is A) a continuation of and claims priority to International Patent Application No. PCT/US2008/080264, entitled “Manipulation of Beads in Droplets,” filed Oct. 17, 2008, which claims priority to provisional U.S. Patent Application No. 60/980,782, entitled “Manipulation of Beads in Droplets,” filed on Oct. 17, 2007; and B) a continuation-in-part of and claims priority to U.S. patent application Ser. No. 11/639,531, entitled “Droplet-Based Washing,” filed Dec. 15, 2006, now U.S. Pat. No. 8,613,889, issued Dec. 24, 2013, which claims priority to provisional U.S. Patent Application Nos. 60/745,058, entitled “Filler Fluids for Droplet-Based Microfluidics” filed on Apr. 18, 2006; 60/745,039, entitled “Apparatus and Methods for Droplet-Based Blood Chemistry,” filed on Apr. 18, 2006; 60/745,043, entitled “Apparatus and Methods for Droplet-Based PCR,” filed on Apr. 18, 2006; 60/745,059, entitled “Apparatus and Methods for Droplet-Based Immunoassay,” filed on Apr. 18, 2006; 60/745,914, entitled “Apparatus and Method for Manipulating Droplets with a Predetermined Number of Cells” filed on Apr. 28, 2006; 60/745,950, entitled “Apparatus and Methods of Sample Preparation for a Droplet Microactuator,” filed on Apr. 28, 2006; 60/746,797 entitled “Portable Analyzer Using Droplet-Based Microfluidics,” filed on May 9, 2006; 60/746,801, entitled “Apparatus and Methods for Droplet-Based Immuno-PCR,” filed on May 9, 2006; 60/806,412, entitled “Systems and Methods for Droplet Microactuator Operations,” filed on Jun. 30, 2006; and 60/807,104, entitled “Method and Apparatus for Droplet-Based Nucleic Acid Amplification,” filed on Jul. 12, 2006, the entire disclosures of which are incorporated herein by reference. 
     
    
     GOVERNMENT INTEREST 
       [0002]    This invention was made with government support under DK066956-02 and CA114993-01A2 awarded by the National Institutes of Health. The government has certain rights in the invention. 
     
    
     BACKGROUND 
       [0003]    Droplet actuators are used to conduct a wide variety of droplet operations. A droplet actuator typically includes two substrates separated by a gap. The substrates include electrodes for conducting droplet operations. The space is typically filled with a filler fluid that is immiscible with the fluid that is to be manipulated on the droplet actuator. The formation and movement of droplets is controlled by electrodes for conducting a variety of droplet operations, such as droplet transport and droplet dispensing. There is a need for improvements to droplet actuators that facilitate handling of droplets with beads. 
       SUMMARY OF THE INVENTION 
       [0004]    The invention provides a method of dispersing or circulating magnetically responsive beads within a droplet in a droplet actuator. The invention, in one embodiment, makes use of a droplet actuator with a plurality of droplet operations electrodes configured to transport the droplet, and a magnet field present at a portion of the plurality of droplet operations electrodes. A bead-containing droplet is provided on the droplet actuator in the presence of the uniform magnetic field. Beads are circulated in the droplet during incubation by conducting droplet operations on the droplet within a uniform region of the magnetic field. Other aspects of the invention will be apparent from the ensuing description of the invention. 
       DEFINITIONS 
       [0005]    As used herein, the following terms have the meanings indicated. 
         [0006]    “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. 
         [0007]    “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 fluids may include one or more magnetically responsive and/or non-magnetically responsive beads. Examples of droplet actuator techniques for immobilizing magnetically responsive beads and/or non-magnetically responsive beads and/or conducting droplet operations protocols using beads are described in U.S. patent application Ser. No. 11/639,566, entitled “Droplet-Based Particle Sorting,” filed on Dec. 15, 2006; U.S. Patent Application No. 61/039,183, entitled “Multiplexing Bead Detection in a Single Droplet,” filed on Mar. 25, 2008; U.S. Patent Application No. 61/047,789, entitled “Droplet Actuator Devices and Droplet Operations Using Beads,” filed on Apr. 25, 2008; U.S. Patent Application No. 61/086,183, entitled “Droplet Actuator Devices and Methods for Manipulating Beads,” filed on Aug. 5, 2008; International Patent Application No. PCT/US2008/053545, entitled “Droplet Actuator Devices and Methods Employing Magnetically responsive beads,” filed on Feb. 11, 2008; International Patent Application No. PCT/US2008/058018, entitled “Bead-based Multiplexed Analytical Methods and Instrumentation,” filed on Mar. 24, 2008; International Patent Application No. PCT/US2008/058047, “Bead Sorting on a Droplet Actuator,” filed on Mar. 23, 2008; and International Patent Application No. PCT/US2006/047486, entitled “Droplet-based Biochemistry,” filed on Dec. 11, 2006; the entire disclosures of which are incorporated herein by reference. 
         [0008]    “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, for example, be aqueous or non-aqueous or may be mixtures or emulsions including aqueous and non-aqueous components. 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. 
         [0009]    “Droplet Actuator” means a device for manipulating droplets. For examples of droplets, 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/US2006/047486, entitled “Droplet-Based Biochemistry,” filed on Dec. 11, 2006, the disclosures of which are incorporated herein by reference. Methods of the invention may be executed using droplet actuator systems, e.g., as described in International Patent Application No. PCT/US2007/009379, entitled “Droplet manipulation systems,” filed on May 9, 2007. In various embodiments, the manipulation of droplets by a droplet actuator may be electrode mediated, e.g., electrowetting mediated or dielectrophoresis mediated. 
         [0010]    “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; condensing a droplet from a vapor; 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. In various embodiments, the droplet operations may be electrode mediated, e.g., electrowetting mediated or dielectrophoresis mediated. 
         [0011]    “Filler fluid” means a fluid associated with a droplet operations substrate of a droplet actuator, which fluid is sufficiently immiscible with a droplet phase to render the droplet phase subject to electrode-mediated droplet operations. 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/US2006/047486, entitled, “Droplet-Based Biochemistry,” filed on Dec. 11, 2006; and in International Patent Application No. PCT/US2008/072604, entitled “Use of additives for enhancing droplet actuation,” filed on Aug. 8, 2008. 
         [0012]    “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, substantially 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. 
         [0013]    “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. 
         [0014]    “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. 
         [0015]    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. 
         [0016]    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. 
         [0017]    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 
         [0018]      FIG. 1  illustrates a top view of a portion of a droplet actuator useful for incubating a droplet including magnetically responsive beads; 
           [0019]      FIG. 2  illustrates a top view of a portion of a droplet actuator useful for incubation of antibodies, wherein a sample and magnetically responsive beads are provided within the magnet field of a magnet; 
           [0020]      FIG. 3  illustrates a top view of a portion of a droplet actuator useful for incubation of magnetically responsive beads within a droplet, wherein a sample and magnetically responsive beads are subjected to droplet operations within the magnet field of a magnet; 
           [0021]      FIG. 4  illustrates a method of shielding the effect of multiple magnets in a droplet actuator by using a magnetic shielding material; 
           [0022]      FIG. 5  illustrates a magnet array for performing multiple immunoassays; 
           [0023]      FIG. 6  illustrates simulation results that show the surface field of 2 columns of the magnet array of  FIG. 5 ; 
           [0024]      FIG. 7  illustrates a top view of a portion of a droplet actuator useful for resuspension of beads (e.g., magnetically-responsive beads) within a reservoir configured with multiple electrodes; 
           [0025]      FIG. 8  illustrates a shows a top view of a portion of a droplet actuator useful for resuspending beads (e.g., magnetically-responsive beads) within a reservoir by pushing out a finger of liquid and then merging back; 
           [0026]      FIG. 9  illustrates a top view of a portion of the droplet actuator of  FIG. 8  including a reservoir in which beads are resuspended by applying high frequency voltage to the reservoir electrode; 
           [0027]      FIG. 10  illustrates a side view of a droplet actuator that includes a top substrate and bottom substrate separated by a gap; 
           [0028]      FIG. 11  illustrates a side view of another embodiment of a droplet actuator including a top substrate and bottom substrate separated by a gap; 
           [0029]      FIG. 12  illustrates a side view of yet another embodiment of a droplet actuator that includes a top substrate and bottom substrate separated by a gap; 
           [0030]      FIG. 13  illustrates a top view of a portion of a droplet actuator useful for a process of asymmetrically splitting a droplet; 
           [0031]      FIG. 14  illustrates a top view of a portion of a droplet actuator useful for a process employing a hydrophilic patch in a droplet splitting operation; 
           [0032]      FIG. 15  illustrates a top view of a portion of a droplet actuator useful for a process of using a magnetic strip that is integrated into the gasket material at the point of bead immobilization; 
           [0033]      FIG. 16  illustrates a side view of a droplet actuator that includes a top substrate and bottom substrate that are separated by a gap useful for facilitating consistent droplet splitting by use of a physical barrier in the droplet actuator; 
           [0034]      FIG. 17  illustrates a side view of the portion of droplet actuator in  FIG. 16  useful for facilitating consistent droplet splitting by use of a magnetic physical barrier in the droplet actuator; 
           [0035]      FIG. 18  illustrates embodiments of electrode configuration for improved droplet splitting; and 
           [0036]      FIG. 19  illustrates detection strategies for quantifying an analyte. 
       
    
    
     DESCRIPTION 
       [0037]    The invention provides droplet actuators having specialized configurations for manipulation of droplets including beads and/or for manipulation of beads in droplets. In certain embodiments, the droplet actuators of the invention include magnets and/or physical barriers manipulation of droplets including beads and/or for manipulation of beads in droplets. The invention also includes methods of manipulating of droplets including beads and/or for manipulation of beads in droplets, as well as methods of making and using the droplet actuators of the invention. The droplet actuators of the invention are useful for, among other things, conducting assays for qualitatively and/or quantitatively analyzing one or more components of a droplet. Examples of such assays include affinity based assays, such as immunoassays; enzymatic assays; and nucleic acid assays. Other aspects of the invention will be apparent from the ensuing discussion. 
       7.1 Incubation of Beads 
       [0038]    In certain embodiments, the invention provides droplet actuators and methods for incubating beads. For example, a sample including bead-containing antibodies may be incubated on the droplet actuator in order to permit one or more target components to bind to the antibodies. Examples of target components include analytes; contaminants; cells, such as bacteria and protozoa; tissues; and organisms, such as multicellular parasites. In the presence of a magnet, magnetic beads in the droplet may be substantially immobilized and may fail to circulate throughout the droplet. The invention provides various droplet manipulations during incubation of droplets on a droplet actuator in order to increase circulation of beads within the droplet and/or circulation of droplet contents surrounding beads. It will be appreciated that in the various embodiments described below employing magnetically responsive beads, beads that are not substantially magnetically responsive may also be included in the droplets. 
         [0039]      FIG. 1  illustrates techniques that are useful process of incubating a droplet including magnetically responsive beads. Among other things, the techniques are useful for enhancing circulation of fluids and beads within the droplet during an incubation step. 
         [0040]    In  FIG. 1 , each step is illustrated on a path of electrodes  110 . A magnet  112  is associated with a subset of electrodes  110 . Magnet  112  is arranged relative to the electrodes  110  such that a subset of electrodes  110  are within a uniform region of the magnetic field produced by magnet  112 . Bead clumping is reduced when the droplet is present in this uniform region. 
         [0041]    In Step  1 , droplet  116  is located atop magnet  112 . Beads  116  are substantially immobilized in a distributed fashion adjacent to the droplet operations surface. The beads are generally less clumped than they would be in the presence of a non-uniform region of the magnetic field. In Step  2  droplet  114  is split using droplet operations into two sub-droplets  114 A,  114 B. During the splitting operation beads and liquid are circulated within the droplets  114 ,  114 A and  114 B. In Step  3  Droplets  114 A and  114 B are merged using droplet operations into a single droplet  114 . This merging operation is accomplished within the uniform region of the magnetic field. During the merging operation beads and liquid are further circulated within the droplets  114 ,  114 A and  114 B. 
         [0042]    In Step  4 , droplet  114  is transported using droplet operations along electrodes  110  away from the magnet  112 . As droplet  116  moves away from magnet  110 , beads  116  are pulled to the edge of droplet  114  that nearest the magnet  112 . Movement of beads  116  within droplet  114  provides further beneficial circulation of beads and liquid within the droplet  114 . In Step  5 , droplet  116  is transported using droplet operations back to the step  1  position. Beads  116  within the droplet  116  are again dispersed in the presence of the uniform magnetic field of magnet  112 . This redistribution of beads, as droplet  114  returns to its position within the uniform region of the magnetic field provides further beneficial circulation of beads and liquid within the droplet  114 . 
         [0043]    These steps may be conducted in any logical order. Each step may be conducted any number of times between the other steps. For example, Steps  1 - 3  may be repeated multiple times before moving onto Step  4 . Similarly, Steps  3 - 5  may be repeated multiple times before returning to Steps  1 - 3 . Moreover, all steps are not required. For example, in one embodiment, an incubation step in an assay is accomplished by repeating Steps  1 - 3 . In another embodiment, an incubation step in an assay is accomplished by repeating Steps  3 - 5 . 
         [0044]    The incubation method of the invention is useful for enhancing circulation of magnetically responsive beads with the liquid in a droplet while the droplet remains in the presence of a magnetic field. Among other advantages, the approach may reduce bead clumping and permit tighter droplet actuator designs making more efficient use of droplet actuator real estate. 
         [0045]    In one embodiment, the invention provides a droplet operations incubation scheme, that does not allow magnetically responsive beads to be introduced into a region of the magnetic field which is sufficiently non-uniform to cause bead clumping. In another embodiment, the invention provides a merge-and-split incubation scheme, that does not allow magnetically responsive beads to be introduced into a region of the magnetic field which is sufficiently non-uniform to cause bead clumping. In yet another embodiment, the invention provides a droplet transport incubation scheme, that does not allow magnetically responsive beads to be introduced into a region of the magnetic field which is sufficiently non-uniform to cause bead clumping. 
         [0046]    Any combination of droplet operations which result in effective mixing (e.g., substantially complete mixing) may be chosen. Mixing is complete when it is sufficient for conducting the analysis being undertaken. The droplet may be oscillated in the presence of the uniform region of the magnetic field by transporting the droplet back and forth within the uniform region. In some cases, electrode sizes used for the oscillation may be varied to increase circulation within the droplet. In some cases, droplet operations electrodes are used to effect droplet operations to transport a droplet back and forth or in one or more looping patterns. Preferably the oscillation pattern does not allow to be introduced into a region of the magnetic field which is sufficiently uniform to cause bead clumping. 
         [0047]    In some cases, droplet operations are performed at an edge of the magnet to more equally redistribute the magnetically responsive beads. In some cases, droplet operations are performed performed away from the magnet, followed by transporting the droplet. 
         [0048]      FIG. 2  illustrates another process of incubation of antibodies, wherein a sample and magnetically responsive beads are provided within the magnet field of a magnet, e.g., within a uniform magnetic field region of a magnet.  FIG. 2  shows a top view of a portion of droplet actuator  100  that is described in  FIG. 1 . 
         [0049]    In Step  1 , beads  116  are substantially immobilized along the surface of the droplet operations electrodes  110  due to the magnetic field of the magnet  112 . I Step  2 , droplet  114  is split using droplet operations into two droplets  118 , both remaining in the uniform region of the magnetic field. In step  4 , the two droplets  118  are transported away from the magnet  112 , thereby attracting the beads  116  to the edge of the two droplets  118  nearest the magnet  112 . This operation causes flow reversal within the droplets  118 , which enhances effective mixing. The two droplets  118  may alternatively be transported away from the magnet in different directions, such as in opposite directions. In Step  4  the two droplets  118  are merged into one droplet  116 . In step  5 , the droplet  116  is transported back to the step  1  position, causing the beads  116  to disperse within the droplet  116 . 
         [0050]      FIG. 3  illustrates another process of incubation of magnetically responsive beads within a droplet, wherein a sample and magnetically responsive beads are subjected to droplet operations within the magnet field of a magnet.  FIG. 3  shows a top view of a portion of a droplet actuator  300  that includes a set of droplet operations electrodes  310  (e.g., electrowetting electrodes) that is arranged in sufficient proximity to a magnet, such that a droplet  314  moving along the droplet operations electrodes  310  is within the magnet field of the magnet, e.g., a region of uniform magnetic field. For example, the set of droplet operations electrodes  310  are arranged in a closed loop and in the presence of two magnets, such as a magnet  312 A and magnet  312 B, as shown in  FIG. 3 . In this embodiment, the droplet  314  may include sample and beads  316 , and some or all of the beads may be magnetically responsive. 
         [0051]    In Step  1 , sample with beads  316  in the droplet  314  is provided on droplet actuator. Beads  316  are substantially immobilized along the surface of the droplet operations electrodes  310  due to the magnetic field of the first magnet  312 A that is located at “lane A” of the electrode loop. In Step  2 , the droplet  314  is split using droplet operations into two droplets  318 , distributing the beads  316  in the two droplets  318  at “lane A” of the electrode loop. In Step  3 , the two droplets  318  are transported using droplet operations in opposite directions away from the first magnet  312 A at “lane A” and toward the second magnet  312 B that is located at “lane B” of the electrode loop. In Step  4 , in the presence of the second magnet  312 B at “lane B,” droplets  318  are merged into one droplet  320 . 
         [0052]    In Steps  5 - 6 , not shown, the process of steps  1 - 3  may be essentially repeated in reverse. In step  5 , droplet  320  may be split into two droplets  318 , distributing the beads  316  in the two droplets  318  at “lane B.” In Step  6 , droplets  318  are transported in opposite directions away from the second magnet  312 B at “lane B” and back to the first magnet  312 A at “lane A.” In Step  7 , in the presence of the first magnet  312 A at “lane A,” droplets  318  are merged into one droplet  320 . 
         [0053]    The droplet split and merge operation as described above provide efficient dispersion of beads in the presence of a magnet, thereby improving the efficiency of the binding of antibodies and the analyte. The various droplet operations may be conducted in primarily or completely in uniform regions of the magnetic fields generated by magnets  312 A,  312 B. Alternatively, the droplet split and merge operation as described above may be performed away from the magnet and/or near the edge of the magnet. 
       7.2 Magnet Configurations 
       [0054]      FIG. 4  illustrates a method of shielding the effect of multiple magnets in a droplet actuator  400  by using a magnetic shielding material, preferably one that has high magnetic permeability. One example of such material is Mu-metal foil. Mu-metal is a nickel-iron alloy (e.g., 75% nickel, 15% iron, plus copper and molybdenum) that has very high magnetic permeability.  FIG. 4  shows a top view of multiple washing lanes  410 , wherein each washing lane  410  includes a string of droplet operations electrodes  412  in the presence of a magnet  414 . An electrode array  416  (e.g., an array of electrowetting electrodes) for performing droplet operations feed the multiple washing lanes  410 . Additionally, the droplets  418  that are transported may include magnetically responsive beads (not shown). Furthermore, this embodiment provides a magnetic shield  420 , provided as a layer that is beneath the electrode array  416 . 
         [0055]    Because of the presence of multiple magnets  414 , which are used to immobilize magnetically responsive beads during washing, the magnetically responsive beads in the reservoir tend to become aggregated, sometimes irreversibly. When bead-containing droplets are dispensed using droplet operations, bead aggregation may cause the number of beads that are present in each dispensed droplet to vary. Variation in bead dispensing may affect the assay result, which is not desirable. The invention, as shown in  FIG. 4 , provides magnetic shield  420  in the area under the electrode array  416  of the droplet actuator  400 . The magnetic shield  420  may be formed of alloys, such as Mu-metal foil, which shields the magnetically responsive beads within the electrode array  416  from stray magnetic fields  422 . 
         [0056]      FIG. 5  illustrates a magnet array  500  for performing multiple immunoassays that has reduced, preferably substantially no, interference due to adjacent magnets within a droplet actuator (not shown) having a substrate associated with droplet operations electrodes. The electrodes are arranged for conducting one or more droplet operations on a droplet operations surface of the substrate. Magnets, such as the magnet array  500  shown in  FIG. 5 , may be arranged with respect to the droplet actuator such that one or more magnets cancels out some portion of a magnetic field of one or more other magnets. In this manner, an area of the surface may have some portions that are subject to magnetic fields and some portions in which the magnetic fields have been cancelled out. For example, magnets may be arranged to cancel the field in areas of the droplet actuator that includes liquid along with magnetically responsive beads. Specifically reservoirs, incubation regions, detection regions are preferably in regions in which the magnetic fields have been cancelled out. 
         [0057]    In one embodiment, the arrangement involves an array of alternately placed magnets, e.g., as shown in  FIG. 5 . In general, magnets may be located in any position which supplies a magnetic field to the vicinity of the droplet operations surface where the magnetic field is desired and eliminates or weakens the magnetic field in other areas where the magnetic field is not desired. In one embodiment, a first magnet produces a first magnetic field where it is desirable to immobilize magnetically responsive beads in a droplet, while a second magnet produces a second magnetic field which cancels or weakens a portion of the first magnetic field. This arrangement produces a device in which a portion of the droplet operations surface that would have otherwise been influenced by the first magnetic field is subjected to a weak or absent field because the first magnetic field has been cancelled or weakened by the second magnetic field. 
         [0058]    In one embodiment, one or more of the magnets is fixed in relation to the droplet operations surface, and the invention comprises conducting one or more droplet operations using droplets that contain magnetically responsive beads, where the droplets are in proximity to one or more magnets and are in the presence or absence of a magnetic field. 
         [0059]    In another embodiment, the magnetic field exerts sufficient influence over magnetically responsive beads that the droplets may be substantially immobile during one droplet operation, such as a splitting operation, and yet not so stable that the droplets are restrained from being transported away from the magnetic field with the magnet. In this embodiment, the droplet may be surrounded by a filler fluid, and yet the droplet with the magnetically responsive beads may be transported away from the magnetic with substantially no loss of magnetically responsive beads to the filler fluid. 
         [0060]      FIG. 6  illustrates simulation results  600  that show the surface field of 2 columns of magnet array  500  of  FIG. 5 . 
         [0000]    7.3 Resuspension of Beads within a Reservoir 
         [0061]      FIG. 7  illustrates a process of resuspension of beads (e.g., magnetically-responsive beads) within a reservoir configured with multiple electrodes within the.  FIG. 7  shows a top view of a portion of a droplet actuator  700  that includes a reservoir  710  that is formed of multiple electrodes (e.g., electrodes  1  through  9  in a 3×3 array), whereby the reservoir  710  feeds a line of droplet operations electrodes  712  (e.g., electrowetting electrodes) to which droplets that contain beads may be dispensed. 
         [0062]    Referring to  FIG. 7 , a process of resuspension of beads within a reservoir by having multiple electrodes within the same reservoir may include, but is not limited to, the following steps. In Step  1 , beads  714  are aggregated within the solution  716  due to the presence of multiple magnets (not shown). In Step  2 , electrodes within the reservoir  710  are used to subject the solution  716  to droplet operations, thereby resuspension of the beads  714 . The electrode activation sequence may be randomized to create more chaotic flow fields for more efficient resuspension. The liquid may be split and merged and subjected to other droplet operations. 
         [0063]    During the above-described process, the electrode activation sequence may be chosen such that the beads are mixed well by means of droplet operations. Additionally, when dispensing (e.g., pulling out a finger of fluid) a bead droplet from the electrode array of the reservoir, all the electrodes within the reservoir may be switched ON and OFF at the same time, depending on the requirement. It should be noted that an almost infinite variety of electrode shapes is possible. Any shape which is capable of facilitating a droplet operation will suffice. 
         [0064]    The resuspension process may be repeated between every 1, 2, 3, 4, 5 or more droplet dispensing operations. The resuspend-and-dispense pattern may be adjusted as required based on the specific characteristics of bead types and droplet compositions. For example, in one embodiment, the process of the invention results in dispensing bead-containing droplets with greater than 95% consistency in bead count. In another embodiment, the process of the invention results in dispensing bead-containing droplets with greater than 99% consistency in bead count. In another embodiment, the process of the invention results in dispensing bead-containing droplets with greater than 99.9% consistency in bead count. In another embodiment, the process of the invention results in dispensing bead-containing droplets with greater than 99.99% consistency in bead count. 
         [0065]      FIG. 8  illustrates a process of resuspending beads (e.g., magnetically-responsive beads) within a reservoir by pushing out a finger of liquid and then merging back.  FIG. 8  shows a top view of a portion of a droplet actuator  800  that includes a reservoir  810  that feeds a line of droplet operations electrodes  812  (e.g., electrowetting electrodes) to which droplets that contain beads may be dispensed. Additionally, the reservoir includes a solution  814  that includes beads  816 . Referring to  FIG. 8 , a process of resuspension of beads within a reservoir by pushing out a finger of liquid and then merging back may include, but is not limited to, the following steps. 
         [0066]    In Step  1 , beads  816  are aggregated within the solution  814  due to the presence of multiple magnets (not shown). In Step  2 , a finger of solution  814  that includes beads  816  is pulled out of the reservoir  810  using droplet operations. In Step  3 , a 2X slug  818  is dispensed by splitting the middle of the finger of solution  814 . In Step  4 , the 2X slug  818  is merged back with the solution  814  that includes magnetically responsive beads  816  within the reservoir  810 . 
         [0067]    Steps  2  through  4  may be repeated until the desired degree of resuspension is achieved, e.g., until substantially completely resuspended beads are obtained within the bead solution of the reservoir  810 . When the desired degree of resuspension is achieved, bead-containing droplets may be dispensed, achieving a target percentage of variation in each droplet. 
         [0068]    The resuspension process may be repeated, between every 1, 2, 3, 4, 5 or more droplet dispensing operations. The resuspend-and-dispense pattern may be adjusted as required based on the specific characteristics of bead types and droplet compositions. For example, in one embodiment, the process of the invention results in dispensing bead-containing droplets with greater than 95% consistency in bead count. In another embodiment, the process of the invention results in dispensing bead-containing droplets with greater than 99% consistency in bead count. In another embodiment, the process of the invention results in dispensing bead-containing droplets with greater than 99.9% consistency in bead count. In another embodiment, the process of the invention results in dispensing bead-containing droplets with greater than 99.99% consistency in bead count. 
         [0069]      FIG. 9  illustrates a reservoir in which beads are resuspended by applying high frequency voltage to the reservoir electrode. The figure shows a top view of a portion of droplet actuator  800  of  FIG. 8 . Reservoir  810  includes a droplet  814  that includes magnetically responsive beads  816 . Beads  816  in a reservoir  810  may tend to become aggregated due to, for example, the presence of nearby magnets (not shown). Aggregation may adversely affect bead count in dispensed beads, adversely impacting reliability of assay results for assays conducted using the dispensed bead-containing droplets. Beads  816  may be resuspended within the magnetically responsive bead solution within the reservoir  810  by applying a high frequency AC voltage to the reservoir electrode  810 , in accordance with the invention. Because of the high frequency AC voltage, the magnetically responsive beads  816  tend to oscillate because of the wetting and dewetting of the contact line of the droplet. This oscillation at the periphery disperses the magnetically responsive beads  816  and resuspends them in the supernatant. In one example, the high frequency AC voltage may be in the range from about 100 volts to about 300 volts with a frequency from about 10 Hz to about 1000 Hz. 
         [0070]    The resuspension process may be repeated between every 1, 2, 3, 4, 5 or more droplet dispensing operations. The resuspend-and-dispense pattern may be adjusted as required based on the specific characteristics of bead types and droplet compositions. For example, in one embodiment, the process of the invention results in dispensing bead-containing droplets with greater that 95% consistency in bead count. In another embodiment, the process of the invention results in dispensing bead-containing droplets with greater than 99% consistency in bead count. In another embodiment, the process of the invention results in dispensing bead-containing droplets with greater than 90.9% consistency in bead count. In another embodiment, the process of the invention results in dispensing bead-containing droplets with greater than 90.99% consistency in bead count. 
         [0000]    7.4 Improving Dispersion of Magnetically Responsive beads by Magnet Configurations 
         [0071]      FIG. 10  illustrates a side view of a droplet actuator  1000  that includes a top substrate  1010  and bottom substrate  1012  that are separated by a gap. A set of droplet operations electrodes  1014  (e.g., electrowetting electrodes) is provided on the bottom substrate  1012 . Additionally, a first electromagnet  1016  is arranged near the top substrate  1010  and a second electromagnet  1018  is arranged near the bottom substrate  1012 . The proximity of the electromagnets  1016  and  1018  to the droplet actuator  1000  is sufficiently close that the gap is within the magnetic fields thereof. A droplet  1020  that includes magnetically responsive beads  1022  is in the gap and may be manipulated along the droplet operations electrodes  1014 . 
         [0072]    Electromagnets  1016  and  1018  may be used to improve dispersion of magnetically responsive beads  1022 . Improved dispersion may, for example, to improve binding efficiency of antibodies and analytes to the surface of the beads. By providing an electromagnet on the top and bottom of the droplet  1020 , the magnetically responsive beads  1022  may be effectively dispersed within the droplet  1020  by switching ON and OFF the magnetic fields of electromagnets  1016  and  1018 . In one example,  FIG. 10A  shows the electromagnet  1016  turned ON and the electromagnet  1018  turned OFF, which causes the beads  1022  to be attracted to the electromagnet  1016  and are, therefore, pulled to the electromagnet  1016  side of the droplet  1020 . Subsequently, electromagnet  1018  is turned ON and electromagnet  1016  is turned OFF, which causes the beads  1022  to be attracted to electromagnet  1018  and are, therefore, pulled to the electromagnet  1018  side of the droplet  1020 . Alternating the activation of electromagnets  1016  and  1018  may be repeated until resuspension of the beads  1022  is substantially achieved.  FIG. 10B  shows both electromagnets  1016  and  1018  turned ON at the same time, which causes a pillar of beads  1022  to form through droplet  1020 . Various changes in the configuration of magnet activation (ON/ON, ON/OFF, OFF/ON, and OFF/OFF) may be used to circulate magnetically responsive beads  1022  within droplet  1020 . In some embodiments, the pattern of magnet activation may be randomized. Examples include ON/OFF, OFF/ON, ON/OFF, OFF/ON, ON/OFF, etc.; ON/ON, ON/OFF, OFF/ON, ON/ON, ON/OFF, OFF/ON, ON/ON, ON/OFF, OFF/ON, etc; ON/ON, ON/OFF, OFF/ON, OFF/OFF, ON/ON, ON/OFF, OFF/ON, OFF/OFF, ON/ON, ON/OFF, OFF/ON, OFF/OFF, etc.; ON/OFF, OFF/OFF, OFF/ON, OFF/OFF, ON/OFF, OFF/OFF, OFF/ON, OFF/OFF, ON/OFF, OFF/OFF, OFF/ON, OFF/OFF, etc. Various other magnet activation patterns will be apparent to one of skill in the art in light of the present specification. 
         [0073]      FIG. 11  illustrates a side view of a droplet actuator  1100  including a top substrate  1110  and bottom substrate  1112  that are separated by a gap. A set of droplet operations electrodes  1114  (e.g., electrowetting electrodes) is provided on the bottom substrate  1112 . Additionally, multiple magnets  1116  are arranged near the top substrate  1110  and multiple magnets  1116  are arranged near the bottom substrate  1112 . In one example, magnets  1116 - 1 ,  1116 - 3 , and  1116 - 5  are arranged near the top substrate  1110  and magnets  1116 - 2 ,  1116 - 4 , and  1116 - 6  are arranged near the bottom substrate  1112 . The proximity of the magnets  1116  to the droplet actuator  1100  is sufficiently close that the gap is within the magnetic fields thereof A slug of liquid  1118  (e.g., antibodies sample mixture) that includes magnetically responsive beads  1120  is in the gap along the droplet operations electrodes  1114 . This aspect of the invention may improve the binding of analytes or other target substances, such as cells, with antibodies that are present on the beads  1120 . 
         [0074]    Referring to  FIG. 11 , a process of providing improved dispersion of magnetically responsive beads by use of a magnet arrangement, such as shown in  FIG. 11 , may include, but is not limited to, the following steps. 
         [0075]    Step  1 : Magnet  1116 - 1 =OFF, magnet  1116 - 2 =ON, magnet  1116 - 3 =OFF, magnet  1116 - 4 =OFF, magnet  1116 - 5 =OFF, and magnet  1116 - 6 =OFF, which causes the magnetically responsive beads  1120  to be attracted toward magnet  1116 - 2 . 
         [0076]    Step  2 : Magnet  1116 - 1 =OFF, magnet  1116 - 2 =OFF, magnet  1116 - 3 =ON, magnet  1116 - 4 =OFF, magnet  1116 - 5 =OFF, and magnet  1116 - 6 =OFF, which causes the magnetically responsive beads  1120  to be attracted toward magnet  1116 - 3 . 
         [0077]    Step  3  (not shown): Magnet  1116 - 1 =OFF, magnet  1116 - 2 =OFF, magnet  1116 - 3 =OFF, magnet  1116 - 4 =OFF, magnet  1116 - 5 =OFF, and magnet  1116 - 6 =ON, which causes the magnetically responsive beads  1120  to be attracted toward magnet  1116 - 6 . 
         [0078]    Step  4  (not shown): Magnet  1116 - 1 =OFF, magnet  1116 - 2 =OFF, magnet  1116 - 3 =OFF, magnet  1116 - 4 =OFF, magnet  1116 - 5 =ON, and magnet  1116 - 6 =OFF, which causes the magnetically responsive beads  1120  to be attracted toward magnet  1116 - 5 . 
         [0079]    Step  5  (not shown): Magnet  1116 - 1 =OFF, magnet  1116 - 2 =OFF, magnet  1116 - 3 =OFF, magnet  1116 - 4 =ON, magnet  1116 - 5 =OFF, and magnet  1116 - 6 =OFF, which causes the magnetically responsive beads  1120  to be attracted toward magnet  1116 - 4 . 
         [0080]    Step  6  (not shown): Magnet  1116 - 1 =ON, magnet  2 =OFF, magnet  3 =OFF, magnet  4 =OFF, magnet  5 =OFF, and magnet  6 =OFF, which causes the magnetically responsive beads to be attracted toward magnet  1 . 
         [0081]    Steps  1  through  6  may be repeated until a desired degree of dispersion or circulation of magnetically responsive beads  1120  and liquid is achieved. 
         [0082]      FIG. 12  illustrates a side view of a droplet actuator  1200  that includes a top substrate  1210  and bottom substrate  1212  that are separated by a gap. A set of droplet operations electrodes  1214  (e.g., electrowetting electrodes) is provided on the bottom substrate  1212 . A droplet  1216  that includes magnetically responsive beads  1218  is provided in the gap and may be manipulated along the droplet operations electrodes  1214 . Additionally, a first magnet  1220 A is arranged near the top substrate  1210  and a second magnet  1220 B is arranged near the bottom substrate  1212 . The proximity of the magnets  1220 A and  1220 B to the droplet actuator  1200  is sufficiently close that the gap is within the magnetic fields thereof. However, the distance of the magnets  1220 A and  1220 B from the droplet actuator  1200  may be adjusted by, for example, a mechanical means, thereby adjusting the influence of the magnetic fields upon the magnetically responsive beads  1218 . 
         [0083]    Mechanical movement of the magnets  1220 A and  1220 B disperses or otherwise circulates magnetically responsive beads and liquids within the droplet. In one example,  FIG. 12A  shows both magnets  1220 A and  1220 B in close proximity to the droplet actuator  1200 , which causes a pillar of beads  1218  to form through the droplet  1216 . In another example,  FIG. 12B  shows the magnet  1220 A only may be moved mechanically by a distance “x” where substantially no magnetic field of magnet  1220 A reaches the magnetically responsive beads  1218  and, thus, the beads  1218  are attracted toward the magnet  1220 B, thereby dispersing the beads  1218 . In like manner, the magnet  1220 B only may be moved mechanically by a distance “x” where substantially no magnetic field of magnet  1220 B reaches the magnetically responsive beads  1218  and, thus, the beads are attracted toward the first magnet  1220 A, thereby dispersing the beads  1218 . By, for example, alternating the mechanical movement of the magnets, effective dispersion of magnetically responsive beads  1218  is substantially ensured. In some embodiments, both magnets are moved. Magnets may be oscillated to rapidly circulate beads and liquids within the droplet. 
       7.5 Improved Droplet Splitting by Magnet Configurations 
       [0084]      FIG. 13  illustrates a process of asymmetrically splitting a droplet.  FIG. 13  shows a top view of a portion of a droplet actuator  1300  that includes a set of droplet operations electrodes  1310  (e.g., electrowetting electrodes) that is arranged in sufficient proximity to a magnet  1312 , such that a droplet  1314  moving along the droplet operations electrodes  1310  is within the magnet field of the magnet  1312 , e.g., a region of uniform magnetic field. In this embodiment, the droplet  1314  may be may include sample and beads  1316 , and some or all of the beads  1316  may be magnetically responsive. 
         [0085]    The process may include, without limitation, the following steps. In step  1 , after immobilizing the magnetically responsive beads  1316  to a localized area in the presence of magnet  1312 , droplet operations electrodes  1310  are activated to extend droplet  1314  into a 4x-slug of liquid that extents beyond the boundary of magnet  1312 . In Step  2 , droplet operations electrode  1310  is deactivated, and the next two droplet operations electrodes  1310  remain on, and a third droplet operations electrode is activated to provide the asymmetric split. The process may, for example, be employed in a merge-and-split bead washing protocol. 
         [0086]      FIG. 14  illustrates a process employing a hydrophilic patch in a droplet splitting operation.  FIG. 14  shows a top view of a portion of a droplet actuator  1400  that includes a set of droplet operations electrodes  1410  (e.g., electrowetting electrodes) arranged in sufficient proximity to a magnet  1412 , such that a droplet moving along the droplet operations electrodes  1410  is within the magnet field of the magnet  1412 , e.g., a region of uniform magnetic field. In this embodiment, the droplet may be may include sample and beads  1414 , and some or all of the beads may be magnetically responsive. 
         [0087]    The process may include, without limitation, the following steps. In Step  1 , a small hydrophilic patch  1416 , which is patterned on the top substrate (not shown) and opposite a certain droplet operations electrode  1410 , immobilizes the aqueous slug  1418 , and the magnet  1412  immobilizes the magnetically responsive beads  1414 . In Step  2 , a droplet splitting operation is executed (e.g., forming droplets  1420  and  1422 ). The hydrophilic patch  1416  ensures droplet splitting at the same point in relation to the droplet operations electrode  1410  that is downstream of the hydrophilic patch  1416 . In this example, the magnetically responsive beads  1414  remain substantially immobilized in droplet  1422  by the magnet  1412  and droplet  1522  is substantially free of beads  1420 . The process may, for example, be employed in a merge-and-split bead washing protocol. 
         [0088]      FIG. 15  illustrates a process of using a magnetic strip that is integrated into the gasket material at the point of bead immobilization.  FIG. 15  shows a top view of a portion of a droplet actuator  1500  that includes a set of droplet operations electrodes  1510  (e.g., electrowetting electrodes) that is arranged in sufficient proximity to a magnetic strip  1512  that is integrated into the gasket material  1514  of the droplet actuator  1500 , such that a droplet moving along the droplet operations electrodes  1510  is within the magnet field of the magnetic strip  1512 , e.g., a region of uniform magnetic field. In this embodiment, the droplet may be may include sample and beads  1516 , and some or all of the beads may be magnetically responsive. 
         [0089]    The process may include, but is not limited to, the following steps. In Step  1 , magnetic strip  1512  immobilizes the magnetically responsive beads  1516  in an aqueous slug  1518 . In Step  2 , a droplet splitting operation occurs (e.g., forming droplets  1520  and  1522 ), whereby the magnetically responsive beads  1516  remain substantially immobilized in droplet  1520  by the magnetic strip  1512  and droplet  1522  is substantially free of beads  1516 . The process may, for example, be employed in a merge-and-split bead washing protocol. 
       7.6 Improved Droplet Splitting by Physical Barrier 
       [0090]      FIG. 16  illustrates a process of facilitating consistent droplet splitting by use of a physical barrier in the droplet actuator.  FIG. 16  shows a side view of a droplet actuator  1600  that includes a top substrate  1610  and bottom substrate  1612  that are separated by a gap. A set of droplet operations electrodes  1614  (e.g., electrowetting electrodes) is provided on the bottom substrate  1612 . Additionally, a magnet  1616  is arranged in sufficient proximity to the droplet operations electrodes  1614 , such that a droplet moving along the droplet operations electrodes  1610  is within the magnet field of the magnet  1616 , e.g., a region of uniform magnetic field. In this embodiment, the droplet may be may include sample and beads  1618 , and some or all of the beads  1618  may be magnetically responsive. Additionally, the droplet actuator  1600  includes a physical barrier  1620  that is arranged as shown in  FIG. 16 . The physical barrier  1620  is used to reduce the gap at the point of splitting, thereby assisting the droplet splitting operation. Additionally, because of the existence of the rigid barrier, consistent splitting may be obtained substantially at the same point. Further, the physical barrier  1620  may in some cases substantially nonmagnetic. 
         [0091]    The process may include, but is not limited to, the following steps. In Step  1 , magnet  1612  immobilizes the magnetically responsive beads  1618  in, for example, an aqueous slug  1622 . The aqueous slug  1622  is intersected by the physical barrier  1620 , which reduces the gap. In Step  2 , a droplet splitting operation occurs (e.g., forming droplets  1624  and  1626 ), whereby the magnetically responsive beads  1618  remain substantially immobilized by the magnet  1616  and the physical barrier  1620  is used to reduce the gap at the point of splitting, thereby assisting the droplet splitting operation. In this example, magnetically responsive beads  1618  remain substantially immobilized in droplet  1624  by the magnet  1612  and droplet  1626  is substantially free of beads  1618 . For example, substantially all of the magnetically responsive beads  1618  may remain in droplet  1618 , while droplet  1610  may be substantially free of magnetically responsive beads  1618 . The process may, for example, be employed in a merge-and-split bead washing protocol. 
         [0092]      FIG. 17  illustrates a process of facilitating consistent droplet splitting by use of a magnetic physical barrier in the droplet actuator.  FIG. 17  shows a side view of the portion of droplet actuator  1600  that is described in  FIG. 16 . However,  FIG. 17  shows that the substantially nonmagnetic physical barrier  1620  of  FIG. 16  is replaced with a magnetic physical barrier  1710 .  FIG. 17  also shows that magnet  1616  of  FIG. 16  is removed from proximity to bottom substrate  1612 . The magnetic physical barrier  1710  is used to (1) immobilize the magnetically responsive beads  1618  and (2) to reduce the gap at the point of splitting, thereby assisting the droplet splitting operation. Additionally, because of the existence of the rigid magnetic physical barrier  1710 , consistent splitting may be obtained substantially at the same point. 
         [0093]    The process may include, but is not limited to, the following steps. In Step 1, the magnetic physical barrier  1710  immobilizes the magnetically responsive beads  1618  in the aqueous slug  1622 . The aqueous slug  1622  is intersected by the magnetic physical barrier  1710 , which reduces the gap. In Step 2, a droplet splitting operation is executed (e.g., forming droplets  1624  and  1626 ), whereby the magnetically responsive beads remain substantially immobilized by the magnetic physical barrier  1710  and the magnetic physical barrier  1710  is used to reduce the gap at the point of splitting, thereby assisting the droplet splitting operation. In this example, magnetically responsive beads  1618  remain substantially immobilized in droplet  1624  by magnetic physical barrier  1710  and droplet  1626  is substantially free of beads  1618 . The process may, for example, be employed in a merge-and-split bead washing protocol. 
       7.7 Electrode Configurations for Improved Droplet Splitting 
       [0094]      FIG. 18  illustrates embodiments of electrode configuration for improved droplet splitting. In one example,  FIG. 18A  shows an electrode path  1810  that includes a splitting region  1812  that includes a segmented electrode  1814 , such as multiple electrode strips. In a splitting operation, electrodes may be activated to extend a slug across the region of electrode strips. The electorode strips may be deactivated starting with the outer strips and continuing to the inner strips in order to cause a controlled split of the droplet at the electrode strip region of the electrode path  1810 . In an alternative embodiment, the electrode strips may be rotated  90  degrees. In this embodiment, deactivation may start from the inner electrodes of the electrode strips and continue to the outer electrodes in order to controllably split the droplet at the electrode strips. 
         [0095]    In another example,  FIG. 18B  shows an electrode path  1820  that includes a splitting region  1822  that includes a tapered electrode  1824  that may span a distance equivalent, for example, to about two standard droplet operations electrodes. In operation, a droplet may be extended along electrodes of the electrode path across tapered electrode  1824 . Electrode  1824  or the adjacent electrode  1825  may be deactivated to controllably split the droplet. 
         [0096]    In yet another example,  FIG. 18C  shows an electrode pattern  1830  that includes a splitting region  1832  that includes a long tapered electrode  1834  and a short tapered electrode  1836 , where the smallest end of the tapered electrodes face one another. The tapered electrode pair may span a distance equivalent, for example, to about three standard droplet operations electrodes. In operation, a droplet may be extended along electrodes of the electrode path across tapered electrodes  1834  and  1836 . Electrode  1834  and/or electrode  1836  may be deactivated to controllably split the droplet. 
         [0097]    In yet another example,  FIG. 18D  shows an electrode pattern  1840  that includes a splitting region  1842  that includes a long tapered electrode  1842  and a short interlocking electrode  1844 , where the smallest end of the tapered electrode  1842  faces the interlocking electrode  1844 . The electrode pair may span a distance equivalent, for example, to about three standard droplet operations electrodes. In operation, a droplet may be extended along electrodes of the electrode path across tapered electrodes  1844  and  1846 . Electrode  1844  and/or electrode  1846  may be deactivated to controllably split the droplet. 
         [0098]    In yet another example,  FIG. 18E  shows an electrode pattern  1850  that includes a splitting region  1852  that includes a segmented electrode  1854 , such as multiple row or columns of electrode strips. n operation, a droplet may be extended along electrodes of the electrode path across splitting region  1852 . Each segment may be independently deactivated as desired to controllably split the droplet. 
       7.8 Improved Detection 
       [0099]    A process for the detection of supernatant after adding a substrate to the assayed magnetically responsive beads is disclosed, in accordance with the invention. After the washing protocol to remove the excess unbound antibody is complete, a chemiluminescent substrate is added to the assayed and washed beads, which produces chemiluminescence as a result of the reaction between the enzyme on the beads and the substrate. 
         [0100]    The substrate may be incubated with the magnetically responsive beads for some fixed time, where the magnetically responsive beads are substantially immobilized and the supernatant is transported away for detection. This approach reduces, preferably entirely eliminates, the need to transport the magnetically responsive bead droplet over long distances to the detector and also reduces, preferably entirely eliminates, the possibility of loss of beads during the transport operation. 
         [0101]    Alternatively the antibody-antigen-enzyme complex can be released from the bead by chemical or other means into the supernatant. The beads may then be substantially immobilized and the supernatant processed further for detection. 
         [0102]    Additionally, the same split, merge, and transport strategies that are explained for incubating beads/antibodies/sample mixture may be employed here also for incubating substrate and assayed beads. 
         [0103]    Bead based sandwich or competitive affinity assays, such as ELISAs, may be performed using the procedures described in this application in conjunction with various steps described in International Patent Application No. PCT/US 06/47486, entitled “Droplet-Based Biochemistry,” filed on Dec. 11, 2006. Further, after incubation, unbound sample material and excess reporter antibody or reporter ligand may be washed away from the bead-antibody-antigen complex using various droplet operations. A droplet of substrate (e.g., alkaline phosphatase substrate, APS-5) may be delivered to the bead-antibody-antigen complex. During incubation, the substrate is converted to product which begins to chemiluminesce. The decay of the product (which generates light) is sufficiently slow that the substrate-product droplet can be separated from the alkaline phosphatase-antibody complex and still retain a measurable signal. After an incubation period of the substrate with the bead-antibody-antigen complex (seconds to minutes), the magnetically responsive bead-antibody-antigen complex may be retained with a magnetic field (e.g., see U.S. Patent Application No. 60/900,653, filed on Feb. 9, 2007, entitled “Immobilization of magnetically-responsive beads during droplet operations,”) or by a physical barrier (e.g., see U.S. Patent Application No. 60/881,674, filed on Jan. 22, 2007, entitled “Surface-assisted fluid loading and droplet dispensing,” the entire disclosure of which is incorporated herein by reference) and only the substrate-product droplet may be presented (using droplet operations) to the sensor (e.g., PMT) for quantitation of the product. 
         [0104]    The substrate-product droplet alone is sufficient to generate a signal proportional to the amount of antigen in the sample. Incubation of the substrate with the magnetically responsive bead-antibody-antigen complex produces enough product that can be quantitated when separated from the enzyme (e.g., alkaline phosphatase). By measuring the product in this manner, the bead-antibody-antigen complex does not have to be presented to the PMT. There are no beads or proteins to “foul” the detector area as they are never moved to this area. Also the product droplet does not have to oscillate over the detector to keep beads in suspension during quantitation. The droplet volume may also be reduced in the absence of beads. Detection of the bead-antibody-antigen complex may employ a slug of liquid (e.g., 4 droplets) to move the complex, whereas with the beadless method the droplet could be smaller (e.g., less than 4 droplets). Time to result may also be shorter with this approach when performing multiplex ELISAs because the product droplet can be moved to the detector more quickly in the absence of beads. 
         [0105]    Bead based sandwich or competitive affinity assays, such as ELISAs, may be performed using droplet operations for one or more steps, such as combining sample, capture beads and reporter antibody or reporter ligand. After incubation, unbound sample material and excess reporter antibody or reporter ligand may be washed away from the bead-antibody-antigen complex using an on-chip washing protocol. After washing, a droplet of substrate (e.g., alkaline phosphatase substrate, APS-5) may be delivered to the bead-antibody-antigen complex. During the incubation, the substrate is converted to product which begins to chemiluminesce. The decay of the product (which generates light) is sufficiently slow that the substrate-product droplet can be separated from the alkaline phosphatase-antibody complex and still retain a measurable signal. After an incubation period of the substrate with the bead-antibody-antigen complex (seconds to minutes), the magnetically responsive bead-antibody-antigen complex may be retained with a magnet or by a physical barrier and only the substrate-product droplet may be presented (using droplet operations) to the sensor (e.g., PMT) for quantitation of the product. 
         [0106]    The substrate-product droplet alone is sufficient to generate a signal proportional to the amount of antigen in the sample. Incubation of the substrate with the magnetically responsive bead-antibody-antigen complex produces enough product that can be quantitated when separated from the enzyme (e.g., alkaline phosphatase). By measuring the product in this manner, the bead-antibody-antigen complex does not have to be presented to the PMT. There are no beads or proteins to “foul” the detector area as they are never moved to this area. Also the product droplet does not have to oscillate over the detector to keep beads in suspension during quantitation. The droplet volume may also be reduced in the absence of beads. Detection of the bead-antibody-antigen complex may employ a slug of liquid (e.g., 4 droplets) to move the complex, whereas with the beadless method the droplet could be smaller (e.g., less than 4 droplets). Time to result may also be shorter with this approach when performing multiplex ELISAs because the product droplet can be moved to the detector more quickly in the absence of beads. 
         [0107]      FIG. 19  illustrates detection strategies for quantifying an analyte. In particular, the immunoassay may be developed without any secondary antibody that is labeled with enzyme, fluorophore, or quantum dots. After binding of the analyte to the antibody that is bound to the magnetically responsive beads, the hydrodynamic diameter of the beads increases due to the immune complex that is bound to the surface of the bead. A superconductive quantum interference device (SQUID) gradiometer system may be used in order to measure the standard magnetization (Ms) of magnetically labeled immune complexes, such as the A 5 -Ag complex shown in  FIG. 19 . 
       7.9 Operation Fluids 
       [0108]    For examples of fluids that may be subjected to droplet operations using the approach of the invention, see the patents listed in section 2, especially International Patent Application No. PCT/US2006/047486, 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, biological washes, liquids with cells, tissues, multicellular organisms, single cellular organisms, protozoa, bacteria, fungal cells, viral particles, organelles. 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. 
         [0109]    The fluids may include one or more magnetically responsive and/or non-magnetically responsive beads. Examples of droplet actuator techniques for immobilizing magnetically responsive beads and/or non-magnetically responsive beads are described in the foregoing international patent applications and in Sista, et al., U.S. Patent Application No. 60/900,653, entitled “Immobilization of Magnetically-responsive Beads During Droplet Operations,” filed on Feb. 9, 2007; Sista et al., U.S. Patent Application No. 60/969,736, entitled “Droplet Actuator Assay Improvements,” filed on Sep. 4, 2007; and Allen et al., U.S. Patent Application No. 60/957,717, entitled “Bead Washing Using Physical Barriers,” filed on Aug. 24, 2007, the entire disclosures of which is incorporated herein by reference. 
       Concluding Remarks 
       [0110]    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. The definitions are intended as a part of the description 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.