Abstract:
The invention provides droplet actuators and droplet actuator cassettes including reagent storage capabilities, as well as methods of making and using the droplet actuators and cassettes. The invention also provides continuous flow channel elements and techniques for using electrodes to manipulate droplets in flowing streams. The invention also discloses methods of separating compounds on a droplet actuator. Various other aspects of the invention are also disclosed.

Description:
RELATED APPLICATIONS 
     This application is a continuation of and claims priority to U.S. patent application Ser. No. 12/990,815, entitled “Method of Loading a Droplet Actuator,” filed on Jun. 6, 2011, the application of which is a National Stage Entry of and claims priority to International Patent Application PCT/US2009/042731, entitled “Reagent and Sample Preparation, Loading, and Storage,” filed on May 4, 2009, the application of which is related and claims priority to the following U.S. Patent Applications: 61/050,207, entitled “Sample collection devices with sample processing and data storage capability,” filed on May 3, 2008; 61/052,215, entitled “Processing Non-liquid Samples on a Droplet Actuator,” filed on May 11, 2008; 61/052,224, entitled “Reagent Storage for Field-based Detection,” filed on May 11, 2008; 61/075,616, entitled “Rapid Detection of Methicillin Resistant  Staphylococcus aureus  (MRSA) Using Digital Microfluidics,” filed on Jun. 25, 2008; 61/085,032, entitled “Rapid Pathogen Detection on a Droplet Actuator,” filed on Jul. 31, 2008; 61/088,555, entitled “Fluidic Systems for and Methods of Loading a Droplet actuator,” filed on Aug. 13, 2008; 61/093,462, entitled Nucleic Acid Sample Preparation and Analysis on a Droplet Actuator,” filed on Sep. 2, 2008; and 61/157,302, entitled “Droplet Actuator Techniques Using Non-liquid Fluids,” filed on Mar. 4, 2009; the entire disclosures of each of these applications is incorporated herein by reference. 
    
    
     GOVERNMENT SUPPORT 
     This invention was made with government support under grant number AI066590-02 awarded by the National Institutes of Health. The government has certain rights in the invention. The foregoing statement applies only to those aspects of the invention described and claimed in this application arising out of U.S. Patent Application Nos. 61/075,616, entitled “Rapid Detection of Methicillin Resistant  Staphylococcus aureus  (MRSA) Using Digital Microfluidics,” filed on Jun. 25, 2008; 61/085,032, entitled “Rapid Pathogen Detection on a Droplet Actuator,” filed on Jul. 31, 2008; and 61/093,462, entitled “Nucleic Acid Sample Preparation and Analysis on a Droplet Actuator filed on Sep. 2, 2008. 
    
    
     BACKGROUND 
     Droplet actuators are used to conduct a wide variety of droplet operations. A droplet actuator typically includes two substrates separated to form a droplet operations gap. The substrates include electrodes for conducting droplet operations. The gap between the substrates is typically filled with a filler fluid that is immiscible with the liquid that is to be subjected to droplet operations. Droplet operations are controlled by electrodes associated with one or both of the substrates. There is a need for droplet actuator devices, techniques and systems for making and using droplet actuators. There is a need for devices, techniques and systems for preparing samples and/or reagents for loading onto a droplet actuator; for loading samples and/or reagents onto a droplet actuator; for storing samples and/or reagents on a droplet actuator and/or for use on a droplet actuator; and/or for conducting droplet operations using samples and/or reagents on a droplet actuator. There is a need for devices, techniques and systems for conducting flow through bead handling and washing. For example, there is a need for techniques for splitting droplets in a flow-through system, compartmentalizing beads in droplets in a flow-through system, and washing droplets in a flow-through system. There is a need for droplet actuator devices, techniques and systems for making and using droplet actuators to process viscous, solid or semi-solid samples. For example, there is a need for a techniques for processing process viscous, semisolid, and/or solid samples. There is a need for droplet actuator devices including a gel for use in gel electrophoresis, along with techniques and systems for conducting gel electrophoresis on a droplet actuator. There is a need for a fluidics system and technique for using the system for loading liquids onto a droplet actuator. There is a need for droplet actuators loaded using the fluidics system and method of the invention and methods of using such droplet actuators to conduct droplet operations. There is a need for droplet actuator devices, techniques and systems for processing samples for use on a droplet actuator device. There is a need for droplet actuator devices, techniques and systems for capturing, concentrating and/or eluting nucleic acids; and sensitively isolating nucleic acids using one or more droplet operations to perform separation protocols. There is a need for kits including droplet actuators of the invention along with various other components suitable for executing the techniques of the invention, such as reagents, sample collection devices, and/or instructions. 
     SUMMARY OF THE INVENTION 
     The invention provides a droplet actuator, and methods of making and using the droplet actuator. The droplet actuator may include two substrates separated to provide a droplet operations gap. One or more electrodes may be associated with one or both substrates and arranged for conducting one or more droplet operations in the droplet operations gap. The droplet actuator may include a reagent storage cassette with one or more reservoirs including one or more liquids. One or more fluid paths may be provided from the one or more reservoirs into the droplet operations gap. The fluid paths may be blocked by a film or other breakable, removable or puncturable material. A plunger may be associated with the reservoir and arranged to force liquid from the reservoir into the fluid path when depressed into the reservoir. A series of the reservoirs and a series of the plungers may be included. Each of the reservoirs may be associated with a corresponding plunger arranged to force liquid from the reservoir into the fluid path. The plungers may be coupled to a common plunger depressor. The film may be selected with physical and/or chemical characteristics which permit it to break upon application of pressure to liquid in the reservoir or reservoirs when the plunger or plungers are depressed. For example, film may be scored or include a thin or weakened region that breaks upon application of pressure to liquid in the reservoir or reservoirs by depressing the plunger or plungers. The reagent storage cassette may include an awl, scribe or other puncturing device arranged to puncture the film and thereby permit liquid to flow through the fluid path. The device may include an awl, point, scribe or other puncturing device slideably inserted within a slot in the plunger and arranged to puncture the film and thereby permit liquid to flow through the fluid path. The droplet actuator may include a series of reservoirs, wherein each reservoir in the series of the reservoirs may be associated with a connecting fluid path extending from the reservoir and into a channel of the fluid path, such that upon depression of the plungers, a series of droplets may be forced through the connecting fluid path and into the channel. The channel may include a liquid filler fluid which may be immiscible with the series of droplets. The channel may be coupled to a pressure or vacuum source for flowing droplets through the channel and into the droplet operations gap. The channel may be associated with one or more electrodes configured for transporting droplets through the channel and into the droplet operations gap. The droplet actuator may include a series of reservoirs, wherein each reservoir in the series of reservoirs is associated with a fluid path from the reservoir into the droplet operations gap. Liquid forced through the fluid path into the droplet operations gap may be subject to one or more droplet operations in the droplet operations gap. In some embodiments, the fluid path from the reservoir into the droplet operations gap passes through an opening in an electrode. The electrode may, for example, be a droplet operations electrode, such as a reservoir electrode. In some embodiments, the fluid path may be fluidly coupled with one or more filler fluid channels arranged for flowing filler fluid around droplets in the fluid path. 
     The invention provides a method of conducting a droplet operation including providing a channel; flowing an immiscible liquid including a droplet through the channel and into proximity with a set of one or more electrodes; using the set of one or more electrodes with the droplet to conduct a droplet operation; and continuing to flow the droplet or one or more daughter droplets formed during the droplet operation through the channel. The droplet operation may be effected without stopping flow of the immiscible liquid through the channel. The droplet operation may include splitting the droplet into two or more daughter droplets. The droplet operation may include interrupting the flow of the droplet through the channel. In some embodiments, the channel splits into first and second branches, and the droplet operation may include splitting the droplet into two or more droplets, one or more droplets flowing into a first of the two or more branches and a second of the two or more droplets flowing into a second of the two or more branches. In some embodiments, the channel splits into first and second branches, and the droplet operation causes the droplet to flow down one or the other of the first and second branches. 
     The invention also provides a method of manipulating a droplet, the method comprising providing a channel; flowing a liquid filler fluid including a magnetically-responsive, bead-containing droplet through the channel and into proximity with a magnetic field to substantially immobilize the magnetically responsive bead and thereby capture the bead-containing droplet; releasing the magnetically responsive bead from the magnetic field, thereby permitting it to continue to flow through the channel. In some cases, substantially all of the liquid volume of the bead-containing droplet remains with the magnetically responsive bead when it may be substantially immobilized by the magnetic field. In other cases, at least a portion of the liquid volume of the bead-containing droplet breaks away from the magnetically responsive bead when it may be substantially immobilized by the magnetic field and continues to flow with the liquid filler fluid through the channel. The method may also include flowing a second droplet in the flowing filler fluid into contact with the captured bead-containing droplet, wherein the second droplet merges with the bead-containing droplet. In some embodiments the flowing filler fluid causes a bead-free droplet to break away from the bead-containing droplet and continue to flow with the liquid filler fluid through the channel. The second droplet may, for example, include a wash buffer. The method may also include repeating the flowing of a second droplet in the flowing filler fluid into contact with the captured bead-containing droplet using a series of two or more of such second droplets to reduce the concentration and/or quantity of a substance present in the liquid volume of the bead-containing droplet. The method may also include repeating the flowing of a second droplet in the flowing filler fluid into contact with the captured bead-containing droplet until the liquid volume of the bead-containing droplet may be substantially replaced. The second droplet may include a sample droplet having a target for which the bead may have affinity. The method may also include repeating the flowing of a second droplet in the flowing filler fluid into contact with the captured bead-containing droplet using a series of two or more of such second droplets to concentrate a target substance on the bead of the bead-containing droplet. The target substance may, for example, include organic molecules, inorganic molecules, peptides, proteins, macromolecules, subcellular components of a biological cell, cells, group of cells, single celled organisms, multicellular organisms. The method may also include flowing a third droplet in the flowing filler fluid into contact with the captured bead-containing droplet, wherein the third droplet merges with the bead-containing droplet; the flowing filler fluid causes a bead-free droplet to break away from the bead-containing droplet and continue to flow with the liquid filler fluid through the channel. The third droplet may, for example, include a wash buffer. The method may also include repeating the flowing of a third droplet in the flowing filler fluid into contact with the captured bead-containing droplet using a series of two or more of such third droplets sufficient to reduce the concentration and/or quantity of a substance present in the liquid volume of the bead-containing droplet. The method may also include repeating the flowing of a third droplet in the flowing filler fluid into contact with the captured bead-containing droplet until the liquid volume of the bead-containing droplet may be substantially replaced. The method may also include releasing the magnetically responsive bead from the magnetic field, e.g., to permit a reconstituted magnetically responsive bead containing droplet to flow in the filler fluid through the channel. In some embodiments the magnetic field may be in proximity with a set of one or more electrodes and the method may include using the set of one or more electrodes with the bead-containing droplet to conduct a droplet operation. The method may include continuing to flow the droplet or one or more daughter droplets formed during the droplet operation through the channel. The method may include flowing the reconstituted magnetically responsive bead containing droplet into a droplet actuator reservoir and/or into a droplet operations gap of a droplet actuator, where the magnetically responsive bead containing droplet may be subjected to one or more droplet operations. The one or more droplet operations may include steps in an assay protocol to analyze a target substance on the magnetically responsive bead. The droplet operation may be effected without stopping flow of the immiscible liquid through the channel. The droplet operation may include splitting the droplet into two or more daughter droplets: one or more of such daughter droplets including the magnetically responsive bead; and one or more of such daughter droplets substantially lacking in magnetically responsive beads. The droplet operation may include interrupting the flow of the droplet through the channel. In some cases, the channel splits into first and second branches; and the droplet operation includes splitting the droplet into two or more droplets, including one or more daughter droplets flowing into a first of the two or more branches and including the magnetically responsive bead; and one or more daughter droplets flowing into a second of the two or more branches and substantially lacking in magnetically responsive beads. 
     The invention also provides a method of encapsulating a magnetically responsive bead in a droplet, the method may include providing a channel; flowing an immiscible liquid including a magnetically responsive bead through the channel and into proximity with a magnetic field; capturing the magnetically responsive bead in the magnetic field; flowing an immiscible liquid including a droplet into contact with the magnetically responsive bead to encapsulate the magnetically responsive bead in the droplet, thereby yielding a bead-containing droplet. The magnetically responsive bead may have affinity for an aqueous medium, the droplet may include an aqueous medium, and the filler fluid may include a non-aqueous liquid. The method may also include releasing the magnetically responsive bead from the magnetic field, thereby permitting the bead-containing droplet to continue to flow with the filler fluid through the channel. 
     The invention provides a method of sampling a non-liquid sample on a droplet actuator, the method may include providing a droplet actuator including a droplet operations and electrodes configured to conduct one or more droplet operations on the droplet operations surface; supplying a non-liquid sample in proximity to or in contact with the droplet operations surface; effecting one or more droplet operations to contact a droplet on the droplet operations surface into contact with the non-liquid sample to dissolve into the droplet one or more components of the non-liquid sample; effecting one or more droplet operations to conduct the droplet away from the non-liquid sample. The non-liquid sample may include a solid sample, a semi-solid sample and/or a viscous sample. The droplet may include one or more beads having affinity for one or more of the components of the non-liquid sample. The droplet may include an enzyme having affinity for a component of the non-liquid sample. The droplet may have pH selected to dissolve the non-liquid sample. The sample may include cells and the droplet may include a lysis buffer solution selected to lyse the cells. The one or more droplet operations may include an electrode-mediated droplet operation. The one or more droplet operations may include an electrowetting-mediated droplet operation. The one or more droplet operations may include a dielectrophoresis-mediated droplet operation. The non-liquid sample sufficiently viscous, semi-solid or solid to permit a droplet to contact the sample and be transported away from the sample without being substantially combined with the sample. The non-liquid sample may be selected from the group consisting of sputum, coagulated blood, animal tissue samples, plant tissue samples, soil samples, and rock samples. The non-liquid sample may include a matrix used to collect the sample. The droplet may include an aqueous droplet. The droplet may include a non-aqueous droplet. The method may include using the droplet to conduct an assay analyzing a component of the sample. In some cases, the assay analyzes a protein or peptide present in the sample. In some cases, the assay may include amplifying a nucleic acid present in the sample. The method may include removing the droplet from the droplet actuator. 
     The invention provides a method of providing a polymerized material on a droplet operations surface. The method may include providing a droplet actuator including a substrate including electrodes arranged for conducting droplet operations on a droplet operations surface of the substrate. The method may include providing on the droplet operations substrate a polymerizable droplet on the droplet operations substrate and a catalyst droplet including a catalyst selected to accelerate polymerization of the polymerizable droplet. The method may also include conducting droplet operations mediated by the electrodes to combine the polymerizable droplet with the catalyst droplet to yield a polymerizing droplet. Further, the method may include permitting the polymerizing droplet to polymerize, thereby yielding a polymerized material on the droplet operations surface. The droplet actuator may include a second substrate separated from the droplet operations surface to provide a droplet operations gap in which the droplet operations may be conducted. In some embodiments the droplet operations may be conducted in a liquid filler fluid which may be immiscible with the polymerizable droplet and the catalyst droplet. In some embodiments the polymerized material may include a gel selected for conducting gel electrophoresis. The polymerized material may, for example, be a polyacrylamide gel or an agarose gel. The method may include activating a series of two or more electrodes underlying the polymerizable droplet to elongate the droplet prior to combining the polymerizable droplet with the catalyst droplet. The method may include activating a series of two or more electrodes underlying the polymerizing droplet to elongate the droplet prior to permitting the polymerizing droplet to polymerize. 
     The invention also provides a method of causing separation of one or more substances. The method may include providing a sample droplet including substances for separation on the droplet operations surface or in a reservoir associated with a fluid path arranged to flow liquid from the reservoir into contact with the polymerized material. The method may include contacting the sample droplet with the polymerized material. The method may include applying current to the polymerized material to cause separation of one or more of the substances provided in the sample droplet. In some cases, the droplet actuator may include a second substrate separated from the droplet operations surface to provide a droplet operations gap, the second substrate including an opening providing a fluid path from an exterior locus into the droplet operations gap; and providing a sample droplet may include supplying a sample droplet through the opening in the second substrate into contact with the polymerized material. The method may include marking one or more target substances for detection. For example, in some cases, the one or more substances for separation may include one or more nucleic acids, and the marking may include staining the one or more nucleic acids. In some embodiments the marking may include providing a marker droplet on the droplet operations surface, and using one or more droplet operations to transport the marker droplet into contact with the polymerized material. 
     The invention also provides a droplet actuator including a substrate including electrodes arranged for conducting droplet operations on a droplet operations surface of the substrate; a polymerized material for conducting gel electrophoresis; negative and positive electrodes in contact with the polymerized material. The droplet actuator may include a second substrate separated from the droplet operations surface to provide a droplet operations gap in which the droplet operations may be conducted. The droplet actuator may include a liquid filler fluid in contact with the droplet operations surface. The polymerized material may, for example, include a gel selected from the group consisting of polyacrylamide gels and agarose gels. The droplet actuator may include a sample droplet including substances for separation on the droplet operations surface or in a reservoir associated with a fluid path arranged to flow liquid from the reservoir into contact with the polymerized material. The substances for separation may, for example, include proteins, peptides and/or nucleic acids. The droplet actuator may include a second substrate separated from the droplet operations surface to provide a droplet operations gap. In some cases, the second substrate including an opening providing a fluid path from an exterior locus into the droplet operations gap. The droplet actuator may include including a marker droplet including reagents for marking one or more target substances in the polymerized material for detection. 
     The invention provides a droplet actuator loading circuit including a primary fluid circuit arranged to flow fluid through a fluid path including a droplet operations gap of a droplet actuator and a an external fluid circuit. The droplet actuator loading circuit may include a reagent fluid path branching from the primary fluid circuit and fluidly connecting the primary fluid path to one or more reservoirs including reagents and/or filler fluid. The droplet actuator loading circuit may include a mechanism for switching the reagent fluid path from one reservoir to another reservoir. The mechanism for switching the reagent fluid path between reagent reservoirs may include a robotic device for moving a terminus of the reagent fluid path from one reservoir to another reservoir. The droplet actuator loading circuit may include one or more valves configured in the primary fluid circuit and/or the reagent fluid path to permit switching between circulating liquid in the primary fluid circuit, and flowing liquid from the one or more reservoirs including reagents and/or filler fluid into the primary fluid circuit. The droplet actuator loading circuit may include a reagent fluid path branching from the primary fluid circuit and fluidly connecting the primary fluid path to one or more reservoirs including reagents, and a filler fluid path branching from the primary fluid circuit and fluidly connecting the primary fluid path to one or more reservoirs including a liquid filler fluid. The droplet actuator loading circuit may include one or more valves configured in the primary fluid circuit and/or the reagent fluid path to permit switching between circulating liquid in the primary fluid circuit, and flowing liquid reagent from the one or more reservoirs including reagents into the primary fluid circuit, flowing filler fluid from the one or more reservoirs including liquid filler fluid into the primary fluid circuit. The droplet actuator loading circuit, wherein the reagent fluid path and/or the filler fluid path branches from the primary fluid circuit at a locus which may be in the external fluid circuit. The droplet actuator loading circuit including a pump disposed to pump liquid through the primary fluid circuit. The pump may, for example, include a reversible pump. The pump may include a peristaltic pump. The pump may be disposed to pump liquid through the primary fluid circuit, wherein the pump may be disposed in the primary fluid circuit at a locus which lies between a locus in the primary fluid circuit at which the reagent fluid path branches from the primary fluid circuit, and a locus in the primary fluid circuit at which the filler fluid path branches from the primary fluid circuit. The droplet actuator loading circuit may also include an overflow fluid path fluidly coupled into the droplet operations gap. The droplet actuator loading circuit may also include a reservoir and a pump disposed to pump liquid from the droplet operations gap through the overflow fluid path and into a reservoir. The reservoir and pump together may comprise a syringe pump. 
     The invention provides a method of loading a droplet actuator. The method may include providing a droplet actuator loading circuit including a primary fluid circuit arranged to flow fluid through a fluid path including a droplet operations gap of a droplet actuator and a an external fluid circuit. The method may include filling the loading circuit, including the droplet operations gap, with a liquid filler fluid and thereby purging the loading circuit of air. The method may include flowing reagent liquid into the external fluid circuit to form droplets in the liquid filler fluid contained therein. The method may include flowing contents of the external fluid circuit into the droplet operations gap of the droplet actuator. Filling the loading circuit, including the droplet operations gap, with a liquid filler fluid may include flowing filler fluid into the primary fluid circuit via a filler fluid branch in the primary fluid circuit. The filler fluid branch in the primary fluid circuit may be situated in the external fluid circuit. Flowing reagent liquid into the external fluid circuit may include flowing reagent into the primary fluid circuit via a reagent branch in the primary fluid circuit. The reagent branch in the primary fluid circuit may be situated in the external fluid circuit. Different kinds of reagent droplets may be loaded into the external fluid circuit. Reagent types may be selected by switching the reagent branch from one reservoir to another reservoir. The switching may be effected by a robotic device configured to move a terminus of the reagent fluid path from one reservoir to another reservoir. Valves configured in the primary fluid circuit and/or the reagent fluid path to switch between circulating liquid in the primary fluid circuit, and flowing liquid from the one or more reservoirs including reagents and/or filler fluid into the primary fluid circuit. The method further may include flowing liquid from the droplet operations gap through an overflow fluid path fluidly coupled into the droplet operations gap. Flowing liquid from the droplet operations gap through an overflow fluid path may include pumping the liquid through the overflow path into a reservoir. The reservoir and pump together may include a syringe pump. 
     The invention provides a method of preparing a sample droplet. The method may include providing a droplet actuator substrate including a droplet operations surface and electrodes configured to conduct droplet operations on the droplet operations surface. The method may include providing a sample droplet including cells including a target substance on the droplet operations surface. The method may include providing a lysis droplet including a lysis buffer on the droplet operations surface. The method may include using one or more droplet operations mediated by the electrodes to combine the lysis droplet with the sample droplet to yield a lysed droplet including lysed cells. The method may include providing in the lysed droplet beads having affinity for the target substance. The sample droplet may include beads. Beads may be added to the sample droplet buffer prior to providing the sample droplet on the droplet operations surface. The sample droplet may be merged with a bead droplet including the beads on the droplet operations surface. The lysis droplet may be provided with the beads. Beads may be added to the lysis buffer prior to providing the lysis droplet on the droplet operations surface. The lysis droplet may be merged with a bead droplet including the beads on the droplet operations surface. The method may include washing the beads to yield a washed bead droplet substantially lacking in unbound material from the sample. The method may include providing an elution droplet on the droplet operations surface, and using one or more droplet operations to combine the elution droplet with the washed bead droplet to yield an eluted droplet in which the target substance may be eluted from the beads. The method may include heating the combined elution droplet and washed bead droplet to accelerate elution of target substance from the beads. The method may include trapping the beads and using one or more droplet operations to transport away from the beads a substantially bead-free droplet on the droplet operations surface. The target substance may include a target protein or target peptide. The target substance may include a target nucleic acid. The method may include supplying the substantially bead-free droplet or the washed bead droplet with reagents for conducting nucleic acid amplification to yield an amplification-ready droplet. One or more droplet operations may be used to combine the substantially bead-free droplet or the washed bead droplet with an amplification reagent droplet including reagents for conducting nucleic acid amplification. The method may include thermal cycling the amplification-ready droplet to amplify the target nucleic acid. The cells may include eukaryotic cells or prokaryotic cells. The cells may include bacterial cells. In some embodiments, the bacterial cells may include cells from  Staphylococcus  species,  Streptococcus  species,  Enterococcus  species,  Pseudomonas  species,  Clostridium  species, and/or  Acinetobacter  species. In some embodiments, the bacterial cells may include cells from  Staphylococcus aureus, Pseudomonas aeruginosa, Clostridium difficile, Acinetobacter baumannii, Bacillus anthracis, Franciscella tularensis, Mycoplasma pneumoniae , and  Eschericia coli.    
     The invention provides a droplet actuator device including a droplet actuator including a electronic storage and/or transmission element. The electronic storage and/or transmission element may be affixed to or incorporated in a droplet actuator. The electronic storage and/or transmission element may be affixed to or incorporated in a droplet actuator cartridge including a droplet actuator. The electronic storage and/or transmission element may include a computer readable data storage element. The computer readable data storage element may include semiconductor memory, magnetic storage, optical storage, volatile memory, non-volatile memory, a radio-frequency identification tag, read-only memory, random access memory, electrically erasable programmable read-only memory, flash memory, and/or a magnetic stripe. The magnetic stripe may be provided on a magnetic stripe card, and the droplet actuator may be mounted on the magnetic stripe card. The droplet actuator mounted on the magnetic stripe card may include electrical contacts arranged to couple with electrical contacts on a droplet actuator instrument when the magnetic stripe card may be inserted in a magnetic card slot of a magnetic card reading instrument. The droplet actuator may be electrically connected to wires on the card. The wires on the card may terminate in contacts arranged to be electrically coupled to electrical contacts on an instrument so that the droplet actuator may be controlled by the instrument. The card may have a shape and size of a standard credit card. The electronic storage and/or transmission element may include a unique identifier for the droplet actuator. The droplet actuator device may be configured with a connect device for connecting the droplet actuator device to a computer as a peripheral device. The connect device may, for example, include a universal serial bus connector. The droplet actuator device may also include a positioning device, such as a global positioning device. 
     The invention also includes a networked system including the droplet actuator device distributed in a target geographical region with communications capabilities for transmitting data to one or more data aggregation centers. The droplet actuators may be installed on fixed bases. The fixed bases may be selected from the group consisting of: buildings, farms, water supply sources, buoys, and weather balloons. The droplet actuators may be installed on fixed bases. The mobile bases may be selected from the group consisting of: mobile robotic devices, airplanes, unmanned drones, vehicles in vehicle fleets. The mobile bases may be selected from the group consisting of: police cars, school buses, ambulances, military vehicles, oceangoing vessels, postal vehicles, and vehicles in commercial vehicle fleets. 
     The invention provides a system including the droplet actuator device and a global position sensor. The invention provides a system including one or more kiosks including a dispenser for dispensing a droplet actuator device. The invention provides a system including one or more kiosks including a receptacle for receiving a droplet actuator device. The kiosk may also include an input device for inputting information associated with a droplet actuator device. 
     DEFINITIONS 
     As used herein, the following terms have the meanings indicated. 
     “Activate” with reference to one or more electrodes means effecting a change in the electrical state of the one or more electrodes which, in the presence of a droplet, results in a droplet operation. 
     “Bead,” with respect to beads on a droplet actuator, means any bead or particle 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 Magnetic 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. 
     “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. For examples of droplet fluids that may be subjected to droplet operations using the approach of the invention, see International Patent Application No. PCT/US 06/47486, entitled, “Droplet-Based Biochemistry,” filed on Dec. 11, 2006. In various embodiments, a droplet may include a biological sample, such as whole blood, lymphatic liquid, serum, plasma, sweat, tear, saliva, sputum, cerebrospinal liquid, amniotic liquid, seminal liquid, vaginal excretion, serous liquid, synovial liquid, pericardial liquid, peritoneal liquid, pleural liquid, transudates, exudates, cystic liquid, bile, urine, gastric liquid, intestinal liquid, fecal samples, liquids containing single or multiple cells, liquids containing organelles, fluidized tissues, fluidized organisms, liquids containing multi-celled organisms, biological swabs and biological washes. Moreover, a droplet may include a reagent, such as water, deionized water, saline solutions, acidic solutions, basic solutions, detergent solutions and/or buffers. Other examples of droplet contents include reagents, such as a reagent for a biochemical protocol, such as a nucleic acid amplification protocol, an affinity-based assay protocol, an enzymatic assay protocol, a sequencing protocol, and/or a protocol for analyses of biological fluids. 
     “Droplet Actuator” means a device for manipulating droplets. For examples of droplet actuators, 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; and Roux et al., U.S. Patent Pub. No. 20050179746, entitled “Device for Controlling the Displacement of a Drop Between two or Several Solid Substrates,” published on Aug. 18, 2005; the disclosures of which are incorporated herein by reference. Certain droplet actuators will include a substrate, droplet operations electrodes associated with the substrate, one or more dielectric and/or hydrophobic layers atop the substrate and/or electrodes forming a droplet operations surface, and optionally, a top substrate separated from the droplet operations surface by a gap. One or more reference electrodes may be provided on the top and/or bottom substrates and/or in the gap. In various embodiments, the manipulation of droplets by a droplet actuator may be electrode mediated, e.g., electrowetting mediated or dielectrophoresis mediated or Coulombic force mediated. Examples of other methods of controlling liquid flow that may be used in the droplet actuators of the invention include devices that induce hydrodynamic fluidic pressure, such as those that operate on the basis of mechanical principles (e.g. external syringe pumps, pneumatic membrane pumps, vibrating membrane pumps, vacuum devices, centrifugal forces, piezoelectric/ultrasonic pumps and acoustic forces); electrical or magnetic principles (e.g. electroosmotic flow, electrokinetic pumps, ferrofluidic plugs, electrohydrodynamic pumps, attraction or repulsion using magnetic forces and magnetohydrodynamic pumps); thermodynamic principles (e.g. gas bubble generation/phase-change-induced volume expansion); other kinds of surface-wetting principles (e.g. electrowetting, and optoelectrowetting, as well as chemically, thermally, structurally and radioactively induced surface-tension gradients); gravity; surface tension (e.g., capillary action); electrostatic forces (e.g., electroosmotic flow); centrifugal flow (substrate disposed on a compact disc and rotated); magnetic forces (e.g., oscillating ions causes flow); magnetohydrodynamic forces; and vacuum or pressure differential. In certain embodiments, combinations of two or more of the foregoing techniques may be employed in droplet actuators of the invention. 
     “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 that are 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 volume of the resulting droplets (i.e., the volume 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. Droplet operations may be electrode-mediated. In some cases, droplet operations are further facilitated by the use of hydrophilic and/or hydrophobic regions on surfaces and/or by physical obstacles. 
     “Filler fluid” means a liquid associated with a droplet operations substrate of a droplet actuator, which liquid 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; International Patent Application No. PCT/US2008/072604, entitled “Use of additives for enhancing droplet actuation,” filed on Aug. 8, 2008; and U.S. Patent Publication No. 20080283414, entitled “Electrowetting Devices,” filed on May 17, 2007; the entire disclosures of which are incorporated herein by reference. The filler fluid may fill the entire gap of the droplet actuator or may coat one or more surfaces of the droplet actuator. Filler fluid may be conductive or non-conductive. 
     “Immobilize” with respect to magnetically responsive beads, means that the beads are substantially restrained in position in a droplet or in filler fluid on a droplet actuator. For example, in one embodiment, immobilized beads are sufficiently restrained in position to permit execution of a splitting operation on a droplet, yielding one droplet with substantially all of the beads and one droplet substantially lacking in the beads. 
     “Magnetically responsive” means responsive to a magnetic field. “Magnetically responsive beads” include or are composed of magnetically responsive materials. Examples of magnetically responsive materials include paramagnetic materials, ferromagnetic materials, ferrimagnetic materials, and metamagnetic materials. Examples of suitable paramagnetic materials include iron, nickel, and cobalt, as well as metal oxides, such as Fe3O4, BaFe12O19, CoO, NiO, Mn2O3, Cr2O3, and CoMnP. 
     “Washing” with respect to washing a magnetically responsive bead means reducing the amount and/or concentration of one or more substances in contact with the magnetically responsive bead or exposed to the magnetically responsive bead from a droplet in contact with the magnetically responsive bead. The reduction in the amount and/or concentration of the substance may be partial, substantially complete, or even complete. The substance may be any of a wide variety of substances; examples include target substances for further analysis, and unwanted substances, such as components of a sample, contaminants, and/or excess reagent. In some embodiments, a washing operation begins with a starting droplet in contact with a magnetically responsive bead, where the droplet includes an initial amount and initial concentration of a substance. The washing operation may proceed using a variety of droplet operations. The washing operation may yield a droplet including the magnetically responsive bead, where the droplet has a total amount and/or concentration of the substance which is less than the initial amount and/or concentration of the substance. Examples of suitable washing techniques are described in Pamula et al., U.S. Pat. No. 7,439,014, entitled “Droplet-Based Surface Modification and Washing,” granted on Oct. 21, 2008, the entire disclosure of which is incorporated herein by reference. 
     The terms “top,” “bottom,” “over,” “under,” and “on” are used throughout the description with reference to the relative positions of components of the droplet actuator, such as relative positions of top and bottom substrates of the droplet actuator. It will be appreciated that the droplet actuator is functional regardless of its orientation in space. 
     When a liquid in any form (e.g., a droplet or a continuous body, whether moving or stationary) is described as being “on”, “at”, or “over” an electrode, array, matrix or surface, such liquid could be either in direct contact with the electrode/array/matrix/surface, or could be in contact with one or more layers or films that are interposed between the liquid and the electrode/array/matrix/surface. 
     When a droplet is described as being “on” or “loaded on” a droplet actuator, it should be understood that the droplet is arranged on the droplet actuator in a manner which facilitates using the droplet actuator to conduct one or more droplet operations on the droplet, the droplet is arranged on the droplet actuator in a manner which facilitates sensing of a property of or a signal from the droplet, and/or the droplet has been subjected to a droplet operation on the droplet actuator. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1A and 1B  illustrate a reagent storage cassette of the invention.  FIG. 1A  shows the reagent storage cassette and an open position.  FIG. 1B  shows the reagent storage cassette in a closed position. 
         FIGS. 2A and 2B  illustrate a reagent storage cassette of the invention including a protective film configured to protect reagents from contamination and/or prevent leaking of reagents.  FIG. 2A  shows the reagent storage cassette and an open position.  FIG. 2B  shows an alternative embodiment of the reagent storage cassette in a closed position where the film includes a pull tab. 
         FIGS. 3A, 3B, 3C, 3D, and 3E  show cross-sectional views of segments of top and bottom members of a reagent storage cassette of the invention that makes use of plungers to force droplets from storage reservoirs.  FIG. 3A  illustrates a segment of the bottom member with the protective film in place.  FIG. 3B  illustrates the segment of the bottom member with plungers fully inserted to force droplets from storage reservoirs.  FIGS. 3C and 3D  illustrate a corresponding segment of a top member of a reagent storage cassette juxtaposed with the cross sectional view of a segment of the bottom member of the reagent storage cassette. In  FIG. 3C , the droplets are present in the reservoirs. In  FIG. 3D , plungers are fully inserted to force droplets from storage reservoirs.  FIG. 3E  illustrates an end-wise cross-sectional view of the reagent storage cassette showing a section of the top member juxtaposed with a corresponding section of the bottom member. 
         FIG. 4  illustrates another embodiment of the invention in which a droplet actuator cartridge is provided with a droplet actuator portion an integral reagent cassette portion. 
         FIGS. 5A and 5B  illustrate a side cross-sectional view and a top view, respectively, of a droplet actuator configured to supply droplets into reservoirs in a droplet operations gap. 
         FIGS. 6A, 6B, 6C, 6D, 6E, and 6F  illustrate another aspect of the invention in which a plunger is used to force liquid into the droplet operations gap of a droplet actuator.  FIG. 6A  shows liquid in a reservoir outside the droplet operations gap, prior to being forced into the droplet operations gap.  FIG. 6B  shows a top view of a reservoir electrode through which liquid is supplied into the droplet operations gap of the droplet actuator.  FIG. 6C  illustrates puncturing the dielectric layer so that liquid may flow into the droplet operations gap.  FIG. 6D  illustrates the plunger fully inserted and liquid having been forced into the droplet operations gap.  FIG. 6E  shows an alternative embodiment in which a single electrode is provided on a top substrate of the droplet actuator.  FIG. 6F  illustrates an alternative embodiment in which a reservoir electrode and droplet operations electrodes are provide on the top substrate and a ground or reference electrode is provided on the bottom substrate. 
         FIG. 7  illustrates an alternative embodiment of the reagent cassette of the invention including a first channel into which droplets are loaded, and a second channel for flowing liquid filler fluid around the droplets in the first channel. 
         FIG. 8  illustrates a flow-through system that makes use of droplet operations for splitting droplets. 
         FIG. 9  illustrates a flow-through system configured for adding beads to droplets. 
         FIG. 10  illustrates a flow-through system that makes use of droplet operations to wash beads in droplets. 
         FIGS. 11A, 11B, and 11C  illustrate a section of a droplet actuator and a method of processing a viscous, solid or semi-solid sample on a droplet actuator. 
         FIGS. 12A, 12B, and 12C  illustrate a section of a droplet actuator and a process of separating and analyzing a sample using gel electrophoresis. 
         FIGS. 13A, 13B, 13C, 13D, 13E, 13F, 13G, 13H, and 13I  are schematic diagrams of fluidics system for loading liquid receptacle, such as a channel or droplet operations gap of a droplet actuator, with liquid.  FIG. 13A  illustrates the system generally, while  FIGS. 13B-13I  each illustrate a specific step in a loading process. 
         FIGS. 14A, 14B, 14C, 14D, 14E, and 14F  are schematic diagrams of another fluidics system for loading liquid receptacle, such as a channel or droplet operations gap of a droplet actuator, with liquid.  FIG. 14A  illustrates the system generally, while  FIGS. 14B-14F  each illustrate a specific step in a loading process. 
         FIG. 15A  shows a plot of real-time PCR data for detection of MRSA using digital microfluidics.  FIG. 15B  shows a plot of real-time PCR data for detection of  Bacillus anthracis  using digital microfluidics. 
         FIG. 16  shows a plot resulting from amplification of MRSA genomic DNA captured, concentrated and eluted on a droplet actuator. 
         FIG. 17  illustrates a droplet actuator device of the invention, including a droplet actuator with an electronic storage and/or transmission element. 
         FIG. 18  illustrates another droplet actuator device of the invention, including a droplet actuator with an electronic storage and/or transmission element, where the electronic storage and/or transmission element includes a magnetic stripe card. 
         FIG. 19  is a functional diagram of a sample collection and analysis system of the invention. 
         FIG. 20  is a functional diagram of another sample collection and analysis system of the invention. 
     
    
    
     DESCRIPTION 
     The invention provides droplet actuator devices, techniques and systems for making and using droplet actuators. The invention provides devices, techniques and systems for preparing samples and/or reagents for loading onto a droplet actuator; for loading samples and/or reagents onto a droplet actuator; for storing samples and/or reagents on a droplet actuator and/or for use on a droplet actuator; and/or for conducting droplet operations using samples and/or reagents on a droplet actuator. The invention also provides devices, techniques and systems for conducting flow through bead handling and washing. For example, the invention provides techniques for splitting droplets in a flow-through system, compartmentalizing beads in droplets in a flow-through system, and washing droplets in a flow-through system. The invention provides droplet actuator devices, techniques and systems for making and using droplet actuators to process viscous, solid or semi-solid samples. For example, the invention provides techniques for processing viscous, semisolid, and/or solid samples. The invention provides droplet actuator devices including a gel for use in gel electrophoresis, along with techniques and systems for conducting gel electrophoresis on a droplet actuator. The invention provides a fluidics system and technique for using the system for loading liquids onto a droplet actuator. The invention also provides droplet actuators loaded using the fluidics system and method of the invention and methods of using such droplet actuators to conduct droplet operations. The invention provides droplet actuator devices, techniques and systems for processing samples for use on a droplet actuator device. In some cases, the processing includes pre-processing steps conducted prior to introduction of the samples onto a droplet actuator. The invention provides droplet actuator devices, techniques and systems for capturing, concentrating and/or eluting nucleic acids; and sensitively isolating nucleic acids using one or more droplet operations to perform separation protocols. The invention also provides kits including droplet actuators of the invention along with various other components suitable for executing the techniques of the invention, such as reagents, sample collection devices, and/or instructions. 
     Liquid Storage and Loading 
     The invention provides devices, techniques and systems for preparing samples and/or reagents for loading onto a droplet actuator; for loading samples and/or reagents onto a droplet actuator; for storing samples and/or reagents on a droplet actuator and/or for use on a droplet actuator; and/or for conducting droplet operations using samples and/or reagents on a droplet actuator. The reagents may be stored on the droplet actuator itself, and/or in reagent storage containers that are provided with a droplet actuator cartridge. In some cases, the droplet actuator cartridge may be provided in a kit along with reagents stored and storage containers. 
     Reagents selected for storage in accordance with the invention may be reagents which are useful in conducting an assay. For example, the reagents may be useful in an assay for assessing the presence or absence of, and/or quantify the amount of, a chemical or a biochemical substance. Examples of suitable assay types include immunoassays, nucleic acid amplification assays, nucleic acid sequencing assays, enzymatic assays, and other forms of assays. Assays may be conducted with various purposes; examples include medical diagnostics, veterinary diagnostics, weapons or explosives detection, chemical weapons detection, biological weapons detection, environmental testing, water testing, air testing, soil testing, food quality testing, forensics, species identification etc. 
     Samples may be collected and tested at a point of sample collection. For example, the point of sample collection may be in a medical care facility, at a subject&#39;s bedside, in a laboratory, or in the field. A sample may be collected, loaded onto the droplet actuator cartridge; the cartridge may be inserted into an instrument, an assay may be run, and results may be provided, all at the point of sample collection. In other embodiments, one or more of these steps may be accomplished remotely from the point of sample collection, e.g., in a central laboratory. A sample may be collected in the field, and transported to a laboratory, where it is loaded into a cartridge which is mounted on an instrument; the assay may be run, and results provided. A sample may be collected in the field and loaded into a cartridge in the field, e.g., loaded into a sample reservoir in a droplet actuator cartridge; the cartridge may be returned to a laboratory, where it is mounted on an instrument, an assay is run, and results are provided. Various other combinations are also possible within the scope the invention. The instrument may include electronic and detection components, as well as a means for mounting the cartridge on the instrument, or otherwise coupling the cartridge to the instrument, in a manner in which aligns electronic and detection components with corresponding components or regions of the droplet actuator. The cartridge may include a droplet actuator, electrical components which correspond to the electrical components of the instrument, and one or more detection regions, which are aligned with detection components on the instrument. In various embodiments, the reagent storage and loading techniques described in this specification may also be used for loading sample, e.g., sample may be collected and loaded into a reservoir, where it is stored. Sample may be loaded from the reservoir into the droplet operations gap of the droplet actuator cartridge in preparation for conducting an assay using the droplet actuator cartridge. 
     The reagent storage and reconstitution techniques of the invention may be useful in a variety of fluidics devices, such as droplet actuator devices. In some cases, the devices of the invention are packaged with or include reagents. The reagents may be provided in a format that is suitable for use in the field. In certain embodiments, the devices are suitable for use without requiring refrigeration and/or specialized dispensing equipment. Reagents may be rapidly reconstituted and used in express testing. Assay results, currently available by in-laboratory testing, may be made available substantially in ‘real-time.’ Decisions made using assay results can be made more quickly. 
       FIG. 1  illustrates a reagent storage cassette  100  of the invention. Cassette  100  includes a bottom member  105  and a top member  110 . Bottom member  105  and a top member  110  may be coupled by a flexible hinge portion or member  115 .  FIG. 1A  shows bottom member  105  and a top member  110  in an open position.  FIG. 1B  shows bottom member  105  and a top member  110  in a closed position. In one embodiment, the storage cassette is provided with bottom member  105  and a top member  110  in an open position, and the cassette is manipulated by a user into the closed position shown in  FIG. 1B . The flexible member  115  may have different electrical and physical properties in the regions overlying bottom member  105 , overlying top member  110 , and at the hinge and thus can comprise of multiple materials or a same material with different properties or a same material with substantially similar properties. The flexible member  115  may be an insulating material that serves dual purpose of insulating the electrodes on substrate  105  that are used for effecting droplet operations and also serve as a tether to the top plate. The flexible member  115  may have different properties in the portions overlying members  105  and  110  so that it is insulating on bottom member  105  but substantially conductive on top member  110  or vice versa. In other embodiments, the flexible member  115  may be rigid over members  105  and  110  but flexible only at the hinge connecting both the members. In another embodiment, the flexible member may also comprise a gasket or standoff material that forms a gap between the members  105  and  110  so that droplets can reside between members  105  and  110 . The flexible member may also be hydrophobized so that it is ready for droplet operations. 
       FIG. 2  illustrates a reagent storage cassette  100  of the invention with protective film  205 . Protective film  205  may be included to provide a seal, enclosing reagent components to protect them from the environment and/or separating reagent components from one another. One embodiment, shown in  FIG. 2A  illustrates a protective film  205  covering facing surfaces of the bottom member  105  and a top member  110  of cassette  100 . In operation, protective film  205  is removed to expose reagent components, and then bottom member  105  and a top member  110  of cassette  100  are sealed together.  FIG. 2B  illustrates a protective film inserted between facing surfaces of the bottom member  105  and a top member  110  of cassette  100 . In operation, protective film  205  is removed to expose reagent components in bottom member  105  to reagent components in top member  110  of cassette  100 . The protective film can also serve as a removable insulating or hydrophobic material. After droplet operations are performed, the members  110  and  105  may be separated and a new film  205  may be attached so that the surfaces of the droplet actuator are clean and can be reused without any concern for cross contamination. 
       FIG. 3  illustrates cross-sectional views  300  of a segment of bottom member  105 . Bottom member  105  includes reservoir  305  formed therein, which serve as reservoirs for droplets  315  of liquid reagents or samples. Openings  310  provide a liquid path for forcing droplets  315  onto a surface of bottom member  105 . During storage, protective film  205 , illustrated in  FIG. 3A , may be maintained in place to seal droplets  315  in reservoirs  310 . Droplets  315  may be pre-metered; however, in some cases, exact premetering is not required, since the droplets will be subject to dispensing operations on the droplet actuator in which dispensed subdroplets will have precise volumes suitable for conducting assays. 
     Reservoir  305  is associated with plungers  325  and plunger depressor  330 . Plunger depressor  330  is configured to force plungers  325  into openings  310 , thereby forcing droplets  315  out of openings  310 . Plunger depressor  330  may be manually operated, such that an operator may, by applying pressure to plunger depressor  330 , force plungers  325  into openings  310 , thereby forcing droplets  315  out of openings  310 . Plunger depressor  330  may be automatically operated, for example, so that when an operator inserts the droplet actuator cartridge into an instrument, the active insertion also forces plunger depressor  330  to move plungers through  25  into openings  310 . While reservoir/plunger assemblies are illustrated in  FIGS. 3A / 3 B, it will be appreciated that in some cases only a single such reservoir/plunger assembly is required. In other cases, more than two reservoir/plunger assemblies may be provided. 
     Prior to forcing plungers  325  into openings  310 , protective film  205  may be removed. Alternatively, protective film  205  may be scored or otherwise weakened in regions atop openings  310  so that by applying pressure to plunger depressor  330 , droplets  315  may be forced out of openings  310  and through protective film  205 . In yet another embodiment, an awl, scribe, needle, or other puncturing component may be used to puncture or weaken protective film  205 . For example, top member  110  may be equipped with an awl, scribe or other component configured to puncture or weaken protective film  205  when bottom member  105  and top member  110  are fitted together. 
     One or more of droplets  315  may be a fully constituted reagent. One or more of droplets  315  may, when forced out of opening  310 , contact and combine with one or more reagents on surface of bottom member  105  and/or top member  110  of cassette  100  to yield a fully constituted reagent. In another embodiment, droplet  315  constitutes a sample. For example, a sample may be loaded in reservoir  305  at a point of sample collection, and may later be loaded in accordance with the reagent loading techniques described herein. In another embodiment, droplet  315  constitutes a standard solution with known amount of material that can either be used as a calibrant or can be diluted using droplet operations to setup a standard curve using multiple concentrations derived from performing dilutions. 
     In some embodiments, protective film  205  also serves as an adhesive and/or dielectric layer. For example, a droplet actuator substrate  105  may include electrodes (not shown) associated with substrate  105 . Protective film  205  may be a dielectric layer atop the electrodes, arranged such that the electrodes may be used to conduct droplet operations atop protective film  205 . Protective film  205  may or may not be bound to substrate  105  using an adhesive layer. A hydrophilic coating (not shown) may, in some cases, be provided atop protective film  205 . 
     Substrate  105  may be any rigid substrate, such as a silicon, PCB, plastic, or other polymeric substrate. Electrodes may be any material which is suitably conductive to permit electrodes to mediate droplet operations atop protective film  205 . Examples include copper, chrome, aluminum, gold, silver, indium tin oxide, and other conductive materials. The adhesive layer, when present, may be any adhesive which is suitable for binding protective film  205  to the underlying layers of substrate  105 . In alternative embodiments, the adhesive layer may be absent. Protective film  205  may be any dielectric material, and hydrophobic coating may be any hydrophobic coating that binds to the underlying layers in a manner which is sufficient to permit one or more droplet operations to be conducted atop droplet actuator substrate  105 . The protective film  205  may be coated with a hydrophobic layer. Examples of suitable hydrophobic coatings include fluoropolymers and perfluoroploymers, such as polytetrafluoroethylenes; perfluoroalkoxy polymer resins; fluorinated ethylene-propylenes; polyethylenetetrafluoroethylenes; polyvinylfluorides; polyethylenechlorotrifluoroethylenes; polyvinylidene fluorides; polychlorotrifluoroethylenes; and perfluoropolyethers. In one embodiment, the hydrophobic coating includes an amorphous Teflon fluoropolymer or a TEFLON® fluoropolymer. In another embodiment, the hydrophobic coating includes a CYTOP™ perfluoropolymer. 
     In one embodiment, an adhesive layer binds protective film  205  to electrodes and substrate  105 . In one example, protective film  205  is a polyimide film. In yet another example, the adhesive layer includes an acrylic adhesive. In still another example, an adhesive-backed polyimide film provides adhesive layer and protective film  205 . For example, adhesive-backed polyimide film may be a PYRALUX® LF flexible composite (DuPont). PYRALUX® LF7013, for example is an approximately 13 μM viscous, solid or semi-solid DuPont KAPTON® polyimide film and 25 μM viscous, solid or semi-solid acrylic adhesive. Other examples of suitable adhesive-backed films include PYRALUX® LF LF0110, LF0120, LF0130, LF0150, LF0210, LF0220, LF0230, LF0250, LF0310, LF7001, LF7082, LF1510, and LF7034. 
     In some embodiments, the adhesive is selected to be releasable, so that the adhesive-backed film may be removed following use and replaced with a fresh adhesive-backed film. In some embodiments, the adhesive may serve as the protective film and the backing may serve as a hydrophobic coating. In other embodiments, the dielectric may be formed as a permanent part of the substrate, and a protective film having a hydrophilic backing may be applied to the permanent dielectric. In yet another embodiment, multiple films may be used and replaced together or separately. For example, a hydrophobic film may be used atop a protective film, and both films may be applied atop a droplet actuator substrate including electrodes. Each of the hydrophobic film and protective film may be replaced together or separately, as needed. 
     In one embodiment, the protective film includes a dielectric film, and the droplet actuator substrate includes the substrate, electrodes and a dielectric atop the substrate. The protective film is placed atop the dielectric, and an adhesive may optionally be included between the dielectric and the protective film. 
     In another embodiment, the protective film includes a dielectric film, and the droplet actuator substrate includes the substrate, electrodes and a dielectric atop the substrate. The protective film may be placed atop the dielectric, and an adhesive may optionally be included between the dielectric and the protective film. Alternatively, the droplet actuator substrate may include the substrate and electrodes with no dielectric atop the substrate. The protective film may be placed atop the substrate and electrodes, and an adhesive may optionally be included between the substrate and electrodes and the film. 
     Top member  110  may be equipped with an awl, scribe or other component configured to puncture or weaken protective film  205  when bottom member  105  and top member  110  are fitted together. Plungers  325  may be equipped with an awl, scribe or other component configured to puncture or weaken protective film  205  when plungers  325  are inserted in reservoirs  305 . Once liquid  315  is forced atop substrate  105 , electrodes associated with top member  110  and/or bottom member  105  may be used to effect droplet operations using droplets  315 . In certain embodiments, the awl, scribe or other component configured to puncture or weaken protective film has a hydrophobic surface. 
     In yet another embodiment, the protective film may double as a hydrophobic layer. For example, the wells may be located in the top substrate and separated from the gap by the protective film, doubling as a hydrophobic layer. The protective film may be punctured during loading of droplets into the gap, e.g., by an awl, scribe or other component configured to puncture or weaken protective film, permitting droplets to flow through the punctured region and into the gap where they are subject to droplet operations. In certain embodiments, the awl, scribe or other component configured to puncture or weaken protective film has a hydrophobic surface. 
     In an alternative embodiment, the top substrate and bottom substrate are provided bound together, and separated to provide a droplet operations gap. In this embodiment, the droplets  315  would be forced by the plungers  325  into the gap, where they would be subject to droplet operations using electrodes associated with the top member  110  and/or the bottom member  105 . 
       FIGS. 3C and 3D  illustrates a length-wise cross sectional view  301  of a segment of top member  110  juxtaposed with a cross sectional view  300  of a segment of bottom member  105  described with reference to  FIG. 3B . As shown in  FIG. 3C , top member  110  includes dried reagent  375  affixed thereto. When droplet  315  is forced out of its opening and into contact with dried reagent  375 , droplet  315  combines with dried reagent  375  to yield a fully constituted reagent, as illustrated in  FIG. 3D . 
       FIG. 3E  illustrates an end-wise cross-sectional view of reagent storage cassette  302  showing top member  110  juxtaposed with bottom member  105 . Top member  110  includes channel  306 , which may be any type of liquid path. Bottom member  105  includes reservoirs  305  formed therein. Film  205  seals channel  306  and reservoir  305 . Reservoir  305  includes a liquid, such as a sample or a reagent or a calibrant. Cassette  302  may include multiple reservoirs. Channel  306  may include one or more dried, concentrated or viscous, solid or semi-solidened reagents  375 . Each dried, concentrated or viscous, solid or semi-solidened reagent  375  may be aligned with a corresponding reservoir  305 , such that when liquid  315  is caused to flow into channel  306 , each dried reagent  375  is combined with a droplet of liquid  315  to yield a constituted reagent. Reservoir  305  is associated with plungers  325  and plunger depressor  330 . Plunger depressor  330  is configured to permit a user to force plungers  325  into reservoir  305 , thereby forcing droplets  315  out of openings  310  and into channel  306 . Reservoir  305  is thus bounded and substantially sealed by substrate  105  along opening  310 , film  205  and plunger  325 . In some embodiments, compressible material  380  may be provided between plunger compressor  330  and bottom member  105  to retain plunger  325  in place during storage and shipment. 
     In operation, film  205  may be removed. Plunger  325  may be forced into reservoir  305 , thereby forcing liquid  315  into channel  306 . A series of such liquids  315  may be forced into channel  306 , thereby forming a series of droplets separated by a filler fluid. In cases where the reagent storage cassette is provided as an integral part of a droplet actuator cartridge, droplets  315  may be transported and from the reagent storage cassette into another region of the cartridge. For example, reagent droplets  315  may be transported from the reagent storage cassette into a droplet operations gap of a droplet actuator. Similarly, droplets  315  may be transported from the reagent storage cassette into a reservoir of a droplet actuator, from where they may be transported through a liquid path into a droplet operations gap of a droplet actuator. In the droplet operations gap, droplets  315  and/or sub-droplets dispensed therefrom may be subjected to droplet operations. For example, the droplet operations may be part of a droplet operations protocol which is designed to use droplets  315  to perform an assay. 
     In some embodiments, a pressure source may provide pressure for forcing droplets from the reagent storage cassette into a droplet actuator, or into another region of a droplet actuator cassette. As illustrated in  FIG. 3D , a pressure source may forced droplets  315  through channel  306  and into the droplet actuator. Channel  306  may be in any configuration, for example, it may be linear or curvilinear. Channel  306  may be provided generally in a common plane with a droplet operations gap, such that droplets  315  may flow along a common plane through channel  306  and into the droplet operations gap. Alternatively, channel  306  may be provided in a different plane than the plane of the droplet operations gap. For example, channel  306  may be located in a plane which is separate, but parallel to the playing of the droplet operations gap. A liquid passage may connect channel  306  to the droplet operations gap, such that the pressure source may cause the droplets  315  to flow through channel  306 , through the connecting liquid passage, and into the droplet operations gap. In yet another embodiment, the channel  306  need not be parallel to the droplet operations gap, e.g., the channel may be in a position relative to the droplet operations gap which establishes an angle which is between 0 and 180°. 
     In one embodiment, pressure may be applied to the contents of channel  306 , thereby forcing droplets and filler fluid into a droplet operations gap of the droplet actuator. Alternatively, a vacuum source may be used to pull the contents of channel  306  into a droplet operations gap of a droplet actuator. Further, channel  306  may itself be associated with electrodes capable of effecting forces suitable for causing the transport of droplets  315  along the path of channel  306 . Such electrodes may, for example, form a path having one or more electrode members which are adjacent to electrode members in a droplet operations gap of a droplet actuator. In this manner, the electrodes may be used to transport one or more droplets through channel  306 , and from channel  306  into a droplet operations gap of a droplet actuator. 
       FIG. 4  illustrates another embodiment of the invention in which a droplet actuator cartridge  400  is provided with a droplet actuator portion  401  and an integral reagent cassette portion  402 . In the embodiment illustrated, droplet actuator portion  401  includes top substrate  410  separated from bottom substrate  415  by droplet operations gap  420 . Bottom substrate  415  includes electrodes  418  arranged for conducting one of more droplet operations in droplet operations gap  420 . It will also be appreciated that one or more droplet operations and/or reference electrodes may be associated with top substrate  410  and/or bottom substrate  415 . Reagent cassette portion  402  includes bottom substrate  410 , which is the same as top substrate  410  of droplet actuator portion  401 . Reagent cassette portion  402  also includes top substrate  405 , which includes reservoirs  435  formed therein. As illustrated, plungers  325  are inserted into reservoirs  435 . Channel  306  is formed in top substrate  405  and/or bottom substrate  410 . Channel  306  is connected to droplet actuator gap  420  by liquid path  440 . Channel  306  may also be coupled to a pressure source  445  configured for providing pressure into channel  306 . Pressure source  445  may be coupled to channel  306  by a liquid path  448  established, for example, by capillary tube  450  and associated fitting  455 . Similarly, an output flow path  460  may be coupled to droplet operations gap  420 ; the coupling may, for example, be established by a capillary tube  465  and associated fitting  470 . In this manner, a liquid path is established from pressure source  445  through liquid path  448 , through channel  306 , through connecting liquid path  440 , through droplet operations gap  420 , and through exit liquid path  460 . In some cases, rather than a pressure source  445 , a vacuum source  480  may be coupled via liquid path  460  to droplet operations gap  420 . In operation, droplets  315  may be stored in reservoirs  435 . A film (not shown) may be provided over openings to reservoirs  435  to retain droplets  315  therein. As noted above, the film may be scored in order to facilitate breaking of the film upon application of pressure thereto by insertion of plungers  325 . Alternatively, a puncturing device may be employed, e.g., as illustrated below with respect to  FIG. 6 . In any case, plungers  325  may be forced into reservoirs  435 , thereby forcing droplets  315  into channel  306 . Pressure from pressure source  445  and/or vacuum from vacuum source  480  may be used to cause droplets  315  to flow through channel  306 , through liquid path  440 , and into droplet operations gap  420  where such droplets may be subject to droplet operations mediated by electrodes  418 . In an alternative embodiment, droplet operations and/or reference electrodes may be associated with surfaces adjacent to channel  306  and/or connecting liquid path  440 , and droplets  315  may be transported into droplet operations gap using one or more droplet operations facilitated by such electrodes. The figure is illustrative, and many other embodiments are possible. For example,  FIG. 4  can be considered as a top view (top plate not shown and  410  serves as only a gasket with no electrodes) where the plungers are inserted within the gap of the droplet actuator and electrodes  418  move to underneath the channel  420  while  415  serves as a gasket. 
       FIGS. 5A and 5B  illustrate a side cross-sectional view and a top view, respectively, of a droplet actuator  500  according to the invention. Droplet actuator  500  is like droplet actuator  400 , except that rather than forcing droplets into a channel, which is used to supply droplets into a droplet operations gap, droplet actuator  500  supplies droplets directly into reservoirs in a droplet operations gap. One or more sub-droplets may be dispensed from the reservoirs. Droplet actuator  500  includes top substrate  505  and bottom substrate  510 , separated by gasket  515  to form gap  520 . Top substrate  505  includes droplet operations electrodes  523 , though it will be appreciated that as described elsewhere herein, in an alternative embodiment, droplet operations electrodes  523  may be supplied on bottom substrate  510  rather than top substrate  505 . Bottom substrate  510  also includes reservoirs  525  into which plungers  530  are inserted. Gasket  515  also forms reservoirs  535  in droplet operations gap  520 . Each reservoir  535  includes an electrode  523  associated with top substrate  505  and aligned with reservoir  535 . Adjacent to each electrode  523  is a path of droplet operations electrodes  524 . The paths of droplet operations electrodes  524  are arranged in a network of paths. It will be appreciated that the network of paths illustrated in  FIG. 5B  is illustrative only, and that a wide variety of similar such networks is possible within the scope of the invention.  FIG. 5A  shows droplets  536 , including one droplet in each reservoir  535 . As illustrated, the droplets have been forced in the place using plungers  530 , i.e., by forcing plungers  530  into reservoirs  525 . In operation, droplet actuator  500  may include a protective film as described herein, which may be removed and/or punctured prior to forcing droplets  536  into place within reservoirs  535 . Ideally, the top surface of each plunger is hydrophobic or is coated with a hydrophobic material in order to facilitate droplet operations conducted using electrodes  523  and  524 . Further, the surface of reservoir  525  and/or reservoir  535  may also be hydrophobic or coated with a hydrophobic material. 
       FIGS. 6A, 6B, 6C, 6D, 6E, and 6F  illustrate another aspect of the invention in which a plunger is used to force liquid into the droplet operations gap of a droplet actuator. As illustrated in  FIG. 6A , droplet actuator  600  includes a top substrate  605  and a bottom substrate  610  separated by gasket  615  the form drop operations gap  620 . Reservoir  625  is formed on bottom substrate  610 . As illustrated, reservoir  625  includes a liquid  630 , which may, for example, include reagent and/or sample. Bottom substrate  610  further includes electrodes  635  arranged for conducting droplet operations in the droplet operations gap. Bottom substrate  610  further includes an electrode  636 , which includes an opening  637  therein.  FIG. 6A  shows liquid  630  in reservoir  625  outside the droplet operations gap  620 , prior to being forced by plunger  650  into the droplet operations gap  620 . 
       FIG. 6B  shows a top view of electrode  636 . Opening  637  is shown as being centrally located, but it will be appreciated that the opening may be provided in any region of electrode  636 . Further, in an alternative embodiment, no opening is provided in electrode  636 , and instead, an opening providing a liquid path into the droplet operations gap is provided adjacent to electrode  636 . It will also be appreciated that while the opening is shown as being generally circular, any shape is suitable. Moreover, while a single opening is shown, multiple openings may be provided. Opening  637  provides a liquid path  645  from reservoir  625  into droplet operations gap  620 . 
     Bottom substrate  610  further includes a dielectric layer  640  atop electrodes  635  and  636 . Dielectric layer  640  blocks the liquid path. Various examples of a dielectric layer are as described above with respect to aspects in which protective film  205  is a dielectric layer. A hydrophobic layer may also be provided atop dielectric layer  640 . Droplet actuator  600  also includes a plunger  650 , which extends into reservoir  625 , and seals liquid  630  therein. Plunger  650  includes an awl  655  arranged for puncturing dielectric layer  640  to open liquid path  645 , thereby permitting liquid  630  to flow through liquid path  645  and into droplet operations gap  620 . As illustrated, awl  655  is inserted through an opening in plunger  650  and aligned to puncture dielectric layer  640  through opening  637 . 
     Puncturing of dielectric layer  640  is illustrated in  FIG. 6C . On puncturing dielectric layer  640 , plunger  650  may be forced into reservoir  625 , thereby forcing liquid  630  through liquid path  645  into droplet operations gap  620 . 
       FIG. 6D  illustrates plunger  650  in a fully inserted position, and shows liquid  630  situated in droplet operations gap  620  atop electrode  636 . From this position, droplets may be dispensed from liquid  630  using electrodes  635  and  636 . Awl  655  is shown in a retracted position in which the tip of awl  655  is removed from the punctured region of dielectric  640  in order to permit liquid to flow with reduced obstruction through liquid path  645 . 
       FIG. 6E  shows droplet actuator  601 , which is like droplet actuator  600 , except that in droplet actuator  601 , a single electrode  660  is provided on top substrate  605 . Electrode  660  may serve as a reference electrode. In one embodiment, top substrate  605  is made from a transparent material, such as glass or plastic, while electrode  660  is also made from a transparent electrode material, such as indium tin oxide. 
       FIG. 6F  shows droplet actuator  602 , which is like droplet actuator  601  in  FIG. 6E , except that in droplet actuator  602 , a reservoir electrode  636  and droplet operations electrodes  635  are provided on top substrate  605 , while ground or reference electrode  660  is provided on bottom substrate  610 . Fluid  630  flows into droplet operations gap  620  through liquid path  645 , which includes an opening  637  in ground electrode  660 . 
     In other embodiments, the plunger  650  is not required and only the awl/needle  655  is utilized. As shown in  FIG. 6D , plunger  650  serves as a fixed element and an integral part of bottom substrate  610  and in some cases they both may be the same element. The needle  655  in this case may be actuated during the action of loading the cartridge. The needle puncturing the dielectric  640  and part of the electrode  636  may be hydrophilic so that upon puncturing the liquid automatically is drawn onto electrode  636 . In another embodiment, the needle and the reservoir arrangement could be on the top plate  605 . 
       FIG. 7  illustrates an alternative embodiment of the reagent cassette of the invention. In addition to the components already described, this embodiment includes a channel  705  for flowing an immiscible liquid filler fluid around droplets  315  and/or droplets  605 . Further, top member includes a top plunger member  710 , which may be used to agitate droplet  315  in the presence of dried reagent  481  to promote mixing. Top plunger member  710  may be associated with a sonicator arranged to vibrate top plunger member  710  and thereby promote mixing of dried reagent  481  in droplet  315 . 
     Steps A-F illustrate the following: Step A shows top member  305  and bottom member  405  with protective film  205  in place, protecting droplets  315  and dried reagent  481 . Step B shows top member  305  and bottom member  405  with protective film  205  removed, and top member  305  and bottom member  405  fitted together. Step C shows plunger  325  compressed to force droplet  315  into channel  505 . Step D shows droplet  315  mixed with dried reagent  418 . Step E shows filler fluid flowed through channels  705  into space in channel  505  surrounding droplets  315 . Step F shows compression of plunger member  710  to compress droplet  315 , e.g., to cause mixing of droplet  315 . 
     Various embodiments may include a filler fluid reservoir in association with channel  705  and/or channel  505  for flowing oil into channel  505 . In other embodiments, droplets  315  may include beads and/or dried reagents may include beads which dissolve into droplets  315 . Beads may, for example, have affinity for target analytes or compounds that interfere with assay chemistry. Some embodiments may include vents from channel  705  and/or channel  505  for venting bubbles prior to loading droplets onto a droplet actuator or other microfluidic device. Protective films may be made from any material which is suitably non-reactive with reagents contacting the films. Examples include aluminum and various polymeric films. Dried reagents for use in the cassette may be prepared using methods known to one of skill in the art, such as commercial off-the-shelf (COTS) equipment and well-established procedures. 
     Flow Through Bead Handling and Washing Techniques 
     The invention also provides devices, techniques and systems for conducting flow through bead handling and washing. For example, the invention provides techniques for splitting droplets in a flow-through system, compartmentalizing beads in droplets in a flow-through system, and washing droplets in a flow-through system. 
       FIG. 8  illustrates a flow-through system  800  that makes use of droplet operations for splitting droplets. Flow-through system  800  includes channel  805  which intersects with channel  810 . A set of electrodes  812  are associated with channel  810  at a position which is approximately opposite to an entry point of channel  805  into channel  810 . The internal walls of channels  805  and  810  are hydrophobic. Channels  805  and  810  are filled with a liquid filler fluid which is substantially immiscible with parent droplets  815 . Parent droplets  815  flow through channel  805  in the direction of arrow A. When electrodes  812  are activated, the internal wall of channel  810  in the region of electrodes  812  behaves in hydrophilic manner. When a parent droplet  815  impacts the electrode-associated region, the droplet spreads to conform to the shape of the activated electrodes. When an intermediate electrode is deactivated, the droplet splits into two sub-droplets  816 . In the embodiment illustrated, the two sub-droplets  816  flow into channel  810  in opposite directions, as illustrated by arrows B and C. In operation, by controlling the flow of filler fluid through channels  805  and  810 , droplets  815  may be sequentially contacted with electrodes  812 , electrode  812  may be used to split each droplet into two sub-droplets  816 , and sub-droplets  816  may be flowed into channel  810 , as indicated. In an alternative embodiment, it will be appreciated that a flow may be established in channel  810  which causes sub-droplets  816  to flow in the same direction, i.e., arrows B and C may indicate a flow in a common direction, rather than opposite directions. 
       FIG. 9  illustrates a flow-through system  900  configured for adding beads to droplets. System  900  includes channel  905  which intersects with channel  910 . A liquid filler fluid in channel  905  flows in the direction of arrow A. A liquid filler fluid in channel  910  flows in the direction of arrow B. Droplets  915  are provided in channel  905 . The liquid filler fluid in channel  905  is substantially immiscible with droplets  915 . Droplets  915  flow through channel  905  into channel  910  at a velocity sufficient to cause them to impact a region on the wall of channel  910 . The various dimensions of channel  905 , channel  910  as it enters the intersection between the two channels, and channel  910  as it exits the intersection between the two channels, as well as the angle of intersection and the velocity of filler fluid flow through the respective channels may be adjusted as needed to achieve the pre-selected droplet impact on the wall of channel  910 . A magnet  920  is associated with channel  910  at a position which is approximately the point of impact of droplets  915  on the wall of channel  910 . The magnet may be adjustable in order to align it with the appropriate location at which droplets  915  impact the wall of channel  910 . The magnet may be an electromagnet, which may be switched on and off. The magnet may be a permanent magnet, which is movable, e.g., generally in the direction of the axis indicated by arrow A. Beads  916  are provided in the filler fluid which flows through channel  910 . Beads  916  may be hydrophilic and may be provided in a hydrophobic filler fluid. As each bead comes into proximity with magnet  920 , the bead is substantially immobilized on magnet  920 . When a droplet  915  impacts a bead  916  immobilized on magnet  920 , bead  916  is engulfed by the droplet, yielding bead containing droplet  917 . The bead-containing droplet  917  may continue to flow through channel  910  in the direction of arrow C. Various techniques may be used to separate bead containing droplet  917  from the magnet  920  to permit bead containing droplet  917  to continue to flow-through channel  910 . For example, the surface tension of droplet  915  may be selected to overcome the attractive force of magnet  920  on the bead, as the bead containing droplet  917  is forced through channel  910  by the flowing filler fluid. In this embodiment, it is not necessary to remove or deactivate magnet  920 . In another embodiment, magnet  920  is an electromagnet, and the electromagnet is switched off to release the bead-containing droplet  917 . In yet another embodiment, magnet  920  is removable, and magnet  920  is physically moved away from channel  910  in order to permit the release of bead containing droplet. The spacing of droplets  915  and beads  916  may be adjusted in order to achieve a pre-selected number of beads and each droplet. For example, several beads may be permitted to collect at magnet  920  between each droplet  915  in order to provide droplets with multiple beads. Droplets potentially containing beads may be tested downstream, and sorted to exclude any droplets which lack beads or which lack the pre-selected number of beads. Sorting may, for example, be based on optical properties and/or electrical properties of the bead-containing droplets. 
       FIG. 10  illustrates a flow-through system  1000  which makes use of droplet operations to wash beads in droplets. Flow-through system  1000  includes channel  1005 , which intersects with channel  1010 . A liquid filler fluid in channel  1005  flows in the direction of arrow A. A liquid filler fluid in channel  1010  flows in the direction of arrow B. Bead-containing droplets  1015  are provided in channel  1005 . Wash droplets  1016  are provided in channel  1010 . Wash droplets  1016  may include a wash buffer. It will also be appreciated that in an alternative embodiment, rather than washing the beads, the method is used to concentrate one or more substances onto the beads. In such other embodiment, wash droplets  1016  may be replaced with sample droplets or other droplets including droplets including one or more target substances for which the beads have affinity. In yet another embodiment, rather than a single magnet  1020  attracting bead-containing droplet  1015  to the wall of channel  1010 , one or more magnets may be provided around channel  1010  and arranged to substantially immobilized the bead within the channel, but away from the wall of the channel. The size of channel  1010  at magnet  1020  may be selected to ensure that wash droplets  1016  impact immobilized bead containing droplet  1015  as they flow past magnet  1020  or other magnet arrangement. The velocity of impact is selected to cause droplets  1016  to impact droplet  1015 , merge with droplet  1015 , followed by a breaking off of a new droplet  1017  moving in the direction of arrow C. In this manner, by sequentially merging the bead containing droplet with a wash droplet in and breaking off a separate droplet, the liquid surrounding the bead-containing droplet maybe be depleted of unwanted substances. Upon completion of the wash cycle, when the depletion of unwanted substances is calculated to have been achieved based on the number of wash droplets passed across the bead, the bead containing droplet may be released to continue to flow-through channel  1010 . Downstream, the bead containing droplets may be separated from the used wash droplets  1017 . Thus, the invention provides a technique for washing beads in a flow-through operation, wherein a bead containing droplet is immobilized using a magnet, and one of more wash droplets are caused to impact and merge with the bead-containing droplet, and wherein the filler fluid flowing through the channel is at a velocity sufficient to cause one or more droplets to break off of the combined droplet, thereby leaving a bead containing droplet with a reduced amount of one or more substances relative to the starting bead-containing droplet. Similarly, the invention provides a technique for concentrating a substance on beads in a flow-through operation, wherein a bead containing droplet is immobilized using a magnet in a channel, and one of more droplets including a target substance are caused to impact and merge with the immobilized bead-containing droplet, thereby causing a bead in the bead-containing droplet having affinity for the target substance to concentrate target substance thereon. As with the washing operation, the filler fluid flowing through the channel may cause one or more droplets to break off of the combined droplet, thereby leaving a bead containing droplet with an increased amount of one or more substances concentrated on the bead relative to the starting bead-containing droplet. The various sizes of channels  1005  and  1010 , as well as the angle of intersection between the two channels, may be adjusted in order to improve efficiency of the washing operation. Multiple beads may also be present in droplets  1015 . The ratio of spacing and velocity of bead containing droplets  1015  flowing through channel  1005  relative to the spacing and velocity of wash droplets or sample droplets flowing through channel  1010  may be adjusted to achieve the pre-selected effect. In yet another embodiment, channel  1010  may include a series of sample droplets for concentrating sample onto the immobilized bead, followed by a series of wash droplets for washing the immobilized bead. In an alternative embodiment, the splitting off of wash droplets following merging of the wash droplets with the immobilized beat-containing droplet may be facilitated by droplet operations mediated by electrodes, e.g. as described above with reference to  FIG. 8 . 
     In the various flow-through embodiments described herein, it is possible for droplets to be sorted to select out a pre-selected subset of droplets from the overall droplet population. For example, droplets may be sorted as described in Link et al., US Patent Publication No. 20080014518, entitled “Microfluidic Devices and Methods of Use Thereof,” published on Jan. 17, 2008, the entire disclosure of which is incorporated herein by reference for its teaching concerning sorting of droplets in microfluidic devices. Further, once droplets of interest are isolated, the droplets may be flowed onto a droplet actuator of the invention for further analysis. For example, a subset of droplets of interest from a flow-through droplet sorting operation may be flowed into a droplet operations gap of a droplet actuator where they are subject to droplet operations mediated by electrodes. Similarly, a subset of droplets of interest from a flow-through droplet sorting operation may be flowed into a reservoir of the droplet actuator, which reservoir is coupled by a liquid path to a droplet operations gap of a droplet actuator, such that the droplets of interest may be transported from the reservoir into the droplet operations gap where they may be subject to droplet operations mediated by electrodes. In one embodiment, multiple droplets of interest are pooled together in a reservoir of a droplet actuator prior to being subjected to droplet operations in a droplet operations gap of the droplet actuator. 
     Techniques Using Viscous, Solid, or Semi-Solid Samples 
     The invention provides droplet actuator devices, techniques and systems for making and using droplet actuators to process viscous, solid or semi-solid samples. For example, the invention provides a technique for processing viscous, semisolid, and/or solid samples. Target substances of interest are extracted from the viscous, solid or semi-solid sample, and then processed using standard droplet operations. 
       FIGS. 11A, 11B, and 11C  illustrate a section of a droplet actuator  1100  and a method of processing a viscous, solid or semi-solid sample on a droplet actuator. Droplet actuator  1100  includes a top substrate  1105  and a bottom substrate  1110  separated by droplet operations gap  1112 . In certain embodiments, top substrate  1105  may be omitted. Droplet operations electrodes  1115  (e.g., electrowetting electrodes) and reference electrodes (not shown) are associated with top substrate  1105  and/or bottom substrate  1110 . Droplet operations electrodes  1115  are configured for conducting one or more droplet operations in droplet operations gap  1112 . Top substrate  1105  includes an opening  1120  therein for loading sample  1125  into droplet operations gap  1112 . Sample  1125  includes one or more target substances  1130 . As illustrated in  FIG. 11A , droplet  1135  is positioned in droplet operations gap  1112  atop one or more droplet operations electrodes  1115 .  FIG. 11B  shows droplet  1135  being transported into contact with sample  1125 , such that one or more target substances  1130  is dissolved into droplet  1135 . Transport of droplet  1135  may be effected using one or more droplet operations. For example, in one embodiment, transport is effected by sequentially activating/deactivating electrodes  1115 . Droplet  1135  may be transported away from sample  1125  via droplet operations as shown in  11 C. Droplet  1135  that potentially included one or more target substances may be used as input for conducting one or more assays to identify and/or quantify one or more target substances  1130 . In one embodiment, sample  1125  is sufficiently viscous, semi-solid, or solid in order to permit droplet  1135  to contact sample  1125  and be transported away from sample  1125  without being substantially mixed with sample  1125 . 
       FIGS. 11A, 11B, and 11C  illustrate a general principle in which a micro or nano liquid is transported into contact with a viscous or solid sample for collection of a target substance and then transported away. As illustrated, using one or more droplet operations, droplet  1135  contacts sample  1125 , which brings droplet  1135  into lateral contact with sample  1125 . However, it will be appreciated that sample  1125  may be positioned at any angle relative to droplet  1135 , e.g., above or below droplet  1135 . For example, sample  1125  may project only slightly into droplet operations gap  1112 , in which case, droplet  1135  may be transported along a path of electrodes underneath sample  1125 . In this example, contact is between the top of droplet  1135  and the bottom of sample  1125 . Alternatively, sample  1125  may be exposed to droplet operations gap  1112  and droplet  1135  via an opening (not shown) in bottom substrate  1110 . 
     The methods of the invention are particularly suitable for tests involving viscous, solid or semi-solid samples. Samples may, for example, be environmental samples, process samples, or biological samples. Examples of suitable samples include sputum, coagulated blood, animal tissue samples, plant tissue samples, soil samples, rock samples, and the like. In some cases, samples are sufficiently viscous, semi-solid or solid to permit a droplet to contact the sample and be transported away from the sample without being substantially mixed with the sample. Further, the sample may include foreign matter, such as a matrix (e.g., a swab) used to collect the sample. For example, when a droplet of sputum is loaded, it may not be readily transportable using droplet operations then a droplet that lyses sputum can be brought in contact with sputum. After incubation and preferably some agitation of the lysis droplet, the sputum will be liquefied rendering it to be transportable using droplet operations. 
     Droplet  1135  may be aqueous or non-aqueous. In one embodiment, droplet  1135  is an aqueous buffer established at a pH which is suitable for dissolving sample  1125 . Droplet  1135  may also include one or more reagents. The chemical characteristics of droplet  1135  may be adjusted to render droplet  1135  suitable for acquiring one or more target substances  1130 . In one example, droplet  1135  includes a lysis buffer solution. A lysis buffer solution is used to lyse cells for use in assays involving target substances, which are sub-components of the cells. In some embodiments, droplet  1135  includes one or more beads, e.g., magnetically responsive or non-magnetically responsive beads. Examples of suitable magnetically responsive beads are described in U.S. Pat. No. 7,205,160, entitled, “Multiplex flow assays preferably with magnetic particles as solid phase,” granted on Apr. 17, 2007. The beads may have an affinity for one or more target substances or contaminants. For example, the beads may have affinity for target cells, protein, DNA, and/or antigens. In one example, the beads may have an affinity for one or more target substances  1130  from the sample  1125  of interest. 
     Examples of droplet actuator techniques for immobilizing magnetic beads and/or non-magnetic beads are described in the foregoing international patent applications and in Sista, et al., U.S. Patent Application Nos. 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 are incorporated herein by reference. 
     Gel Electrophoresis Techniques 
     The invention provides droplet actuator devices including a gel for use in gel electrophoresis, along with techniques and systems for conducting gel electrophoresis on a droplet actuator. The gel electrophoresis techniques of the invention are useful for separating substances present in a droplet on a droplet actuator. For example, the invention is useful for separating complex biomolecules (e.g., proteins and/or nucleic acids) using an electric current applied to a gel matrix on a droplet actuator. The gel matrix may, for example, be a cross-linked polymer whose composition and porosity are selected based on the specific weight (e.g., molecular weight) and composition of the substances being analyzed. In one embodiment, the gel may be composed of different concentrations of acrylamide and a cross-linker to produce different-sized mesh networks of polyacrylamide. Polyacrylamide may be used to separate and analyze proteins or small nucleic acids (e.g., DNA, RNA, or oligonucleotides). In another embodiment, the gel may be composed of a purified agarose matrix. Agarose gels may be used to separate larger nucleic acids and/or complex biomolecules. 
     The methods of the invention make use of gel electrophoresis on a droplet actuator for analytical purposes (e.g., separation and quantitation of a specific target(s)). In another embodiment, gel electrophoresis may be used as a preparative technique (e.g., for isolation of a specific target(s)) prior to use of other assay techniques for further characterization of a substance. Other assay techniques may, for example, include PCR, cloning, nucleic acid sequencing, immunoassays, enzymatic assays, exposure to sensors, etc. In another embodiment, a “capture” droplet may be used to capture a target droplet as it elutes off the gel slug as a fraction collector, e.g. using the techniques described with reference to  FIG. 11 . 
       FIGS. 12A, 12B, and 12C  illustrate a section of droplet actuator  1200  and a process of separating and analyzing a sample using gel electrophoresis. Droplet actuator  1200  may include bottom substrate  1210  separated from top substrate  1214  by droplet operations gap  1213 . Path  1216  of droplet operations electrodes  1217  is arranged on bottom substrate  1210 ; however, it will be appreciated that droplet operations electrodes and/or ground electrodes may be associated with top substrate  1214  and/or bottom substrate  1210 . Droplet operations electrodes  1217  may, for example, be electrowetting electrodes. Electrophoresis electrodes  1218   a  and  1218   b , are arranged on top substrate  1214 , but may be on either or both substrates. One of electrodes  1218   a  and  1218   b  may be a negative electrode, while the other may be a positive electrode. 
     Droplet operations gap  1213  may be provided with one or more gel droplets  1226  and one or more catalyst droplets  1230 , although in some cases neither a catalyst nor a catalyst droplet are required. In some cases, the catalyst may just be photoinitiation. Gel droplets  1226  may typically be from about 1× to about 5× or larger droplets. A 3× droplet, for example, has a footprint that is approximately 3 times the area of one droplet operations electrode  1217 . Gel droplet  1226  may, for example, include reagents suitable for forming a polyacrylamide gel, such as acrylamide, bis-acrylamide, and buffer. Gel droplet  1226  remains in a liquid form until polymerization of the acrylamide is initiated by the addition of a catalyst. Catalyst droplet  1230  contains the chemical reagents required to accelerate polymerization of gel droplet  1226 . For example, catalyst droplet  1230  may include N,N,N,N-Tetramethyl-Ethylenediamine (TEMED) and ammonium persulfate to accelerate polymerization of the acrylamide in gel droplet  1226 . 
     Droplet operations gap  1213  may be provided with one or more sample droplets, e.g., sample droplet  1234 . Sample droplet  1234  includes one or more target substances  1242  to be evaluated. Target substances  1242  may, for example, be fluorescently labeled proteins or nucleic acids. To evaluate target substances  1242 , an imaging device  1240  is associated with droplet actuator  1200 . Imaging device  1240  may be used to capture digital images of substances separated in gel droplet  1226 , such as labeled proteins or nucleic acids. In some cases, imaging device  1240  may capture images through top substrate  1214 , which may be, for example, a glass or a plastic plate that is substantially transparent. 
       FIG. 12A  shows a first step in which droplet operations may be executed in order to form a gel for conducting gel electrophoresis on droplet actuator  1200 . Activated electrodes are shown in black. Using one or more droplet operations, gel droplet  1226  may be elongated along several droplet operations electrodes in contact with electrophoresis electrodes  1218   a  and  1218   b . Catalyst droplet  1230  may be transported using one or more droplet operations into contact with gel droplet  1226 . Catalyst droplet  1230  and gel droplet  1226  merge, initiating polymerization in gel droplet  1226  to form the gel matrix for electrophoresis.  FIG. 12B  shows a second step in which sample droplet  1234  is transported into contact with the polymerized gel droplet  1226 .  FIG. 12C  shows a third step in which an electrical potential (e.g., about 40 to about 100 volts) may be applied to gel droplet  1226  via electrophoresis electrodes  1218   a  and  1218   b . In some cases, electrode  1218   a  might directly contact droplet  1234 . The electrical current causes target substances  1242  in sample droplet  1234  to electrophorese into and through gel droplet  1226 . Separation of target substances  1242  in gel droplet  1226  is typically determined by charge such that different molecules will move at different rates. As an example, target substances  1242  may be negatively charged (e.g., nucleic acids) and migrate from electrophoresis electrode  1218   a  (i.e., negative electrode) toward electrophoresis electrode  1218   b  (i.e., positive electrode). Imaging device  1240  may be used to capture an image of separated target substances  1242  in gel droplet  1226  and/or as they elute off the end of gel droplet  1226 . The captured image may be used to identify and/or quantitate different target substances  1242  in sample  1234 . 
     It will be appreciated that the method of the invention also provides a generic method of forming a polymerized structure in a droplet operations gap of a droplet actuator. The method may include using one or more droplet operations to form a first droplet into a pre-selected shape, and to contact the first droplet with a second droplet to cause polymerization of the combined droplets. One of the first droplet and/or second droplet may be a polymer droplet, while the other of the first droplet and/or second droplet may be a catalyst droplet. In addition to use for forming gels for electrophoresis, the method may be used to provide a physical obstacle on a droplet actuator. The physical obstacle may, for example, be useful for sealing off a region of the droplet actuator. In one embodiment a droplet actuator is provided that includes a barrier in the droplet operations gap establishing two regions on the droplet actuator. An opening is provided in the barrier, and electrodes are arranged for transporting droplets through the opening. When it is desirable to close the opening, a polymer droplet is polymerized in the opening. For example, a polymer droplet may be transported into the opening. A catalyst droplet may be combined with the polymer droplet in the opening. Upon polymerization, the opening may be substantially closed. 
     Fluidics System for Loading Droplet Actuator 
     The invention provides a fluidics system and technique for using the system for loading liquids onto a droplet actuator. The invention also provides droplet actuators loaded using the fluidics system and method of the invention and methods of using such droplet actuators to conduct droplet operations. In some embodiments, the loading provides a droplet actuator in which the droplet operations gap or a reagent storage channel is fully filled with filler fluid and reagents that is substantially lacking in air bubbles. 
       FIG. 13A-13I  are schematic diagrams of fluidics system  1300  for loading liquid receptacle, such as a channel or droplet operations gap of a droplet actuator, with liquid. Fluidics system  1300  may include an arrangement of one or more valves, one or more pumps, one or more capillaries, and one or more liquid supply vessels; all fluidly connected. Additionally, a droplet actuator may be fluidly connected to fluidics system  1300 , such that liquid may be flowed from fluidics system  1300  into a liquid receptacle of droplet actuator  1320 . 
     Fluidics system  1300  includes a plurality of valves, illustrated here as pinch valves (PV): PV1, PV2, PV3, PV4, and PV5. A pinch valve is a valve in which a flexible tube is pinched between one or two moving external elements in order to stop the flow through the tube. 
     Fluidics system  1300  includes one or more pumps, illustrated here as peristaltic pump P1. In a peristaltic pump, liquid is contained within a flexible tube fitted inside a circular pump casing. A rotor with one or more of rollers, shoes, or wipers that are attached to the external circumference compresses the flexible tube. As the rotor turns, the part of tube under compression closes, which forces the liquid to be pumped to move through the tube. Referring to  FIG. 13A , peristaltic pump P1 may controlled to operate in a clockwise (CW) and counter clockwise (CCW) direction. The peristaltic pump may be replaced with any suitable pump type, such as gear pumps, progressing cavity pumps, roots-type pumps, reciprocating-type pumps, double-diaphragm pumps, peristaltic pumps, kinetic pumps, centrifugal pumps, eductor-jet pumps, etc. 
     Fluidics system  1300  also includes a pump, such as syringe pump P2. Syringe pump includes a cylinder that holds a quantity of liquid, such as filler fluid (e.g., silicone oil), which is expelled by a piston. The piston may be advanced or retracted by a motor (not shown) connected thereto, in order to provide smooth pulseless flow. 
     Fluidics system  1300  includes a liquid supply vessel V1, which is, for example, any vessel for holding a quantity of liquid, such as filler fluid (e.g., silicone oil). 
     Fluidics system  1300  includes another liquid supply, illustrated here as a multi-well plate (MWP1). MWP1 contains, for example, multiple reservoirs including reagents  1310  (and/or sample) under a layer of filler fluid  1314  (e.g., silicone oil). A mechanically or robotically controlled supply line  1318  may be manipulated in the X, Y, and Z directions in order to access a certain one of the multiple fluids that are contained in MWP1. In an alternative embodiment, multiple supply lines may be provided extending into the MWP1 reservoirs from the top, or through an opening in the reservoirs, such as opening in the bottom of the reservoirs. 
     Fluidics system  1300  includes a capillary CP1, which is a small diameter tube of any pre-selected length, depending on the pre-selected quantity of liquid to be contained therein. Various liquid lines L fluidly connect the parts of the invention. 
     Fluidics system  1300  may include droplet actuator  1320 , which is the droplet actuator to be loaded by fluidics system  1300 . Droplet actuator  1320  is fluidly connected to fluidics system  1300  via one or more liquid input/output ports. The ports provide a fluid path from an exterior of the droplet actuator into a droplet operations gap of the droplet actuator or into another reservoir in the droplet actuator, such as a channel reservoir. In one example, the droplet operations gap of droplet actuator  1320  is fluidly connected to fluidics system  1300  via ports C1, C2, and C3. In some cases, the ports may provide access to one or more channels within droplet actuator  1320 , and the one or more channels are used to supply filler fluids and/or reagents into a droplet operations gap of droplet actuator  1320 . 
     Referring again to  FIG. 13A , the elements of fluidics system  1300  are fluidly connected as follows. A liquid line L fluidly connects vessel V1 to one opening of valve PV1. A liquid line L fluidly connects to the opposite opening of valve PV1 to an opening of T-connection T1. A liquid line L fluidly connects a first branch of T1 to an opening of valve PV2. A liquid line L fluidly connects the opposite opening of valve PV2 to port C1 of droplet actuator  1320 . A liquid line L fluidly connects a second branch of T1 to one opening of peristaltic pump P1. A liquid line L fluidly connects to the opposite opening of peristaltic pump P1 to one opening of capillary CP1. A liquid line L fluidly connects the opposite opening of capillary CP1 to a T-connection, T2. A liquid line L fluidly connects a first branch of T2 to one opening of valve PV4. A liquid line L fluidly connects the opposite opening of valve PV4 to port C2 of droplet actuator  1320 . A liquid line L fluidly connects a second branch of T2 to one opening of valve PV5. A liquid line L fluidly connects the opposite opening of valve PV5 to supply line  1318  that fluidly connects to MWP1. An input/output port of syringe pump P2 fluidly connects to port C3 of droplet actuator  1320  through valve PV3. Note that all liquid lines of fluidics system  1300  may be capillaries and that capillary CP1 may be formed of an extended length of capillary that couples peristaltic pump P1 and junction T2. 
     Fluidics system  1300  of  FIG. 13A  is exemplary only, other system variations are possible. For example, syringe pump P2 may be replaced with other types of pumps. Alternatively, fluidics system  1300  may include a single pump only. An exemplary method of purging air from fluidics system  1300  and droplet actuator  1320  and filling fluidics system  1300  and droplet actuator  1320  with substantially bubble-free liquid is described with reference to  FIGS. 13B-13I . 
     Purging Fluidics System—Step 1 
       FIG. 13B , with reference to Table 1 below, illustrates a purging step in which valves PV1 and PV5 are open, valves PV2, PV3, and PV4 are closed, peristaltic pump P1 is activated in the CW direction, and syringe pump P2 is stopped. This arrangement establishes a flow of liquid from vessel V1, through valve PV1, through peristaltic pump P1, and into capillary CP1. Liquid displaces air in the path from vessel V1 to capillary CP1. Air is vented through valve PV5 to supply line  1318 . Upon completion of this step, a quantity of liquid from vessel V1 that is sufficient to fill the liquid line between T1 and port C1 of droplet actuator  1320  is contained in capillary CP1. 
                                             TABLE 1               PV1   PV2   PV3   PV4   PV5   P1   P2                   OPEN   CLOSED   CLOSED   CLOSED   OPEN   CW   STOP                    
Purging Fluidics System—Step 2
 
       FIG. 13C , with reference to Table 2 below, illustrates a second purging step in which valves PV2 and PV4 are open, valves PV1, PV3, and PV5 are closed, peristaltic pump P1 is activated in the CCW direction, and syringe pump P2 is stopped. This arrangement establishes a flow of liquid from capillary CP1, through peristaltic pump P1, through valve PV2, and into port C1 of droplet actuator  1320 . Liquid displaces air in the path from T1 to port C1 of droplet actuator  1320 . Air is vented through port C2 of droplet actuator  1320  and through valve PV4. 
                                             TABLE 2               PV1    PV2   PV3   PV4   PV5   P1   P2                   CLOSED   OPEN   CLOSED   OPEN   CLOSED   CCW    STOP                    
Purging Fluidics System—Step 3
 
       FIG. 13D , with reference to Table 3 below, illustrates a third purging step in which valves PV1 and PV5 are open, valves PV2, PV3, and PV4 are closed, peristaltic pump P1 is activated in the CW direction, and syringe pump P2 is stopped. This arrangement establishes a flow of liquid from vessel V1, through valve PV1, through peristaltic pump P1, through capillary CP1, through one branch of T2, through valve PV5, and through supply line  1318  to MWP1. Liquid displaces air in the path from vessel V1 to MWP1. Air is vented through supply line  1318  to MWP1. 
                                             TABLE 3               PV1   PV2   PV3   PV4   PV5   P1   P2                   OPEN    CLOSED   CLOSED   CLOSED   OPEN   CW   STOP                    
Purging Fluidics System—Step 4
 
       FIG. 13E , with reference to Table 4 below, illustrates a fourth purging step in which valves PV1, PV2, and PV3 are open, valves PV4 and PV5 are closed, peristaltic pump P1 is stopped (i.e., pump P1 acts as a closed valve), and syringe pump P2 is activated in a direction selected to pull liquid from fluidics system  1300 . This arrangement establishes a flow of liquid from vessel V1, through valve PV1, through T1, through valve PV2, through droplet actuator  1320  from port C1 to port C3, through valve PV3, and into the cylinder of syringe pump P2. Liquid displaces air in the path from vessel V1 to syringe pump P2. Droplet actuator  1320  is purged of air. Air is drawn into syringe pump P2. 
                                             TABLE 4               PV1   PV2   PV3   PV4   PV5   P1   P2                   OPEN   OPEN   OPEN   CLOSED   CLOSED   STOP   PULL                    
Purging Fluidics System—Step 5
 
       FIG. 13F , with reference to Table 5 below, illustrates a fifth purging step in which valves PV3, PV4, and PV5 are open, valves PV1 and PV2 are closed, peristaltic pump P1 is stopped (i.e., pump P1 acts as a closed valve), and syringe pump P2 is activated in a direction to push liquid into fluidics system  1300 . This arrangement establishes a flow of liquid from syringe pump P2, through valve PV3, through droplet actuator  1320  from port C3 to port C2, through valve PV4, through T2, through valve PV5, and through supply line  1318  to MWP1. Liquid displaces air in the path from syringe pump P2 to MWP1. Air is vented through supply line  1318  to MWP1. 
     
       
         
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
               
                   
               
               
                 PV1 
                 PV2 
                 PV3 
                 PV4 
                 PV5 
                 P1 
                 P2 
               
               
                   
               
             
             
               
                 CLOSED 
                 CLOSED 
                 OPEN  
                 OPEN  
                 OPEN 
                 STOP 
                 PUSH 
               
               
                   
               
             
          
         
       
     
     At the completion of this step, all air has been purged from fluidics system  1300 , and droplet actuator  1320 . All liquid lines and elements of fluidics system  1300  and all channels of droplet actuator  1320  are filled with liquid and substantially free of air bubbles. 
     Loading Droplet Actuator—Step 1 
       FIG. 13G , with reference to Table 6 below, illustrates an exemplary first step in a method of loading a droplet actuator. Fluidics system  1300  has two pumps, peristaltic pump P1 and syringe pump P2, that are available for loading reagents into droplet actuator  1320 . Peristaltic pump P1 of fluidics system  1300  is used for loading reagents into droplet actuator  1320 . Valves PV1 and PV5 are open, valves PV2, PV3, and PV4 are closed, peristaltic pump P1 is activated in the CCW direction, and syringe pump P2 is stopped. Additionally, using the xyz-motion, supply line  1318  is inserted into a well of MWP1 that contains the pre-selected reagent  1310 . A certain amount of reagent  1310  is drawn from MWP1 in a flow loop through peristaltic pump P1 and toward vessel V1, as indicated in  FIG. 13G . Subsequently, supply line  1318  is lifted out of reagent  1310  and into filler fluid  1314  and a certain amount of filler fluid  1314  is drawn from MWP1. In some embodiments, supply line  1318  may oscillate up and down in the well to create multiple slugs. A train of reagent slugs that are separated by filler fluid flows toward capillary CP1. When the entire train of reagent slugs is present within CP1, peristaltic pump P1 is stopped. 
                                             TABLE 6               PV1   PV2   PV3   PV4   PV5   P1   P2                   OPEN   CLOSED   CLOSED   CLOSED   OPEN   CCW   STOP                    
Loading Droplet Actuator—Step 2
 
       FIG. 13H , with reference to Table 7 below, illustrates an exemplary next step in a method of loading a droplet actuator. Fluidics system  1300  has two pumps, peristaltic pump P1 and syringe pump P2, that are available for loading reagents into droplet actuator  1320 . Peristaltic pump P1 of fluidics system  1300  is used for loading reagents into droplet actuator  1320 . Valves PV2 and PV4 are open, valves PV1, PV3, and PV5 are closed, peristaltic pump P1 is activated in the CW direction, and syringe pump P2 is stopped. This arrangement establishes a flow loop through droplet actuator  1320  that includes peristaltic pump P1 and capillary CP1, as indicated in  FIG. 13H . The train of reagent slugs within capillary CP1 flows into droplet actuator  1320 , from port C2 toward port C1, and droplet actuator  1320  is, thus, loaded with the pre-selected reagent and ready for operation. 
                                             TABLE 7               PV1   PV2   PV3   PV4   PV5   P1   P2                   CLOSED   OPEN   CLOSED   OPEN   CLOSED   CW   STOP                    
Direct Dispensing Method of Loading a Droplet Actuator
 
       FIG. 13I , with reference to Table 8 below illustrates another step in a method of loading a droplet actuator. Fluidics system  1300  has two pumps, peristaltic pump P1 and syringe pump P2, that are available for loading reagents into droplet actuator  1320 . Syringe pump P2 of fluidics system  100  is used for loading reagents into droplet actuator  1320 . Valves PV3, PV4, and PV5 are open, valve PV1 is closed, PV2 is optionally closed, peristaltic pump P1 is stopped (i.e., pump P1 acts as a closed valve), and syringe pump P2 is activated in a direction to pull liquid from fluidics system  1300 . Additionally, using the xyz motion, supply line  1318  is inserted into a pre-selected well of MWP1 that contains the pre-selected reagent  1310 . A certain amount of reagent  1310  is drawn from MWP1 in a flow loop through droplet actuator  1320  from port C2 to port C3 and toward syringe pump P2, as indicated in  FIG. 13I . In one example, syringe pump P2 is used for loading a large volume reagent slug from MWP1 into droplet actuator  1320 . 
     
       
         
               
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
               
                   
               
               
                 PV1 
                 PV2 
                 PV3 
                 PV4 
                 PV5 
                 P1 
                 P2 
               
               
                   
               
             
             
               
                 CLOSED 
                 CLOSED 
                 OPEN  
                 OPEN  
                 OPEN 
                 STOP 
                 PULL 
               
               
                   
               
             
          
         
       
     
       FIGS. 14A-14F  are schematic diagrams of an example of another fluidics system  1400  for loading a droplet actuator with liquid. With reference to  FIG. 14A , fluidics system  1400  may include any arrangement of one or more valves, one or more pumps, one or more capillaries, and one or more liquid supply vessels; all fluidly connected. A droplet actuator to be loaded is fluidly connected to fluidics system  1400 . In one example, fluidics system  1400  is substantially the same as fluidics system  1300 , except that a vent path that includes a pinch valve PV6 is provided between peristaltic pump P1 and capillary CP1, and MWP1 is replaced with a capillary CP2, which is preloaded with a certain train of reagent slugs. Note that, like fluidics system  1300 , all liquid lines L of fluidics system  1400  may be capillaries or other tubes and that capillary CP1 may be an extended length of capillary that couples peristaltic pump P1 and junction T2. Similarly, capillary CP2 may be formed of an extended length of capillary coupled to pinch valve P5. 
     Fluidics system  1400  is exemplary only, other system variations are possible within the scope of the invention. For example, syringe pump P2 may be replaced with other types of pumps. Alternatively, fluidics system  1400  may include a single pump only. 
     Purging Air from Fluidics System—Step 1 
       FIG. 14B , with reference to Table 9 below, illustrates a first purging step, in which valve PV1 is optionally open, valves PV3 and PV4 are open, valves PV2, PV5, and PV6 are closed, peristaltic pump P1 is activated in the CW direction, and syringe pump P2 is activated in a direction to pull liquid from fluidics system  1400 . By using peristaltic pump P1 and syringe pump P2 simultaneously, liquid is drawn from vessel V1, through valve PV1, through peristaltic pump P1, through capillary CP1, through valve PV4, through droplet actuator  1420 , through valve PV3, and into syringe pump P2. Liquid displaces air in the path from vessel V1 to syringe pump P2. Air is drawn into syringe pump P2. 
                                                 TABLE 9               PV1   PV2   PV3   PV4   PV5   PV6   P1   P2                   OPEN   CLOSED   OPEN    OPEN   CLOSED   CLOSED   CW   PULL                    
Purging air from Fluidics System—Step 2
 
       FIG. 14C , with reference to Table 10 below, illustrates a second purging step, in which valves PV1, PV2, PV3, PV5, and PV6 are open, valve PV4 is closed, peristaltic pump P1 is stopped (i.e., pump P1 acts as a closed valve), and syringe pump P2 is activated in a direction to push liquid into fluidics system  1400 . This arrangement establishes a flow of liquid from syringe pump P2, through droplet actuator  1420  from port C3 to port C1, through valve PV2, through T1, through valve PV1, and into vessel V1, as indicated in  FIG. 14C . Liquid displaces air in the path from syringe pump P2 to vessel V1. Air is vented at vessel V1. 
                                                 TABLE 10               PV1   PV2   PV3   PV4   PV5   PV6   P1   P2                   OPEN   OPEN   OPEN    CLOSED   OPEN   OPEN   STOP   PUSH                    
Purging Air from Fluidics System—Step 3
 
       FIG. 14D , with reference to Table 11 below, illustrates a third purging step, in which valves PV1 and PV5 are open, valves PV2, PV3, PV4, and PV6 are closed, peristaltic pump P1 is activated in the CCW direction, and syringe pump P2 is stopped. Peristaltic pump P1 pumps liquid from capillary CP2, through valve PV5, and through T2. Peristaltic pump P1 is operated until such time that any air that precedes the train of reagent slugs from preloaded capillary CP2 is trapped between peristaltic pump P1 and T3. 
                                                 TABLE 11               PV1   PV2   PV3   PV4   PV5   PV6   P1   P2                   OPEN   CLOSED   CLOSED   CLOSED   OPEN   CLOSED   CCW   STOP                    
Purging Air from Fluidics System—Step 4
 
       FIG. 14E , with reference to Table 12 below, illustrates a fourth purging step, in which valves PV1 and PV6 are open, valves PV2, PV3, PV4, and PV5 are closed, peristaltic pump P1 is activated in the CW direction, and syringe pump P2 is stopped. This arrangement establishes a flow of liquid between vessel V1 and valve PV6, which is the vent path. Pump P1 pushes air trapped between peristaltic pump P1 and T3 through PV6, through which air is vented from fluidics system  1400 . 
     
       
         
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 12 
               
               
                   
               
               
                 PV1 
                 PV2 
                 PV3 
                 PV4 
                 PV5 
                 PV6 
                 P1 
                 P2 
               
               
                   
               
             
             
               
                 OPEN 
                 CLOSED 
                 CLOSED 
                 CLOSED 
                 CLOSED 
                 OPEN 
                 CW 
                 STOP 
               
               
                   
               
             
          
         
       
     
     At the completion of this step, all air has been purged from fluidics system  1400  and droplet actuator  1420 , as all liquid lines and elements of fluidics system  1400  and all channels of droplet actuator  1420  are filled with liquid and substantially free of air bubbles. 
     Loading a Droplet Actuator 
       FIG. 14F , with reference to Table 13 below illustrates a step in a method of loading a droplet actuator. Valves PV2 and PV4 are open, and valves PV1, PV3, PV5, and PV6 are closed, peristaltic pump P1 is activated in the CW direction, and syringe pump P2 is stopped. Because fluidics system  1400  contains all filler fluid and reagent slugs necessary for the operation of droplet actuator  1420 , the pumping action of peristaltic pump P1 moves the train of reagent slugs into droplet actuator  1420 , from port C2 to port C1, as indicated in  FIG. 14F . 
                                                 TABLE 13               PV1   PV2   PV3   PV4   PV5   PV6   P1   P2                   CLOSED   OPEN   CLOSED   OPEN   CLOSED   CLOSED   CW   STOP                    
Sample Processing
 
     The invention provides a droplet actuator device and methods for processing samples for use on a droplet actuator device. For example, the invention provides methods of processing samples for conducting genetic analysis of microbiological organisms in a biological sample. The device and methods of the invention may be used to detect and identify microorganisms such as bacteria, viruses, and/or fungi in a biological sample. Examples of biological samples include, blood, plasma, serum, isolated microorganisms, nucleic acid spiked into an assay buffer, other samples described herein, and other known sample types. In various embodiments, the invention provides for droplet actuator-based sample preparation and nucleic acid analysis. The device and methods of the invention may, in one embodiment, be used for rapid and accurate identification of atypical bacteria that have specific treatment implications, such as selection of effective antibiotics and length of therapy. For example, in the immunosuppressed population the ability to distinguish between bacteria, viruses, and fungi both rapidly and accurately will be life-saving. 
     Sample Preprocessing 
     The invention provides a droplet actuator device and methods for pre-processing samples prior to introduction of the samples onto a droplet actuator. Prior to transfer of sample to the droplet actuator, the sample may be combined with magnetic beads having affinity for analytes (e.g., DNA and/or RNA) of interest. The analytes of interest may be bound to the magnetically responsive capture beads. The magnetically responsive beads may be concentrated in a small part of the processed sample volume. The reduced sample volume that contains the magnetically responsive beads may be loaded onto the droplet actuator. For example, volume reduction may be from about ≧1 milliliter (mL) to about ≦10 microliters (μL). 
     The droplet actuator may be provided as part of a system which is programmed to execute analysis protocols using electrical fields to perform droplet operations. For example, in a real-time PCR assay, thermocycling is accomplished by transporting reaction droplets through isothermal temperature zones within the droplet actuator rather than by cycling the heaters (“flow-through” PCR). This and other PCR approaches are described in Pollack et al., International Patent Application No. PCT/US 06/47486, entitled “Droplet-Based Biochemistry,” filed on Dec. 11, 2006, the entire disclosure of which is incorporated herein by reference. 
     The droplet actuator may be electrically coupled with the system using mating alignment features to ensure proper positioning. The mating alignment features align the droplet actuator with various functional elements, such as heaters, magnets, and detection elements, that are aligned with specific regions of droplet actuator. A sample is loaded into the sample well. The sample well may be sealed before the analysis protocol can be started. Once the analysis protocol is started, it proceeds to completion without requiring operator intervention. Using one or more droplet operations, the sample is combined in the cassette with appropriate reagents, such as lysis buffer, capture buffer, and capture beads, as required by the analysis protocol. Meanwhile the droplet actuator is primed for performing the final assay (e.g., real-time PCR). 
     The low thermal mass of the droplets combined with the speed and agility with which they can be positioned using one or more droplet operations enables extremely rapid and precise thermal profiles to be achieved. The inventors have successfully implemented real-time PCR in microfluidic format, which includes tests for bacterial and fungal pathogens ( Bacillus anthracis, Franciscella tularensis, Candida albicans, Mycoplasma pneumoniae, Eschericia coli , Methicillin-resistant  Staphylococcus aureus  (MRSA)), human gene targets (RPL4, CFTR, PCNA) and RNA. 
       FIG. 15A  shows a plot of real-time PCR data for detection of MRSA using digital microfluidics.  FIG. 15B  shows a plot of real-time PCR data for detection of  Bacillus anthracis  using digital microfluidics. 
     Referring to  FIG. 15A , for detection of MRSA by real-time PCR, a forward primer mecii574 (5′-GTC AAA AAT CAT GAA CCT CAT TAC TTA TG-3′) and reverse primer Xsau325 (5′-GGA TCA AAC GGC CTG CAC A-3′) were used to amplify a 176 bp fragment of  Staphylococcus aureus  genomic DNA (ATCC #700699D-5). The 50 μl PCR mix was comprised of 20 mM Tris HCl (pH 8.4), 50 mM KCl, 200 μM dNTPs, 1 μM of each primer, 2× Evagreen (Biotium), 6.125U of KAPA2G Fast DNA polymerase (Kapa Biosystems). This mix was adjusted to 50 μl with H20 and approximately 1-2 μL of this mixture was loaded in one of the droplet actuator reservoirs. 
     The protocol performed on the droplet actuator was to dispense two (450 nL) droplets from the reservoir and combine them to form a single (900 nL) reaction droplet. When sample and reagent are provided separately one droplet would be for the sample and the other droplet would be for the 2× reaction mixture. The droplets are then transported to the 95° C. zone and, following an initial activation step, the droplets are cycled between the 60° C. and 95° C. zones 40 times. A fluorescence reading was taken at the end of each extension cycle within the 60° C. zone. The two positions were spanned by 16 electrodes and the droplets were typically transferred at a rate of 20 electrodes per second, thus the time to transfer the droplet between the two thermal zones was approximately 750 milliseconds (ms). Real-time PCR curves obtained for 10-fold dilutions of MRSA genomic DNA concentration exhibited roughly the expected 3.3 cycle separation. The results were confirmed by gel analysis of the amplified product collected from the droplet actuator (not shown). In all cases the amplified product was of the expected length and no by-products were observed. 
     Referring to  FIG. 15B , an experiment was also conducted to evaluate detection of  Bacillus anthracis  (anthrax) using digital microfluidic PCR. These experiments were performed using an early version of a droplet actuator and instrument and were not optimized for speed. Genomic DNA (chromosomal &amp; plasmids) and primers targeted against  B. anthracis  protective antigen were provided from a commercially available kit (Idaho Technology, Salt Lake City, Utah) and combined with a similar reaction mixture to that described above for detection of MRSA. These experiments were performed with varying amounts of the DNA (i.e., 1 ng, 100 pg, 10 pg, 1 pg genomic DNA) added into the reactions which were amplified on the droplet actuator. Cycling conditions were 10 sec at 95° C. and 60 sec at 60° C. times 40 cycles. The data demonstrate the expected quantitation with detection down to 1 pg of genomic DNA. 
     Capture, Concentration and Elution of Nucleic Acids 
     The invention provides droplet actuator devices, techniques and systems for capturing, concentrating and/or eluting nucleic acids. 
       FIG. 16  shows a plot resulting from amplification of MRSA genomic DNA captured, concentrated and eluted on a droplet actuator. In operation, a droplet actuator is electrically coupled to the instrument (not shown). A suspension of magnetically responsive beads that contain captured DNA in a lysis solution was loaded into a sample reservoir of the droplet actuator. In an alternative embodiment, the lysis solution that contains MRSA genomic DNA may be provided as a droplet on a droplet actuator and combined with the bead-containing droplet on the droplet actuator. A permanent magnet located in close proximity to the droplet actuator is used to collect the magnetically responsive beads at the bottom of the well. A single droplet is dispensed from the sample reservoir. The single droplet contains substantially all of the magnetically responsive beads from the original sample, effectively concentrating the beads by a factor of about 50 or more. The droplet is then transported to a wash station where the magnetically responsive beads are magnetically immobilized and repeatedly washed. For about the last several washes, the wash fluid is switched to an elution buffer. The droplets that contain eluted DNA are accumulated within another reservoir. The purified DNA droplet is subsequently dispensed from the reservoir and mixed with multiple sets of PCR reagent droplets. The droplets are transported to the heater zone of the deck and flow-through real-time PCR is performed. 
     As a proof of concept, genomic MRSA DNA was added to several mL of cell lysis solution that contained DNA-capture magnetically responsive beads. The beads were then concentrated off-actuator and transferred in 15 μL of solution to the sample well of the droplet actuator. The beads were further concentrated into a single (˜300 nL) DNA capture droplet. The DNA capture droplet was washed using a merge-and-split protocol with 8 droplets of TE buffer (pH 7.0) and then eluted with 12 droplets of TE buffer (pH 8.5) into a reservoir. Droplets of purified DNA were then dispensed and mixed in a 1:1 ratio with a real-time PCR mix. 
     Data indicate that sample concentration, elution, and detection were successfully performed on a droplet actuator. 
     Sample Preparation on a Droplet Actuator 
     On-actuator preparation of biological samples provides a method for sensitive isolation of nucleic acids using one or more droplet operations to perform separation protocols. Droplet actuator-based sample preparation includes lysis (when necessary) of a sample, capture of nucleic acids (e.g., on magnetically responsive beads), pre-concentration of nucleic acids, a washing of captured nucleic acids to remove unbound material prior to analysis. The flexibility and programmability of the droplet actuator provides for variation in the order in which sample and reagents may be combined during sample preparation. 
     In one embodiment, a sample droplet may be combined using one or more droplet operations with a lysis buffer droplet in order to yield a lysed sample droplet in which nucleic acid has been released. A droplet that includes magnetically responsive capture beads may be combined with the lysed sample droplet in order to bind nucleic acid, yielding a nucleic acid capture droplet in which nucleic acid is bound to the magnetically responsive beads. The nucleic acid capture droplet may be transported using one or more droplet operations into the presence of a magnet and washed using a merge-and-split wash protocol to remove unbound material, yielding a washed bead-containing droplet substantially lacking in unbound material. In some applications, the washed bead-containing droplet may be merged with an elution buffer droplet to elute the nucleic acid, yielding a bead-containing elution droplet. The bead-containing elution droplet may be transported using one or more droplet operations into a thermal zone in order to promote release of the nucleic acid. In other applications, the washed bead-containing droplet may be transported using one or more droplet operations into a thermal zone to promote release of the nucleic acid. The eluted nucleic acid contained in the droplet surrounding the magnetically responsive beads may then be transported away from the magnetically responsive beads for further processing, e.g., PCR analysis. 
     In an alternative embodiment, a lysis buffer droplet that includes magnetically responsive beads may be combined using one or more droplet operations with a sample droplet to yield a nucleic acid capture droplet in which nucleic acid is bound to the magnetically responsive beads. 
     In yet another embodiment, a sample droplet that includes magnetically responsive beads may be combined using one or more droplet operations with a lysis buffer droplet to yield a nucleic acid capture droplet in which nucleic acid is bound to the magnetically responsive beads. 
     In yet another embodiment, a sample droplet may be combined using one or more droplet operations with a lysis buffer droplet in order to yield a lysed sample droplet. A wash buffer droplet that includes magnetically responsive beads may be combined with the lysed sample droplet in order to yield a nucleic acid capture droplet in which nucleic acid is bound to the magnetically responsive beads. 
     In yet another embodiment, magnetically responsive beads may be pre-concentrated prior to being loaded on the droplet actuator. For example, as the result of off-actuator processing, analytes in (e.g., nucleic acid) may be captured on magnetically responsive beads. The magnetically responsive beads may, for example, be provided in the sample, a lysis solution, or a wash solution. This approach permits the beads to be assembled into a volume which is a small part of the total sample volume. This small volume of beads may then be loaded onto the droplet actuator, e.g., into a reservoir for on-actuator dispensing. Dispensing may result in the production of a number of unit-sized, bead-containing droplets. The magnetic capture beads may be further consolidated, as needed, on the droplet actuator for conducting a droplet-based assay protocol. 
     Preparation of Viral RNA 
     Viral RNA may be prepared using, for example, Dynabeads SILANE viral NA from Dynal. A droplet including Proteinase K and a viral sample may be combined using one or more droplet operations with a lysis buffer droplet to yield a lysed sample droplet in which RNA has been released. A droplet including magnetically responsive Dynabeads may be combined with the lysed sample droplet to bind RNA, yielding an RNA capture droplet in which RNA is bound to the Dynabeads. The RNA capture droplet may be transported using one or more droplet operations into the presence of a magnet and washed using a merge-and-split wash protocol to remove unbound material, yielding a washed bead-containing droplet substantially lacking in unbound material. A droplet including elution buffer may be merged with the washed bead-containing droplet to elute RNA, yielding a bead-containing elution droplet. The bead-containing elution droplet may be transported using one or more droplet operations into a thermal zone to promote release of RNA from the Dynabeads, e.g., by heating to approximately 70° C. The eluted RNA contained in the droplet surrounding the Dynabeads may then be transported away from the Dynabeads for further processing, e.g., for execution of a droplet based RT-PCR protocol. Viral DNA may be prepared using, for example, Dynabeads SILANE viral NA from Dynal. 
     Preparation of Bacterial Genomic DNA 
     Bacterial genomic DNA, such as genomic DNA from  Bacillus anthracis , may be prepared using beads having an affinity for DNA. For example, Dynabeads DNA DIRECT from Dynal may be used. A droplet including lysis buffer and magnetically responsive Dynabeads may be combined using one or more droplet operations with a bacterial sample to yield a lysed sample droplet in which released DNA is bound to the Dynabeads. The DNA capture droplet may be transported using one or more droplet operations into the presence of a magnet and washed using a merge-and-split wash protocol to remove unbound material, yielding a washed bead-containing droplet substantially lacking in unbound material. A droplet including resuspension buffer may be merged with the washed bead-containing droplet, yielding a DNA/bead-containing droplet. The DNA/bead-containing droplet is ready for further processing, e.g., for execution of a droplet based PCR protocol. Alternatively, the DNA/bead-containing droplet may be transported using one or more droplet operations into a thermal zone to promote release of DNA from the Dynabeads, e.g., by heating to approximately 65° C. The eluted DNA contained in the droplet surrounding the Dynabeads may then be transported away from the Dynabeads for further processing, e.g., for execution of a droplet based PCR protocol. 
     Droplet Actuator Systems 
     The invention provides droplet actuators with storage and/or transmission devices useful for controlling and/or monitoring distribution and/or use of droplet actuators. The invention also provides networked systems and methods of using such networked systems for controlling and/or monitoring distribution and/or use of droplet actuators. 
       FIG. 17  illustrates a droplet actuator device  1700  of the invention. Droplet actuator device  1700  includes droplet actuator  1724 . Droplet actuator  1724  includes an electronic storage and/or transmission element  1714 . Electronic storage and/or transmission element  1714  may be affixed to and/or incorporated in droplet actuator  1724  or affixed to and/or incorporated in a cartridge incorporating droplet actuator  1724 . Storage and/or transmission element  1714  may, for example, include semiconductor memory, magnetic storage, optical storage, and/or other available forms of computer readable data storage. Storage and/or transmission element  1714  may be volatile or non-volatile. Examples of specific storage and/or transmission elements  1714  include radio-frequency identification (RFID) tags, read-only memory (ROM), random access memory (RAM), electrically erasable programmable read-only memory (EEPROM) (such as flash memory), and magnetic stripes. 
     In one embodiment, the storage and/or transmission element includes an RFID tag. The RFID tag may be affixed to and/or incorporated in the droplet actuator or droplet actuator cartridge. For example, where the substrate of the droplet actuator is made from a printed circuit board (PCB), the RFID tag may also be mounted on the PCB. The RFID tag may provide for wireless identification of the droplet actuator. For example, the RFID tag may transmit a unique identifier for each droplet actuator. RFID monitors, such as those manufactured by Texas Instruments (Dallas, Tex.), may track the location and use of the droplet actuator. In one embodiment, the invention provides a system in which a subject&#39;s RFID and a droplet actuator&#39;s RFID are scanned at a subject&#39;s bedside into a system which matches the subject with the droplet actuator. The subject&#39;s sample may be loaded onto the droplet actuator, for example, into a droplet actuator reservoir and/or into the droplet operations gap of the droplet actuator. The droplet actuator may be mounted on an instrument and used to execute an assay using the sample. Upon completion of the assay, assay results may be automatically associated with the subject. In some embodiments, the information may be automatically added to the laboratory information system or hospital information system or the subject&#39;s electronic medical records. Similar methods may be used in testing applications outside of the medical field. 
     In another example, the storage and/or transmission element includes a memory device, such as a random access memory (RAM) device, read-only memory (ROM) device, or a flash drive. For example, such a droplet actuator may be provided in the form of a peripheral connect device, such as a USB device, that plugs into a computer to power the droplet actuator and permit data exchange between the computer and the device. As another example, the droplet actuator can also be connected and powered by a personal digital assistant (PDA) or a smartphone or a mobile phone. Identifying information from the droplet actuator may be read by the computer, and output information from the assay may be stored on the computer and/or the USB device. 
     In another embodiment, the invention provides a system for conducting environmental studies, such as studies relating to pollution and/or biological or chemical warfare agents. The device may include a droplet actuator in an instrument associated with the droplet actuator including elements required to power the droplet actuator and/or control the droplet actuator. The system may also include elements for gathering other information, such as temperature, humidity, GPS location, and the like. Information including the results of the assay and the other information may be transmitted to a networked computer. Information including the results the assay and the other information may alternatively be stored on the droplet actuator device and information from multiple devices may be transported to an uploading station where the information may be aggregated onto a computer or a computer network. 
     In one embodiment, the invention provides a system for detecting and tracking the extent of a chemical or biological attack or release of a dangerous chemical. Droplet actuators may be installed at various locations throughout a target region, for example, on buildings, farms, water supply sources, buoys, weather balloons, etc. Droplet actuators may be installed on mobile devices, such as mobile robotic devices, airplanes, unmanned drones, and vehicle fleets (such as police cars, school buses, ambulances, military vehicles, oceangoing vessels, postal vehicles, commercial vehicles, etc.). Droplet actuators may be associated with GPS systems for determining coordinates of the droplet actuators when samples are taken. Tests may be executed using the droplet actuator devices, and results may be transmitted back to a central location, along with sample collection location information, for aggregation and analysis. 
       FIG. 18  illustrates another embodiment, droplet actuator  1724  is provided as part of a magnetic stripe card device  1800 . Card device  1800  includes a card  1805  with a magnetic stripe  1810  affixed thereto for receiving and storing data. A droplet actuator  1724  is also affixed to the card. Droplet actuator  1724  may include electrical contacts  1815  for electrically coupling droplet actuator  1724  to an instrument. Alternatively, droplet actuator  1724  may be electrically connected to wires on card  1805 . Wires on card  1805  may terminate in contacts, and these contacts may be electrically coupled to electrical contacts on the instrument so that the droplet actuator may be controlled by the instrument. Droplet actuator  1724  may include an opening  1820  or loading mechanism for loading a sample into droplet actuator  1724  in a manner which subjects the sample to droplet operations mediated by electrodes coupled to the electrical contacts and controlled by the instrument to which the card/droplet actuator is electrically coupled. 
     In some embodiments, the card may have the shape and size of a standard credit card, and magnetic stripe  1810  may have a location on card  1805  which is similar to the location of the magnetic stripe on a standard credit card. Magnetic stripe  1810  may be, for example, any magnetic stripe capable of storing data, such as those commonly used on magnetic stripe cards (e.g., credit cards, identity cards, and transportation tickets). Magnetic stripe  1810  may be read by physical contact and swiping past a reading head (not shown), as is well known. In one embodiment, the instrument is configured so that magnetic stripe  1810  may be read as the card is inserted into the instrument. Further, information from the assay may be written to magnetic stripe  1810  during and/or following the completion of the assay. 
     The instrument may be configured in a manner similar to an automated teller machine, in which the card is inserted by a user, a card reader device transports the card into an operational position in which the card electrical contacts are coupled to the instrument. Establishing the card in operational position may be controlled by a card insertion device and/or may be manually controlled. Further, in operational position, any detection region or window on the droplet actuator may be aligned with a detector on the instrument. In operational position, the instrument may control the execution of an assay on the droplet actuator, and then read and store information to and from the magnetic stripe. Information from magnetic stripe  1810  may be read by a magnetic stripe reader. An assay may be conducted, and information pertaining to assay results may be written to magnetic stripe  1810 . The instrument may be coupled to a network and may upload results from the assay to the network, e.g., into an electronic medical record system. The instrument may include an output device, such as a display and/or printer, which outputs information pertaining to assay results. In another embodiment, a magnetic stripe reader/writer at a subject&#39;s bedside is used to associate card  1800  with a specific subject, e.g., by reading a card identifier from magnetic stripe  1810  and copying the identifier into a subject record and/or by writing a subject identifier onto card  1800 . Magnetic stripe  1810  may also include information about the expiration date of card device  1800 , information about the assay type, instructions for a user for electronic display by the instrument, and software instructions for controlling the assay or selecting a software protocol on the instrument for controlling the assay. Further, printed material on card device  1800  may also include information about the expiration date of card device  1800 , information about the assay type, instructions for a user. In an alternative embodiment, the card is a smart card containing an integrated circuit actuator. The card may have metal contacts connecting the card physically to the reader. Similarly, the card may be a contactless card that uses a magnetic field or radio frequency (RFID) for proximity reading. A battery supply may be included on the card for self-contained execution of an assay. 
     As noted, the invention provides a droplet actuator with electronic storage and/or transmission element. Information that may be stored and/or transmitted by the electronic storage and/or transmission element includes, for example, sample identification information, test identification information (such as assay type), and subject identification information. Examples of subject identification information include medical history information, subject contact information, insurance information, and test results information. In short, the information may include any data of interest suited for the application in which it is used. Systems that use the droplet actuators of the invention that have data storage capability may, for example, provide the advantage of automated tracking, automated distribution, reduction in medical errors, and/or improved anonymity. 
       FIG. 19  is a functional diagram of a sample collection and analysis system  1900  of the invention. System  1900  utilizes droplet actuators  1724  of the invention that have data storage components  1714 . In this embodiment, sample collection system  1900  includes one or more kiosks  1915  for dispensing one or more droplet actuators  1724 . Kiosks  1915  may be standalone kiosks or may be provided as components of a computer network, such as a wide area network (WAN) or local area network (LAN). Kiosk  1915  may be located, for example, in a pharmacy, grocery store, mall, gas station, doctor&#39;s office, hospital, clinic, and/or any convenient location suited for collecting samples. An example method of using system  1900  may include, but is not limited to, the following steps: 
     Step 1: Using kiosk  1915 , a subject obtains a droplet actuator  1724 . For example, droplet actuator  1724  may be purchased by the subject using a credit card transaction. Droplet actuator  1724  is dispensed from kiosk  1915 . The subject may input identifying information into system  1900  using kiosk  1915 . Kiosk  1915  may include a keypad for inputting information, information may be collected from the subject&#39;s credit card or insurance card, and/or the subject may be provided with an identification card with information that is readable by kiosk  1915 . User information may, for example, include name, address, telephone, insurance information, physician information, etc. System  1900  may associate the subject&#39;s identifying information with identifying information from droplet actuator  1724 . A user-generated code or a kiosk-generated code may be provided during the transaction. In this way, the purchased droplet actuator  1724  may be associated with subject. This association may be stored locally within kiosk  1915  or, alternatively, the information is transferred to a networked computer via the networked system. 
     Step 2: The subject or a medical care provider may load a sample on droplet actuator  1724 . For example, a urine sample, blood sample, saliva sample, or stool sample may be loaded into a reservoir of droplet actuator  1724 . Non-medical samples may also be used, e.g., a drinking water sample, aquarium water sample, a swimming pool water sample, a pond water sample, a plant sample, and the like. Kiosk  1915  may dispense instructions and or sample collection devices for collecting and handling the sample. Droplet actuator  1724  may be sealed to prevent leakage of the sample. Droplet actuator  1724  may be placed in a sealed container to prevent leakage of the sample. 
     Step 3: The subject may return the droplet actuator  1724  that has a sample therein to the site of kiosk  1915 . Droplet actuator  1724  may be inserted into kiosk  1915  via any kind of receiving mechanism, such as a secure slot. Droplet actuator  1724  may be stored in a secure manner within kiosk  1915  until such time that it may be removed by an attendant. Droplet actuator  1724  may be stored in a temperature controlled environment within kiosk  1915 . Alternatively, droplet actuator  1724  may be provided to an attendant at the site of the kiosk, at a physician&#39;s office or elsewhere. In another embodiment, a mailing label, package, and/or instructions may be dispensed with droplet actuator  1724 , and droplet actuator may be mailed to a laboratory for processing. 
     Step 4: Droplet actuator  1724  is removed from kiosk  1915  by an attendant. 
     Step 5: Data storage device  1714  of droplet actuator  1724  may be scanned or otherwise read in order to extract the unique identification number and subject information. Alternatively, data storage device  1714  may include only a unique serial number, and the patient information may be stored at the local kiosk  1915  or at a centralized computer electronically coupled to the kiosk. The subject information may thus be matched to the serial number of the droplet actuator  1724 . In this way, the droplet actuator  1724  is automatically associated with the certain subject that has provided the sample. 
     Step 6: Sample within droplet actuator  1724  may be analyzed and the results automatically reported via, for example, telephone, email, and/or the subject accessing the results via kiosk  1915  (e.g., using a code). For example, results may be reported to the subject or the subject&#39;s medical care provider. 
     A similar process may be used in a hospital environment. For example, a subject identifier and droplet actuator identifier may be associated at a subject&#39;s bedside or via a hospital supply system. Associated information may be centrally stored and/or stored on storage and/or transmission element  1714  of droplet actuator  1724 . 
     A similar approach may be used for environmental studies, such as testing for contaminants in drinking water. Sample collection devices with unique serial numbers may be mailed to participants in the study. The serial numbers may, for example, be stored in an electronic format, such as an RFID actuator or in a physical format, such as a bar code. Users may load samples into the sample collection devices, and drop off the samples at local collection points (or ship them to a collection point) for analysis. The identifying information may be associated with the user&#39;s address. In this manner, certain geographical distributions of drinking water contamination may be identified. In a related embodiment, the users may take the sample collection devices to a kiosk analyzer, which controls droplet operations on the sample collection device and provides an output directly to the user. For example, a user may purchase a drinking water analysis collection device at a store, take it home and load it with a drinking water sample, bring it to a kiosk where it can be plugged in, permit the kiosk to run tests on the drinking water using a droplet actuator device that is part of the sample collection device, and provide the user with an output indicative of certain drinking water contaminants. 
     In yet another embodiment, kiosk  1915  may be reader instrument, and the user may insert droplet actuator  1724  into a reader slot, and kiosk  1915  may execute an assay using the droplet actuator. For example, a user may bring a water sample from home, obtain a droplet actuator from kiosk  1915 , load a droplet of water onto the droplet actuator, electronically couple the droplet actuator to kiosk, whereupon kiosk  1915  executes and assay and provides the user with results. As another example, a sample collection container may be mailed to a user, the user may collect the sample, such as a water sample, take the sample to kiosk  1915 , obtain a droplet actuator from kiosk  1915 , load a droplet of water onto the droplet actuator, couple the droplet actuator to kiosk, whereupon kiosk  1915  executes and assay and provides the user with results. Results may also be centrally stored for further analysis. In some cases, kiosk  1915  may be set up to receive biohazardous materials. 
       FIG. 20  is a functional diagram of a sample collection system  2000  of the invention. System  2000  utilizes droplet actuators  1724  with data storage components  1714  (see  FIG. 17 ). Sample collection system  2000  includes distribution center  2010 , which may be, for example, a manufacturer facility or a warehouse distribution facility. Distribution center  2010  maintains an inventory of droplet actuators  1724 . 
     A server is provided by which one or more subjects or medical providers may place orders for droplet actuators  1724  via, for example, a computer in a subject&#39;s home, a medical care provider&#39;s office, pharmacy, clinic, and so on. Using any standard web browser or network interface application, the server facilitates the order placement and payment operations. For each transaction by which droplet actuator  1724  is ordered, an association may be made between droplet actuator identifying information and an ordering party&#39;s or subject&#39;s identifying information. This association may be provided via credit card information, purchase order number, other subject information such as name, address, email, and telephone, and/or a subject-generated or system-generated code. In this manner, droplet actuator  1724  may be associated with a certain subject. This association may be stored on web server or other networked computer and associated with assay results. 
     As will be appreciated by one of skill in the art, aspects of the invention may be embodied as a method, system, or computer program product. Accordingly, various aspects of the invention may take the form of hardware embodiments, software embodiments (including firmware, resident software, micro-code, etc.), or embodiments combining software and hardware aspects that may all generally be referred to herein as a “circuit,” “module” or “system.” Furthermore, the methods of the invention may take the form of a computer program product on a computer-usable storage medium having computer-usable program code embodied in the medium. 
     Any suitable computer useable medium may be utilized for software aspects of the invention. The computer-usable or computer-readable medium may be, for example but not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, device, or propagation medium. More specific examples (a non-exhaustive list) of the computer-readable medium would include some or all of the following: an electrical connection having one or more wires, a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), an optical fiber, a portable compact disc read-only memory (CD-ROM), an optical storage device, a transmission medium such as those supporting the Internet or an intranet, or a magnetic storage device. Note that the computer-usable or computer-readable medium could even be paper or another suitable medium upon which the program is printed, as the program can be electronically captured, via, for instance, optical scanning of the paper or other medium, then compiled, interpreted, or otherwise processed in a suitable manner, if necessary, and then stored in a computer memory. In the context of this document, a computer-usable or computer-readable medium may be any medium that can contain, store, communicate, propagate, or transport the program for use by or in connection with the instruction execution system, apparatus, or device. 
     Computer program code for carrying out operations of the invention may be written in an object oriented programming language such as Python, Java, Smalltalk, C++ or the like. However, the computer program code for carrying out operations of the invention may also be written in conventional procedural programming languages, such as the “C” programming language or similar programming languages. The program code may execute entirely on the user&#39;s computer, partly on the user&#39;s computer, as a stand-alone software package, partly on the user&#39;s computer and partly on a remote computer or entirely on the remote computer or server. In the latter scenario, the remote computer may be connected to the user&#39;s computer through a local area network (LAN) or a wide area network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet Service Provider). 
     Certain aspects of invention are described with reference to various methods and method steps. It will be understood that each method step can be implemented by computer program instructions. These computer program instructions may be provided to a processor of a general purpose computer, special purpose computer, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, create means for implementing the functions/acts specified in the methods. 
     The computer program instructions may also be stored in a computer-readable memory that can direct a computer or other programmable data processing apparatus to function in a particular manner, such that the instructions stored in the computer-readable memory produce an article of manufacture including instruction means which implement various aspects of the method steps. 
     The computer program instructions may also be loaded onto a computer or other programmable data processing apparatus to cause a series of operational steps to be performed on the computer or other programmable apparatus to produce a computer implemented process such that the instructions which execute on the computer or other programmable apparatus provide steps for implementing various functions/acts specified in the methods of the invention. 
     CONCLUDING REMARKS 
     The foregoing detailed description of embodiments refers to the accompanying drawings, which illustrate specific embodiments of the invention. Other embodiments having different structures and operations do not depart from the scope of the present invention. The term “the invention” or the like is used with reference to certain specific examples of the many alternative aspects or embodiments of the applicants&#39; invention set forth in this specification, and neither its use nor its absence is intended to limit the scope of the applicants&#39; invention or the scope of the claims. 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.