Abstract:
Droplet actuators and techniques for droplet-based assays are provided. A method of conducting an assay comprises: incubating a droplet in oil, the droplet comprising an umbelliferone substrate, a sample potentially comprising an enzyme which cleaves the umbelliferone substrate, and a zwitterionic surfactant; and detecting a signal emitted from the droplet.

Description:
RELATED APPLICATIONS 
       [0001]    This application claims the benefit of provisional U.S. Patent Application Nos. 61/506,369 and 61/506,359, both filed on Jul. 11, 2011. 
     
    
     GRANT INFORMATION 
       [0002]    This invention was made with government support under HG004354 awarded by the National Institutes of Health of the United States. 
     
    
     FIELD OF THE INVENTION 
       [0003]    The invention relates to a droplet actuator with a modified droplet operations surface. 
       BACKGROUND 
       [0004]    A droplet actuator typically includes one or more substrates configured to form a surface or gap for conducting droplet operations. The one or more substrates establish a droplet operations surface or gap for conducting droplet operations and may also include electrodes arrange to conduct the droplet operations. The droplet operations substrate or the gap between the substrates may be coated or filled with a filler fluid that is immiscible with the liquid that forms the droplets. 
         [0005]    Droplet actuators are used in a variety of applications, including molecular diagnostic assays, such as enzymatic assays. In one example, lysosomal enzyme tests used in newborn testing assays (NBS) may be performed on a droplet actuator. The NBS assays are fluorescent based tests which measure the release of 4-methylumbelliferone (4-MU) or other umbelliferyl derivatives (e.g., 6-hexadecanoylamido-4-methylumbelliferone; HMU) after enzymatic hydrolysis of the substrates. In the droplet operations environment of a droplet actuator, partitioning of 4-MU (or derivatives) between the aqueous phase (i.e., droplet) and the organic phase (filler fluid) may result in a reduction in the assay signal and potential contamination of neighboring samples. Therefore, there is a need for improved methods for reducing partitioning of 4-MU (or derivatives) in droplet-based bioassays on a droplet actuator. 
       SUMMARY OF THE INVENTION 
       [0006]    The invention provides a method of conducting an assay. The method may, for example, include incubating a droplet in oil, the droplet including an umbelliferone substrate, a sample potentially including an enzyme which cleaves the umbelliferone substrate, and a zwitterionic surfactant, and detecting a signal emitted from the droplet. 
         [0007]    In certain embodiments, the droplet further includes a cyclodextrin compound. In certain embodiments, the cyclodextrin compound is selected from the group consisting of α-cyclodextrins, β-cyclodextrins, and γ-cyclodextrins, and analogs and derivatives of the foregoing. In some cases, the umbilliferone substrate is selected from the group consisting of alkylumbelliferyl-α-L-iduronides, 4-methylumbelliferyl-α-L-iduronide, 4-methylumbelliferyl-α-L-iduronide-2-sulfate, 4-methylumbelliferyl-α-L-idopyranosiduronic acid, 4-methylumbelliferyl-α-L-fucoside, 4-methylumbelliferyl-α-L-mannoside, 4-methylumbelliferyl-β-D-mannoside, 4-methylumbelliferyl β-D-N-acetylglucosaminide, 4-methylumbelliferyl β-D-N-acetylglucosaminide sulfate, alkylumbelliferyl-β-D-glycosides, methylumbelliferyl-β-D-glycosides, 4-methylumbelliferyl-α-D-galactoside, 4-methylumbelliferyl-β-D-galactoside, 4-methylumbelliferyl-β-D-glucouronic acid, phenolphthalein-β-D-glucuronic acid, ethylumbelliferyl-β-D-glycosides, multrifluoroethylumbelliferyl-β-D-glycosides, pentafluoroethylumbelliferyl-β-D-glycosides, pentafluoroethylumbelliferyl-β-D-glucoside, umbelliferylchiotriosides, 4-alkyumbelliferylchiotrioside, 4-methylumbelliferylchiotrioside, 4-methylumbelliferyl-β-galactose, 4-alkyumbeliferrone phosphates, 4-methylumbeliferrone phosphate, 6-alkanoylamido-4-methylumbelliferones, substrates including a 4-methyllumbelliferyl group, 6-hexadecanoylamido-4-methylumbelliferone, 4-methyllumbelliferyl-β-D-glucosaminide, 4-methylumbelliferyl-α-neuraminic acid, 4-methylumbelliferyl-α-D-N-acetylgalactosaminide, and their functional analogs and derivatives. In certain embodiments, the steps are performed within droplets controlled by a droplet actuator. In certain embodiments, the droplet actuator controls the steps using electrode mediated droplet operations. In certain embodiments, the droplet actuator controls the steps using electrowetting mediated droplet operations. In certain embodiments, the droplet actuator controls the steps using dielectrophoresis mediated droplet operations. 
         [0008]    The invention provides a method of conducting a droplet-based enzyme assay. For example, the method may include providing an immiscible fluid including a sample droplet including an enzyme of interest, and one or more reagent droplets including a substrate which is potentially modified in the presence of the enzyme yielding one or more signal-producing products, a zwitterionic surfactant, and optionally, other reagents sufficient to produce the activity of the target enzyme under ordinary conditions. The method may include combining the sample droplet and the one or more reagent droplets in the immiscible fluid to yield a reaction droplet effecting an enzyme reaction in the immiscible fluid, and measuring any signal produced by the one or more signal producing products. In certain embodiments, the zwitterionic surfactant is selected form the group consisting of n-Octylphosphocholine, n-Nonylphosphocholine, n-decylphosphocholine, n-dodecylphosphocholine, 3-cyclohexyl-1-propylphosphocholine, decylphospho-N-methylethanolamine, n-decyl-N,N-dimethylglycine, n-octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, dimethylbenzylammonium propane sulfonate, 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane sulfonate, and 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate. 
         [0009]    The invention provides a method of providing diagnostic information, the method including conducting an assay according to a method of the invention, wherein the sample droplet includes a clinical sample from a subject, the sample including the enzyme of interest, and providing to the subject diagnostic information based on the activity of the enzyme of interest from the human clinical sample. In certain embodiments, the diagnostic information includes information diagnostically relevant to a glycogen storage disease. In certain embodiments, the assay is conducted and the diagnostic information is provided at a point of sample collection. In certain embodiments, the point of sample collection is in the presence of the subject. In certain embodiments, the clinical sample includes a sample substance selected from the group consisting of: blood, plasma, serum, tears, saliva, and urine. In certain embodiments, the clinical sample includes a dried blood sample. In certain embodiments, the clinical sample includes a fresh blood sample. In certain embodiments, the fresh blood sample is collected from the subject and immediately loaded onto a droplet actuator for conducting the assay. In certain embodiments, time from collection of the blood sample to providing diagnostic information is less than about 12 hours. In certain embodiments, time from collection of the blood sample to providing diagnostic information is less than about 6 hours. In certain embodiments, the clinical sample includes a human clinical sample. In certain embodiments, the clinical sample includes a non-human animal clinical sample. In certain embodiments, the substrate includes a glycoside substrate. In certain embodiments, the substrate releases a fluorophore upon contact with the enzyme of interest. In certain embodiments, two or more assays are conducted simultaneously using different fluorophores for each enzyme tested. In certain embodiments, the fluorophore includes 4-methylumbelliferyl. In certain embodiments, the substrate includes a glycoside substrate which releases a fluorophore upon contact with the enzyme of interest. In certain embodiments, the substrate includes a glycoside substrate including glucose, galactose, fucose, mannose, sialic acid, hexose, hexosamine and/or N-acetylated hexosamine. In certain embodiments, the substrate includes a 4-methylumbelliferyl glycoside. In certain embodiments, the method further includes reducing or eliminating reaction contaminants associated with the substrate prior to yielding the assay droplet. In certain embodiments, the reducing or eliminating reaction contaminants includes photobleaching the substrate prior to yielding the assay droplet. In certain embodiments, the photobleaching is effected prior to providing the droplet including the substrate on the droplet actuator. In certain embodiments, the photobleaching is effected after to providing the droplet including the substrate on the droplet actuator. In certain embodiments, the substrate includes a 4-methylumbelliferyl glycoside substrate. In certain embodiments, the method includes photobleaching the substrate prior to yielding the assay droplet. In certain embodiments, the immiscible liquid includes a filler fluid. In certain embodiments, the immiscible liquid includes a silicone oil. In certain embodiments, the filler fluid includes a surfactant. In certain embodiments, the surfactant includes nonionic low hydrophile-lipophile balanced (HLB) surfactant. In certain embodiments, the HLB is less than about 10. In certain embodiments, the HLB is less than about 5. In certain embodiments, the surfactant is selected from the group consisting of Triton X-15, Span 85, Span 65, Span 83, Span 80, Span 60, and fluorinated surfactants. In certain embodiments, the sample droplet includes a reconstituted blood sample, the blood sample is reconstituted using a single universal reconstitution solution, the blood sample is divided to yield two or more reaction droplets, and two or more of the reaction droplets are each combined with one or more sets of one or more reagent droplets, each such set including reagents selected for establishing reaction conditions for a different enzyme assay. In certain embodiments, the universal reconstitution solution includes a saline solution. In certain embodiments, the universal reconstitution solution includes water. In certain embodiments, the enzyme assay is selected to provide diagnostic information about an enzyme deficiency. In certain embodiments, the enzyme deficiency is selected from lysosomal storage diseases. In certain embodiments, the enzyme deficiency is selected from the group consisting of Pompe, Niemann-Pick, Fabry, Krabbe, and Gaucher. In certain embodiments, the method also includes providing therapeutic treatment to a subject based on the diagnostic information. In certain embodiments, the sample droplet including an enzyme of interest includes cultured cells and/or supernatant from a cell culture. In certain embodiments, the substrate is selected from the group consisting of 4-methylumbelliferyl-α-L-iduronide, 4-methylumbelliferyl-β-D-galactoside, 4-methylumbelliferyl-β-D-glucuronic acid, 4-methylumbelliferyl-α-L-fucoside, 4-methylumbelliferyl-α-mannoside, 4-methylumbelliferyl-β-D-mannoside, 4-nitrocathecol sulfate, 4-methylumbelliferyl-β-D-N-acetylglucosaminide, 4-methylumbelliferyl-β-D-N-acetylglucosaminide sulfate, 4-methylumbelliferyl-β-D-glucosaminide, 4-methylumbelliferyl-α-D-galactoside, 4-methylumbelliferyl-α-D-neuraminic acid, 4-methylumbelliferyl-α-D-N-acetylgalactosaminide, phenolphthalein β-D-glucuronic acid, and mixtures and derivatives thereof. In certain embodiments, the substrate includes a fluorophoric moiety. In certain embodiments, the fluorophoric moiety includes 4-methyllumbelliferyl. In certain embodiments, the substrate includes a chromophoric moiety. In certain embodiments, the chromophoric moiety includes 4-nitrocathecol or phenolphthalein. In certain embodiments, the substrate includes a radioactive moiety. In certain embodiments, the radioactive moiety includes 14C sphingomyelin or 3H galactosylceramide. In certain embodiments, incubating the reaction droplet for a period of less than about 12 hours. 
         [0010]    The invention provides a method of conducting an assay in a droplet in oil, the droplet including a lipophilic moiety, the method including in the droplet a nonionic surfactant selected from the group consisting of n-hexyl-β-D-glucopyranoside, 2-cyclohexyl-1-ethyl-β-D-maltoside, 3-cyclohexyl-1-propyl-β-D-maltoside, octanoyl-N-methylglucamide, nonanoyl-N-methylglucamide, octanoyl-N-hydroxyethylglucamide, nonanoyl-N-hydroxyethylglucamide, n-hexyl-β-D-glucopyrano side, and α-[4-(1,1,3,3-tetramethylbutyl)phenyl]-ω-hydroxy-poly(oxy-1,2-ethanediyl). In certain embodiments, the lipophilic moiety includes an umbilliferone substrate. In certain embodiments, umbilliferone substrate is selected from the group consisting of alkylumbelliferyl-α-L-iduronides, 4-methylumbelliferyl-α-L-iduronide, 4-methylumbelliferyl-α-L-iduronide-2-sulfate, 4-methylumbelliferyl-α-L-idopyranosiduronic acid, 4-methylumbelliferyl-α-L-fucoside, 4-methylumbelliferyl-α-L-mannoside, 4-methylumbelliferyl-β-D-mannoside, 4-methylumbelliferyl β-D-N-acetylglucosaminide, 4-methylumbelliferyl β-D-N-acetylglucosaminide sulfate, alkylumbelliferyl-β-D-glycosides, methylumbelliferyl-β-D-glycosides, 4-methylumbelliferyl-α-D-galactoside, 4-methylumbelliferyl-β-D-galactoside, 4-methylumbelliferyl-β-D-glucouronic acid, phenolphthalein-β-D-glucuronic acid, ethylumbelliferyl-β-D-glycosides, multrifluoroethylumbelliferyl-β-D-glycosides, pentafluoroethylumbelliferyl-β-D-glycosides, pentafluoroethylumbelliferyl-β-D-glucoside, umbelliferylchiotriosides, 4-alkyumbelliferylchiotrioside, 4-methylumbelliferylchiotrioside, 4-methylumbelliferyl-β-galactose, 4-alkyumbeliferrone phosphates, 4-methylumbeliferrone phosphate, 6-alkanoylamido-4-methylumbelliferones, substrates including a 4-methyllumbelliferyl group, 6-hexadecanoylamido-4-methylumbelliferone, 4-methyllumbelliferyl-β-D-glucosaminide, 4-methylumbelliferyl-α-neuraminic acid, 4-methylumbelliferyl-α-D-N-acetylgalactosaminide, and their functional analogs and derivatives. 
         [0011]    The invention provides a droplet actuator including one or more substrates arranged to form a droplet operations gap, a fluoropolymer surface on one or more droplet operations gap-facing surfaces of the one or more substrates, where the fluoropolymer surface is deposited using a plasma-enhanced chemical vapor deposition process, a perfluorinated oil filler fluid in the droplet operations gap. In certain embodiments, the fluoropolymer surface includes an amorphous fluoropolymer. In certain embodiments, the fluoropolymer surface includes an amorphous fluoropolymer including carboxyl end-groups. In certain embodiments, the fluoropolymer surface includes an amorphous fluoropolymer including amino-silane coupling agents. In certain embodiments, the fluoropolymer surface includes an amorphous fluoropolymer including perfluoro groups. In certain embodiments, the fluoropolymer surface includes CYTOP® Type A. In certain embodiments, the fluoropolymer surface includes CYTOP® Type M. In certain embodiments, the fluoropolymer surface includes CYTOP® or Type S. In certain embodiments, the one or more substrates comprise one or more electrodes arranged for conducting droplet operations. In certain embodiments, the one or more substrates comprise an arrangement of electrodes arranged for conducting electrowetting-mediated droplet operations. In certain embodiments, the one or more substrates includes a printed circuit board substrate. In certain embodiments, the one or more substrates includes a silicone substrate. In certain embodiments, the one or more substrates includes a glass substrate. In certain embodiments, a droplet in the droplet operations gap, the droplet including a lipophilic substance. In certain embodiments, the water contact angle (θ) of the surface is greater than about 100. 
       DEFINITIONS 
       [0012]    As used herein, the following terms have the meanings indicated. 
         [0013]    “Activate,” with reference to one or more electrodes, means affecting a change in the electrical state of the one or more electrodes which, in the presence of a droplet, results in a droplet operation. Activation of an electrode can be accomplished using alternating or direct current. 
         [0014]    Any suitable voltage may be used. For example, an electrode may be activated using a voltage which is greater than about 150 V, or greater than about 200 V, or greater than about 250 V, or from about 275 V to about 1000 V, or about 300 V. Where alternating current is used, any suitable frequency may be employed. For example, an electrode may be activated using alternating current having a frequency from about 1 Hz to about 10 MHz, or from about 10 Hz to about 60 Hz, or from about 20 Hz to about 40 Hz, or about 30 Hz. 
         [0015]    “Bead,” with respect to beads on a droplet actuator, means any bead or particle that is capable of interacting with a droplet on or in proximity with a droplet actuator. Beads may be any of a wide variety of shapes, such as spherical, generally spherical, egg shaped, disc shaped, cubical, amorphous and other three dimensional shapes. The bead may, for example, be capable of being subjected to a droplet operation 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 provided in a droplet, in a droplet operations gap, or on a droplet operations surface. Beads may be provided in a reservoir that is external to a droplet operations gap or situated apart from a droplet operations surface, and the reservoir may be associated with a flow path that permits a droplet including the beads to be brought into a droplet operations gap or into contact with a droplet operations surface. 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, a portion of a bead, or only one component 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 beads include flow cytometry microbeads, polystyrene microparticles and nanoparticles, functionalized polystyrene microparticles and nanoparticles, coated polystyrene microparticles and nanoparticles, silica microbeads, fluorescent microspheres and nanospheres, functionalized fluorescent microspheres and nanospheres, coated fluorescent microspheres and nanospheres, color dyed microparticles and nanoparticles, magnetic microparticles and nanoparticles, superparamagnetic microparticles and nanoparticles (e.g., DYNABEADS® particles, available from Invitrogen Group, Carlsbad, Calif.), fluorescent microparticles and nanoparticles, coated magnetic microparticles and nanoparticles, ferromagnetic microparticles and nanoparticles, coated ferromagnetic microparticles and nanoparticles, and those described in U.S. Patent Publication Nos. 20050260686, entitled “Multiplex flow assays preferably with magnetic particles as solid phase,” published on Nov. 24, 2005; 20030132538, entitled “Encapsulation of discrete quanta of fluorescent particles,” published on Jul. 17, 2003; 20050118574, entitled “Multiplexed Analysis of Clinical Specimens Apparatus and Method,” published on Jun. 2, 2005; 20050277197. Entitled “Microparticles with Multiple Fluorescent Signals and Methods of Using Same,” published on Dec. 15, 2005; 20060159962, entitled “Magnetic Microspheres for use in Fluorescence-based Applications,” published on Jul. 20, 2006; the entire disclosures of which are incorporated herein by reference for their teaching concerning beads and magnetically responsive materials and beads. Beads may be pre-coupled with a biomolecule or other substance that is able to bind to and form a complex with a biomolecule. Beads may be pre-coupled with an antibody, protein or antigen, DNA/RNA probe or any other molecule with an affinity for a desired target. 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. Bead characteristics may be employed in the multiplexing aspects of the invention. Examples of beads having characteristics suitable for multiplexing, as well as methods of detecting and analyzing signals emitted from such beads, may be found in U.S. Patent Publication No. 20080305481, entitled “Systems and Methods for Multiplex Analysis of PCR in Real Time,” published on Dec. 11, 2008; U.S. Patent Publication No. 20080151240, “Methods and Systems for Dynamic Range Expansion,” published on Jun. 26, 2008; U.S. Patent Publication No. 20070207513, entitled “Methods, Products, and Kits for Identifying an Analyte in a Sample,” published on Sep. 6, 2007; U.S. Patent Publication No. 20070064990, entitled “Methods and Systems for Image Data Processing,” published on Mar. 22, 2007; U.S. Patent Publication No. 20060159962, entitled “Magnetic Microspheres for use in Fluorescence-based Applications,” published on Jul. 20, 2006; U.S. Patent Publication No. 20050277197, entitled “Microparticles with Multiple Fluorescent Signals and Methods of Using Same,” published on Dec. 15, 2005; and U.S. Patent Publication No. 20050118574, entitled “Multiplexed Analysis of Clinical Specimens Apparatus and Method,” published on Jun. 2, 2005. 
         [0016]    “Droplet” means a volume of liquid on a droplet actuator. Typically, a droplet is at least partially bounded by a filler fluid. For example, a droplet may be completely surrounded by a filler fluid or may be bounded by filler fluid and one or more surfaces of the droplet actuator. As another example, a droplet may be bounded by filler fluid, one or more surfaces of the droplet actuator, and/or the atmosphere. As yet another example, a droplet may be bounded by filler fluid and the atmosphere. 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, combinations of such shapes, 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 fluid, serum, plasma, sweat, tear, saliva, sputum, cerebrospinal fluid, amniotic fluid, seminal fluid, vaginal excretion, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudates, exudates, cystic fluid, bile, urine, gastric fluid, intestinal fluid, fecal samples, 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. A droplet may include one or more beads. 
         [0017]    “Droplet Actuator” means a device for manipulating droplets. For examples of droplet actuators, see Pamula et al., U.S. Pat. No. 6,911,132, entitled “Apparatus for Manipulating Droplets by Electrowetting-Based Techniques,” issued on Jun. 28, 2005; 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; Pollack et al., International Patent Application No. PCT/US2006/047486, entitled “Droplet-Based Biochemistry,” filed on Dec. 11, 2006; Shenderov, 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; Kim and/or Shah et al., U.S. patent application Ser. No. 10/343,261, entitled “Electrowetting-driven Micropumping,” filed on Jan. 27, 2003, Ser. No. 11/275,668, entitled “Method and Apparatus for Promoting the Complete Transfer of Liquid Drops from a Nozzle,” filed on Jan. 23, 2006, Ser. No. 11/460,188, entitled “Small Object Moving on Printed Circuit Board,” filed on Jan. 23, 2006, Ser. No. 12/465,935, entitled “Method for Using Magnetic Particles in Droplet Microfluidics,” filed on May 14, 2009, and Ser. No. 12/513,157, entitled “Method and Apparatus for Real-time Feedback Control of Electrical Manipulation of Droplets on Chip,” filed on Apr. 30, 2009; Velev, U.S. Pat. No. 7,547,380, entitled “Droplet Transportation Devices and Methods Having a Fluid Surface,” issued on Jun. 16, 2009; Sterling et al., U.S. Pat. No. 7,163,612, entitled “Method, Apparatus and Article for Microfluidic Control via Electrowetting, for Chemical, Biochemical and Biological Assays and the Like,” issued on Jan. 16, 2007; Becker and Gascoyne et al., U.S. Pat. No. 7,641,779, entitled “Method and Apparatus for Programmable fluidic Processing,” issued on Jan. 5, 2010, and U.S. Pat. No. 6,977,033, entitled “Method and Apparatus for Programmable fluidic Processing,” issued on Dec. 20, 2005; Decre et al., U.S. Pat. No. 7,328,979, entitled “System for Manipulation of a Body of Fluid,” issued on Feb. 12, 2008; Yamakawa et al., U.S. Patent Pub. No. 20060039823, entitled “Chemical Analysis Apparatus,” published on Feb. 23, 2006; Wu, International Patent Pub. No. WO/2009/003184, entitled “Digital Microfluidics Based Apparatus for Heat-exchanging Chemical Processes,” published on Dec. 31, 2008; Fouillet et al., U.S. Patent Pub. No. 20090192044, entitled “Electrode Addressing Method,” published on Jul. 30, 2009; Fouillet et al., U.S. Pat. No. 7,052,244, entitled “Device for Displacement of Small Liquid Volumes Along a Micro-catenary Line by Electrostatic Forces,” issued on May 30, 2006; Marchand et al., U.S. Patent Pub. No. 20080124252, entitled “Droplet Microreactor,” published on May 29, 2008; Adachi et al., U.S. Patent Pub. No. 20090321262, entitled “Liquid Transfer Device,” published on Dec. 31, 2009; 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; Dhindsa et al., “Virtual Electrowetting Channels: Electronic Liquid Transport with Continuous Channel Functionality,” Lab Chip, 10:832-836 (2010); the entire disclosures of which are incorporated herein by reference, along with their priority documents. Certain droplet actuators will include one or more substrates arranged with a droplet operations gap therebetween and electrodes associated with (e.g., layered on, attached to, and/or embedded in) the one or more substrates and arranged to conduct one or more droplet operations. For example, certain droplet actuators will include a base (or bottom) substrate, droplet operations electrodes associated with the substrate, one or more dielectric layers atop the substrate and/or electrodes, and optionally one or more hydrophobic layers atop the substrate, dielectric layers and/or the electrodes forming a droplet operations surface. A top substrate may also be provided, which is separated from the droplet operations surface by a gap, commonly referred to as a droplet operations gap. Various electrode arrangements on the top and/or bottom substrates are discussed in the above-referenced patents and applications and certain novel electrode arrangements are discussed in the description of the invention. During droplet operations it is preferred that droplets remain in continuous contact or frequent contact with a ground or reference electrode. A ground or reference electrode may be associated with the top substrate facing the gap, the bottom substrate facing the gap, in the gap. Where electrodes are provided on both substrates, electrical contacts for coupling the electrodes to a droplet actuator instrument for controlling or monitoring the electrodes may be associated with one or both plates. In some cases, electrodes on one substrate are electrically coupled to the other substrate so that only one substrate is in contact with the droplet actuator. In one embodiment, a conductive material (e.g., an epoxy, such as MASTER BOND™ Polymer System EP79, available from Master Bond, Inc., Hackensack, N.J.) provides the electrical connection between electrodes on one substrate and electrical paths on the other substrates, e.g., a ground electrode on a top substrate may be coupled to an electrical path on a bottom substrate by such a conductive material. Where multiple substrates are used, a spacer may be provided between the substrates to determine the height of the gap therebetween and define dispensing reservoirs. The spacer height may, for example, be from about 5 μm to about 600 μm, or about 100 μm to about 400 μm, or about 200 μm to about 350 μm, or about 250 μm to about 300 μm, or about 275 μm. The spacer may, for example, be formed of a layer of projections form the top or bottom substrates, and/or a material inserted between the top and bottom substrates. One or more openings may be provided in the one or more substrates for forming a fluid path through which liquid may be delivered into the droplet operations gap. The one or more openings may in some cases be aligned for interaction with one or more electrodes, e.g., aligned such that liquid flowed through the opening will come into sufficient proximity with one or more droplet operations electrodes to permit a droplet operation to be effected by the droplet operations electrodes using the liquid. The base (or bottom) and top substrates may in some cases be formed as one integral component. One or more reference electrodes may be provided on the base (or bottom) and/or top substrates and/or in the gap. Examples of reference electrode arrangements are provided in the above referenced patents and patent applications. 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 techniques for controlling droplet operations that may be used in the droplet actuators of the invention include using 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 to conduct a droplet operation in a droplet actuator of the invention. Similarly, one or more of the foregoing may be used to deliver liquid into a droplet operations gap, e.g., from a reservoir in another device or from an external reservoir of the droplet actuator (e.g., a reservoir associated with a droplet actuator substrate and a flow path from the reservoir into the droplet operations gap). Droplet operations surfaces of certain droplet actuators of the invention may be made from hydrophobic materials or may be coated or treated to make them hydrophobic. For example, in some cases some portion or all of the droplet operations surfaces may be derivatized with low surface-energy materials or chemistries, e.g., by deposition or using in situ synthesis using compounds such as poly- or per-fluorinated compounds in solution or polymerizable monomers. Examples include TEFLON® AF (available from DuPont, Wilmington, Del.), members of the cytop family of materials, coatings in the FLUOROPEL® family of hydrophobic and superhydrophobic coatings (available from Cytonix Corporation, Beltsville, Md.), silane coatings, fluorosilane coatings, hydrophobic phosphonate derivatives (e.g., those sold by Aculon, Inc), and NOVEC™ electronic coatings (available from 3M Company, St. Paul, Minn.), other fluorinated monomers for plasma-enhanced chemical vapor deposition (PECVD), and organosiloxane (e.g., SiOC) for PECVD. In some cases, the droplet operations surface may include a hydrophobic coating having a thickness ranging from about 10 nm to about 1,000 nm. Moreover, in some embodiments, the top substrate of the droplet actuator includes an electrically conducting organic polymer, which is then coated with a hydrophobic coating or otherwise treated to make the droplet operations surface hydrophobic. For example, the electrically conducting organic polymer that is deposited onto a plastic substrate may be poly(3,4-ethylenedioxythiophene)poly(styrenesulfonate) (PEDOT:PSS). Other examples of electrically conducting organic polymers and alternative conductive layers are described in Pollack et al., International Patent Application No. PCT/US2010/040705, entitled “Droplet Actuator Devices and Methods,” the entire disclosure of which is incorporated herein by reference. One or both substrates may be fabricated using a printed circuit board (PCB), glass, indium tin oxide (ITO)-coated glass, and/or semiconductor materials as the substrate. When the substrate is ITO-coated glass, the ITO coating is preferably a thickness in the range of about 20 to about 200 nm, preferably about 50 to about 150 nm, or about 75 to about 125 nm, or about 100 nm. In some cases, the top and/or bottom substrate includes a PCB substrate that is coated with a dielectric, such as a polyimide dielectric, which may in some cases also be coated or otherwise treated to make the droplet operations surface hydrophobic. When the substrate includes a PCB, the following materials are examples of suitable materials: MITSUI™ BN-300 (available from MITSUI Chemicals America, Inc., San Jose Calif.); ARLON™ 11N (available from Arlon, Inc, Santa Ana, Calif.).; NELCO® N4000-6 and N5000-30/32 (available from Park Electrochemical Corp., Melville, N.Y.); ISOLA™ FR406 (available from Isola Group, Chandler, Ariz.), especially IS620; fluoropolymer family (suitable for fluorescence detection since it has low background fluorescence); polyimide family; polyester; polyethylene naphthalate; polycarbonate; polyetheretherketone; liquid crystal polymer; cyclo-olefin copolymer (COC); cyclo-olefin polymer (COP); aramid; THERMOUNT® nonwoven aramid reinforcement (available from DuPont, Wilmington, Del.); NOMEX® brand fiber (available from DuPont, Wilmington, Del.); and paper. Various materials are also suitable for use as the dielectric component of the substrate. Examples include: vapor deposited dielectric, such as PARYLENE™ C (especially on glass), PARYLENE™ N, and PARYLENE™ HT (for high temperature, ˜300° C.) (available from Parylene Coating Services, Inc., Katy, Tex.); TEFLON® AF coatings; cytop; soldermasks, such as liquid photoimageable soldermasks (e.g., on PCB) like TAIYO™ PSR4000 series, TAIYO™ PSR and AUS series (available from Taiyo America, Inc. Carson City, Nev.) (good thermal characteristics for applications involving thermal control), and PROBIMER™ 8165 (good thermal characteristics for applications involving thermal control (available from Huntsman Advanced Materials Americas Inc., Los Angeles, Calif.); dry film soldermask, such as those in the VACREL® dry film soldermask line (available from DuPont, Wilmington, Del.); film dielectrics, such as polyimide film (e.g., KAPTON® polyimide film, available from DuPont, Wilmington, Del.), polyethylene, and fluoropolymers (e.g., FEP), polytetrafluoroethylene; polyester; polyethylene naphthalate; cyclo-olefin copolymer (COC); cyclo-olefin polymer (COP); any other PCB substrate material listed above; black matrix resin; and polypropylene. Droplet transport voltage and frequency may be selected for performance with reagents used in specific assay protocols. Design parameters may be varied, e.g., number and placement of on-actuator reservoirs, number of independent electrode connections, size (volume) of different reservoirs, placement of magnets/bead washing zones, electrode size, inter-electrode pitch, and gap height (between top and bottom substrates) may be varied for use with specific reagents, protocols, droplet volumes, etc. In some cases, a substrate of the invention may derivatized with low surface-energy materials or chemistries, e.g., using deposition or in situ synthesis using poly- or per-fluorinated compounds in solution or polymerizable monomers. Examples include TEFLON® AF coatings and FLUOROPEL® coatings for dip or spray coating, other fluorinated monomers for plasma-enhanced chemical vapor deposition (PECVD), and organosiloxane (e.g., SiOC) for PECVD. Additionally, in some cases, some portion or all of the droplet operations surface may be coated with a substance for reducing background noise, such as background fluorescence from a PCB substrate. For example, the noise-reducing coating may include a black matrix resin, such as the black matrix resins available from Toray industries, Inc., Japan. Electrodes of a droplet actuator are typically controlled by a controller or a processor, which is itself provided as part of a system, which may include processing functions as well as data and software storage and input and output capabilities. Reagents may be provided on the droplet actuator in the droplet operations gap or in a reservoir fluidly coupled to the droplet operations gap. The reagents may be in liquid form, e.g., droplets, or they may be provided in a reconstitutable form in the droplet operations gap or in a reservoir fluidly coupled to the droplet operations gap. Reconstitutable reagents may typically be combined with liquids for reconstitution. An example of reconstitutable reagents suitable for use with the invention includes those described in Meathrel, et al., U.S. Pat. No. 7,727,466, entitled “Disintegratable films for diagnostic devices,” granted on Jun. 1, 2010. 
         [0018]    “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. For examples of droplet operations, see the patents and patent applications cited above under the definition of “droplet actuator.” Impedance or capacitance sensing or imaging techniques may sometimes be used to determine or confirm the outcome of a droplet operation. Examples of such techniques are described in Sturmer et al., International Patent Pub. No. WO/2008/101194, entitled “Capacitance Detection in a Droplet Actuator,” published on Aug. 21, 2008, the entire disclosure of which is incorporated herein by reference. Generally speaking, the sensing or imaging techniques may be used to confirm the presence or absence of a droplet at a specific electrode. For example, the presence of a dispensed droplet at the destination electrode following a droplet dispensing operation confirms that the droplet dispensing operation was effective. Similarly, the presence of a droplet at a detection spot at an appropriate step in an assay protocol may confirm that a previous set of droplet operations has successfully produced a droplet for detection. Droplet transport time can be quite fast. For example, in various embodiments, transport of a droplet from one electrode to the next may exceed about 1 sec, or about 0.1 sec, or about 0.01 sec, or about 0.001 sec. In one embodiment, the electrode is operated in AC mode but is switched to DC mode for imaging. It is helpful for conducting droplet operations for the footprint area of droplet to be similar to electrowetting area; in other words, 1×-, 2×- 3×-droplets are usefully controlled operated using 1, 2, and 3 electrodes, respectively. If the droplet footprint is greater than the number of electrodes available for conducting a droplet operation at a given time, the difference between the droplet size and the number of electrodes should typically not be greater than 1; in other words, a 2× droplet is usefully controlled using 1 electrode and a 3× droplet is usefully controlled using 2 electrodes. When droplets include beads, it is useful for droplet size to be equal to the number of electrodes controlling the droplet, e.g., transporting the droplet. 
         [0019]    “Filler fluid” means a fluid associated with a droplet operations substrate of a droplet actuator, which fluid is sufficiently immiscible with a droplet phase to render the droplet phase subject to electrode-mediated droplet operations. For example, the droplet operations gap of a droplet actuator is typically filled with a filler fluid. The filler fluid may, for example, be a low-viscosity oil, such as silicone oil or hexadecane filler fluid. The filler fluid may fill the entire gap of the droplet actuator or may coat one or more surfaces of the droplet actuator. Filler fluids may be conductive or non-conductive. Filler fluids may, for example, be doped with surfactants or other additives. For example, additives may be selected to improve droplet operations and/or reduce loss of reagent or target substances from droplets, formation of microdroplets, cross contamination between droplets, contamination of droplet actuator surfaces, degradation of droplet actuator materials, etc. Composition of the filler fluid, including surfactant doping, may be selected for performance with reagents used in the specific assay protocols and effective interaction or non-interaction with droplet actuator materials. Examples of filler fluids and filler fluid formulations suitable for use with the invention are provided in Srinivasan et al, International Patent Pub. Nos. WO/2010/027894, entitled “Droplet Actuators, Modified Fluids and Methods,” published on Mar. 11, 2010, and WO/2009/021173, entitled “Use of Additives for Enhancing Droplet Operations,” published on Feb. 12, 2009; Sista et al., International Patent Pub. No. WO/2008/098236, entitled “Droplet Actuator Devices and Methods Employing Magnetic Beads,” published on Aug. 14, 2008; and Monroe et al., U.S. Patent Publication No. 20080283414, entitled “Electrowetting Devices,” filed on May 17, 2007; the entire disclosures of which are incorporated herein by reference, as well as the other patents and patent applications cited herein. 
         [0020]    “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 in a droplet to permit execution of a droplet splitting operation, yielding one droplet with substantially all of the beads and one droplet substantially lacking in the beads. 
         [0021]    “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. 
         [0022]    “Reservoir” means an enclosure or partial enclosure configured for holding, storing, or supplying liquid. A droplet actuator system of the invention may include on-cartridge reservoirs and/or off-cartridge reservoirs. On-cartridge reservoirs may be (1) on-actuator reservoirs, which are reservoirs in the droplet operations gap or on the droplet operations surface; (2) off-actuator reservoirs, which are reservoirs on the droplet actuator cartridge, but outside the droplet operations gap, and not in contact with the droplet operations surface; or (3) hybrid reservoirs which have on-actuator regions and off-actuator regions. An example of an off-actuator reservoir is a reservoir in the top substrate. An off-actuator reservoir is typically in fluid communication with an opening or flow path arranged for flowing liquid from the off-actuator reservoir into the droplet operations gap, such as into an on-actuator reservoir. An off-cartridge reservoir may be a reservoir that is not part of the droplet actuator cartridge at all, but which flows liquid to some portion of the droplet actuator cartridge. For example, an off-cartridge reservoir may be part of a system or docking station to which the droplet actuator cartridge is coupled during operation. Similarly, an off-cartridge reservoir may be a reagent storage container or syringe which is used to force fluid into an on-cartridge reservoir or into a droplet operations gap. A system using an off-cartridge reservoir will typically include a fluid passage means whereby liquid may be transferred from the off-cartridge reservoir into an on-cartridge reservoir or into a droplet operations gap. 
         [0023]    “Transporting into the magnetic field of a magnet,” “transporting towards a magnet,” and the like, as used herein to refer to droplets and/or magnetically responsive beads within droplets, is intended to refer to transporting into a region of a magnetic field capable of substantially attracting magnetically responsive beads in the droplet. Similarly, “transporting away from a magnet or magnetic field,” “transporting out of the magnetic field of a magnet,” and the like, as used herein to refer to droplets and/or magnetically responsive beads within droplets, is intended to refer to transporting away from a region of a magnetic field capable of substantially attracting magnetically responsive beads in the droplet, whether or not the droplet or magnetically responsive beads is completely removed from the magnetic field. It will be appreciated that in any of such cases described herein, the droplet may be transported towards or away from the desired region of the magnetic field, and/or the desired region of the magnetic field may be moved towards or away from the droplet. Reference to an electrode, a droplet, or magnetically responsive beads being “within” or “in” a magnetic field, or the like, is intended to describe a situation in which the electrode is situated in a manner which permits the electrode to transport a droplet into and/or away from a desired region of a magnetic field, or the droplet or magnetically responsive beads is/are situated in a desired region of the magnetic field, in each case where the magnetic field in the desired region is capable of substantially attracting any magnetically responsive beads in the droplet. Similarly, reference to an electrode, a droplet, or magnetically responsive beads being “outside of” or “away from” a magnetic field, and the like, is intended to describe a situation in which the electrode is situated in a manner which permits the electrode to transport a droplet away from a certain region of a magnetic field, or the droplet or magnetically responsive beads is/are situated away from a certain region of the magnetic field, in each case where the magnetic field in such region is not capable of substantially attracting any magnetically responsive beads in the droplet or in which any remaining attraction does not eliminate the effectiveness of droplet operations conducted in the region. In various aspects of the invention, a system, a droplet actuator, or another component of a system may include a magnet, such as one or more permanent magnets (e.g., a single cylindrical or bar magnet or an array of such magnets, such as a Halbach array) or an electromagnet or array of electromagnets, to form a magnetic field for interacting with magnetically responsive beads or other components on chip. Such interactions may, for example, include substantially immobilizing or restraining movement or flow of magnetically responsive beads during storage or in a droplet during a droplet operation or pulling magnetically responsive beads out of a droplet. 
         [0024]    “Washing” with respect to washing a bead means reducing the amount and/or concentration of one or more substances in contact with the bead or exposed to the bead from a droplet in contact with the 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. 
         [0025]    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. 
         [0026]    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. In one example, filler fluid can be considered as a film between such liquid and the electrode/array/matrix/surface. 
         [0027]    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 
         [0028]      FIG. 1  shows a diagram of an example of an on-bench assay protocol for evaluating the effect of aqueous surfactants on 4-MU partitioning; 
           [0029]      FIG. 2  shows an example of a plot of relative fluorescence readings for backward transfer partitioning (backward extraction; BE) of a 4-MU partitioning assay used to evaluate the effect of aqueous phase surfactants on 4-MU containment; 
           [0030]      FIG. 3  illustrates a side view of a portion of an example of a droplet actuator that has hydrophobic coatings on the droplet operations surfaces thereof and the gap may be filled with perfluorinated oil; 
           [0031]      FIGS. 4A and 4B  illustrate water contact angle (θ) with respect to hydrophilicity and hydrophobicity, respectively; 
           [0032]      FIGS. 5A and 5B  show top down views of a portion of a droplet actuator that includes M-type CYTOP® coatings applied using the liquid deposition process; 
           [0033]      FIG. 6  shows a top down view of a portion of a droplet actuator that includes hydrophobic coatings applied using the Plasmatreat PECVD process; 
           [0034]      FIG. 7  shows a top down view of a portion of a droplet actuator that includes hydrophobic coatings applied using the Triton PECVD process; 
           [0035]      FIG. 8  shows a top down view of a portion of a droplet actuator that includes hydrophobic coatings applied using the GVD PECVD process at room temperature; 
           [0036]      FIG. 9  shows a top down view of a portion of a droplet actuator that includes hydrophobic coatings applied using the GVD PECVD process at high temperature; and 
           [0037]      FIG. 10  illustrates a functional block diagram of an example of a microfluidics system that includes a droplet actuator. 
       
    
    
     DESCRIPTION 
       [0038]    The present invention provides methods to reduce, preferably substantially or entirely eliminate, partitioning of 4-MU (or derivatives) in droplet-based bioassays on a droplet actuator. In one embodiment, surfactants (detergents) may be used to retain 4-MU (or derivatives) within an aqueous phase droplet. The methods of the invention provide significantly improved signal retention and substantially reduced cross-contamination between droplets. The methods of the invention also provide significantly improved discrimination between a positive signal and a negative signal in 4-MU-based bioassays. 
         [0039]    Further, the invention provides a droplet actuator with a droplet operations surface having a hydrophobic coating that is chemically compatible with the perfluorinated oil (or solvents) and that also ensure suitable droplets operations performance. The invention provides methods of depositing hydrophobic coatings on the droplet operations surfaces of a droplet actuator to render them suitable for use with perfluorinated solvents. The invention also provides hydrophobic coatings for improving operations performance. For example, the invention provides droplet actuators with coatings applied by liquid deposition processes as well as coatings applied by plasma-enhanced chemical vapor deposition (PECVD)-based processes. 
         [0040]    Reducing Partitioning of 4-MU 
         [0041]    4-MU-containing substrates (a.k.a 7-hydroxy 4-methylcoumarin) are used in a number of fluorometric enzyme assays (e.g., enzymatic assays for the detection of lysosomal storage disorders in newborns). The fluorometric enzyme assays are based on the hydrolysis of a 4-MU-containing substrate by a specific enzyme to yield the fluorescent molecule 4-MU. In the droplet operations environment of a droplet actuator, partitioning of 4-MU between the aqueous phase (i.e., droplet) and the organic phase (filler fluid) may result in a reduction in the assay signal and potential contamination of neighboring samples. The enzymatic turnover of the 4-MU substrate requires a low-pH environment (acidic environment). At low pH (pK of 4-MU=7.9), 4-MU is non-ionic and hydrophobic and partitions preferentially from the aqueous droplet phase into the oil filler phase (100:1). Droplets subsequently prepared for the detection step of the bioassay are at a high pH. Fluorescence of 4-MU is optimal at elevated pH (pH&gt;10). A high pH (pH&gt;10) facilitates reverse partitioning of 4-MU from the oil phase back into an aqueous phase droplet. The potential for droplet cross-contamination occurs when an acidic droplet with elevated enzyme concentration (producing significant amounts of 4-MU product) is in proximity of a basic droplet with substantially lower 4-MU concentrations. 
         [0042]    The efficacy of different surfactants (detergents) in containing 4-MU (or derivatives) within an aqueous phase may, for example, be evaluated using a partitioning assay. Parameters that may be varied in the assay for evaluation of surfactants in aqueous containment of 4-MU (or derivatives) include, but are not limited to, the pH of the aqueous phase solution, and the critical micelle concentration of the surfactant. 
         [0043]      FIG. 1  shows a diagram of an example of an on-bench assay protocol  100  for evaluating the effect of aqueous surfactants on 4-MU partitioning. The assay format includes forward transfer partitioning (forward extraction; FE) of 4-MU from an aqueous phase to an oil phase and backward transfer partitioning (backward extraction; BE) of 4-MU from an oil phase to an aqueous phase. The assay is performed in 96-well microtiter substrates; clear 96-well substrates (e.g., Costar 3631) for evaluation of forward transfer partitioning with bottom probe fluorescence detection and solid black 96-well substrates (e.g., Costar 3915) for evaluation of backward transfer partitioning with top probe fluorescence detection. A BioTek Synergy HT instrument with 3 mm top probe and 5 mm bottom probe, may, for example, be used for fluorescence measurements. 
         [0044]    An example of an assay format used for testing the effect of surfactants on contamination through 4-MU partitioning includes, but is not limited to, the following steps: Pipette an aliquot (20 μL) of an aqueous phase solution (e.g., at pH 2 to pH 10.5) containing 0.01% Tween® 20 in a well of a 96-well clear microtiter plate. The aqueous phase solution may also include 4-MU (e.g., 100 μM), NaCl (e.g., 50 mM), and BSA (e.g., 1 mg/mL). Add 130 μL of oil (e.g., silicone oil 5 cSt, 0.1% Triton X-15) to each well that contains an aqueous phase droplet. Seal the plate with aluminum foil and shake using a bench top shaker (e.g., Thermofisher shaker at speed setting 5) for 30 min at room temperature. Carefully remove the aluminum foil and observe each well to note and record any defects in droplet quality (minimize light exposure during this step). Measure the fluorescence of each well using a bottom probe at gains 40, 45, and 50. Transfer, without disturbing the aqueous droplet, 75 μL of the oil phase (FE oil) from each well into the respective well of a solid black microtiter plate that contains 75 μL of 200 mM NaHCO 3  in each well. Seal the plate with aluminum foil and shake using, for example, a Thermofisher bench top shaker (e.g., speed setting 5) for 60 min at 40° C. Remove the aluminum foil and measure the fluorescence of each well using a top probe at gains 50, 60, 70, and 80. 
         [0045]      FIG. 2  shows a bar graph  200  of relative fluorescence readings for backward transfer partitioning (backward extraction; BE) of a 4-MU partitioning assay used to evaluate the effect of aqueous phase surfactants on 4-MU containment. In this example, surfactants were selected from an array of different surfactants available in the detergent screening kit HR2-408 from Hampton Research, Inc. The experiment was performed using 5 cSt silicone oil with 0.1% w/v Triton X-15 as the organic phase (oil phase). Each surfactant was used at 1.5 times the surfactant&#39;s critical micellar concentration (CMC). The identity of each surfactant is listed in Tables 1 and 2. AS17 (ANAPOE®-20 or Tween® 20) was used as a reference signal (43,000-44,000 RFU) and reflects an example of the level of droplet cross-contamination that may be observed in a 4-MU-based bioassay (e.g., newborn screening assay) performed on a droplet actuator. Surfactants that retained 4-MU more efficiently than AS17 (ANAPOE®-20 or Tween® 20), i.e., to the left of the arrow in  FIG. 2 , are listed in Table 1. Surfactants that were less efficient than AS17 in retaining 4-MU, i.e., to the right of the arrow in  FIG. 2 , are listed in Table 2. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Aqueous surfactants (AS) in 4-MU retention 
               
               
                 assay of FIG. 2 (left of arrow) 
               
             
          
           
               
                 AS # 
                 Surfactant 
                 AS # 
                 Surfactant 
               
               
                   
               
               
                 AS 93 
                 FOS-Choline ®-8 
                 AS 64 
                 HEGA ®-9 
               
               
                 AS 91 
                 ZWITTERGENT ® 3-10 
                 AS 63 
                 C-HEGA ®-10 
               
               
                 AS 1 
                 BAM 
                 AS 84 
                 DDMAB 
               
               
                 AS 90 
                 FOS-Choline ®-9 
                 AS 81 
                 FOS-Choline ®-8, 
               
               
                   
                   
                   
                 fluorinated 
               
               
                 AS 70 
                 n-Hexyl-β-D- 
                 AS 95 
                 LysoFos ™ Choline 12 
               
               
                   
                 glucopyranoside 
               
               
                 AS 69 
                 CYMAL ®-2 
                 AS 2 
                 n-Dodecyl-β- 
               
               
                   
                   
                   
                 iminodipropionic acid, 
               
               
                 AS 66 
                 MEGA-8 
                 AS 79 
                 n-Dodecyl-N,N- 
               
               
                   
                   
                   
                 dimemylglycine 
               
               
                 AS 68 
                 HEGA ®-8 
                 AS 82 
                 n-Undecyl-N,N- 
               
               
                   
                   
                   
                 Dimethlamine-oxide 
               
               
                 AS 89 
                 n-Decyl-N,N- 
                 AS 60 
                 n-Heptyl-β-D- 
               
               
                   
                 dimethylglycine 
                   
                 thioglucopyranoside 
               
               
                 AS 67 
                 HEGA ®-9 
                 AS 25 
                 ANAPOE ®-C 12 E 10   
               
               
                 AS 88 
                 FOS-Choline ®-10 
                 AS 54 
                 Pluronic ® F-68 
               
               
                 AS 83 
                 ZWITTERGENT ® 3-12 
                 AS 53 
                 C-HEGA ®-11 
               
               
                 AS 7 
                 Sodium cholate 
                 AS 55 
                 HECAMEG ® 
               
               
                 AS 9 
                 ANAPOE ®-X-305 
                 AS 29 
                 ANAPOE ®-X-405 
               
               
                 AS 92 
                 CYCLOFOS ™-3 
                 AS 17 
                 ANAPOE ®-20 
               
               
                   
                   
                   
                 (Tween ® 20) 
               
               
                 AS 62 
                 CYMAL ®-3 
                 AS 56 
                 n-Octyl-β-D- 
               
               
                   
                   
                   
                 glucoside 
               
               
                 AS 86 
                 CHAPS 
                 AS 96 
                 LysoFos ™ 
               
               
                   
                   
                   
                 Choline 10 
               
               
                 AS 94 
                 ZWITTERGENT ® 3-08 
                 AS 59 
                 2,6-Dimethyl-4- 
               
               
                   
                   
                   
                 heptyl-β-D-malto- 
               
               
                 AS 77 
                 NDSB-256 
                 AS 57 
                 n-Octanoylsucrose 
               
               
                 AS 80 
                 FOS-Choline ®-12 
                 AS 31 
                 ANAPOE ®-C 10 E 6   
               
               
                 AS 58 
                 MEGA-9 
                 AS 74 
                 NDSB-201 
               
               
                 AS 87 
                 CHAPSO 
                 AS 12 
                 ANAPOE ®-58 
               
               
                 AS 85 
                 FOS-MEA ®-10 
               
               
                 AS 78 
                 ZWITTERGENT ® 3-14 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Aqueous surfactants (AS) in 4-MU retention 
               
               
                 assay of FIG. 2 (right of arrow) 
               
             
          
           
               
                 AS # 
                 Surfactant 
                 AS # 
                 Surfactant 
               
               
                   
               
               
                 AS 17 
                 ANAPOE ®-20 
                 AS 21 
                 ANAPOE ®-C 12 E 8   
               
               
                   
                 (Tween ® 20) 
               
               
                 AS 34 
                 ANAPOE ®-C 10 E 9   
                 AS 22 
                 n-Dodecyl-β-D- 
               
               
                   
                   
                   
                 maltoside 
               
               
                 AS 35 
                 ANAPOE ®-35 
                 AS 75 
                 NDSB-211 
               
               
                 AS 24 
                 ANAPOE ®-X-114 
                 AS 36 
                 n-Decyl-β-D- 
               
               
                   
                   
                   
                 maltoside 
               
               
                 AS 20 
                 ANAPOE ®-C 13 E 8   
                 AS 11 
                 n-Hexadecyl-β-D- 
               
               
                   
                   
                   
                 maltoside 
               
               
                 AS 76 
                 NDSB-221 
                 AS 42 
                 n-Nonyl-β-D- 
               
               
                   
                   
                   
                 maltoside 
               
               
                 AS 49 
                 n-Octyl-β-D 
                 AS 50 
                 n-Octyl-β-D- 
               
               
                   
                 thiomaltoside 
                   
                 thioglucoside 
               
               
                 AS 10 
                 IPTG 
                 AS 61 
                 n-Octyl-β-D- 
               
               
                   
                   
                   
                 galactopyranoside 
               
               
                 AS 73 
                 NDSB-195 
                 AS 37 
                 LDAO 
               
               
                 AS 71 
                 C-HEGA ®-8 
                 AS 41 
                 CYMAL ®-5 
               
               
                 AS 14 
                 ANAPOE ®-80 
                 AS 38 
                 n-Decanoylsucrose 
               
               
                 AS 4 
                 CTAB 
                 AS 35 
                 Big CHAP, deoxy 
               
               
                 AS 52 
                 DDAO 
                 AS 6 
                 Sodium dodecyl sulfate 
               
               
                 AS 72 
                 CYMAL ®-2 
                 AS 32 
                 n-Decyl-β-D- 
               
               
                   
                   
                   
                 thiomaltoside 
               
               
                 AS 47 
                 CYMAL ®-4 
                 AS 18 
                 Thesit ® 
               
               
                 AS 16 
                 ANAPOE ®-C 12 E 9   
                 AS 44 
                 HEGA ®-10 
               
               
                 AS 5 
                 Deoxycholic acid, 
                 AS 28 
                 n-Undecyl-β-D- 
               
               
                   
                 sodium salt 
                   
                 maltoside 
               
               
                 AS 45 
                 MEGA-10 
                 AS 33 
                 Octyl maltoside, 
               
               
                   
                   
                   
                 fluorinated 
               
               
                 AS 40 
                 n-Nonyl-β-D- 
                 AS 23 
                 CYMAL ®-7 
               
               
                   
                 thiomaltoside 
               
               
                 AS 15 
                 n-Tridecyl-β-D- 
                 AS 26 
                 Sucrose monolaurate 
               
               
                   
                 maltoside 
               
               
                 AS 8 
                 Sodium dodecanoyl 
                 AS 27 
                 CYMAL ®-6 
               
               
                   
                 sarcosine 
               
               
                 AS 43 
                 n-Nonyl-β-D- 
                 AS 3 
                 Dodecyltrimethylammonium 
               
               
                   
                 glucoside 
                   
                 chloride 
               
               
                 AS 30 
                 TRITON ® X-100 
                 AS 46 
                 C 8 E 5   
               
               
                 AS 13 
                 n-Tetradecyl-β-D- 
                 AS 51 
                 Hexaethylene glycol 
               
               
                   
                 maltoside 
                   
                 monooctyl ether 
               
               
                 AS 39 
                 n-Nonyl-β-D- 
                 AS 48 
                 C 8 E 4   
               
               
                   
                 thioglucoside 
               
               
                   
               
             
          
         
       
     
         [0046]    Classification of the most efficient surfactants (detergents) in retaining 4-MU in an aqueous droplet is shown in Table 3. The zwitterionic surfactant group (14) includes sulfobetaines, betaine and lipid-like phosphocholine and phosphoethanolamine. The non-ionic surfactant group (9) includes sugar-based surfactants (glycosides, glucamides). 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Classification of top 25 aqueous surfactants in 4-MU retention assay (&lt;32,000 RFU) 
               
             
          
           
               
                 AS # 
                 Surfactant 
                 Type 
                 Chemical Class 
               
               
                   
               
               
                 AS 93 
                 FOS-Chorine ®-8 
                 Zwitterionic 
                 Alkyl phosphocholine 
               
               
                 AS 91 
                 ZWITTERGENT ® 3-10 
                 Zwitterionic 
                 Alkyl sulfobetaine 
               
               
                 AS 1 
                 BAM 
                 Cationic 
                 Quaternary ammonium 
               
               
                 AS 90 
                 FOS-Chorine ®-9 
                 Zwitterionic 
                 Alkyl phosphocholine 
               
               
                 AS 70 
                 n-Hexyl-β-D-glucopyranoside 
                 Non-Ionic 
                 Alkyl glycoside †   
               
               
                 AS 69 
                 CYMAL ®-2 
                 Non-Ionic 
                 Cycloalkyl glycoside †   
               
               
                 AS 66 
                 MEGA-8 
                 Non-Ionic 
                 Alkanoyl-N-methylglucamide* †   
               
               
                 AS 68 
                 HEGA ®-8 
                 Non-Ionic 
                 Alkanoyl-N-hydroxyethylglucamide* †   
               
               
                 AS 89 
                 n-Decyl-N,N-dimethylglycine 
                 Zwitterionic 
                 Alkyl betaine 
               
               
                 AS 67 
                 C-HEGA ®-9 
                 Non-Ionic 
                 Cycloalkanoyl hydroxyethylglucamide †   
               
               
                 AS 88 
                 FOS-Choline ®-10 
                 Zwitterionic 
                 Alkyl phosphocholine 
               
               
                 AS 83 
                 ZWITTERGENT ® 3-12 
                 Zwitterionic 
                 Alkyl sulfobetaine 
               
               
                 AS 7 
                 Sodium cholate 
                 Anionic 
                 Bile salts 
               
               
                 AS 9 
                 ANAPOE ®-X-305 
                 Non-Ionic 
                 Polyoxyethylene 
               
               
                 AS 92 
                 CYCLOFOS ™-3 
                 Zwitterionic 
                 Cycloalkyl phosphocholine 
               
               
                 AS 62 
                 CYMAL ®-3 
                 Non-Ionic 
                 Cycloalkyl glycoside †   
               
               
                 AS 86 
                 CHAPS 
                 Zwitterionic 
                 Sulfobetaine 
               
               
                 AS 94 
                 ZWITTERGENT ® 3-08 
                 Zwitterionic 
                 Alkyl sulfobetaine 
               
               
                 AS 77 
                 NDSB-256 
                 Zwitterionic 
                 Non-detergent sulfobetaine 
               
               
                 AS 80 
                 FOS-Choline ®-12 
                 Zwitterionic 
                 Alkyl phosphocholine 
               
               
                 AS 58 
                 MEGA-9 
                 Non-Ionic 
                 Alkanoyl-N-methylglucamide* †   
               
               
                 AS 87 
                 CHAPSO 
                 Zwitterionic 
                 Sulfobetaine 
               
               
                 AS 85 
                 FOS-MEA ®-10 
                 Zwitterionic 
                 Lipid-like 
               
               
                   
                   
                   
                 (phosphoethanolamine) 
               
               
                 AS 78 
                 ZWITTERGENT ® 3-14 
                 Zwitterionic 
                 Alkyl sulfobetaine 
               
               
                 AS 64 
                 HEGA ®-9 
                 Non-Ionic 
                 Alkanoyl-N-hydroxyethylglucamide* †   
               
               
                   
               
               
                 *Fatty acid glucamide or alkanoyl glucamide; 
               
               
                   † Sugar-based surfactants 
               
             
          
         
       
     
         [0047]    Assay Methods 
         [0048]    Enzymatic indicators of lysosomal storage diseases (LSDs) can be identified using droplet based assays on a droplet actuator. In one embodiment, assays of the appropriate glycosidase activity may be used to detect altered activity of a particular glycosidase, which may be an indicator of a particular lysosomal storage disease. Examples of enzyme deficiencies and LSDs include, but are not limited to, the following: a deficiency in iduronate-2-sulfae sulphatase is a diagnostic indicator of Hunter disease; a deficiency in acid β-D-glucosidase or chitotriosidase is a diagnostic indicator of Gaucher disease; a deficiency in acid sphingomyelinase or chitotriosidase is a diagnostic indicator of Niemann-Pick disease; a deficiency in α-glucosidase activity is a diagnostic indicator of Pompe disease; a deficiency in α-galactosidase activity is a diagnostic indicator of Fabry disease; a deficiency in α-L-iduronidase is a diagnostic indicator of Hurler disease; a deficiency in heparan sulfate sulfamidase is a diagnostic indicator of Sanfilippo A (MPS IIIA); a deficiency in alpha-N-acetylglucosaminidase is a diagnostic indicator of Sanfilippo B (MPS IIIB); and a deficiency in arylsulfatase A is a diagnositic indicator of metachromatic leukodystrophy. Multiple diseases and/or multiple samples can be tested simultaneously on a single droplet actuator. 
         [0049]    The lysosomal enzyme tests are performed in aqueous droplets within an oil filled gap of the droplet actuator. Samples and assay reagents are manipulated as discrete droplets upon an electrode array (digital electrowetting). Sample droplets are blood or blood-derived samples, such as plasma, serum, tissue, cell fractions, and treated, fractionated, concentrated and/or diluted forms of the foregoing. For example, diagnosis for Pompe disease is performed on fibroblasts. Other biological fluids may be used as samples; nonlimiting examples include tears, semen, urine, saliva, amniotic liquid and cerebrospinal fluid. For example, in the testing to diagnose Fabry disease, tears may be used as the input sample droplet. Biological fluids may be treated as necessary to prepare them for being subjected to the protocols of the invention. For example, samples may be diluted or buffered, heated or cooled; pH may be adjusted; and/or blood samples may be treated with one or more anticoagulants. In some embodiments, the sample includes a reconstituted dried blood spot and/or dried plasma spot. Samples may be loaded into a reservoir associated with a droplet actuator, and may be dispensed into one or more subsamples. In some cases, the subsamples are unit-sized subsamples. The subsamples may be in contact with or surrounded with one or more filler fluids. 
         [0050]    Assay reagents for testing for lysosomal storage disorders (e.g., LSDs) on a droplet actuator may include any one or more of the following: reaction buffer, 4-MU enzyme substrate, supplemented secondary enzyme, assay-specific inhibitor, and stop buffer (e.g., 0.2M Sodium bicarbonate pH 10.0 with 0.01% Tween® 20). Examples of 4-MU substrates include, but are not limited to,  4 -Methylumbelliferyl-α-L-Iduronide-2-Sulfate (4-MU-αIdoA-2S), Hunter substrate; 4-Methylumbelliferyl α-D-Galactopyranoside (4-MU-α Gal), Fabry substrate; 4-MU-α-D-glucopyranoside (4-MU-α-Glue), Pompe substrate; 4-Methyumbelliferyl-β-D-Glucopyranoside (4-MU-β-Glue), Gaucher substrate; 4-Methylumbelliferyl-α-L-Iduronide Sodium Salt (4-MU-α-Idu), Hurler substrate; 4-Trifluoromethylumbelliferylchitroside, Gaucher and Niemann-Pick substrate; 4-Methylumbelliferyl-β-Galactose (4-MU-β-Galactose), Morquio B substrate; 4-Methylumbelliferyl-α-N-Sulpho-D-Glucosaminide (MU-αGlcNS), Sanfilippo A (MPS IIIA) substrate; 4-Methylumbelliferyl-α-D-N-Acetylglucosamine, Sanfilippo B (MPS IIIB); and 3-O-Sulfate-β-D-Galactosyl-4-Methylumbelliferyl), metachromatic leukodystrophy (MLD) substrate. 
         [0051]    In one embodiment, the invention provides a 4-MU assay in which a droplet comprising assay reagents and a zwitterionic surfactant is dispensed and merged using droplet operations with a sample droplet in a droplet operations gap or on a droplet operations surface. The combined reaction droplet is split using droplet operations into 2 reaction droplets. One reaction droplet is combined using droplet operations with a stop buffer droplet. Fluorescence of the combined droplet is measured (t=0 h). The second reaction droplet is incubated for a predetermined time and then the reaction droplet is combined with a stop buffer droplet. End point fluorescence is measured (t=END h). In this example, a single sample droplet is dispensed and analyzed. However, any number of sample droplets may be dispensed and analyzed. The concentration of zwitterionic surfactant is preferably about in the range of 1.5 times the surfactant&#39;s critical micellar concentration (CMC). Examples of suitable zwitterionic surfactants (detergents) include n-octylphosphocholine (FOS-Choline®- 8 ), n-nonylphosphocholine (FOS-Choline®-9), n-decylphosphocholine (FOS-Choline®-10), n-dodecylphosphocholine (FOS-Choline®-12), 3-Cyclohexyl-1-propylphosphocholine (CYCLOFOS™-3), decylphospho-N-methylethanolamine (FOS-MEA®-10), n-Decyl-N,N-dimethylglycine, n-Octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (ZWITTERGENT® 3-8), n-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (ZWITTERGENT® 3-10), n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (ZWITTERGENT® 3-12), n-Tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (ZWITTERGENT® 3-14), dimethylbenzylammonium propane sulfonate (NDSB-256), 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), and 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO). The droplet operations gap or surface may be coated, filled or partially filled with a filler fluid. For example, the filler fluid may be 5 cSt Silicone oil with 0.1% Triton X15. 
         [0052]    In another embodiment, the invention provides a 4-MU assay in which a droplet comprising assay reagents and a non-ionic surfactant is dispensed and merged using droplet operations with a sample droplet in a droplet operations gap or on a droplet operations surface. The combined reaction droplet is split using droplet operations into 2 reaction droplets. One reaction droplet is combined using droplet operations with a stop buffer droplet. Fluorescence of the combined droplet is measured (t=0 h). The second reaction droplet is incubated for a predetermined time and then the reaction droplet is combined with a stop buffer droplet. End point fluorescence is measured (t=END h). In this example, a single sample droplet is dispensed and analyzed. However, any number of sample droplets may be dispensed and analyzed. The concentration of non-ionic surfactant is preferably about in the range of 1.5 times the surfactant&#39;s critical micellar concentration (CMC). Examples of suitable non-ionic surfactants (detergents) include n-hexyl-β-D-glucopyranoside, 2-cyclohexyl-1-ethyl-β-D-maltoside (CYMAL®-2), 3-cyclohexyl-1-propyl-β-D-maltoside (CYMAL®-3), octanoyl-N-methylglucamide (MEGA-8), nonanoyl-N-methylglucamide (MEGA-9), octanoyl-N-hydroxyethylglucamide (HEGA®-8), nonanoyl-N-hydroxyethylglucamide (HEGA®-9), n-hexyl-β-D-glucopyranoside, and α-[4-(1,1,3,3-tetramethylbutyl)phenyl]-δ-hydroxy-poly(oxy-1,2-ethanediyl) (ANAPOE®X-305). The droplet operations gap or surface may be coated, filled or partially filled with a filler fluid. For example, the filler fluid may be 5 cSt Silicone oil with 0.1% Triton X15. 
         [0053]    In yet another embodiment, the invention provides a 4-MU assay in which a droplet comprising assay reagents and an ionic surfactant is dispensed and merged using droplet operations with a sample droplet in a droplet operations gap or on a droplet operations surface. The combined reaction droplet is split using droplet operations into 2 reaction droplets. One reaction droplet is combined using droplet operations with a stop buffer droplet. Fluorescence of the combined droplet is measured (t=0 h). The second reaction droplet is incubated for a predetermined time and then the reaction droplet is combined with a stop buffer droplet. End point fluorescence is measured (t=END h). In this example, a single sample droplet is dispensed and analyzed. However, any number of sample droplets may be dispensed and analyzed. The concentration of ionic surfactant is preferably about in the range of 1.5 times the surfactant&#39;s critical micellar concentration (CMC). Examples of suitable ionic surfactants (detergents) include cationic Benzyldimethyldodecylammonium bromide (BAM) and anionic 3α,7α,12α-Trihydroxy-5β-cholan-24-oic acid, monosodium salt (Sodium cholate). The droplet operations gap or surface may be coated, filled or partially filled with a filler fluid. For example, the filler fluid may be 5 cSt Silicone oil with 0.1% Triton X15. 
         [0054]    Droplet Actuators and Hydrophobic Coatings 
         [0055]      FIG. 3  illustrates cross section of a portion of an example of a droplet actuator  300  that has hydrophobic coatings on the droplet operations surfaces. The gap in droplet actuator  300  may be filled with perfluorinated oil. Droplet actuator  300  may include a bottom substrate  310  and a top substrate  312  that are separated by a gap  314 . Bottom substrate  310  may, for example, be a printed circuit board (PCB), plastic, silicon, glass, or other suitable material. Top substrate  312  may, for example, be formed of glass, injection-molded plastic, silicon, or other suitable material. Bottom substrate  310  may include an arrangement of droplet operations electrodes  316  (e.g., electrowetting electrodes). Droplet operations electrodes  316  are arranged in a manner that permits them to be used to mediate droplet operations using droplets in the droplet operations gap. Top substrate  312  may include a conductive layer  318 . Conductive layer  318  is on the side of top substrate  312  that is facing gap  314 . In one example, conductive layer  318  is formed of indium tin oxide (ITO), which is a material that is electrically conductive and substantially transparent to light. Droplet operations are conducted between droplet operations electrodes  316  and conductive layer  318 . Droplet operations electrodes  316  of bottom substrate  310  are coated with a dielectric material  328 . A hydrophobic layer  320  is provided atop dielectric material  328  of bottom substrate  310 . A hydrophobic layer  322  is provided atop conductive layer  318  of top substrate  312 . 
         [0056]    In operation, gap  314  of droplet actuator  300  is filled or partially filled with a filler fluid  324 . Filler fluid  324  may be a perfluorinated oil or solvent, such as perfluorinated silicone oil. One or more droplets  326  are provided in gap  314 . Droplet  326  may be subjected to droplet operations the filler fluid  324 . These droplet operations are mediated by droplet operations electrodes  316 . 
         [0057]    Water Contact Angle 
         [0058]      FIGS. 4A and 4B  illustrate water contact angle (θ) with respect to hydrophilicity and hydrophilicity, respectively. Referring to  FIG. 4A , a water droplet tends to spread on a hydrophilic surface. The evaluation of hydrophilicity is made through water contact angle (θ) measurements. Referring to  FIG. 4B , hydrophobicity refers to the physical property of a material that repels a mass of water. Because a water droplet is repelled by hydrophobic material, a water droplet tends to contact only a small area of the surface and the shape of the droplet is spherical. 
         [0059]    Evaluation of CYTOP®Liquid Deposition and Other PECVD Processes 
         [0060]    Details of examples of evaluating various chemistries and various methods of depositing hydrophobic coatings are described with reference to Tables 4, 5, and 6 and  FIGS. 5A through 9  below. Water contact angle (θ) and hysteresis evaluations as well as droplet operations tests were performed on M-type CYTOP® coating (an amorphous fluoropolymer) that is deposited on a substrate via a liquid deposition process. Additionally, water contact angle (θ) and hysteresis evaluations as well as droplet operations tests were performed on hydrophobic coatings deposited on a substrate via various plasma-enhanced chemical vapor deposition (PECVD) processes. PECVD is a process used to deposit thin films from a gas state (vapor) to a solid state on a substrate. For example, tests were performed using the PECVD system from Plasmatreat US LP (Elgin, Ill.). Other tests were performed using the PECVD system from Triton Systems, Inc. (Chelmsford, Mass.), which is a spray-based process. Yet other tests were performed using the PECVD system from GVD Corporation (Cambridge, Mass.), which is a chamber-based process. The M-type CYTOP® coating evaluation may be considered the standard against which the PECVD processes are evaluated. In all cases, the droplet operations performance is evaluated with respect to perfluorinated oil or solvent, such as silicone oil, in the gap of a droplet actuator. 
         [0061]    Water contact angle (θ) and respective hysteresis values were determined for various treatments of a glass surface. For example, the surface of a glass slide (e.g., 25 mm×25 mm×1 mm thick) was treated and evaluated with respect to water contact angle (θ) and hysteresis. For each sample, a water droplet of known volume is deposited on the glass and then the water contact angle (θ) is measured using a goniometer. When liquid is added to the droplet, the volume of the droplet increases and, therefore, the water contact angle (θ) also increases. This is referred to as theta advancing (θa). Theta advancing (θa) is greater than the static water contact angle (θs), or θa&gt;θs. By contrast, when liquid is removed from the droplet, the volume of the droplet decreases and, therefore, the water contact angle (θ) also decreases. This is referred to as theta receding (θr). Theta receding (θr) is less than the static water contact angle (θs), or θr&lt;θs. A hysteresis value is determined by θa minus θr, or hysteresis=θa−θr. 
         [0062]    Table 4 shows an example of evaluation results for five initial screening tests. The five tests are summarized as follows.
       Test #1 is M-type CYTOP® coating liquid deposition process on glass at high temperature (i.e., 120° C.).  FIGS. 3A and 3B  show droplet operations tests related to Test #1.   Test #2 is hydrophobic coatings deposited on glass using the Plasmatreat PECVD process.  FIG. 4  shows a droplet operations test related Test #2.   Test #3 is hydrophobic coatings deposited on glass using the Triton PECVD process.  FIG. 5  shows a droplet operations test related Test #3.   Test #4 is hydrophobic coatings deposited on glass using the GVD PECVD process at room temperature.  FIG. 6  shows a droplet operations test related Test #4.   Test #5 is hydrophobic coatings deposited on glass using the GVD PECVD process at high temperature (i.e., 125° C.).  FIG. 7  shows a droplet operations test related Test #5.       
 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Evaluation results: Water contact angle (θ) and Hysteresis values 
               
             
          
           
               
                   
                   
                 Water Contact 
                   
               
               
                 Test 
                 Process 
                 Angle (θ) 
                 Hysteresis 
               
               
                   
               
             
          
           
               
                 #1 
                 M-type CYTOP ® coating 
                 114 
                 15 
               
               
                   
                 (120° C.) 
               
               
                 #2 
                 Plasmatreat PECVD 
                 85 
                 12 
               
               
                 #3 
                 Triton PECVD 
                 104 
                 20 
               
               
                 #4 
                 GVD PECVD (room temp) 
                 122 
                 65 
               
               
                 #5 
                 GVD PECVD (125° C.) 
                 150 
                 30 
               
               
                   
               
             
          
         
       
     
         [0068]    Following the aforementioned tests, droplet operations performance was tested on five droplet actuators. The five droplet actuators had bottom substrates coated with M-type CYTOP® coating per the liquid deposition process. However, the respective top substrates are unique in each test, as described with reference to  FIGS. 5A through 9 . This evaluation is performed to determine whether the hydrophobic coatings perform well under standard droplet operations conditions. 
         [0069]      FIGS. 5A and 5B  show top down views of a portion of a droplet actuator  500  that includes M-type CYTOP® coatings applied using the liquid deposition process, which substantially corresponds to the process used in Test #1 of Table 4. Droplet actuator  500  includes bottom substrate  510 . An electrode arrangement  520  is patterned on bottom substrate  510 . Electrode arrangement  520  may include, for example, various reservoir electrodes (of on-actuator reservoirs) and droplet operations electrodes. In the top down view of droplet actuator  500 , bottom substrate  510  is visible through a substantially transparent top substrate, which is present but not visible. Bottom substrate  510  and the top substrate (not visible) are separated by a gap. 
         [0070]    In this example, bottom substrate  510  is a PCB that is coated with M-type CYTOP® coating per the liquid deposition process. The top substrate (not visible) is formed of ITO-coated glass that is also coated with M-type CYTOP® coating per the liquid deposition process. The gap between bottom substrate  510  and the top substrate is filled with silicone oil. 
         [0071]    In a test performed on droplet actuator  500 , droplet operations are conducted atop the various electrodes to determine the droplet operations performance of M-type CYTOP® coating with respect to silicone oil.  FIGS. 5A and 5B  show a volume of fluid  522  at a reservoir electrode, which is the electrode of an on-actuator reservoir. A droplet  524  is dispensed from the fluid  522  at the on-actuator reservoir and transported along a line of droplet operations electrodes.  FIGS. 5A and 5B  show that a droplet of fluid is successfully dispensed from the on-actuator reservoir. Further, the size of the droplet is suitable for use in assay protocols and the droplet transported well. 
         [0072]      FIG. 6  shows a top down view of a portion of a droplet actuator  600  that includes hydrophobic coatings applied using the Plasmatreat PECVD process, which substantially corresponds to the process used in Test #2 of Table 4. Droplet actuator  600  includes bottom substrate  610 . An electrode arrangement  620  is patterned on bottom substrate  610 . Electrode arrangement  620  may include, for example, various reservoir electrodes (of on-actuator reservoirs) and droplet operations electrodes. In the top down view of droplet actuator  600 , bottom substrate  610  is visible through a substantially transparent top substrate, which is present but not visible. Bottom substrate  610  and the top substrate (not visible) are separated by a gap. 
         [0073]    In this example, bottom substrate  610  is a PCB that is coated with M-type CYTOP® coating per the liquid deposition process. However, the top substrate (not visible) is formed of ITO-coated glass that is coated with hydrophobic material per the Plasmatreat PECVD process. The gap between bottom substrate  610  and the top substrate is filled with silicone oil. 
         [0074]    In a test performed on droplet actuator  600 , droplet operations are conducted atop the various electrodes to determine the droplet operations performance of the hydrophobic material deposited via the Plasmatreat PECVD process with respect to silicone oil.  FIG. 6  shows a volume of fluid  622  at a reservoir electrode, which is the electrode of an on-actuator reservoir. A droplet  624  is dispensed from the fluid  622  at the on-actuator reservoir and transported along a line of droplet operations electrodes.  FIG. 6  shows that a droplet of fluid is successfully dispensed from the on-actuator reservoir. However, the size of the droplet is not suitable for use in assay protocols and the droplet did not transport well. 
         [0075]      FIG. 7  shows a top down view of a portion of a droplet actuator  700  that includes hydrophobic coatings applied using the Triton PECVD process, which substantially corresponds to the process used in Test #3 of Table 4. Droplet actuator  700  includes bottom substrate  710 . An electrode arrangement  720  is patterned on bottom substrate  710 . Electrode arrangement  720  may include, for example, various reservoir electrodes (of on-actuator reservoirs) and droplet operations electrodes. In the top down view of droplet actuator  700 , bottom substrate  710  is visible through a substantially transparent top substrate, which is present but not visible. Bottom substrate  710  and the top substrate (not visible) are separated by a gap. 
         [0076]    In this example, bottom substrate  710  is a PCB that is coated with M-type CYTOP® coating per the liquid deposition process. However, the top substrate (not visible) is formed of ITO-coated glass that is also coated with hydrophobic material per the Triton PECVD process. The gap between bottom substrate  710  and the top substrate is filled with silicone oil. 
         [0077]    In a test performed on droplet actuator  700 , droplet operations are conducted atop the various electrodes to determine the droplet operations performance of the hydrophobic material deposited via the Triton PECVD process with respect to silicone oil.  FIG. 7  shows a volume of fluid  722  at a reservoir electrode, which is the electrode of an on-actuator reservoir. A droplet  724  is dispensed from the fluid  722  at the on-actuator reservoir and transported along a line of droplet operations electrodes.  FIG. 7  shows that a droplet of fluid is successfully dispensed from the on-actuator reservoir. Further, the size of the droplet is suitable for use in assay protocols and the droplet transported well. 
         [0078]      FIG. 8  shows a top down view of a portion of a droplet actuator  800  that includes hydrophobic coatings applied using the GVD PECVD process at room temperature, which substantially corresponds to the process used in Test #4 of Table 4. Droplet actuator  800  includes bottom substrate  810 . An electrode arrangement  820  is patterned on bottom substrate  810 . Electrode arrangement  820  may include, for example, various reservoir electrodes (of on-actuator reservoirs) and droplet operations electrodes. In the top down view of droplet actuator  800 , bottom substrate  810  is visible through a substantially transparent top substrate, which is present but not visible. Bottom substrate  810  and the top substrate (not visible) are separated by a gap. 
         [0079]    In this example, bottom substrate  810  is a PCB that is coated with M-type CYTOP® coating per the liquid deposition process. However, the top substrate (not visible) is formed of ITO-coated glass that is also coated with hydrophobic material per the GVD PECVD process at room temperature. The gap between bottom substrate  810  and the top substrate is filled with silicone oil. 
         [0080]    In a test performed on droplet actuator  800 , droplet operations are conducted atop the various electrodes to determine the droplet operations performance of the hydrophobic material deposited via the GVD PECVD process at room temperature with respect to silicone oil.  FIG. 8  shows a volume of fluid  822  at a reservoir electrode, which is the electrode of an on-actuator reservoir. A droplet  824  is dispensed from the fluid  822  at the on-actuator reservoir and transported along a line of droplet operations electrodes.  FIG. 8  shows that a droplet of fluid is successfully dispensed from the on-actuator reservoir. Further, the size of the droplet is suitable for use in assay protocols and the droplet transported well. 
         [0081]      FIG. 9  shows a top down view of a portion of a droplet actuator  900  that includes hydrophobic coatings applied using the GVD PECVD process at high temperature (e.g., about 125° C.), which substantially corresponds to the process used in Test #5 of Table 4. Droplet actuator  900  includes bottom substrate  910 . An electrode arrangement  920  is patterned on bottom substrate  910 . Electrode arrangement  920  may include, for example, various reservoir electrodes (of on-actuator reservoirs) and droplet operations electrodes. In the top down view of droplet actuator  900 , bottom substrate  910  is visible through a substantially transparent top substrate, which is present but not visible. Bottom substrate  910  and the top substrate (not visible) are separated by a gap. 
         [0082]    In this example, bottom substrate  910  is a PCB that is coated with M-type CYTOP® coating per the liquid deposition process. However, the top substrate (not visible) is formed of ITO-coated glass that is also coated with hydrophobic material per the GVD PECVD process at high temperature. The gap between bottom substrate  910  and the top substrate is filled with silicone oil. 
         [0083]    In a test performed on droplet actuator  900 , droplet operations are conducted atop the various electrodes to determine the droplet operations performance of the hydrophobic material deposited via the GVD PECVD process at high temperature with respect to silicone oil.  FIG. 9  shows a volume of fluid  922  at a reservoir electrode, which is the electrode of an on-actuator reservoir. A droplet  924  is dispensed from the fluid  922  at the on-actuator reservoir and transported along a line of droplet operations electrodes.  FIG. 9  shows that a droplet of fluid is successfully dispensed from the on-actuator reservoir. Further, the size of the droplet is suitable for use in assay protocols and the droplet transported well. 
         [0084]    Referring again to Table 4 and  FIGS. 5A through 9 , the samples having a water contact angle (θ) greater than about 100 correlate to the samples having the most suitable droplet operations performance. For example, the samples of Tests #1, #3, #4, and #5 have water contact angles (θ) greater than about 100, while the corresponding droplet actuators  300 ,  500 ,  600 , and  700 , respectively, demonstrated suitable droplet operations performance By contrast, the sample of Test #2 has a water contact angle (θ) of about 85 and the corresponding droplet actuator  400  did not demonstrate suitable droplet operations performance. Therefore, in certain circumstances, the Plasmatreat PECVD process may be a less desirable process for depositing hydrophobic material on a droplet actuator. 
         [0085]    Additionally, while the hydrophobic material deposited via the GVD PECVD process at room temperature and at high temperature provides a good hydrophobic surface, the hydrophobic surface is brittle. Therefore, in certain circumstances, the GVD PECVD process may not be a preferred process for depositing hydrophobic material on a droplet actuator. As a result, the M-type CYTOP® coating liquid deposition process and the Triton PECVD process may be a less desirable processes for depositing hydrophobic material on a droplet actuator. 
         [0086]    Evaluation of Solubility 
         [0087]    With respect to the M-type CYTOP® coating liquid deposition process, currently the M-type CYTOP® coating is cured at about 93° C. for 1 hour. Tests were performed to evaluate the water contact angle (θ) and hysteresis when M-type CYTOP® coating samples are cured at up to about 200° C., before and after exposure to Galden® HT170 fluid (a perfluoropolyether) for about 2 hours. The Galden® HT170 fluid is an example of a specific brand of perfluorinated solvent that is available from Solvay Solexis (West Deptford, N.J.). M-type CYTOP® coating samples were prepared and cured at various temperatures in order to evaluate the chemical stability to Galden® HT170 fluid. The preference is that the high temperature cure results in little or no change in water contact angle (θ) and hysteresis. Table 5 shows chemical stability results of samples treated with M-type CYTOP® coating process vs. GVD PECVD process vs. Triton PECVD process before and after exposure to Galden® HT170 fluid. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Chemical stability results of samples treated with M-type CYTOP ® coating process vs. GVD 
               
               
                 process vs. Triton process before and after exposure to Galden ® HT170 fluid 
               
             
          
           
               
                   
                 M-type 
                 M-type 
                 M-type 
                 M-type 
                 GVD 
                   
               
               
                   
                 CYTOP ® (LT) 
                 CYTOP ® (MT) 
                 CYTOP ® (HT) 
                 CYTOP ® (XHT) 
                 (HT) 
                 Triton 
               
               
                   
                   
               
             
          
           
               
                 Cure Temp 
                 93/60 
                 120/60 
                 80/30 + 160/30 
                 80/30 + 200/60 
                 125° C. 
                 N/A 
               
               
                 (° C.)/Time 
               
               
                 (minutes) 
               
               
                 Thickness 
                 200 
                 200 
                 400 
                 200 
                 250 
                 200 
               
               
                 (nanometers) 
               
               
                 *Water 
                 113/1 
                 113/1 
                 112/1 
                 113/2 
                 146/3 
                 104/1 
               
               
                 contact 
               
               
                 angle (θ) 
               
               
                 before 
               
               
                 exposure to 
               
               
                 HT170 @ 60° C. 
               
               
                 for 2 hrs 
               
               
                 *Hysteresis 
                  12/1 
                  12/1 
                  12/1 
                  13/1 
                  35/2 
                  22/2 
               
               
                 before 
               
               
                 exposure to 
               
               
                 HT170 @ 60° C. 
               
               
                 for 2 hrs 
               
               
                 *Water 
                 107/4 
                 113/1 
                 112/1 
                 110/5 
                 138/3 
                 104/1 
               
               
                 contact 
               
               
                 angle (θ) 
               
               
                 after 
               
               
                 exposure to 
               
               
                 HT170 @ 60° C. 
               
               
                 for 2 hrs 
               
               
                 *Hysteresis 
                  12/1 
                  18/2 
                  15/3 
                  15/2 
                  37/6 
                  23/0 
               
               
                 after 
               
               
                 exposure to 
               
               
                 HT170 @ 60° C. 
               
               
                 for 2 hrs 
               
               
                   
               
               
                 *xx/x means value/standard deviation 
               
             
          
         
       
     
         [0088]    In each test, a droplet was deposited on the glass slide and the water contact angle (θ) and hysteresis were measured. In the process of testing the samples before and after exposure to Galden® HT170 fluid, the samples were washed with isopropyl alcohol between runs. 
         [0089]    Referring to Table 5, the test results show a slight decrease in water contact angle (θ) after exposure to Galden® HT170 fluid. The HT170 did not affect the droplet operations performance for M-type CYTOP® coating cured at low temperature (93° C.), mid-temperature (120° C.), and high temperature (160° C.). 
         [0090]    Referring now to Table 6, tests were performed before and after exposure to Novec™ 7500 engineered fluid (available from 3M of St. Paul, Minn.) for about 2 hours. M-type CYTOP® coating samples were prepared and cured at various temperatures in order to evaluate the chemical stability to the Novec™ 7500 fluid. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Chemical stability results of samples treated with M-type CYTOP ® coating 
               
               
                 process before and after exposure to Novec ™ 7500 fluid 
               
             
          
           
               
                   
                 M-type 
                 M-type 
                 M-type 
                 M-type 
               
               
                   
                 CYTOP ®(LT) 
                 CYTOP ®(MT) 
                 CYTOP ®(HT) 
                 CYTOP ®(XHT) 
               
               
                   
                   
               
             
          
           
               
                 Cure Temp (° C.)/ 
                  93/60 
                 120/60 
                 80/30 + 160/30 
                 80/30 + 200/60 
               
               
                 Time (minutes) 
               
               
                 Thickness 
                 200 
                 200 
                 400 
                 200 
               
               
                 (nanometers) 
               
               
                 *Water contact 
                 113/1  
                 113/1  
                 112/1 
                 113/2 
               
               
                 angle (Θ) 
               
               
                 before exposure to 
               
               
                 Novec ™ 7500 @ 
               
               
                 60° C. for 2 hrs 
               
               
                 *Hysteresis before 
                 12/1 
                 12/1 
                  12/1 
                  13/1 
               
               
                 exposure to Novec ™ 
               
               
                 7500 @ 60° 
               
               
                 C. for 2 hrs 
               
               
                 *Water contact 
                 102/1  
                 102/3  
                 109/3 
                 111/2 
               
               
                 angle (Θ) 
               
               
                 after exposure to 
               
               
                 Novec ™ 7500 @ 
               
               
                 60° C. for 2 hrs 
               
               
                 *Hysteresis after 
                 41/6 
                 37/7 
                  15/4 
                  13/4 
               
               
                 exposure to Novec ™ 
               
               
                 7500 @ 60° 
               
               
                 C. for 2 hrs 
               
               
                   
               
               
                 *xx/x means value/standard deviation 
               
             
          
         
       
     
         [0091]    Referring again to Tables 4 and 5, with respect to the M-type CYTOP® coating liquid deposition process, the extra-high temperature process (i.e., 160° C. to 200° C.) for about 1 hour may be the preferred process. Optionally, the high temperature cure step and/or the extra-high temperature cure step may be preceded by a prebake step at 80° C. for about 30 minutes. 
         [0092]    Systems 
         [0093]      FIG. 10  illustrates a functional block diagram of an example of a microfluidics system  1000  that includes a droplet actuator  1005 . Digital microfluidic technology conducts droplet operations on discrete droplets in a droplet actuator, such as droplet actuator  1005 , by electrical control of their surface tension (electrowetting). The droplets may be sandwiched between two substrates of droplet actuator  1005 , a bottom substrate and a top substrate separated by a droplet operations gap. The bottom substrate may include an arrangement of electrically addressable electrodes. The top substrate may include a reference electrode plane made, for example, from conductive ink or indium tin oxide (ITO). The bottom substrate and the top substrate may be coated with a hydrophobic material. Droplet operations are conducted in the droplet operations gap. The space around the droplets (i.e., the gap between bottom and top substrates) may be filled with an immiscible inert fluid, such as silicone oil, to prevent evaporation of the droplets and to facilitate their transport within the device. Other droplet operations may be effected by varying the patterns of voltage activation; examples include merging, splitting, mixing, and dispensing of droplets. 
         [0094]    Droplet actuator  1005  may be designed to fit onto an instrument deck (not shown) of microfluidics system  1000 . The instrument deck may hold droplet actuator  1005  and house other droplet actuator features, such as, but not limited to, one or more magnets and one or more heating devices. For example, the instrument deck may house one or more magnets  1010 , which may be permanent magnets. Optionally, the instrument deck may house one or more electromagnets  1015 . Magnets  1010  and/or electromagnets  1015  are positioned in relation to droplet actuator  1005  for immobilization of magnetically responsive beads. Optionally, the positions of magnets  1010  and/or electromagnets  1015  may be controlled by a motor  1020 . Additionally, the instrument deck may house one or more heating devices  1025  for controlling the temperature within, for example, certain reaction and/or washing zones of droplet actuator  1005 . In one example, heating devices  1025  may be heater bars that are positioned in relation to droplet actuator  1005  for providing thermal control thereof. 
         [0095]    A controller  1030  of microfluidics system  1000  is electrically coupled to various hardware components of the invention, such as droplet actuator  1005 , electromagnets  1015 , motor  1020 , and heating devices  1025 , as well as to a detector  1035 , an impedance sensing system  1040 , and any other input and/or output devices (not shown). Controller  1030  controls the overall operation of microfluidics system  1000 . Controller  1030  may, for example, be a general purpose computer, special purpose computer, personal computer, or other programmable data processing apparatus. Controller  1030  serves to provide processing capabilities, such as storing, interpreting, and/or executing software instructions, as well as controlling the overall operation of the system. Controller  1030  may be configured and programmed to control data and/or power aspects of these devices. For example, in one aspect, with respect to droplet actuator  1005 , controller  1030  controls droplet manipulation by activating/deactivating electrodes. 
         [0096]    In one example, detector  1035  may be an imaging system that is positioned in relation to droplet actuator  1005 . In one example, the imaging system may include one or more light-emitting diodes (LEDs) (i.e., an illumination source) and a digital image capture device, such as a charge-coupled device (CCD) camera. 
         [0097]    Impedance sensing system  1040  may be any circuitry for detecting impedance at a specific electrode of droplet actuator  1005 . In one example, impedance sensing system  1040  may be an impedance spectrometer. Impedance sensing system  1040  may be used to monitor the capacitive loading of any electrode, such as any droplet operations electrode, with or without a droplet thereon. For examples of suitable capacitance detection techniques, see Sturmer et al., International Patent Publication No. WO/2008/101194, entitled “Capacitance Detection in a Droplet Actuator,” published on Aug. 21, 2008; and Kale et al., International Patent Publication No. WO/2002/080822, entitled “System and Method for Dispensing Liquids,” published on Oct. 17, 2002; the entire disclosures of which are incorporated herein by reference. 
         [0098]    Droplet actuator  1005  may include disruption device  1045 . Disruption device  1045  may include any device that promotes disruption (lysis) of materials, such as tissues, cells and spores in a droplet actuator. Disruption device  1045  may, for example, be a sonication mechanism, a heating mechanism, a mechanical shearing mechanism, a bead beating mechanism, physical features incorporated into the droplet actuator  1005 , an electric field generating mechanism, a thermal cycling mechanism, and any combinations thereof. Disruption device  1045  may be controlled by controller  1030 . 
         [0099]    It will be appreciated that various aspects of the invention may be embodied as a method, system, computer readable medium, and/or computer program product. 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. 
         [0100]    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. The computer readable medium may include transitory and/or non-transitory embodiments. 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. 
         [0101]    Program code for carrying out operations of the invention may be written in an object oriented programming language such as Java, Smalltalk, C++ or the like. However, the 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 be executed by a processor, application specific integrated circuit (ASIC), or other component that executes the program code. The program code may be simply referred to as a software application that is stored in memory (such as the computer readable medium discussed above). The program code may cause the processor (or any processor-controlled device) to produce a graphical user interface (“GUI”). The graphical user interface may be visually produced on a display device, yet the graphical user interface may also have audible features. The program code, however, may operate in any processor-controlled device, such as a computer, server, personal digital assistant, phone, television, or any processor-controlled device utilizing the processor and/or a digital signal processor. 
         [0102]    The program code may locally and/or remotely execute. The program code, for example, may be entirely or partially stored in local memory of the processor-controlled device. The program code, however, may also be at least partially remotely stored, accessed, and downloaded to the processor-controlled device. A user&#39;s computer, for example, may entirely execute the program code or only partly execute the program code. The program code may be a stand-alone software package that is at least partly on the user&#39;s computer and/or partly executed on a remote computer or entirely on a remote computer or server. In the latter scenario, the remote computer may be connected to the user&#39;s computer through a communications network. 
         [0103]    The invention may be applied regardless of networking environment. The communications network may be a cable network operating in the radio-frequency domain and/or the Internet Protocol (IP) domain. The communications network, however, may also include a distributed computing network, such as the Internet (sometimes alternatively known as the “World Wide Web”), an intranet, a local-area network (LAN), and/or a wide-area network (WAN). The communications network may include coaxial cables, copper wires, fiber optic lines, and/or hybrid-coaxial lines. The communications network may even include wireless portions utilizing any portion of the electromagnetic spectrum and any signaling standard (such as the IEEE 802 family of standards, GSM/CDMA/TDMA or any cellular standard, and/or the ISM band). The communications network may even include powerline portions, in which signals are communicated via electrical wiring. The invention may be applied to any wireless/wireline communications network, regardless of physical componentry, physical configuration, or communications standard(s). 
         [0104]    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 the program code and/or by machine instructions. The program code and/or the machine instructions may create means for implementing the functions/acts specified in the methods. 
         [0105]    The program code may also be stored in a computer-readable memory that can direct the processor, computer, or other programmable data processing apparatus to function in a particular manner, such that the program code stored in the computer-readable memory produce or transform an article of manufacture including instruction means which implement various aspects of the method steps. 
         [0106]    The program code may also be loaded onto a computer or other programmable data processing apparatus to cause a series of operational steps to be performed to produce a processor/computer implemented process such that the program code provides steps for implementing various functions/acts specified in the methods of the invention. 
       CONCLUDING REMARKS 
       [0107]    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.