Patent Application
20140161686
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 arranged 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. Because of the small size of droplet actuators and the small and precise volumes of liquids that are used when performing assays, it can be difficult to load liquids into droplet actuators. Therefore, there is a need for new approaches to loading liquids into droplet actuators.
In an embodiment, a microfluidic system is provided that includes a droplet actuator having an interior cavity and a series of electrodes arranged along the interior cavity for forming a droplet-operation path therethrough. The droplet actuator has a module-engaging side including an opening that is in flow communication with the interior cavity. The microfluidic system also includes a reservoir module configured to be coupled to the droplet actuator. The reservoir module includes a plurality of liquid compartments having respective outlets and at least one seal positioned along the outlets to retain liquid within the liquid compartments. The reservoir module is movable along the module-engaging side of the droplet actuator to position the outlets relative to the opening. The microfluidic system also includes a piercer having a tip configured to penetrate the seal thereby permitting the liquid within the corresponding liquid compartment to flow into the opening.
In an embodiment, a method of dispensing liquid is provided. The method includes providing a microfluidic device having an interior cavity and a module-engaging side. The module-engaging side has an opening that is in fluid communication with the interior cavity. The method also includes positioning a reservoir module along the module-engaging side of the microfluidic device. The reservoir module includes first and second liquid compartments having respective outlets and at least one seal positioned along the outlets to retain liquid within the first and second liquid compartments. The method also includes piercing the seal along the outlet of the first liquid compartment to permit the liquid from the first liquid compartment to flow through the opening of the microfluidic device. The method also includes sliding the reservoir module along the module-engaging side of the microfluidic device. The method also includes piercing the seal along the outlet of the second liquid compartment to permit the liquid from the second liquid compartment to flow through the opening of the microfluidic device.
In an embodiment, a reservoir module is provided that includes a module body having a mounting side configured to interface with a microfluidic device. The module body includes a plurality of liquid compartments that have corresponding liquids preloaded therein. The reservoir module also includes at least one seal extending along the mounting side and covering respective outlets of the liquid compartments. The liquids are separately stored within the corresponding liquid compartments. The seal is configured to be at least one of penetrated or ruptured to permit the liquids to exit the corresponding liquid compartments through the seal and the mounting side.
In an embodiment, a droplet actuator is provided that includes an actuator housing having an interior cavity and a series of electrodes arranged along the interior cavity for forming a droplet-operation path therethrough. The actuator housing has a module-engaging side including an opening that is in flow communication with the interior cavity. The droplet actuator also includes a piercing mechanism having a body that is coupled to the substrate and positioned within or proximate to the opening. The body of the piercing mechanism is configured to at least one of penetrate or rupture a seal of a reservoir along the module-engaging side of the substrate.
As used herein, the following terms have the meanings indicated: “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. 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.
“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.
“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.
“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.
“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.
“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 or include a low-viscosity oil, such as silicone oil or hexadecane filler fluid. The filler fluid may be or include a halogenated oil, such as a fluorinated or perfluorinated oil. 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 be selected to improve droplet operations and/or reduce loss of reagent or target substances from droplets, improve formation of microdroplets, reduce cross contamination between droplets, reduce contamination of droplet actuator surfaces, reduce degradation of droplet actuator materials, etc. For example, filler fluids may be selected for compatibility with droplet actuator materials. As an example, fluorinated filler fluids may be usefully employed with fluorinated surface coatings. Fluorinated filler fluids are useful to reduce loss of lipophilic compounds, such as umbelliferone substrates like 6-hexadecanoylamido-4-methylumbelliferone substrates (e.g., for use in Krabbe, Niemann-Pick, or other assays); other umbelliferone substrates are described in U.S. Patent Pub. No. 20110118132, published on May 19, 2011, the entire disclosure of which is incorporated herein by reference. Examples of suitable fluorinated oils include those in the Galden line, such as Galden HT170 (bp=170° C., viscosity=1.8 cSt, density=1.77), Galden HT200 (bp=200 C, viscosity=2.4 cSt, d=1.79), Galden HT230 (bp=230 C, viscosity=4.4 cSt, d=1.82) (all from Solvay Solexis); those in the Novec line, such as Novec 7500 (bp=128 C, viscosity=0.8 cSt, d=1.61), Fluorinert FC-40 (bp=155° C., viscosity=1.8 cSt, d=1.85), Fluorinert FC-43 (bp=174° C., viscosity=2.5 cSt, d=1.86) (both from 3M). In general, selection of perfluorinated filler fluids is based on kinematic viscosity (<7 cSt is preferred, but not required), and on boiling point (>150° C. is preferred, but not required, for use in DNA/RNA-based applications (PCR, etc.)). 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. Fluorinated oils may in some cases be doped with fluorinated surfactants, e.g., Zonyl FSO-100 (Sigma-Aldrich) and/or others.
“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.
“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.
“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.
“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.
“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.
The terms “top,” “bottom,” “over,” “under,” and “on” are used throughout the description with reference to the relative positions of components of the droplet actuator, such as relative positions of top and bottom substrates of the droplet actuator. It will be appreciated that the droplet actuator is functional regardless of its orientation in space.
When a liquid in any form (e.g., a droplet or a continuous body, whether moving or stationary) is described as being “on”, “at”, or “over” an electrode, array, matrix or surface, such liquid could be either in direct contact with the electrode/array/matrix/surface, or could be in contact with one or more layers or films that are interposed between the liquid and the electrode/array/matrix/surface. In one example, filler fluid can be considered as a film between such liquid and the electrode/array/matrix/surface.
When a droplet is described as being “on” or “loaded on” a droplet actuator, it should be understood that the droplet is arranged on the droplet actuator in a manner which facilitates using the droplet actuator to conduct one or more droplet operations on the droplet, the droplet is arranged on the droplet actuator in a manner which facilitates sensing of a property of or a signal from the droplet, and/or the droplet has been subjected to a droplet operation on the droplet actuator.
The invention is mechanisms for and methods of dispensing liquids in a droplet actuator. For example, various types of reservoirs for use with droplet actuators are disclosed, wherein the reservoirs are preloaded with, for example, sample fluid, liquid reagent, or filler fluid and sealed. In some embodiments, the preloaded reservoir is integrated directly into, for example, the top substrate of the droplet actuator. In other embodiments, the preloaded reservoir is a separate and disposable component with respect to the droplet actuator that can be mechanically and fluidly coupled to the droplet actuator.
Additionally, various types of piercing mechanisms are disclosed for rupturing the seals of the preloaded reservoirs, wherein rupturing the seals causes the liquid to be dispensed into the droplet actuator. In some embodiments, the piercing mechanism is integrated directly into the droplet actuator. Namely, a piercing mechanism is provided in the droplet operations gap of the droplet actuator or protruding from the top substrate. In other embodiments, the piercing mechanism is integrated into the preloaded reservoir, which may be a separate and disposable component with respect to the droplet actuator.
Further, dispensing mechanisms are disclosed for precisely metering the amount of liquid that is dispensed into the droplet actuator. For example, dispensing mechanisms include bladders and weirs for controlling the amount of liquid that is dispensed.
Further, dispensing mechanisms and systems are disclosed that include multiple preloaded reservoirs and mechanisms for rupturing the seals of the multiple preloaded reservoirs. For example, rotatable dispenser systems are disclosed that include multiple preloaded reservoirs and mechanisms for rupturing the seals thereof.
A reservoir 120 is integrated into top substrate 112 for holding a quantity of liquid 122. Liquid 122 is, for example, sample fluid or liquid reagent. A seal 124 is provided at the outlet of reservoir 120, which is facing droplet operations gap 114 of droplet actuator 100. Similarly, a seal 126 is provided at the inlet of reservoir 120. Seal 124 and seal 126 are used to retain liquid 122 inside of reservoir 120 until liquid 122 is ready for use. Seal 124 and seal 126 are, for example, foil seals or cellophane seals. Optionally, reservoir 120 can be vacuum-sealed.
Piercer 150 is installed through an opening in bottom substrate 110. More details of an example of how piercer 150 is formed and installed are described with reference to
Piercer 150, and in particular pointed tip 152, can be any shape, geometry, or length as long as it provides a piercing mechanism.
A reservoir 820 is integrated into top substrate 812 for holding a volume of liquid 822. Liquid 822 is, for example, sample fluid, liquid reagent, or filler fluid. A seal 824 is provided at the outlet of reservoir 820, which is facing droplet operations gap 814 of droplet actuator 800. Similarly, a seal 826 is provided at the inlet of reservoir 820. Seal 824 and seal 826 are used to retain liquid 822 inside of reservoir 820 until liquid 822 is ready for use. Seal 824 and seal 826 are, for example, foil seals or cellophane seals. Optionally, reservoir 820 can be vacuum-sealed. A piercer 850 is installed in bottom substrate 810. A pointed tip 852 of piercer 850 is disposed in droplet operations gap 814 and in close proximity to seal 824 at the outlet of reservoir 820. In one example, piercer 850 is formed of molded plastic. Additionally, the surface of piercer 850 is hydrophilic. Namely, the surface of piercer 850 has a hydrophilic coating (not shown) thereon. Examples of hydrophilic coatings are HYDAK® hydrophilic coatings available from Biocoat, Inc (Horsham, Pa.).
In one example, an electrical connection 840 is provided between the electrically conductive piercer 850 and one of the droplet operations electrodes 816. A voltage source 842 supplies the droplet operations electrode 816 and therefore supplies piercer 850. Namely, by activating the voltage source 842 of the droplet operations electrode 816, both the droplet operations electrode 816 and the electrically conductive piercer 850 are activated. Optionally, the electrically conductive piercer 850 can be split into two or more electrically isolated and individually controlled components.
In operation, at substantially the same time as or just after the seal 824 is punctured using piercer 850, the electrically conductive piercer 850 is activated. The electrowetting forces that are present due to the electrified piercer 850 assist to pull liquid 822 out of reservoir 820 and into droplet operations gap 814. The presence of electrowetting forces due to the electrified piercer 850 provides a higher flow rate of liquid 822 from reservoir 820 than a piercer 850 that is not electrified.
An off-actuator reservoir 1220 is integrated into top substrate 1212 for holding a quantity of liquid 1222. Liquid 1222 is, for example, sample fluid, liquid reagent, or filler fluid. Off-actuator reservoir 1220 is provided to supply liquid 1222 into the droplet operations gap 1214 of droplet actuator 1200. Off-actuator reservoir 1220 is, for example, a bowl-shaped reservoir. Off-actuator reservoir 1220 is sealed until liquid 1222 is ready for use. For example, an outlet of off-actuator reservoir 1220 has a seal 1224 and an inlet of off-actuator reservoir 1220 has a seal 1226. Seal 1224 at the outlet is, for example, a foil seal or cellophane seal. Seal 1226 at the inlet of off-actuator reservoir 1220 is, for example, a versapor oleophobic membrane, or the combination of a versapor oleophobic membrane and foil. If the latter, seal 1226 must include a small portion that is absent foil so that off-actuator reservoir 1220 can vent through versapor oleophobic membrane, which is porous, when liquid 1222 is dispensed therefrom.
A piercer 1228 is affixed to seal 1226 on the side of seal 1226 that is facing liquid 1222. Piercer 1228 has a pointed tip for puncturing seal 1224 at the outlet of off-actuator reservoir 1220. The length of piercer 1228 is such that when seal 1226 is tautly stretched across off-actuator reservoir 1220 the pointed tip of piercer 1228 is not in contact with seal 1224 and therefore does not puncture seal 1224. However, to dispense liquid 1222 the droplet operations gap 1214, the user applies gentle pressure to seal 1226, which causes seal 1226 to flex slightly toward the droplet operations gap 1214. In so doing, the pointed tip of piercer 1228 comes into contact with seal 1224 and punctures seal 1224, which allows liquid 1222 to flow out of the outlet and into the droplet operations gap 1214 of droplet actuator 1200. Off-actuator reservoir 1220 vents through the versapor oleophobic membrane of seal 1226, which is porous, as liquid 1222 dispenses therefrom.
Pipette-style dispenser 1350 includes a barrel 1352 for holding a quantity of liquid 1354. Liquid 1354 is, for example, sample fluid, liquid reagent, or filler fluid. Barrel 1352 is a tapered barrel, meaning that an inlet of barrel 1352 has a larger diameter than an outlet of barrel 1352. A seal (not shown) at the outlet of barrel 1352 and a seal 1356 at the inlet of barrel 1352 are used to retain liquid 1354 inside of pipette-style dispenser 1350 until liquid 1354 is ready for use. The seal (not shown) at the outlet of barrel 1352 and seal 1356 are, for example, foil seals or cellophane seals. In another example, a removable cap is provided at the outlet of barrel 1352 instead of a seal. Optionally, pipette-style dispenser 1350 can be vacuum-sealed. A piercing mechanism 1360 is associated with pipette-style dispenser 1350. Piercing mechanism 1360 includes, for example, a thumbtack-style piercer 1362 that is embedded in a compressible material 1364. Compressible material 1364 is, for example, silicone rubber or foam. When compressible material 1364 is in a relaxed state the pointed tip of thumbtack-style piercer 1362 is hidden inside of compressible material 1364.
A loading port 1320 is integrated into top substrate 1312 for loading liquid into the droplet operations gap 1314 of droplet actuator 1300. Further, loading port 1320 is designed to receive pipette-style dispenser 1350. A port is an entrance/exit (opening) to the droplet operations gap of a droplet actuator. Liquid may flow through the port into and/or from any portion of the droplet operations gap. In droplet actuator 1300, loading port 1320 provides a fluid path through top substrate 1312 to the droplet operations gap 1314 between bottom substrate 1310 and top substrate 1312. In this example, loading port 1320 is tapered to receive pipette-style dispenser 1350. Namely, an inlet 1322 of loading port 1320 has a larger diameter than an outlet 1324 of loading port 1320. The taper of loading port 1320 substantially corresponds to the taper of barrel 1352 of pipette-style dispenser 1350. A seal 1326 is provided at outlet 1324 of loading port 1320. Seal 1326 is, for example, a foil seal or cellophane seal. The position of seal 1326 is such that it is at the same level as the filler fluid (not shown) in droplet operations gap 1314 and therefore air is not trapped near outlet 1324 of loading port 1320.
The operation of pipette-style dispenser 1350 for loading liquid 1354 into droplet actuator 1300 is as follows. First, the user removes the seal (not shown) at the outlet of barrel 1352 of pipette-style dispenser 1350. Because seal 1356 at the inlet of barrel 1352 is still intact, pipette-style dispenser 1350 is not vented and therefore liquid 1354 will not flow out of the outlet of barrel 1352. Next, the user seats the barrel 1352 of pipette-style dispenser 1350 into loading port 1320 of droplet actuator 1300. In so doing, the tip of barrel 1352 breaks seal 1326 of loading port 1320, thereby readying droplet actuator 1300 to receive liquid 1354. Next, the user places piercing mechanism 1360 against seal 1356 at the inlet of barrel 1352 of pipette-style dispenser 1350. Next, the user applies force to thumbtack-style piercer 1362 of piercing mechanism 1360, which compresses compressible material 1364. In so doing, the pointed tip of thumbtack-style piercer 1362 extended out of compressible material 1364 and pierces or punctures seal 1356 of pipette-style dispenser 1350. Next, the user removes piercing mechanism 1360 from pipette-style dispenser 1350, which allows pipette-style dispenser 1350 to vent. Having vented pipette-style dispenser 1350, liquid 1354 flows out of pipette-style dispenser 1350 and into the droplet operations gap 1314 of droplet actuator 1300. The design of loading port 1320 is such that air (if present in the droplet operations gap 1314) can vent out between the walls of loading port 1320 and pipette-style dispenser 1350 while liquid 1354 is flowing into the droplet operations gap 1314. Once pipette-style dispenser 1350 is empty of liquid 1354, the user may remove pipette-style dispenser 1350 from loading port 1320 of droplet actuator 1300. The empty pipette-style dispenser 1350 can be discarded or reloaded with liquid 1354 and resealed for another use.
Pipette-style dispenser 1450 includes a barrel 1452 for holding a quantity of liquid 1454. Liquid 1454 is, for example, sample fluid, liquid reagent, or filler fluid. Barrel 1452 is, for example, an hourglass-shaped or cylinder-shaped barrel that has a flared outlet 1456. A seal 1458 at flared outlet 1456 and a seal 1460 at the inlet of barrel 1452 are used to retain liquid 1454 inside of pipette-style dispenser 1450 until liquid 1454 is ready for use. Seal 1458 and seal 1460 are, for example, foil seals or cellophane seals. In another example, a removable cap is provided at flared outlet 1456 of barrel 1452 instead of seal 1458. Optionally, pipette-style dispenser 1450 can be vacuum-sealed. Additionally, pipette-style dispenser 1450 includes a versapor oleophobic membrane 1462 atop seal 1460 at the inlet of barrel 1452. Versapor oleophobic membrane 1462 is an acrylic copolymer membrane cast on a non-woven nylon support. In one example, versapor oleophobic membrane 1462 is the Versapor® membrane available from Pall Corporation (Port Washington, N.Y.). The Versapor® membrane is available in a variety of pore sizes ranging, for example, from 0.2 μm to 5.0 μm.
A piercer 1470 is associated with pipette-style dispenser 1450. Piercer 1470 is, for example, a fine tip needle. When using pipette-style dispenser 1450, the user uses piercer 1470 to puncture seal 1460. Namely, the user pushes the tip of piercer 1470 through both the versapor oleophobic membrane 1462 and the seal 1460. The size of the tip of piercer 1470 is selected to be less than or equal to the pore size of versapor oleophobic membrane 1462. In one example, if the pore size of versapor oleophobic membrane 1462 is 3.0 μm, then the size of the tip of piercer 1470 is >3.0 μm. In this way, the tip of piercer 1470 can penetrate versapor oleophobic membrane 1462 without damaging it and therefore without compromising its sealing capabilities. As a result, seal 1460 can be punctured using piercer 1470, at the same time the inlet of pipette-style dispenser 1450 can remain sealed by versapor oleophobic membrane 1462.
A loading port 1420 is integrated into top substrate 1412 for loading liquid into the droplet operations gap 1414 of droplet actuator 1400. Further, loading port 1420 is designed to receive pipette-style dispenser 1450. In this example, a piercing edge 1422 is provided at the inlet of loading port 1420. That is, the inlet of loading port 1420 is designed to provide a hollow piercing mechanism for piercing seal 1458 at flared outlet 1456 of pipette-style dispenser 1450. Additionally, the shape of piercing edge 1422 substantially corresponds to the taper in flared outlet 1456 of pipette-style dispenser 1450. An outlet 1424 of loading port 1420 faces droplet operations gap 1414.
The operation of pipette-style dispenser 1450 for loading liquid 1454 into droplet actuator 1400 is as follows. First, the user seats flared outlet 1456 of pipette-style dispenser 1450 onto piercing edge 1422 of loading port 1420 of droplet actuator 1400. In so doing, piercing edge 1422 breaks seal 1458 of pipette-style dispenser 1450. Additionally, when flared outlet 1456 of pipette-style dispenser 1450 is seated onto piercing edge 1422 of loading port 1420, the outer surface of piercing edge 1422 seals against the inner surface of flared outlet 1456. Pipette-style dispenser 1450 is now ready to dispense liquid 1454 into droplet actuator 1400. Next, the user pushes the tip of piercer 1470 through both the versapor oleophobic membrane 1462 and seal 1460 of pipette-style dispenser 1450 in order to puncture seal 1460. Next, the user removes piercer 1470 from pipette-style dispenser 1450, leaving a puncture in seal 1460 that allows pipette-style dispenser 1450 to vent; namely, versapor oleophobic membrane 1462 is suitably porous that air will pass therethrough. Having vented pipette-style dispenser 1450, liquid 1454 flows out of pipette-style dispenser 1450 and into the droplet operations gap 1414 of droplet actuator 1400. Once, pipette-style dispenser 1450 is empty of liquid 1454, the user may remove pipette-style dispenser 1450 from loading port 1420 of droplet actuator 1400. The empty pipette-style dispenser 1450 can be discarded or reloaded with liquid 1454 and resealed for another use.
An off-actuator reservoir 1520 is integrated into top substrate 1512 for holding a quantity of liquid 1522. Liquid 1522 is, for example, sample fluid, liquid reagent, or filler fluid. Off-actuator reservoir 1520 is sealed until liquid 1522 is ready for use. For example, an outlet of off-actuator reservoir 1520 has a seal 1524 and an inlet of off-actuator reservoir 1520 has a seal 1526. Seal 1524 at the outlet is, for example, a foil seal or cellophane seal. Seal 1524 is arranged in or near the droplet operations gap 1514, as shown. Seal 1526 at the inlet of off-actuator reservoir 1520 is, for example, a versapor oleophobic membrane, or the combination of a versapor oleophobic membrane and foil. If the latter, seal 1526 must include a small portion that is absent foil so that off-actuator reservoir 1520 can vent through versapor oleophobic membrane, which is porous.
Droplet actuator 1500 further includes a wire 1530 for rupturing seal 1524 that is arranged in or near the droplet operations gap 1514. For example, a loop of wire 1530 is arranged between two electrical connections 1532 in bottom substrate 110. A voltage source 1534 that is controlled by a switch 1536 supplies the two electrical connections 1532 of nitinol wire 1530.
Namely, wire 1530 loops between the two electrical connections 1532 and across droplet operations gap 1514 in an arching fashion. A center portion of the arching wire 1530 is bonded to seal 1524, as shown. In one example, if seal 1524 is a foil seal then wire 1530 can be soldered to seal 1524. In another aspect of an embodiment, if seal 1524 is a foil seal then wire 1530 can be adhered to seal 1524 with at least one adhesive. In yet another aspect of an embodiment, if seal 1524 is a foil seal then wire 1530 can be induction welded to seal 1524. In a further aspect of an embodiment, if seal 1524 is a foil seal then wire 1530 can be swaged to seal 1524. Wire 1530 an electrically conductive wire formed of nickel titanium (aka nitinol). Nitinol alloys exhibit two closely related and unique properties: shape memory and superelasticity. Shape memory refers to the ability of nitinol to undergo deformation at one temperature, then recover its original, undeformed shape at another temperature. In droplet actuator 1500, nitinol wire 1530 is heated by passing an electric current therethrough. Consequently, nitinol wire 1530 has one arching shape when no electric current is present therein and deforms to a slightly different arching shape when an electric current is present therein.
In operation and referring now to
The plug 1540 can be ruptured by heating in order to dispense liquid 1522 into droplet operations gap 1514. In one example and referring now to
In another example and referring now to
In yet another example, plug 1540 is a silicone-oil-soluble wax, such as 1-2% TRIACONTYLMETHYLSILOXANE)-(DIMETHYLSILOXANE) COPOLYMER having a viscosity (cSt)=2,000-4,000@room temperature. In this example, when droplet operations gap 1514 of droplet actuator 1500 is filled with silicone oil, the silicone oil dissolves plug 1540 and liquid 1522 is released into droplet operations gap 1514.
A loading port 1920 is integrated into top substrate 1912 for loading filler fluid, such as silicone oil, into the droplet operations gap 1914 of droplet actuator 1900. An inlet of loading port 1920 may be sealed with a seal 1922 (e.g., a foil seal or cellophane seal or versapor oleophobic membrane) until ready for use. A bead 1930 is retained in droplet operations gap 1914 using silicone-oil-soluble wax 1932. For example, before droplet operations gap 1914 is filled with filler fluid, a smear of silicone-oil-soluble wax 1932 is provided in a softened or melted state on the surface of bottom substrate 1910. While in the softened or melted state, bead 1930 is stuck into silicone-oil-soluble wax 1932. Then, silicone-oil-soluble wax 1932 is allowed to harden and thereby retain bead 1930 therein. In one example, bead 1930 is a lyophilized bead. In another example, bead 1930 is an encapsulated liquid reagent. According to aspects of embodiments, one or more encapsulants may be formed of one or more of oil or water. Additional aspects of embodiments include an encapsulant that may be soluble at about room, a temperature above room temperature, and/or a temperature in the range about 25 degrees Celsius to about 100 degrees Celsius.
In operation, seal 1922 is removed and loading port 1920 is used to load the droplet operations gap 1914 of droplet actuator 1900 with filler fluid, such as silicon oil. Once silicon oil enters the droplet operations gap 1914, the silicon oil dissolves silicone-oil-soluble wax 1932 and releases bead 1930. Bead 1930 is now free to be manipulated in the droplet operations gap 1914. Those skilled in the art will recognize that multiple beads 1930 can be retained in the droplet operations gap 1914 using silicone-oil-soluble wax 1932. This technique may be useful for preloading and storing beads in a droplet actuator until ready for use. In other embodiment, the wax is not silicone-oil-soluble. Instead, the wax is a low-melting-point silicone wax that can be melted by heating to release the beads.
An off-actuator reservoir 2020 is integrated into top substrate 2012 for holding a quantity of liquid 2022. Liquid 2022 is, for example, sample fluid, liquid reagent, or filler fluid. Off-actuator reservoir 2020 has an outlet 2024, which has a seal 2026 for retaining liquid 2022 inside of off-actuator reservoir 2020 until ready for use. Seal 2026 at the outlet is, for example, a foil seal or cellophane seal. Piercer 150 is installed in bottom substrate 2010 such that pointed tip 152 of piercer 150 is disposed in droplet operations gap 2014 and in close proximity to seal 2026.
Off-actuator reservoir 2020 is designed for metering a certain volume of liquid 2022 into droplet actuator 2000. For example, a weir 2028 is installed inside of off-actuator reservoir 2020 and surrounding outlet 2024. Weir 2028 is used to control the maximum amount of liquid 2022 that is allowed into the workspace of droplet actuator 2000. More specifically, weir 2028 is designed to hold an amount of liquid 2022 that substantially corresponds to the amount of liquid 2022 that droplet actuator 2000 is designed to receive. In one example, if droplet actuator 2000 is designed to receive 400 μl of liquid 2022, then weir 2028 is designed to hold 400 μl of liquid. In another example, if droplet actuator 2000 is designed to receive 600 μl of liquid 2022, then weir 2028 is designed to hold 600 μl of liquid.
In operation, if a user loads off-actuator reservoir 2020 with a quantity of liquid 2022 that exceeds the amount that droplet actuator 2000 is designed to receive, the excess liquid 2022 overflows weir 2028 and is retained inside of off-actuator reservoir 2020 but outside of weir 2028, as shown in
Additionally, the inlet of off-actuator reservoir 2020 may be capped, covered, or otherwise sealed. Further, a cap or cover (not shown) of off-actuator reservoir 2020 may include a loading port (not shown) for guiding liquid 2022 into weir 2028 when off-actuator reservoir 2020 is being loaded.
A loading port 2216 is integrated into top substrate 2212. An inlet of loading port 2216 has a seal 2218. Seal 2218 is, for example, a foil seal or cellophane seal. Loading port 2216 is designed to receive shroud 2116 of syringe 2100. Namely, loading port 2216 is designed to be press fitted inside of shroud 2116 of syringe 2100.
In order to dispense liquid 2120 from syringe 2100 into droplet operations gap 2214 of droplet actuator 2200, first, the user removes seal 2118 from syringe 2100. Next, the user press fits shroud 2116 of syringe 2100 onto the corresponding receptacle of loading port 2216 of droplet actuator 2200, as shown in
An off-actuator reservoir 2720 is integrated into top substrate 2712 for holding a quantity of liquid 2722. Liquid 2722 is, for example, sample fluid, liquid reagent, or filler fluid. An inlet of off-actuator reservoir 2720 is enclosed using a cover 2724. Cover 2724 may be any type of removable or non-removable cap, cover, or seal. For example, cover 2724 can be a hinged cap, a snap-fitted cap, a foil seal, or a cellophane seal. A seal 2726 is provided at an outlet of off-actuator reservoir 2720, which faces droplet operations gap 2714. Seal 2726 is, for example, a foil seal or cellophane seal that can be punctured using piercer 150 that is installed in bottom substrate 2710 of droplet actuator 2700.
Off-actuator reservoir 2720 further includes a bladder 2728 that is squeezable. Namely, squeezing bladder 2728 collapses the walls of bladder 2728 together and forces out any air or liquid 2722 that is present therein. In one example, bladder 2728 is a hollow plastic tube that is closed (i.e., sealed) on one end and open on the end that is coupled to the sidewall of off-actuator reservoir 2720. A hollow plastic tube is but one example of implementing bladder 2728; other methods of implementing bladder 2728 are possible.
Referring now to
A mechanical mechanism can be provided for squeezing bladder 2728. In one example and referring now to
In
Storage reservoir 3010 can be sized to hold any quantity of liquid 3012. Likewise, bladder 3014 can be sized to dispense any quantity of liquid 3012. In this way, bladder 3014 is used to control the amount of liquid 3012 that is dispensed from disposable storage module 3000. Additionally, the proportion of liquid 3012 stored in storage reservoir 3010 versus bladder 3014 can vary. In the example shown in
A seal 3016 is provided at an outlet of storage reservoir 3010 for sealing liquid 3012 inside of disposable storage module 3000 until is ready for use. Seal 3016 is, for example, a foil seal or cellophane seal that can be ruptured using, for example, piercer 150 of
Referring now to
A portion of body 3122 houses one or more reservoirs. For example, body 3122 includes a single reservoir 3128 and a set of four reservoirs 3130. The single reservoir 3128 has a hinged cover 3132 and the set of four reservoirs 3130 has a cover 3134. The reservoir 3128 and reservoirs 3130 can vary in size, holding volumes of liquid ranging, for example, from about 100 μl to about 500 μl. In this example, dispensing system 3120 includes five reservoirs. However, this is exemplary only. Dispensing system 3120 can include any number of reservoirs.
Another portion of body 3122 houses one or more bladders 3136 (not visible) that are associated with reservoir 3128 and reservoirs 3130. A cover 3138 covers the portion of body 3122 that houses the one or more bladders 3136 (not visible). Integrated into cover 3138 are, for example, two dispensing levers 3140. Dispensing levers 3140 are the mechanisms for squeezing the one or more bladders 3136 that are associated with reservoir 3128 and reservoirs 3130. Namely, dispensing levers 3140 and bladders 3136 are used for pumping liquid out of reservoir 3128 and reservoirs 3130 and into the droplet operations gap of droplet actuator 3100. More details of dispensing system 3120 are shown and described with reference to
Referring now to
Referring again to
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Referring now to
Referring to
Referring now to
In a first step and referring again to
In another step and referring now to
In another step and referring now to
In another step and referring now to
Referring now to
Reservoir module 3928 is, for example, a cylinder-shaped module that is partitioned into multiple compartments, whereas the multiple compartments serve as reservoirs 3930. Each of the reservoirs 3930 holds a volume of liquid, such as sample fluid, liquid reagents, or filler fluid. A seal (not shown) is provided on the outlet-side of reservoirs 3930, reservoirs 3930 are then filled with liquid. In one example, the inlet-side of reservoirs 3930 is left open. In another example, the inlet-side of reservoirs 3930 is sealed. The seals (not shown) are, for example, foil seals or cellophane seals. In particular, the seal at the outlet-side of reservoirs 3930 is the type of seal that can be ruptured using piercer 3926.
The size and/or shape of the individual reservoirs 3930 in reservoir module 3928 can be substantially the same or can vary from one to another. Additionally, the overall shape of reservoir module 3928 can vary. More details of other examples of reservoir modules 3928 and reservoirs 3930 are shown and described with reference to
Reservoir module 3928 includes a center hole 3932 that is sized to fit over spindle 3924 of base plate 3922. A lip 3934 is provided at the base of reservoir module 3928. An O-ring 3936 sits atop lip 3934. Additionally, reservoir module 3928 includes an opening or hole 3938 into which a duckbill valve 3940 is installed.
Rotary dispensing system 3920 further includes a retaining cap 3942 for securing reservoir module 3928 to base plate 3922. Retaining cap 3942 includes a base plate 3944 that has a circular opening through which reservoir module 3928 is fitted. Retaining cap 3942 also includes a ring feature 3946 around the opening in base plate 3944. The footprint of base plate 3944 is substantially the same as the footprint of base plate 3922. Rotary dispensing system 3920 further includes a handle or knob 3948 that is fitted onto retaining cap 3942. When assembled, an opening 3850 in handle or knob 3948 is aligned with and mechanically coupled to duckbill valve 3940. Except for the seals (not shown), the components of rotary dispensing system 3920 can be formed, for example, or molded plastic.
Referring now to
Once reservoir module 3928 is on spindle 3924 of base plate 3922 (in the “park” position), retaining cap 3942 is fitted over reservoir module 3928 and base plate 3944 is secured to base plate 3922. For example, base plate 3944 can be snap-fitted to base plate 3922 or fastened to base plate 3922 using screws or adhesive. In so doing, the surface of ring feature 3946 of retaining cap 3942 is fitted snuggly against O-ring 3936 that is atop lip 3934 at the base of reservoir module 3928, which creates a seal between reservoir module 3928 and retaining cap 3942. Then, handle or knob 3948 is, for example, snap-fitted to the top of reservoir module 3928. Features in handle or knob 3948 align with and mechanically secure to the top of duckbill valve 3940 in reservoir module 3928, as shown in
Continuing the example and referring now to
Referring now to
Top substrate 4520 includes, for example, three loading ports 4522 (e.g., loading ports 4522a, 4522b, and 4522c). The locations of loading ports 4522a, 4522b, and 4522c substantially correspond to the locations of reservoir electrodes 4514a, 4514b, and 4514c, respectively. Loading ports 4522a, 4522b, and 4522c include outlets 4524a, 4524b, and 4524c, respectively. Additionally, a piercer 4526 is integrated into top substrate 4520 inside of each of the loading ports 4522. For example, loading ports 4522a, 4522b, and 4522c include piercers 4526a, 4526b, and 4526c, respectively. Each piercer 4526 is, for example, a pointed spike, as shown in
Rotary dispensing module 4540 includes, for example, a body 4542 that is, for example, cylinder-shaped. Body 4542 is partitioned into, for example, three compartments, thereby forming three reservoirs 4544 (e.g., reservoirs 4544a, 4544b, and 4544c). Rotary dispensing module 4540 includes a center hole 4546 that is sized to fit over spindle 4528 of top substrate 4520. When rotary dispensing module 4540 is installed on spindle 4528 of top substrate 4520, reservoirs 4544a, 4544b, and 4544c substantially align with loading ports 4522a, 4522b, and 4522c, respectively, and with piercers 4526a, 4526b, and 4526c, respectively. Additionally, the outlet-side of rotary dispensing module 4540 includes a seal 4548 for sealing the outlets of reservoirs 4544a, 4544b, and 4544c. That is, one continuous seal 4548 can span all three reservoirs 4544. Seal 4548 is, for example, a foil or cellophane seal. Reservoirs 4544a, 4544b, and 4544c hold liquid 4550. Liquid 4550 is, for example, sample fluid, liquid reagent, or filler fluid. Further, reservoirs 4544a, 4544b, and 4544c can be loaded with the same or different types of liquid 4550. For example, reservoir 4544a can be loaded with sample fluid, while reservoirs 4544b and 4544c are loaded with liquid reagent.
In the example shown in
Referring now to
Whereas rotary dispensing module 4540 of
When in use, rotary dispensing module 4700 is installed atop a droplet actuator (not shown), wherein the droplet actuator includes, in this example, three piercers 4720 (e.g., piercers 4720a, 4720b, and 4720c). The locations of piercers 4720a, 4720b, and 4720c substantially correspond to the locations of three loading ports (not shown) or three reservoirs (not shown) of the droplet actuator. A spindle (not shown) on which rotary dispensing module 4700 is mounted is provided with respect to piercers 4720 so that rotary dispensing module 4700 can rotate with respect to piercers 4720.
In operation and referring now to
Next and referring to
Next and referring to
A loading port 4820 is integrated into top substrate 4812. Loading port 4820 has an outlet 4822 facing droplet operations gap 4814. A piercer 4824 protrudes from top substrate 4812 and is inside of loading port 4820. The length of piercer 4824 is greater than the height of loading port 4820. Therefore, the pointed tip of piercer 4824 extends loading port 4820 as shown.
Slidable dispensing reservoir 4830 includes a reservoir 4832 for holding a quantity of liquid 4834. Liquid 4834 is, for example, sample fluid, liquid reagent, or filler fluid. Additionally, the outlet-side of slidable dispensing reservoir 4830 includes a seal 4836 for sealing the outlet of reservoir 4832. Seal 4836 is, for example, a foil or cellophane seal.
In operation, slidable dispensing reservoir 4830 is provided separately from droplet actuator 4800. Slidable dispensing reservoir 4830 is sealed via seal 4836 and reservoir 4832 is loaded with liquid 4834. The user visually aligns reservoir 4832 with loading port 4820 and a places slidable dispensing reservoir 4830 atop top substrate 4520. In so doing, slidable dispensing reservoir 4830 comes to rest against loading port 4820. Because the length of piercer 4824 is greater than the height of loading port 4820, the tip of piercer 4824 punctures or ruptures seal 4836 of reservoir 4832. Then, the user slightly slides slidable dispensing reservoir 4830 so that piercer 4824 can create a larger tear in seal 4836. Liquid 4834 then flows out of reservoir 4832, through loading port 4820, through outlet 4822, and into droplet operations gap 4814 of droplet actuator 4800.
Referring now to
Droplet actuator 4905 may be designed to fit onto an instrument deck (not shown) of microfluidics system 4900. The instrument deck may hold droplet actuator 4905 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 4910, which may be permanent magnets. Optionally, the instrument deck may house one or more electromagnets 4915. Magnets 4910 and/or electromagnets 4915 are positioned in relation to droplet actuator 4905 for immobilization of magnetically responsive beads. Optionally, the positions of magnets 4910 and/or electromagnets 4915 may be controlled by a motor 4920. Additionally, the instrument deck may house one or more heating devices 4925 for controlling the temperature within, for example, certain reaction and/or washing zones of droplet actuator 4905. In one example, heating devices 4925 may be heater bars that are positioned in relation to droplet actuator 4905 for providing thermal control thereof.
A controller 4930 of microfluidics system 4900 is electrically coupled to various hardware components of the invention, such as droplet actuator 4905, electromagnets 4915, motor 4920, and heating devices 4925, as well as to a detector 4935, an impedance sensing system 4940, and any other input and/or output devices (not shown). Controller 4930 controls the overall operation of microfluidics system 4900. Controller 4930 may, for example, be a general purpose computer, special purpose computer, personal computer, or other programmable data processing apparatus. Controller 4930 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 4930 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 4905, controller 4930 controls droplet manipulation by activating/deactivating electrodes.
In one example, detector 4935 may be an imaging system that is positioned in relation to droplet actuator 4905. 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.
Impedance sensing system 4940 may be any circuitry for detecting impedance at a specific electrode of droplet actuator 4905. In one example, impedance sensing system 4940 may be an impedance spectrometer. Impedance sensing system 4940 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.
Droplet actuator 4905 may include disruption device 4945. Disruption device 4945 may include any device that promotes disruption (lysis) of materials, such as tissues, cells and spores in a droplet actuator. Disruption device 4945 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 4905, an electric field generating mechanism, a thermal cycling mechanism, and any combinations thereof. Disruption device 4945 may be controlled by controller 4930.
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.
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.
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.
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'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'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's computer through a communications network.
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).
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.
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.
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.
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' invention set forth in this specification, and neither its use nor its absence is intended to limit the scope of the applicants' 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.
The present application claims the benefit of U.S. Provisional Application No. 61/735,298, filed on Dec. 10, 2012, which is hereby incorporated by reference in its entirety.
This invention was made with government support under HHSN272200900030C awarded by the National Institutes of Health. The United States Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61735298 | Dec 2012 | US |
