WO2013090889A1 - Préparation d'échantillon sur actionneur de gouttelettes - Google Patents

Préparation d'échantillon sur actionneur de gouttelettes Download PDF

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Publication number
WO2013090889A1
WO2013090889A1 PCT/US2012/070055 US2012070055W WO2013090889A1 WO 2013090889 A1 WO2013090889 A1 WO 2013090889A1 US 2012070055 W US2012070055 W US 2012070055W WO 2013090889 A1 WO2013090889 A1 WO 2013090889A1
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WO
WIPO (PCT)
Prior art keywords
droplet
sample
fluid reservoir
droplet actuator
magnetically responsive
Prior art date
Application number
PCT/US2012/070055
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English (en)
Inventor
Allen Eckhardt
Original Assignee
Advanced Liquid Logic Inc
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Publication date
Application filed by Advanced Liquid Logic Inc filed Critical Advanced Liquid Logic Inc
Publication of WO2013090889A1 publication Critical patent/WO2013090889A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • B01L3/502792—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics for moving individual droplets on a plate, e.g. by locally altering surface tension
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B01L2400/0427—Electrowetting
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N2035/1027—General features of the devices
    • G01N2035/1048—General features of the devices using the transfer device for another function
    • G01N2035/1053—General features of the devices using the transfer device for another function for separating part of the liquid, e.g. filters, extraction phase

Definitions

  • the present invention generally relates to sample preparation on droplet actuators.
  • the present invention is directed to simplified apparatuses and methods of sample preparation on droplet actuators.
  • 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.
  • the present invention is directed to simplified apparatuses and methods of sample preparation on droplet actuators.
  • a droplet actuator in one embodiment, includess: a bottom substrate and a top substrate that are separated by a gap; a fluid reservoir integrated into the top substrate; and an opening in the top substrate which is substantially aligned with a reservoir electrode on the bottom substrate and provides a fluid path from the fluid reservoir into the gap; where the fluid reservoir comprises a chemically modified surface.
  • the chemically modified surface of the droplet actuator is modified by chemical conjugation of antibodies, particularly where the antibodies are anti-erythrocytes, leukocytes, or platelets antibodies.
  • the bottom substrate of the droplet actuator is a printed circuit board.
  • the top substrate of the droplet actuator is formed of at least one of glass, injection-molded plastic, silicon, and indium tin oxide.
  • the bottom substrate of the droplet actuator comprises a configuration of droplet operations electrodes.
  • a method of plasma sample preparation on a droplet actuator by immunocapture includes: loading a quantity of a blood sample into a fluid reservoir, where the fluid reservoir comprises a chemically modified surface; incubating the blood sample in the fluid reservoir, where blood cells in the blood sample bind to the chemically modified surface to produce a plasma sample; and dispensing the plasma sample from the fluid reservoir into a droplet operations gap of the droplet actuator.
  • the chemically modified surface is modified by chemical conjugation of antibodies, particularly where the antibodies are anti-erythrocytes, leukocytes, or platelets antibodies.
  • the plasma sample is dispensed atop a reservoir electrode into the droplet operations gap of the droplet actuator.
  • the plasma sample is substantially cell free.
  • the method comprises processing the plasma sample in the droplet actuator via electrowetting droplet operations.
  • a method of preparing a plasma sample from a whole blood sample on a droplet actuator includes: loading a quantity of the whole blood sample into a fluid reservoir; adding a lectin to the whole blood sample, causing cells in the whole blood sample to agglutinate and fall out of solution to produce a plasma sample; and dispensing the plasma sample from the fluid reservoir onto the droplet actuator.
  • the whole blood sample and plasma sample contain human immunodeficiency virus (HIV).
  • the plasma sample is dispensed from the fluid reservoir into a droplet operations gap of the droplet actuator.
  • the plasma sample is dispensed atop a reservoir electrode in the droplet operations gap of the droplet actuator.
  • the plasma sample is combined with a lysis reagent to produce an HIV RNA prior to the step of dispensing the plasma sample from the fluid reservoir into a droplet operations gap of the droplet actuator.
  • the lectin is lyophilized and stored on the droplet actuator.
  • a method of preparing a substantially purified sample on a droplet actuator using hydrophobic chromatography includes: loading a quantity of the sample into a fluid reservoir, where the fluid reservoir contains magnetically responsive beads; washing and eluting the magnetically responsive beads to produce a substantially purified sample; and dispensing the substantially purified sample from the fluid reservoir.
  • the magnetically responsive beads serve as a matrix for hydrophobic chromatography.
  • the magnetically responsive beads are washed and eluted with a gradient of increasing hydrophobicity to produce the substantially purified sample.
  • the magnetically responsive beads serve as a matrix for hydrophobic chromatography.
  • the magnetically responsive beads are washed and eluted with a gradient of increasing hydrophobicity to produce the substantially purified sample.
  • the substantially purified sample comprises a purified protein or small molecule analyte.
  • the substantially purified sample is dispensed from the fluid reservoir into a droplet operations gap of a droplet actuator, particularly where the substantially purified sample is dispensed atop a reservoir electrode in the droplet operations gap of the droplet actuator.
  • the hydrophobic material is removed from the sample.
  • a method of preparing a substantially purified sample on a droplet actuator using ion exchange chromatography includes: loading a quantity of the sample into a fluid reservoir, where the fluid reservoir contains magnetically responsive ion exchange beads; washing and eluting the magnetically responsive beads to produce a substantially purified sample; and dispensing the substantially purified sample from the fluid reservoir.
  • the magnetically responsive ion exchange beads comprise cations and anions.
  • the magnetically responsive ion exchange beads serve as a matrix for ion exchange chromatography.
  • the magnetically responsive ion exchange beads are washed and eluted with a gradient to produce the substantially purified sample, particularly where the the gradient comprises increasing ionic strength, or comprises increasing or decreasing pH.
  • the gradient is a step gradient or a linear gradient.
  • the substantially purified sample comprises a substantially purified protein or small molecule analyte.
  • the substantially purified sample is dispensed from the fluid reservoir into a droplet operations gap of a droplet actuator, particularly where the substantially purified sample is dispensed atop a reservoir electrode in the droplet operations gap of the droplet actuator.
  • a droplet actuator in another embodiment, includes: a bottom substrate and a top substrate that are separated by a gap; an opening in the top substrate which is substantially aligned with a reservoir electrode on the bottom substrate and provides a fluid path from the fluid reservoir into the gap; and a fluid reservoir integrated into the top substrate; where the fluid reservoir comprises a hollow tube extending through a portion of the fluid reservoir, where one end of the hollow tube is located outside of the fluid reservoir and the opposite end of the hollow tube is located inside of the fluid reservoir, and where the end of the hollow tube that is located inside of the fluid reservoir is held at a height with respect to a plane of the opening in the top substrate.
  • the fluid reservoir is enclosed.
  • the end of the hollow tube that is located inside of the fluid reservoir is substantially aligned with the opening.
  • the fluid reservoir maintains a substantially constant and low head pressure at the opening in the top substrate.
  • the bottom substrate comprises a configuration of droplet operations electrodes.
  • the fluid reservoir is filled by threading a smaller sample input tube through the hollow tube.
  • a method of processing cells on a droplet actuator includes: loading a quantity of a sample into a fluid reservoir, wherein the sample includes cells; adding magnetically responsive beads to the sample, wherein the magnetically responsive beads are coated with a biopolymer; and growing the cells on the coated magnetically responsive beads.
  • the biopolymer is a cell attachment promoter, particulary where the cell attachment promoter is collagen or polylysine.
  • processing the cells comprises at least one of growing and testing of cells.
  • the cells comprise mammalian cells.
  • Activate 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.
  • 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 375 V, or about 300 V.
  • any suitable frequency may be employed.
  • an electrode may be activated using alternating current having a frequency from about 1 Hz to about 100 Hz, 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 fluid 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.
  • 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.
  • 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, CA), fluorescent microparticles and nanoparticles, coated magnetic microparticles and nanoparticles, ferromagnetic microparticles and nanoparticles, coated ferromagnetic microparticles and nanoparticles, and those described in U.S.
  • DYNABEADS® particles available from Invitrogen Group, Carlsbad,
  • Beads for use in Fluorescence-based Applications," published on July 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.
  • 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 No. 11/639,566, entitled “Droplet-Based Particle Sorting," filed on December 15, 2006; U.S. Patent Application No. 61/039,183, entitled "Multiplexing Bead Detection in a
  • Beads filed on February 11, 2008; International Patent Application No. PCT/US2008/058018, entitled “Bead-based Multiplexed Analytical Methods and Instrumentation,” filed on March 24, 2008; International Patent Application No. PCT/US2008/058047, “Bead Sorting on a Droplet Actuator,” filed on March 23, 2008; and International Patent Application No. PCT/US2006/047486, entitled “Droplet-based Biochemistry,” filed on December 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 December 11, 2008; U.S. Patent Publication No. 20080151240, “Methods and Systems for Dynamic Range Expansion,” published on
  • Droplet means a volume of liquid on a droplet actuator.
  • a droplet is at least partially bounded by a filler fluid.
  • 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.
  • a droplet may be bounded by filler fluid, one or more surfaces of the droplet actuator, and/or the atmosphere.
  • 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.
  • 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.
  • 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, ex
  • a droplet may include a reagent, such as water, deionized water, saline solutions, acidic solutions, basic solutions, detergent solutions and/or buffers.
  • reagents such as a reagent for a biochemical protocol, such as a nucleic acid amplification protocol, an affinity-based assay protocol, an enzymatic assay protocol, a sequencing protocol, and/or a protocol for analyses of biological fluids.
  • Droplet Actuator means a device for manipulating droplets. For examples of droplet actuators, see Pamula et al., U.S.
  • Patent 6,911,132 entitled “Apparatus for Manipulating Droplets by Electrowetting-Based Techniques,” issued on June 28, 2005; Pamula et al., U.S. Patent Application No. 11/343,284, entitled “Apparatuses and Methods for Manipulating Droplets on a Printed Circuit Board,” filed on filed on January 30, 2006; Pollack et al., International Patent Application No. PCT/US2006/047486, entitled "Droplet-Based
  • Certain droplet actuators will include one or more substrates arranged with a 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.
  • 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.
  • 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.
  • droplets 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.
  • 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.
  • electrodes on one substrate are electrically coupled to the other substrate so that only one substrate is in contact with the droplet actuator.
  • a conductive material e.g., an epoxy, such as MASTER BONDTM Polymer System EP79, available from Master Bond, Inc., Hackensack, NJ
  • 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 ⁇ to about 600 ⁇ , or about 100 ⁇ to about 400 ⁇ , or about 200 ⁇ to about 350 ⁇ , or about 250 ⁇ to about 300 ⁇ , or about 275 ⁇ .
  • 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.
  • the manipulation of droplets by a droplet actuator may be electrode mediated, e.g., electrowetting mediated or dielectrophoresis mediated or Coulombic force mediated.
  • electrode mediated e.g., electrowetting mediated or dielectrophoresis mediated or Coulombic force mediated.
  • other techniques for controlling droplet operations 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.
  • thermodynamic principles e.g. gas bubble generation/phase-change-induced volume expansion
  • surface-wetting principles e.g. electrowetting, and optoelectrowetting, as well as chemically, thermally, structurally and radioactively induced surface-tension gradients
  • gravity surface tension
  • 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.
  • 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.
  • the droplet operations surface may include a hydrophobic coating having a thickness ranging from about 10 nm to about 1,000 nm.
  • 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.
  • the electrically conducting organic polymer that is deposited onto a plastic substrate may be poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT:PSS).
  • PDOT:PSS poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate)
  • 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.
  • 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.
  • 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.
  • the substrate includes a PCB
  • suitable materials are examples of suitable materials: MITSUITM BN-300 (available from MITSUI Chemicals America, Inc., San Jose CA); ARLONTM 1 IN (available from Arlon, Inc, Santa Ana, CA).; NELCO® N4000-6 and N5000-30/32 (available from Park Electrochemical Corp., Melville, NY); ISOLATM FR406 (available from Isola Group, Chandler, AZ), 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-olefm copolymer (COC); cyclo-olefm polymer (COP); aramid; THERMOUNT® nonwoven aramid reinforcement (available from DuPont, Wilmington, DE); NOMEX® brand fiber (available from DuPont, Wilmington, DE); and paper.
  • MITSUITM BN-300
  • Various materials are also suitable for use as the dielectric component of the substrate. Examples include: vapor deposited dielectric, such as PARYLENETM C (especially on glass) and PARYLENETM N (available from Parylene Coating Services, Inc., Katy, TX); TEFLON® AF coatings; cytop; soldermasks, such as liquid photoimageable soldermasks (e.g., on PCB) like TAIYOTM PSR4000 series, TAIYOTM PSR and AUS series (available from Taiyo America, Inc. Carson City, NV) (good thermal characteristics for applications involving thermal control), and PROBIMERTM 8165 (good thermal characteristics for applications involving thermal control (available from Huntsman
  • dry film soldermask such as those in the VACREL® dry film soldermask line (available from DuPont, Wilmington, DE); film dielectrics, such as polyimide film (e.g., KAPTON® polyimide film, available from DuPont, Wilmington, DE), polyethylene, and fluoropolymers (e.g., FEP), polytetrafluoroethylene; polyester; polyethylene naphthalate; cyclo-olefm copolymer (COC); cyclo-olefm polymer
  • 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-chip reservoirs, number of independent electrode connections, size (volume) of different reservoirs, placement of magnets ead 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.
  • 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, and other fluorinated monomers for plasma-enhanced chemical vapor deposition (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.
  • the noise-reducing coating may include a black matrix resin, such as the black matrix resins available from Toray industries, Inc.,
  • 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.
  • 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.
  • merge “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.
  • splitting is 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).
  • 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.
  • 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 Stunner et al.,
  • the sensing or imaging techniques may be used to confirm the presence or absence of a droplet at a specific electrode.
  • the presence of a dispensed droplet at the destination electrode following a droplet dispensing operation confirms that the droplet dispensing operation was effective.
  • 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.
  • 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.
  • 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, lx-, 2x- 3x-droplets are usefully controlled operated using 1, 2, and 3 electrodes, respectively.
  • the difference between the droplet size and the number of electrodes should typically not be greater than 1; in other words, a 2x droplet is usefully controlled using 1 electrode and a 3x droplet is usefully controlled using 2 electrodes.
  • 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.
  • the 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.
  • 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.
  • 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.
  • 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 Fe 3 0 4 , BaFei 2 Oi 9 , CoO, NiO, Mn 2 0 3 , Cr 2 0 3 , and CoMnP.
  • Transporting into the magnetic field of a magnet is intended to refer to transporting into a region of a magnetic field capable of substantially attracting magnetically responsive beads in the droplet.
  • transporting away from a magnet or magnetic field 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • suitable washing techniques are described in Pamula et al., U.S. Patent 7,439,014, entitled “Droplet-Based Surface Modification and Washing,” granted on October 21, 2008, the entire disclosure of which is incorporated herein by reference.
  • top bottom
  • over under
  • under on
  • 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.
  • a liquid in any form e.g., a droplet or a continuous body, whether moving or stationary
  • a liquid in any form e.g., a droplet or a continuous body, whether moving or stationary
  • 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.
  • a droplet 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.
  • Figures 1A through ID illustrate side views of a portion of an example of a droplet actuator and a process of plasma sample preparation on a droplet actuator by immunocapture;
  • Figure 2 illustrates a flow diagram of an example of a method of preparing plasma that contains HIV from whole blood without the need for filtration or centrifugation;
  • Figure 3 illustrates a flow diagram of an example of a method of sample preparation that uses hydrophobic chromatography for purifying and/or removing unwanted materials from the sample;
  • Figure 4 illustrates a flow diagram of another example of a method of sample preparation that uses hydrophobic chromatography for purifying and/or removing unwanted materials from the sample;
  • Figures 5A through 5D illustrate side views of a portion of an example of a droplet actuator and a process of loading solutions of large volume into a droplet actuator under controlled conditions
  • Figure 6 illustrates a flow diagram of an example of a method of processing cells on a droplet actuator.
  • the invention provides methods of sample preparation on a droplet actuator.
  • the invention provides a method of plasma sample preparation on a droplet actuator by immunocapture.
  • the invention provides a method of preparing plasma that contains HIV from whole blood without the need for filtration or centrifugation.
  • the invention provides methods of sample preparation that use hydrophobic chromatography for purifying and/or removing unwanted materials from the sample.
  • the invention also provides a droplet actuator and method of loading solutions of large volume into a droplet actuator under controlled conditions. Further, the invention provides a method of processing cells on a droplet actuator. 7.1 Sample Preparation on a Droplet Actuator
  • Droplet actuator 100 may include a bottom substrate 110 and a top substrate 112 that are separated by a gap 114.
  • Bottom substrate 110 may, for example, be a printed circuit board (PCB).
  • Top substrate 112 may, for example, be formed of glass, injection-molded plastic, silicon, and/or indium tin oxide (ITO).
  • Bottom substrate 110 may include a configuration of droplet operations electrodes 116 (e.g., electrowetting electrodes). Droplet operations are conducted atop droplet operations electrodes 116 on a droplet operations surface.
  • Bottom substrate 110 may also include a reservoir electrode 118, which may be arranged in the configuration of droplet operations electrodes 116.
  • a fluid reservoir 120 may be integrated into top substrate 112 for holding a certain amount of sample fluid to be loaded into droplet actuator 100.
  • Figures 1A through ID show a method of removing blood cells from a blood sample in order to prepare plasma.
  • the method of removing blood cells from a blood sample in order to prepare plasma may include, but is not limited to, the following steps.
  • fluid reservoir 120 is shown in its original manufactured state and with no sample fluid therein.
  • Figure IB shows a chemically modified surface 124 of fluid reservoir 120.
  • the surface of fluid reservoir 120 may be modified by chemical conjugation of anti-erythrocytes, leukocytes, and platelets antibodies.
  • Blood sample 126 is loaded into fluid reservoir 120 and allowed to incubate for a certain amount of time.
  • Blood sample 126 includes blood cells 128.
  • substantially cell free-plasma 130 may be dispensed atop reservoir electrode 118 and into gap 114 of droplet actuator 100.
  • the substantially cell free-plasma 130 may then be processed in droplet actuator 100 via droplet operations, e.g., electrowetting droplet operations.
  • the invention provides a simple method to prepare plasma on a droplet actuator that requires no magnets, no filters, and no centrifugation. Additionally, the method of the invention is very gentle to avoid cell lysis. Further, the method provides maximum recovery of plasma.
  • Figure 2 illustrates a flow diagram of an example of a method 200 of preparing plasma from whole blood without the need for filtration or centrifugation.
  • the method of the invention is dependent on the differential expression of complex carbohydrates on the plasma membrane of cells that are found in blood and HIV. All cells in blood contain on their surface a mixture of N- and O-linked oligosaccharide chains that have unique carbohydrate structures. HIV contains glycoproteins, which are made of only N-linked carbohydrate chains.
  • the method 200 may include, but is not limited to, the following steps.
  • a lectin which is specific for the sugars found in O-linked oligosaccharide chains, is added to the whole blood sample. This causes the cells to rapidly agglutinate.
  • the agglutinated cells condense into a massive cell pellet and fall out of solution without the need for centrifugation.
  • the plasma may be dispensed onto the droplet actuator for use in immunoassays.
  • the plasma contains human immunodeficiency virus (HIV).
  • HIV human immunodeficiency virus
  • Step 212 may leave HIV virus in the remaining liquid plasma fraction.
  • the plasma may be combined with a lysis reagent (on or off the droplet actuator) for the preparation of HIV RNA.
  • the invention provides a method of preparing plasma that contains HIV from whole blood without the need for centrifugation or filtration through small pore membranes. Further, the method is fast, inexpensive, and simple. Lectins can be lyophilized for long term storage stability on a droplet actuator.
  • Figure 3 illustrates a flow diagram of a method 300, which is one example of a sample preparation process that uses hydrophobic chromatography for purifying and/or removing unwanted materials from the sample.
  • Method 300 may be used to enrich and/or purify a protein and/or small molecule analyte. Method 300 may also be used to remove hydrophobic material from the sample that may interfere with some downstream process or assay. Method 300 may include, but is not limited to, the following steps.
  • magnetically responsive beads are modified with, for example, C-4, C-8, and/or C-18 alkyl chains, which may serve as a matrix for hydrophobic chromatography on a droplet actuator.
  • the magnetically responsive beads may be washed and eluted with a gradient of increasing hydrophobicity, such as going from about 0% to about 70% acetonitrile.
  • a gradient of increasing hydrophobicity such as going from about 0% to about 70% acetonitrile.
  • the invention provides a chromatography/extraction method on a droplet actuator that is compatible with electrowetting up to the upper limit of tolerance for hydrophobic solvents.
  • Figure 4 illustrates a flow diagram of a method 400, which is a sample preparation process that uses ion exchange chromatography for purifying and/or removing unwanted materials from the sample.
  • Method 400 may be used to enrich and/or purify a protein and/or small molecule analyte. Method 400 may also be used to remove contaminating and/or unwanted material from the sample that may interfere with some downstream process or assay. Method 400 may include, but is not limited to, the following steps.
  • magnetically responsive ion exchange beads e.g., cation and anion
  • the beads may serve as a matrix for ion exchange chromatography on a droplet actuator.
  • the magnetically responsive ion exchange beads may be washed or eluted with a gradient of increasing ionic strength or increasing or decreasing pH. Thereby enriching and/or purifying a protein and/or small molecule analyte; and thereby removing contaminating and/or unwanted material from the sample that may interfere with some downstream process or assay.
  • An aspect of the method of the invention is that it is easy to use with magnetically responsive beads on a droplet actuator. Step or linear gradients of salt or pH may be readily set up on a droplet actuator for washing and elution of material from the beads. 7.2 Fluid Loading on a Droplet Actuator
  • Droplet actuator 500 may include a bottom substrate 510 and a top substrate 512 that are separated by a gap 514.
  • Bottom substrate 510 may include droplet operations electrodes 516 (e.g., electrowetting electrodes) arranged for conducting droplet operations. Droplet operations are conducted atop droplet operations electrodes 516 on a droplet operations surface.
  • Bottom substrate 510 may also include a reservoir electrode 518, which may be arranged in the configuration of droplet operations electrodes 516.
  • a fluid reservoir 520 may be integrated into top substrate 512 for holding a certain amount of sample fluid to be loaded into droplet actuator 500.
  • An opening 522 in top substrate 512 provides a fluid path from fluid reservoir 520 to gap 514 of droplet actuator 500.
  • Fluid reservoir 520 is an enclosed fluid reservoir.
  • a hollow tube 524 extends through, for example, the top portion of fluid reservoir 520. One end of tube 524 is outside of the enclosed fluid reservoir 520. The opposite end of tube 524 is located inside of fluid reservoir 520. The end of tube 524 that is inside of fluid reservoir 520 is held at a height hi with respect to, for example, the plane of opening 522.
  • Fluid reservoir 520 is designed to hold a large volume of fluid. Without the presence of tube 524 in fluid reservoir 520, the head pressure at opening 522 will vary depending on the amount of fluid in fluid reservoir 520. More specifically, without tube 524 the head pressure is directly proportional to the amount of fluid in fluid reservoir 520. That is, the greater the volume of fluid in fluid reservoir 520, the greater the head pressure. Consequently, without the presence of tube 524 in fluid reservoir 520, there may be a risk of "flooding" of the droplet actuator. However, fluid reservoir 520 is designed to deliver large volumes of fluids to a droplet actuator under controlled conditions. For example, fluid reservoir 520 is designed to deliver large volumes of fluids to a droplet actuator while maintaining a substantially constant and suitably low head pressure at opening 522 for reducing, preferably entirely eliminating, the risk of "flooding" the droplet actuator.
  • the presence of tube 524 in fluid reservoir 520 is used to reduce head pressures at opening 522 to suitably low levels that prevent "flooding" of the droplet actuator.
  • the head pressure on the fluid that is in fluid reservoir 520 is only the pressure that is present from the base of fluid reservoir 520 (at opening 522) to the end of tube 524 that is near opening 522 and set at height hi.
  • the head pressure on the fluid in fluid reservoir 520 remains constant even when the fluid level changes as long as the end of tube 524 is in the liquid (see Figures 5B, 5C, and 5D).
  • Fluid reservoir 520 may be filled by threading a smaller sample input tube (not shown) through the hollow tube 524.
  • Figure 5B shows fluid reservoir 520 that is substantially full of fluid 526. Yet there is a certain head pressure at opening 522 of top substrate 512 that is dependent on height hi of tube 524.
  • Figure 5C shows fluid reservoir 520 with a lesser amount of fluid 526 therein as compared with the amount of fluid 526 shown in Figure 5B. Yet the head pressure at opening 522 of top substrate 512, which is dependent on height hi of tube 524, remains substantially unchanged.
  • Figure 5D shows fluid reservoir 520 with a yet lesser amount of fluid 526 therein as compared with the amount of fluid 526 shown in Figures 5B and 5C. Yet the head pressure at opening 522 of top substrate 512, which is dependent on height hi of tube 524, remains substantially unchanged.
  • An aspect of the droplet actuator and fluid reservoir of the invention is that it provides a simple structure that delivers solutions of large volumes to a droplet actuator under controlled conditions.
  • Figure 6 illustrates a flow diagram of a method 600 of processing cells on a droplet actuator.
  • Growth and testing of mammalian cells in a droplet actuator has widespread utility. The cells must be fed fresh media at regular intervals and many cells are anchorage dependent and require a surface on which to grow.
  • Method 600 provides a surface for cells to grow on in a droplet actuator, wherein the surface is not a surface of the bottom or top substrate. Method 600 may include, but is not limited to, the following steps.
  • magnetically responsive beads which are coated with biopolymers, such as collagen or other cell attachment promoters (e.g., polylysine), are provided in the sample fluid in a droplet actuator.
  • biopolymers such as collagen or other cell attachment promoters (e.g., polylysine).
  • step 612 because the surface of the magnetically responsive beads is favorable for the anchoring and growth of cells on a droplet actuator, cells may be seeded and allowed to grow on the magnetically responsive beads. This greatly simplifies cell washing and replacing media for nutrients and oxygen.
  • An aspect of the method of the invention is that it provides a cell attachment strategy to support growth on a droplet actuator, wherein the surface on which the cells grow is not a surface of the bottom or top substrate.
  • 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.”
  • 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.
  • 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.
  • 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.
  • RAM random access memory
  • ROM read-only memory
  • EPROM or Flash memory erasable programmable read-only memory
  • CD-ROM compact disc read-only memory
  • CD-ROM compact disc read-only memory
  • a transmission medium such as those supporting the Internet or an intranet, or a magnetic storage device.
  • 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.
  • 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").
  • GUI graphical user interface
  • 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 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 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.
  • IP Internet Protocol
  • the communications network 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.

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Abstract

La présente invention porte sur des procédés de préparation d'échantillon sur un actionneur de gouttelettes. L'invention concerne en outre des procédés de préparation d'échantillon de plasma sur un actionneur de gouttelettes par immuno-capture. L'invention concerne également des procédés de préparation de plasma qui contient un VIH à partir de sang total sans le besoin de filtration ou de centrifugation. De plus, l'invention concerne des procédés de préparation d'échantillon qui utilisent une chromatographie hydrophobe pour purifier ou éliminer des matières non désirées de l'échantillon. L'invention concerne en outre un actionneur de gouttelettes et des procédés de cargement de solutions de grand volume à l'intérieur d'un actionneur de gouttelettes dans des conditions contrôlées. L'invention concerne également des procédés de traitement de cellules sur un actionneur de gouttelettes.
PCT/US2012/070055 2011-12-16 2012-12-17 Préparation d'échantillon sur actionneur de gouttelettes WO2013090889A1 (fr)

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