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WO2010042637A2 - Incubation et lavage de billes sur un actionneur à gouttelettes - Google Patents

Incubation et lavage de billes sur un actionneur à gouttelettes Download PDF

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Publication number
WO2010042637A2
WO2010042637A2 PCT/US2009/059868 US2009059868W WO2010042637A2 WO 2010042637 A2 WO2010042637 A2 WO 2010042637A2 US 2009059868 W US2009059868 W US 2009059868W WO 2010042637 A2 WO2010042637 A2 WO 2010042637A2
Authority
WO
WIPO (PCT)
Prior art keywords
droplet
magnet
magnetically responsive
beads
responsive beads
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2009/059868
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English (en)
Other versions
WO2010042637A3 (fr
Inventor
Ramakrishna Sista
Arjun Sudarsan
Vamsee Pamula
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Advanced Liquid Logic Inc
Original Assignee
Advanced Liquid Logic Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Advanced Liquid Logic Inc filed Critical Advanced Liquid Logic Inc
Publication of WO2010042637A2 publication Critical patent/WO2010042637A2/fr
Publication of WO2010042637A3 publication Critical patent/WO2010042637A3/fr
Priority to US13/081,927 priority Critical patent/US8637324B2/en
Anticipated expiration legal-status Critical
Priority to US14/081,376 priority patent/US8846410B2/en
Priority to US14/466,193 priority patent/US9081007B2/en
Priority to US14/731,740 priority patent/US9395361B2/en
Priority to US15/210,634 priority patent/US10078078B2/en
Priority to US16/132,175 priority patent/US10585090B2/en
Priority to US16/811,789 priority patent/US11525827B2/en
Priority to US18/063,874 priority patent/US20230128722A1/en
Ceased legal-status Critical Current

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Classifications

    • 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
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Definitions

  • the present invention provides methods and apparatuses for incubating and washing magnetically responsive beads on a droplet actuator. More specifically the present invention provides methods for incubating magnetically responsive beads that are labeled with primary antibody, a sample (i.e., analyte), and secondary reporter antibodies on a magnet, on and off a magnet, and completely off a magnet. The invention also provides methods for washing magnetically responsive beads using shape-assisted merging of droplets. The invention also provides methods for shape-mediated splitting, transporting, and dispensing of a sample droplet that contains magnetically responsive beads. The methods of the invention provide for rapid time to result and optimum detection of an analyte in an immunoassay.
  • Droplet actuators are used to conduct a wide variety of droplet operations.
  • a droplet actuator typically includes two substrates separated by a gap.
  • the substrates include electrodes for conducting droplet operations.
  • the gap between the substrates is typically filled with a filler fluid that is immiscible with the fluid that is to be subjected to droplet operations.
  • Droplet operations are controlled by electrodes associated with one or both of the substrates.
  • Droplet actuators are used in a variety of applications, including molecular diagnostic assays, such as immunoassays where time to result is directly affected by the protocols used for each step of the assay. The most time consuming steps in an immunoassay are incubation and washing.
  • Time to result is directly affected by the protocols used for incubation, the duration of time for incubating the antibodies and the antigens, and the duration of time for incubating the substrate with sandwich beads, all of which may depend on the mixing efficiency within the droplets and the reaction and binding kinetics.
  • the amount of washing required to obtain the required sensitivity may also influence the total time to result for immunoassays.
  • the present invention is directed to bead incubation and washing on a droplet actuator.
  • the methods described herein may include providing a droplet including one or more magnetically responsive beads.
  • the methods may include exposing the magnetically responsive beads in the droplet to a first region of a magnetic field capable of substantially attracting magnetically responsive beads in the droplet.
  • the methods may include separating the droplet from the first region of the magnetic field, the magnetically responsive beads remaining in the magnetic field.
  • the magnetically responsive beads may be separated from the droplet while exposing the magnetically responsive beads in the droplet to a first region of a magnetic field and/or while the droplet is being separated from the first region of the magnetic field.
  • a method of incubating droplets having magnetically responsive beads comprises providing a droplet actuator comprising droplet operations electrodes arranged for conducting droplet operations on a droplet operations surface and a magnet positioned relative to the droplet operations surface such that a droplet controlled by one or more of the droplet operations electrodes may be positioned within or away from a first region of the magnet's magnetic field capable of substantially attracting magnetically responsive beads in the droplet.
  • the method also comprises positioning a droplet having magnetically responsive beads therein at a location on the droplet operations surface within the first region of the magnetic field to form a concentration of beads in the droplet; transporting the droplet through activation of selected droplet operations electrodes away from the first region of the magnetic field, thereby resuspending the magnetically responsive beads in the droplet; operating the droplet operations electrodes to cause the droplet to split into two droplets, thereby redistributing the magnetically responsive beads; and operating the droplet operations electrodes to merge the two droplets into a single droplet.
  • a method of incubating droplets having magnetically responsive beads therein comprises providing a droplet actuator comprising droplet operations electrodes arranged for conducting droplet operations on a droplet operations surface and a magnet positioned relative to the droplet operations surface such that a droplet controlled by one or more of the droplet operations electrodes may be positioned within or away from a first region of the magnet's magnetic field capable of substantially attracting magnetically responsive beads in the droplet.
  • the method also comprises positioning a droplet having magnetically responsive beads therein at a location within the first region of the magnetic field of the magnet to form a concentration of beads in the droplet; transporting the droplet through activation of selected droplet operations electrodes away from the first region of the magnetic field of the magnet to resuspend the magnetically responsive beads in the droplet; operating the droplet operations electrodes to cause the droplet to elongate and then split into two droplets at a location away from the magnet; and operating the droplet operations electrodes to merge the two droplets into a single droplet at a location away from the magnet, whereby the transporting, splitting, and merging comprise an incubation cycle.
  • a method of incubating droplets having magnetically responsive beads therein comprises providing a droplet actuator comprising droplet operations electrodes arranged for conducting droplet operations on a droplet operations surface and a magnet positioned relative to the droplet operations surface such that a droplet controlled by one or more of the droplet operations electrodes may be positioned within or away from a first region of the magnet's magnetic field capable of substantially attracting magnetically responsive beads in the droplet.
  • the method further comprises positioning a droplet having magnetically responsive beads therein on a droplet operations electrode, the droplet having a footprint approximately two times the area of a single droplet operations electrode; transporting the droplet through activation of selected droplet operations electrodes in one direction in a manner elongating the droplet; and operating the droplet operations electrodes in a manner to cause the droplet to be transported in an opposite direction to cause mixing and incubation within the droplet.
  • a method of washing magnetically responsive beads for separating and removing unbound material comprises providing a droplet actuator comprising droplet operations electrodes arranged for conducting droplet operations on a droplet operations surface and a magnet positioned relative to the droplet operations surface such that a droplet controlled by one or more of the droplet operations electrodes may be positioned within or away from a first region of the magnet's magnetic field capable of substantially attracting magnetically responsive beads in the droplet.
  • the method further comprises positioning a droplet having magnetically responsive beads therein to have a first region of the droplet within the first region of the magnetic field to form a concentration of beads; at another end of the magnetic field, positioning a wash buffer droplet such that a first region of the wash buffer droplet is within the first region of the magnetic field; operating the droplet operations electrodes to merge the droplet and the wash droplet to cause redistribution of beads; operating the droplet operation electrodes to cause the merged droplet to partially move away from the magnet, and to cause beads in the droplet to concentrate in the merged droplet; and operating the droplet operations electrodes to split the merged droplet to form a supernatant droplet containing unbound reagents.
  • a method of resuspending magnetically responsive beads between wash cycles comprises providing a droplet actuator comprising droplet operations electrodes arranged for conducting droplet operations on a droplet operations surface and a magnet positioned relative to the droplet operations surface such that a droplet controlled by one or more of the droplet operations electrodes may be positioned within or away from a first region of the magnet's magnetic field capable of substantially attracting magnetically responsive beads in the droplet.
  • the method further comprises positioning a droplet having magnetically responsive beads therein at a location partially overlapping the first region of the magnetic field; transporting the droplet through activation of selected droplet operations electrodes away from the first region of the magnetic field; operating the droplet operations electrodes to cause the droplet to move towards the first region of the magnetic field; and repeating the transporting and operating steps to cause sufficient resuspension of beads such that unbound material may be effectively removed in subsequent wash cycles.
  • a method of separating magnetically responsive beads from a droplet comprises providing a droplet actuator comprising droplet operations electrodes arranged for conducting droplet operations on a droplet operations surface and a magnet positioned relative to the droplet operations surface such that a droplet controlled by one or more of the droplet operations electrodes may be positioned within or away from a first region of the magnet's magnetic field capable of substantially attracting magnetically responsive beads in the droplet.
  • the method further comprises positioning a droplet having magnetically responsive beads therein within the first region of the magnetic field of the magnet to cause the magnetically responsive beads to be attracted to the magnet, and activating the droplet operations surface to cause the droplet to be circular in shape; operating the droplet operations surface to cause the droplet to move away from the first region of the magnetic field to form a concentration of magnetically responsive beads in the droplet, and the droplet operations surface being operated to cause the droplet to be transported away from the magnet one droplet operations electrode at a time, to cause the geometry of the droplet to be distorted; and continuing to transport the droplet away from the magnet to cause the concentration of magnetically responsive beads to break away from the droplet to result in a relatively small and highly concentrated magnetically responsive bead droplet left behind and held immobilized by the magnet.
  • a method of transporting magnetically responsive beads within droplets comprises providing a droplet actuator comprising droplet operations electrodes arranged for conducting droplet operations on a droplet operations surface and a magnet positioned relative to the droplet operations surface such that a droplet controlled by one or more of the droplet operations electrodes may be positioned within or away from a first region of the magnet's magnetic field capable of substantially attracting magnetically responsive beads in the droplet.
  • the method further comprises positioning a droplet having magnetically responsive beads located therein at a location wherein the droplet partially overlaps the magnet; operating the droplet operations electrodes to subject an edge of the droplet nearest the magnet to both a magnetic force from the first region of the magnetic field and an electrowetting force from the droplet operations electrodes, and to subject an edge of the droplet furthest from the magnet only to an electrowetting force, to cause the droplet to be transported away from the magnet while retaining the magnetically responsive beads within the droplet; and continuing to transport the droplet away from the magnet to cause the magnetically responsive beads to be redistributed within the droplet.
  • a method of separating beads from a droplet onto a magnet comprises providing a droplet actuator comprising droplet operations electrodes arranged for conducting droplet operations on a droplet operations surface and a magnet positioned relative to the droplet operations surface such that a droplet controlled by one or more of the droplet operations electrodes may be positioned within or away from a first region of the magnet's magnetic field capable of substantially attracting magnetically responsive beads in the droplet.
  • the method further comprises positioning a droplet having magnetically responsive beads therein within the first region of the magnetic field of the magnet to cause the beads to be attracted to the magnet, and activating the droplet operations surface in a manner to cause the droplet to take an elongate shape; operating the droplet operations surface to activate one electrode at a time, to cause the droplet to move away from the magnet, and thereby cause the geometry of the droplet to be distorted; and continuing to operate the droplet operations surface to transport the droplet further away from the magnet and inactivating an electrode intermediate to the droplet to cause the droplet to split into a supernatant droplet and a smaller droplet that has the magnetically responsive beads therein.
  • a droplet actuator structure for extracting DNA from a sample comprises at least six on-actuator reservoirs interconnected for storing and dispensing different reagents onto the droplet actuator; and the reservoirs interconnected through paths of droplet operations electrodes, including at least two paths having magnets associated therewith, and a bead collection reservoir connected to the six on-actuator reservoirs through the droplet operations electrodes paths.
  • a method of extracting DNA from whole blood comprises using a droplet actuator comprising at least six on-actuator reservoirs interconnected for storing and dispensing different reagents onto the droplet actuator; and the reservoirs interconnected through paths of droplet operations electrodes, including at least two paths having magnets associated therewith, and a bead collection reservoir connected to the six on-actuator reservoirs through the droplet operations electrodes paths.
  • the method further comprises dispensing a droplet of magnetically responsive beads suspended in a lysis buffer from a first of the six on-actuator reservoirs, and transporting the droplet through the droplet operations electrodes to a specific location having one of the magnets associated with the location, to hold the magnetically responsive beads within the droplet thereon; dispensing droplets of whole blood from a second reservoir and lysis buffer from a third reservoir into a fourth mixing reservoir to be mixed therein to form a cell lysate; dispensing droplets of the cell lysate across the magnetically responsive beads in succession and removing supernatant from the droplets while holding the magnetically responsive beads; dispensing wash droplets from at least a fifth reservoir to wash the magnetically responsive beads to remove cell debris; and eluting and collecting DNA captured on the magnetically responsive beads at the bead collection reservoir.
  • a method of detecting a component in a sample comprises providing a droplet actuator comprising droplet operations electrodes arranged for conducting droplet operations on a droplet operations surface; a magnet positioned related to the droplet operations surface such that a droplet controlled by one of more droplet operations electrodes may be positioned within or away from a first region of the magnet's magnetic field capable of substantially attracting magnetically responsive beads in the droplet; and a wash reservoir at one end of the arrangement of droplet operations electrodes.
  • the method further comprises positioning a droplet having magnetically responsive beads located therein, the magnetically responsive beads being coated with an antibody having an affinity for a specific target antigen, away from the magnet; operating the droplet operations surface in a manner to repeatedly transport the droplet back and forth, away from the magnet, in a manner to provide sufficient resuspension and mixing of the magnetically responsive beads for antibody and antigen binding; operating the droplet operations surface in a manner to transport the droplet to a location within the first region of the magnetic field, and splitting off a supernatant droplet from the droplet by selectively operating the droplet operations surface, and retaining the magnetically responsive beads at the magnet; operating the droplet operations electrodes to transport a reagent droplet to the droplet operations electrode in the first region of the magnetic field to merge the reagent droplet with the droplet containing the magnetically responsive beads, and transporting the merged droplet back and forth, at a location away from the magnet, to cause incubation; and transporting the merged droplet through operation of the droplet operations
  • Activate with reference to one or more electrodes means effecting a change in the electrical state of the one or more electrodes which results in a droplet operation.
  • Bead with respect to beads on a droplet actuator, means any bead or particle that is capable of interacting with a droplet on or in proximity with a droplet actuator. Beads may be any of a wide variety of shapes, such as spherical, generally spherical, egg shaped, disc shaped, cubical and other three dimensional shapes. The bead may, for example, be capable of being transported in a droplet on a droplet actuator or otherwise configured with respect to a droplet actuator in a manner which permits a droplet on the droplet actuator to be brought into contact with the bead, on the droplet actuator and/or off the droplet actuator.
  • Beads may be manufactured using a wide variety of materials, including for example, resins, and polymers.
  • the beads may be any suitable size, including for example, microbeads, microparticles, nanobeads and nanoparticles.
  • beads are magnetically responsive; in other cases beads are not significantly magnetically responsive.
  • the magnetically responsive material may constitute substantially all of a bead or one component only of a bead. The remainder of the bead may include, among other things, polymeric material, coatings, and moieties which permit attachment of an assay reagent. Examples of suitable magnetically responsive beads are described in U.S. Patent Publication No. 2005-0260686, entitled,
  • Multiplex flow assays preferably with magnetic particles as solid phase published on November 24, 2005, the entire disclosure of which is incorporated herein by reference for its teaching concerning magnetically responsive materials and beads.
  • the fluids may include one or more magnetically responsive and/or non-magnetically responsive beads.
  • 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 Single Droplet,” filed on March 25, 2008; U.S. 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.
  • Droplet means a volume of liquid on a droplet actuator that is at least partially bounded by filler fluid.
  • a droplet may be completely surrounded by filler fluid or may be bounded by filler fluid and one or more surfaces of the droplet actuator.
  • Droplets may, for example, be aqueous or non-aqueous or may be mixtures or emulsions including aqueous and non-aqueous components.
  • Droplets may take a wide variety of shapes; nonlimiting examples include generally disc shaped, slug shaped, truncated sphere, ellipsoid, spherical, partially compressed sphere, hemispherical, ovoid, cylindrical, and various shapes formed during droplet operations, such as merging or splitting or formed as a result of contact of such shapes with one or more surfaces of a droplet actuator.
  • Droplet Actuator means a device for manipulating droplets.
  • droplets see U.S. Patent 6,911,132, entitled “Apparatus for Manipulating Droplets by Electrowetting-Based Techniques,” issued on June 28, 2005 to Pamula et al.; U.S. Patent 6,911,132, entitled “Apparatus for Manipulating Droplets by Electrowetting-Based Techniques,” issued on June 28, 2005 to Pamula et al.; U.S. Patent 6,911,132, entitled “Apparatus for Manipulating Droplets by Electrowetting-Based Techniques,” issued on June 28, 2005 to Pamula et al.; U.S. Patent 6,911,132, entitled “Apparatus for Manipulating Droplets by Electrowetting-Based Techniques,” issued on June 28, 2005 to Pamula et al.; U.S. Patent 6,911,132, entitled “Apparatus for Manipulating Droplets by Electrowetting-Based Techniques,” issued on June 28, 2005 to Pamula et al.
  • Methods of the invention may be executed using droplet actuator systems, e.g., as described in International Patent Application No. PCT/US2007/009379, entitled “Droplet manipulation systems,” filed on May 9, 2007.
  • the manipulation of droplets by a droplet actuator may be electrode mediated, e.g., electrowetting mediated or dielectrophoresis mediated.
  • Droplet operation means any manipulation of a droplet on a droplet actuator.
  • a droplet operation may, for example, include: loading a droplet into the droplet actuator; dispensing one or more droplets from a source droplet; splitting, separating or dividing a droplet into two or more droplets; transporting a droplet from one location to another in any direction; merging or combining two or more droplets into a single droplet; diluting a droplet; mixing a droplet; agitating a droplet; deforming a droplet; retaining a droplet in position; incubating a droplet; heating a droplet; vaporizing a droplet; condensing a droplet from a vapor; cooling a droplet; disposing of a droplet; transporting a droplet out of a droplet actuator; other droplet operations described herein; and/or any combination of the foregoing.
  • any combination of droplet operations sufficient to result in the combination of the two or more droplets into one droplet may be used.
  • “merging droplet A with droplet B” can be achieved by transporting droplet A into contact with a stationary droplet B, transporting droplet B into contact with a stationary droplet A, or transporting droplets A and B into contact with each other.
  • the terms “splitting,” “separating” and “dividing” are not intended to imply any particular outcome with respect to size of the resulting droplets (i.e., the size of the resulting droplets can be the same or different) or number of resulting droplets (the number of resulting droplets may be 2, 3, 4, 5 or more).
  • the term “mixing” refers to droplet operations which result in more homogenous distribution of one or more components within a droplet. Examples of “loading" droplet operations include microdialysis loading, pressure assisted loading, robotic loading, passive loading, and pipette loading.
  • the droplet operations may be electrode mediated, e.g., electrowetting mediated or dielectrophoresis mediated.
  • Filler fluid means a fluid associated with a droplet operations substrate of a droplet actuator, which fluid is sufficiently immiscible with a droplet phase to render the droplet phase subject to electrode-mediated droplet operations.
  • the filler fluid may, for example, be a low- viscosity oil, such as silicone oil.
  • Other examples of filler fluids are provided in International Patent Application No. PCT/US2006/047486, entitled, “Droplet-Based Biochemistry,” filed on December 11, 2006; and in International Patent Application No.
  • PCT/US2008/072604 entitled “Use of additives for enhancing droplet actuation,” filed on August 8, 2008.
  • 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 to permit execution of a splitting operation on a droplet, yielding one droplet with substantially all of the beads and one droplet substantially lacking in the beads.
  • Magnetically responsive means responsive to a magnetic field.
  • Magnetically responsive beads include or are composed of magnetically responsive materials. Examples of magnetically responsive materials include paramagnetic materials, ferromagnetic materials, ferrimagnetic materials, and metamagnetic materials. Examples of suitable paramagnetic materials include iron, nickel, and cobalt, as well as metal oxides, such as Fe 3 O 4 , BaFeI 2 Oi 9 , CoO, NiO, Mn 2 O 3 , Cr 2 O 3 , and CoMnP.
  • Washing with respect to washing a magnetically responsive bead means reducing the amount and/or concentration of one or more substances in contact with the magnetically responsive bead or exposed to the magnetically responsive bead from a droplet in contact with the magnetically responsive bead.
  • the reduction in the amount and/or concentration of the substance may be partial, substantially complete, or even complete.
  • the substance may be any of a wide variety of substances; examples include target substances for further analysis, and unwanted substances, such as components of a sample, contaminants, and/or excess reagent.
  • 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.
  • top and bottom are used throughout the description with reference to the top and bottom substrates of the droplet actuator for convenience only, since the droplet actuator is functional regardless of its position in space.
  • 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 capable of substantially attracting any magnetically responsive beads in the droplet.
  • 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 IA, IB, 1C, ID, and IE illustrate top views of an example of a region of a droplet actuator and show a process of incubating beads on a magnet;
  • Figures 2A, 2B, 2C, 2D, and 2E illustrate top views of an example of a region of a droplet actuator of Figures IA through IE and show a process of incubating droplets that include magnetically responsive beads on and off a magnet;
  • Figures 3A, 3B, 3C, 3D, and 3E illustrate top views of an example of a region of a droplet actuator and show a process of incubating droplets that include magnetically responsive beads completely off a magnet;
  • Figure 4 shows a plot of a comparison of incubation time between on-magnet and off- magnet incubation protocols of Figure 1 and Figure 2, respectively, on immunoassay performance measured in chemiluminescence;
  • Figures 5A, 5B, 5C, 5D, and 5E show a top view of a region of a droplet actuator and a process of washing magnetically responsive beads using shape-assisted merging of droplets;
  • Figure 6 shows a plot of a comparison of washing protocols between slug shaped and circular shaped wash droplets on immunoassay performance measured in chemiluminescence
  • Figures 7 A, 7B, 1C, and 7D illustrate top views of an example of a region of a droplet actuator of Figures 5A through 5E and show a process of resuspending magnetically responsive beads between wash cycles;
  • Figures 8A and 8B show plots of a comparison of washing protocols of Figure 5 without resuspension cycles and with resuspension cycles, respectively;
  • Figure 9 illustrates a top view of a region of a droplet actuator 900 that includes multiple waste wells
  • Figures 1OA, 1OB, and 1OC show a top view of a region of a droplet actuator and a process of separating beads from a sample droplet;
  • Figures 1 IA, 1 IB, and 11C show a top view of a region of a droplet actuator of Figures 1OA through 1OC and a process of transporting beads within a droplet;
  • Figures 12A, 12B, and 12C show a top view of a region of a droplet actuator of Figures 1 OA through 1 OC and another process of separating beads from a sample droplet onto a magnet;
  • Figures 13A and 13B show a comparison of bench top and droplet actuator immunoassay reagent ratios and a plot of reagent concentration versus signal strength, respectively, that provide for optimum droplet based immunoassay performance;
  • Figure 14 shows a plot of the kinetics of a reaction between a chemiluminescent substrate and ALP reporter on magnetically responsive beads for Troponin I (TnI);
  • Figure 15 is a top view of a droplet actuator that may be used for extracting DNA from a whole blood sample
  • Figures 16A and 16B illustrate top views of an example of a portion of a droplet actuator and show a process of cytokine detection on a droplet actuator;
  • Figure 17 shows a plot of two 5-point standard curves for cytokine IL-6.
  • Figure 18 shows a plot of two 6-point standard curves for cytokine TNF- ⁇ .
  • the present invention provides methods and apparatuses for incubating and washing magnetically responsive beads on a droplet actuator. More specifically the present invention provides methods for incubating magnetically responsive beads that are labeled with primary antibody, a sample (i.e., analyte), and secondary reporter antibodies on a magnet, on and off a magnet, and completely off a magnet. The invention also provides methods for washing magnetically responsive beads using shape-assisted merging of droplets. The invention also provides methods for shape-mediated splitting, transporting, and dispensing of a sample droplet that contains magnetically responsive beads. The methods of the invention provide for rapid time to result and optimum detection of an analyte in an immunoassay.
  • a droplet actuator may be used to extract human genomic DNA from a sample.
  • Incubation protocols on a droplet actuator are generally comprised of transporting a droplet (e.g., a droplet that includes an antigen, primary capture antibodies conjugated to magnetically responsive beads, and secondary reporter antibodies) along a path of electrodes by use of splitting and merging operations that are inserted between transport cycles. Transporting, splitting, and merging the droplet ensures that the beads are well distributed (i.e., mixed) within the droplet.
  • An incubation cycle e.g., transport, split, and merge
  • the high mixing efficiency provided by a series of incubation cycles provides for sufficient antigen-antibody binding.
  • Magnetically responsive beads have a tendency to settle and form aggregates due to gravity and/or continued exposure to strong magnetic forces. These aggregates reduce the available surface area for binding and slow down reaction kinetics and, consequently, the time to result and sensitivity of the assay. Moreover, interstices in magnetically responsive bead aggregates can hold unbound species that leads to ineffective washing. This may result in less sensitive assays and inaccuracies between assays due to differing amounts of unbound species held in the interstices. Therefore, it is useful to keep the beads dispersed or resuspended during incubation and in the steps immediately following separation for further processing of the droplets away from the magnets. Resuspension of magnetically responsive beads within droplets, akin to rigorous vortexing of bench scale systems, may be achieved by moving the bead droplet back and forth and exploiting the inherent circulatory flow patterns that are developed during droplet transport.
  • Figures IA, IB, 1C, ID, and IE illustrate top views of an example of a region of a droplet actuator 100 and show a process of incubating droplets that include magnetically responsive beads on a magnet.
  • the method of the invention of Figures IA through IE is an example of an incubation method wherein a droplet is transported into the magnetic field of a magnet and a series of split and merge droplet operations are performed to resuspend the beads within the droplet. Because of the magnet field, resuspension of the beads is primarily in the X-Y direction.
  • Droplet actuator 100 may include a path or array of droplet operations electrodes 110 (e.g., electrowetting electrodes).
  • a magnet 114 is arranged in close proximity to droplet operations electrodes 110.
  • magnet 114 is arranged such that certain droplet operations electrodes 100 (e.g., 3 droplet operations electrodes 11 OM) are within the magnetic field of magnet 114.
  • Magnet 114 may, for example, be a permanent magnet or an electromagnet.
  • Droplet actuator 100 may contain a droplet 118 that may be transported along droplet operations electrodes 110 via electrowetting and upon which droplet operations may be performed.
  • Droplet 118 may, for example, be a 3X droplet, meaning that its footprint is approximately 3 times the area of one droplet operations electrode 110.
  • Droplet 118 may, for example, include 1 part magnetically responsive beads and 2 parts sample.
  • Droplet 118 may, for example, be a sample droplet that includes an analyte (e.g., an antigen) to be evaluated.
  • Droplet 118 may include one or more beads 122, which may be magnetically responsive beads. Beads 122 may have an affinity for certain target substances, such as, for example, a certain type of cell, protein, nucleic acid and/or antigen. In one example, beads 122 are coated with a primary antibody with affinity for a specific target antigen.
  • Figure IA shows a first step in a process of incubating droplets that includes magnetically responsive beads on a magnet.
  • droplet 118 that has beads 122 therein is positioned adjacent to and overlapping droplet operations electrodes HOM, which is within the magnetic field of magnet 114. Because beads 122 are magnetically responsive, a concentration of beads is formed at the side of droplet 118 that is closest to magnet 114.
  • Figure IB shows another step in the process of incubating droplets that include magnetically responsive beads on a magnet.
  • droplet 118 is transported via electrowetting to adjacent electrodes HOM and takes on a slug-shaped geometry.
  • two droplet operations electrodes 110 may be used to transport a 3X droplet 118. Because beads 122 are magnetically responsive, a concentration of beads 122 is formed at the bottom of droplet 118 (e.g., on the surface of droplet actuator 100) and at the center of magnet 114. Elongation of droplet 118 to a slug geometry provides for sufficient flow of fluid within droplet 118 to resuspend beads 122 in droplet 118.
  • Figure 1C shows yet another step in the process of incubating droplets that include magnetically responsive beads on a magnet.
  • droplet 118 is split near the central region of magnet 114 using droplet operations to form, for example, two sample droplets. Splitting of droplet 118 provides for redistribution of beads 122 within the sample droplets.
  • Figure ID shows yet another step in the process of incubating droplets that include magnetically responsive beads on a magnet.
  • split droplet 118 is merged on magnet 114 using droplet operations to form a single droplet 118.
  • the transporting, splitting, and merging operations of Figures IB, 1C, and ID comprise an incubation cycle. Several incubation cycles may be performed to provide for resuspension and redistribution (i.e., mixing) of beads 122 in droplet 118.
  • Figure IE shows yet another step in the process of incubating droplets that include magnetically responsive beads on a magnet.
  • magnet 114 which is, for example, an electromagnet
  • magnet 114 is not activated. Therefore, no magnetic field is generated by magnet 114 and beads 122 of droplet 118 have no attraction to magnet 114.
  • Droplet 118 is transported via electrowetting to adjacent electrodes 110. Beads 122 are resuspended in droplet 118.
  • Figures 2A, 2B, 2C, 2D, and 2E illustrate top views of an example of a region of a droplet actuator 100 of Figures IA through IE and show a process of incubating, on and off a magnet, droplets that include magnetically responsive beads.
  • the method of the invention of Figures 2A through 2E is an example of an incubation method wherein a droplet is transported near the magnetic field of a magnet and then away from the magnet to perform a series of split and merge droplet operations that are used to resuspend the beads within the droplet. Because the split and merge operations are performed away from the magnet, resuspension of the beads is in the lateral X-Y, and vertical Z directions.
  • Figure 2A shows a first step in a process of incubating droplets that include magnetically responsive beads on and off a magnet.
  • droplet 118 that has beads 122 therein is positioned adjacent to droplet operations electrodes HOM, which is within the magnetic field of magnet 114. Because beads 122 are magnetically responsive, a concentration of beads is formed at the side of droplet 118 that is closest to magnet 114.
  • Figure 2B shows another step in a process of incubating, on and off a magnet, droplets that include magnetically responsive beads.
  • droplet 118 is transported via electrowetting away from the magnetic field of magnet 114.
  • Beads 122 are sufficiently resuspended in droplet 118.
  • Figure 2C, 2D, and 2E show the process steps of droplet elongation (i.e., formation of slug-shaped geometry), droplet splitting and droplet merging, respectively, that are used to provide for sufficient flow of fluid within droplet 118 to resuspend and redistribute beads 122 in droplet 118. Because the split and merge operations are performed away from the magnet, resuspension of the beads is in the X, Y, and Z directions.
  • Figures 3A, 3B, 3C, 3D, and 3E illustrate top views of an example of a region of a droplet actuator 300 and show a process of incubating droplets that include magnetically responsive beads substantially out of the magnetic field of a magnet.
  • the method of the invention of Figures 3 A through 3E is an example of an incubation method wherein a series of droplet transport operations are used to resuspend the beads within the droplet. Because the droplet operations are performed a sufficient distance away from a magnet, resuspension of the beads is in the lateral X-Y, and vertical Z directions.
  • Droplet actuator 300 is substantially the same as droplet actuator 100 of Figures IA through IE except that it is configured to support droplet operations on a 2X droplet.
  • droplet 118 is a 2X droplet, meaning that its footprint is approximately 2 times the area of one droplet operations electrode.
  • Droplet 118 may, for example, include 1 part magnetically responsive beads and 1 part sample. Alternatively, droplet 118 may include 1 part sample plus magnetically responsive beads and 1 part secondary reporter antibody.
  • Figures 3 A through 3E show the process steps of transporting droplet 118 along a linear path of droplet operation electrodes 110 to provide mixing of magnetically responsive beads 122.
  • the use of a 2X droplet provides several advantages over the use of a 3X droplet in a droplet actuator-based immunoassay. For example, mixing a 2X droplet using two droplet operations electrodes is more efficient than mixing a 3X droplet using two droplet operations electrodes.
  • the concentration of magnetically responsive beads is also higher in a 2X droplet than a 3X droplet. A higher concentration of magnetically responsive beads provides for increased binding rate. Because the incubation cycle of a 2X droplet is substantially out of the magnetic field of magnet 114, the binding efficiency is also increased.
  • FIG. 4 shows a plot 400 of a comparison of incubation time between on-magnet and off-magnet incubation protocols on immunoassay performance measured in chemiluminescence. Data was generated using the incubation protocols of Figure 1 (on- magnet) and Figure 2 (off-magnet). Immunoassays were performed on a 300 nanoliter
  • nL droplet that contained capture antibody magnetically responsive beads, alkaline phosphatase (ALP)-labeled reporter antibodies and 5 ng/mL Troponin I (TnI).
  • ALP alkaline phosphatase
  • TnI Troponin I
  • an incubation protocol may include merging of a circular bead droplet on a magnet with two circular sample droplets. Mixing in the merged droplet is provided by moving the merged droplet back and forth on droplet operations electrodes while the magnetically responsive beads are immobilized on the magnet.
  • an incubation protocol may include merging of a circular bead droplet on a magnet with a 4X, 5X elongated (slug-shaped) sample droplet. Mixing in the merged slug-shaped droplet is provided by moving the merged droplet back and forth on droplet operations electrodes while the magnetically responsive beads are immobilized on the magnet. 8.2 Washing Protocols
  • washing of magnetically responsive beads, where unbound molecules are separated and removed is one of the most critical steps in implementing an immunoassay in a digital microfluidic system.
  • washing is performed using a merge-and- split protocol, which is repeated until the unbound material is sufficiently depleted from the supernatant to permit accurate and precise detection.
  • Figures 5A, 5B, 5C, 5D, and 5E show a top view of a region of a droplet actuator 500 and a process of washing magnetically responsive beads using shape-assisted merging of droplets.
  • the method of the invention of Figures 5A through 5E is an example of a wash method wherein a wash buffer droplet and a magnetically responsive bead droplet are elongated in a slug-shaped geometry and series of merge and split operations are used to remove unbound material from a bead droplet.
  • the merge and split operations provide for substantially complete fluid replacement of unbound supernatant with wash buffer in the absence of mixing.
  • Droplet actuator 500 may include a path or array of droplet operations electrodes 510
  • a magnet 512 is arranged in close proximity to droplet operations electrodes 510. In particular, magnet 512 is arranged such that certain droplet operations electrodes 510 (e.g., 3 droplet operations electrodes 510M) are within the magnetic field of magnet 512. Magnet 512 may, for example, be a permanent magnet or an electromagnet.
  • Droplet actuator 500 may contain a wash buffer droplet 516 and a bead droplet 514 that may be transported along droplet operations electrodes 510 via electrowetting and upon which droplet operations may be performed.
  • Bead droplet 514 may, for example, include a quantity of magnetically responsive beads 518 that includes bound antigen and reporter antibody (i.e., antigen-antibody-reporter complex), and unbound material such as excess unbound reporter antibody.
  • Bead droplet 514 and wash buffer droplet 516 may, for example, be 2X droplets, meaning that their footprint is approximately 2 times the area of one droplet operations electrode 510.
  • Bead droplet 514 and wash buffer droplet 516 may be configured as slug-shaped droplets (i.e., elongated droplets) by performing droplet operations on the 2X droplets using two active droplet operations electrodes 510. Because the excess droplet volume is now spread over a second active droplet operations electrode 510, the droplets are elongated and conform to the shape of two electrodes.
  • Figure 5A shows a first step in a process of washing beads using shape-assisted merging of droplets.
  • bead droplet 514 that has beads 518 therein is positioned such that one region of droplet 514 is on a droplet operations electrodes 510M which is within the magnetic field of magnet 512 and a second region of droplet 514 is on an adjacent droplet operations electrode 510. Because beads 518 are magnetically responsive, a concentration of beads is formed at the side of bead droplet 514 that is closest to magnet 512.
  • wash buffer droplet 516 is similarly positioned such that one region of wash buffer droplet 516 is on a droplet operations electrodes 510M which is within the magnetic field of magnet 512 and a second region of droplet 516 is on an adjacent droplet operations electrode 510.
  • the timing of the sequence of droplet operations for merging bead droplet 514 and wash buffer 516 is such that bead droplet 514 is elongated to a slug-shaped geometry just as wash buffer droplet is positioned via electrowetting for merging with bead droplet 514.
  • Figures 5B shows another step in a process of washing beads using shape-assisted merging of droplets. In this step, bead droplet 514 and washed droplet 516 are merged.
  • Figures 5C and 5D show another step in a process of washing beads using shape-assisted merging of droplets.
  • a slug of fluid 520 is extended away from magnet 512 by activating the contiguous droplet operations electrodes 510 and inactivating the intermediate droplet operations electrodes 510 outside the magnetic field of magnet 512.
  • Fluid 520 includes wash buffer from wash buffer droplet 516 and unbound reagents from bead droplet 514. As fluid 520 is extended, beads 518 remain concentrated on magnet 512.
  • Figure 5E shows yet another step in a process of washing beads using shape-assisted merging of droplets.
  • a fluid 520 is split using droplet operations to form supernatant droplet 522.
  • Supernatant droplet 522 includes unbound reagents such as unbound reporter antibody from bead droplet 514.
  • Supernatant droplet 522 is typically discarded in a waste well (not shown).
  • Figures 5A through 5E show the set of droplet operations that comprise a wash cycle. Several wash cycles may be performed to provide for sufficient removal of unbound material.
  • a washing protocol may use a wash droplet and a bead droplet that are circular in shape.
  • a circular shape of a droplet may, for example, be obtained by performing droplet operations on a 2X wash droplet and a 2X bead droplet using only one droplet operations electrode each. Because a 2X droplet (i.e., footprint is approximately 2 times the area of one droplet operations electrode) is much larger than a single droplet operations electrode, the droplet takes a more rounded shape.
  • Figure 6 shows a plot 600 of a comparison of washing protocols between slug shaped and circular shaped wash droplets on immunoassay performance measured in chemiluminescence.
  • incubation was performed using the off-magnet incubation protocol of Figure 2 for 3 minutes.
  • Each wash cycle takes about 10 seconds in a slug-based protocol of Figure 5 and about 14 seconds in a circular droplet protocol.
  • FIG. 6 a washing protocol performed using slugs of fluid (or elongated droplets as shown in Figure 5), wherein a 2x wash buffer droplet and a 2x bead droplet were operated using two electrodes, a sufficient wash level was achieved using fewer wash cycles when compared to washing using circular shaped droplets.
  • a slug-based washing protocol mixing was minimized and the bulk of the unbound material from the supernatant was replaced with fresh wash buffer at each cycle.
  • a circular-droplet based protocol mixing was ensured by operating the 2x droplets using only one electrode each.
  • the dispersion of magnetically responsive beads in the lateral plane was higher in the slug-based protocol when a fresh wash buffer droplet was merged with a bead droplet. Greater dispersion of magnetically responsive beads in the merged droplet enables any unbound antibody trapped in the interstices to diffuse into the supernatant and be washed away in subsequent washes.
  • sufficient wash levels were achieved in about 10 wash cycles using a slug-based washing protocol compared to >18 wash cycles in a circular droplet based wash protocol.
  • the washing behavior has two distinct regimes, one regime where washing may be very pronounced and the second where the washing may be subtle.
  • the washing is pronounced with each wash cycle up to about 9 cycles and after that the effect of washing is almost negligible.
  • the washing effect is pronounced until about the 15th wash; although the wash efficiency is less than that observed for the slug-based protocol. Washing is only marginally effective for the circular droplet protocol between about the 15th and 18th washes with only a slight reduction in signal with each cycle. This could happen because all the free unbound material may be washed away in the first few cycles and after that washing only removes the unbound material trapped between the beads.
  • Figures 7 A, 7B, 7C, and 7D illustrate top views of an example of a region of a droplet actuator 500 of Figures 5A through 5E and show a process of resuspending magnetically responsive beads between wash cycles.
  • the method of the invention of Figures 7A through 7D is an example of a sequence in a wash method that uses a series of droplet resuspension cycles to resuspend the magnetically responsive beads between wash cycles such as a wash cycle shown in Figure 5.
  • the resuspension cycles provide sufficient resuspension of the beads such that unbound material from the interstices of bead aggregates may be effectively removed.
  • Figure 7A shows the first step in a process of resuspending magnetically responsive beads between wash cycles.
  • bead droplet 514 that includes magnetically responsive beads 518 is transported via electrowetting away from magnet 512 in the direction of arrow A.
  • Figures 7B, 1C, and 7D show the process steps of transporting bead droplet 514 along a path of droplet operation electrodes 510 in the direction of arrow A. Three transport operations are shown in Figures 7B, 7C, and 7D, but any number of transport operations may be used to comprise a resuspension cycle. Transporting of bead droplet 514 provides for sufficient resuspension of beads 518 such that unbound material from the interstices of bead aggregates may be effectively removed in subsequent wash cycles.
  • a complete wash protocol may include a series of wash cycles, such as the slug based wash cycles of Figure 5, interspersed with a one or more resuspension cycles of Figure 7.
  • any number of wash cycles may be interspersed with any number of resuspension cycles.
  • a complete wash protocol sequence may include, for example, four wash cycles, four resuspension cycles, and four wash cycles.
  • a complete wash protocol sequence ends at a wash cycle.
  • Figures 8A and 8B show plots of a comparison of washing protocols of Figure 5 without resuspension cycles and with resuspension cycles, respectively.
  • a washing protocol in the absence of one or more resuspension cycles provides an initial drop in signal after a number of wash cycles (A).
  • A the number of wash cycles
  • B the number of wash cycles increase
  • a washing protocol that includes one or more resuspension cycles provides more efficient removal of unbound material to a near zero level using fewer numbers of wash cycles (A).
  • Figure 9 illustrates a top view of a region of a droplet actuator 900 that includes multiple waste wells.
  • multiple waste wells are provided to improve the efficiency (e.g., time to result) of a bead washing protocol such as a bead washing protocol shown in Figure 5.
  • droplet actuator 900 includes an array of droplet operations electrodes 910 (e.g., electrowetting electrodes) configured to provide wash lanes 912a, 912b, 912c, and 912d, and a single waste lane 916.
  • Wash lanes 912a, 912b, 912c, and 912d may include magnets 914a, 914b, 914c, and 914d, respectively, and waste wells 920a, 920b, 920c, and
  • Waste lane 916 may include a waste well 918.
  • droplet actuator 900 may be used to conduct a bead washing protocol on four different samples in wash lanes 912a through 912d.
  • a bead washing protocol the supernatant droplet(s) that contain unbound material, such as unbound antigen and secondary reporter antibody, is typically discarded in a waste well.
  • a bead washing protocol may use a single waste well 918.
  • a single waste lane 916 that includes waste well 918 may be used to transport supernatant (i.e., waste) droplets from wash lanes 912a through 912d.
  • supernatant (i.e., waste) droplets from wash lanes 912a through 912d may be transported via electrowetting in the direction of Arrow A to wash lane 916.
  • waste lane 916 may then be transported in waste lane 916 in the direction of Arrow B to waste well 918. Because waste lane 916 is common to wash lanes 912a through 912d, supernatant droplets must be transported serially (i.e., one after another).
  • individual waste wells 920a through 92Od may be provided for each wash lane 912a through 912d, respectively.
  • supernatant droplets may be transported simultaneously in the direction of arrow C to individual waste wells 920a through 92Od. Multiple, individual waste wells provide for increased efficiency (e.g., time to result) in a washing protocol.
  • Multiple waste wells also provide for a reduction in the number of droplet operations electrodes 910 that are required to shuttle a supernatant droplet to a waste well.
  • a reduction in the number of operations electrodes 910 that may be used to transport a supernatant droplet also provides for a reduction in the potential for cross-contamination of subsequent droplets used in a protocol.
  • Figures IQA, IQB, and IQC show a top view of a region of a droplet actuator 1000 and a process of separating beads from a sample droplet.
  • the method of the invention of Figures 1 OA through 1 OC is an example of a method wherein magnetically responsive beads are split from a circular shaped droplet.
  • Droplet actuator 1000 may include a path or array of droplet operations electrodes 1010
  • a magnet 1014 is arranged in close proximity to droplet operations electrodes 1010. In particular, magnet 1014 is arranged such that certain droplet operations electrodes 1010 (e.g., 3 droplet operations electrodes 1010M) are within the magnetic field of magnet 1014. Magnet 1014 may, for example, be a permanent magnet or an electromagnet.
  • Droplet actuator 1000 may contain a droplet 1016 that may be transported along droplet operations electrodes 1010 via electrowetting and upon which droplet operations may be performed.
  • Droplet 1016 may include a quantity of beads 1020, which may be magnetically responsive beads.
  • An example of a process of separating beads from a circular droplet may include, but is not limited to, the following steps.
  • Figure 1OA shows a first step in a process of separating beads from a circular droplet.
  • droplet 1016 that has beads 1020 therein is positioned at a droplet operations electrode 101OM, which is within the magnetic field of magnet 1014. Because beads 1020 are magnetically responsive, beads 1020 are attracted to magnet 1014. Because a single droplet operations electrode 101OM is active, droplet 1016 is circular in shape.
  • Figure 1 OB shows another step in a process of separating beads from a circular droplet. In this step, droplet 1016 is transported via electrowetting away from droplet operations electrode 101OM and to the adjacent droplet operations electrode 1010.
  • a concentration of beads 1020 is formed at the side of droplet 1016 that is closest to magnet 1014. Because droplet 1016 is transported away from magnet 1014 one droplet operations electrode 1010 at a time, the geometry of droplet 1016 may be distorted (e.g., formation of a neck) by the concentration of beads 1020 as droplet 1016 pulls away from magnet 1020.
  • Figure 1OC shows yet another step in the process of separating beads from a circular droplet.
  • droplet 1016 is transported via electrowetting further away from droplet operations electrode 101OM and to a droplet operations electrode 1010 that is yet further away.
  • the concentration of beads 1020 breaks away (snaps off) from droplet 1016. This occurs because one side (e.g., the side nearest magnet 1014) of droplet
  • a similar result can be achieved using a barrier that permits a bead-containing droplet to be transported while restraining transport of the beads with the main body of the droplet.
  • Figures HA, HB, and HC show a top view of a region of a droplet actuator 1000 of
  • Figures 1OA through 1OC and a process of transporting beads within a droplet The method of the invention of Figures 1 IA through 11 C is an example of a method wherein magnetically responsive beads are transported within an elongated droplet away from a magnetic force.
  • Figures HA, HB, and HC are substantially the same as those that are described in Figures 1OA, 1OB, and 1OC except that, instead of processing a IX droplet via electrowetting using one active electrode at a time, droplet 1016 is a slug- shaped 3X droplet that is processed using three active electrodes for each droplet operation.
  • Figures HA, HB, and HC show the process steps of transporting beads 1020 within an elongated droplet 1016 away from magnet 1014.
  • one side (e.g., the side nearest magnet 1014) of droplet 1016 is subjected to both a magnetic force and electrowetting force and the opposite side (e.g., the side farthest from magnet 1014) is subjected to electrowetting force. Because electrowetting force occurs on both sides of droplet 1016, all of the fluid within the droplet is electrowetted and beads 1020 are retained within droplet 1016 during droplet transport away from magnet 1014.
  • Figures 12A, 12B, and 12C show a top view of a region of a droplet actuator 1000 and a process of separating beads from a sample droplet onto a magnet.
  • the method of the invention of Figures 12A through 12C is an example of a method wherein magnetically responsive beads are split from an elongated droplet (e.g., 3X droplet) onto a magnet.
  • the method of the invention of Figures 12A through 12C may be used to dispense magnetically responsive beads onto a magnet.
  • Figure 12A shows a first step in a process of dispensing beads from an elongated droplet.
  • droplet 1016 that has beads 1020 therein is positioned at a droplet operations electrode 101OM, which is within the magnetic field of magnet 1014. Because beads
  • droplet 1020 are magnetically responsive, beads 1020 are attracted to magnet 1014. Because three droplet operations electrodes 1010 are active, droplet 1016 is elongated in shape.
  • Figure 12B shows another step in a process of dispensing beads from an elongated droplet.
  • droplet 1016 is transported away from magnet 1014 via electrowetting using one active droplet operations electrode 1010 at a time.
  • FIG. 1016 moves away from droplet operations electrode 101 OM, a concentration of beads 1020 is formed at the side of droplet 1016 that is closest to magnet 1014. Because droplet 1016 is transported away from magnet 1014 one droplet operations electrode 1010 at a time, the geometry of droplet 1016 may be distorted.
  • Figure 12C shows yet another step in process of dispensing beads from an elongated droplet. In this step, droplet 1016 is transported via electrowetting further away from droplet operations electrode 101OM and to a droplet operations electrode 1010 that is yet further away. Once the sample droplet overlaps the droplet operation electrode 1010 on which droplet 1022 is to be formed, the intermediate electrode 1010 is deactivated. In doing so, droplet 1016 is split into a supernatant droplet 1022 and a smaller droplet 1016 that has beads 1020 therein.
  • Figures 13A and 13B show a comparison of bench top and droplet actuator immunoassay reagent ratios and a plot of reagent concentration versus signal strength, respectively, that provide for optimum droplet based immunoassay performance.
  • the ratio of three components of an immunoassay, beads (i.e., capture antibody conjugated to beads), sample (e.g., serum, plasma), and secondary antibody (II ° Ab) are provided.
  • beads i.e., capture antibody conjugated to beads
  • sample e.g., serum, plasma
  • secondary antibody II ° Ab
  • a typical ratio is 1 part beads (60 ⁇ L): 1 A part sample (30 ⁇ L): 1 part II ° Ab (60 ⁇ L).
  • a reagent ratio for a droplet actuator based immunoassay is typically 1 A bead droplet (150 nL): 1 sample droplet (300 nL): 2 II ° Ab droplets (600 nL).
  • V 2 bead droplet or 1 A concentration of beads V 2 bead droplet or 1 A concentration of beads
  • concentration of secondary antibody is the same in both bench top and droplet actuator immunoassays, but the volume of secondary antibody solution is double in the droplet actuator assay.
  • Figure 13B shows the improvement in detection signal that is provided by the use of 2 droplets of secondary antibody and 2 droplets of detection substrate in a droplet actuator immunoassay.
  • Another parameter which may influence the time to result in an immunoassay is the generation of a signal during the incubation of a chemiluminescent substrate with the washed magnetically responsive beads that contain the antigen-antibody complex.
  • FIG 14 shows a plot 1400 of the kinetics of a reaction between a chemiluminescent substrate and the ALP on magnetically responsive beads for Troponin I (TnI). Immunoassays were performed on TnI (100 ng/mL) using an on-magnet incubation protocol and a circular shaped droplet washing protocol. As shown in Figure 14, about 90% of the end point signal was obtained in about 120 to about 130 seconds. For a lower concentration of the analyte, maximum signal was achieved in about ⁇ 120 seconds. Based on this data, for the type of substrate used, 2 minutes may be selected as an optimum incubation time to generate maximum signal for the chemiluminescence reaction. However, if the chemiluminescence reaction is observed to behave as a flash signal instead of a glow reaction, the 2 minute incubation may be reduced to about a few seconds.
  • TnI Troponin I
  • a droplet actuator may be used to extract human genomic DNA from a sample.
  • Figure 15 is a top view of a droplet actuator 1500 that may be used for extracting DNA from a whole blood sample.
  • the droplet actuator 1500 includes six on-actuator reservoirs, each with a capacity of 2 ⁇ L, which may be used for storing and dispensing different reagents.
  • a typical protocol for DNA extraction on a droplet actuator may include the following steps.
  • a droplet of magnetically responsive beads such as paramagnetic
  • Dynabeads® DNA Direct Universal from Dynal Biotech (1.05 ⁇ m diameter), suspended in a lysis buffer are dispensed from an on-chip reservoir and transported via electrowetting to a specific location on the chip.
  • the beads, which are magnetically responsive, are held by a permanent magnet placed underneath the chip.
  • droplets of whole blood are dispensed from a reservoir and mixed with droplets of lysis buffer (containing 10 M NaOH) dispensed from another on-chip reservoir, into a mixing reservoir in the ratio of 1 :6 and mixed for about 10 seconds.
  • Mixing was performed by dispensing a droplet and then merging the droplet back into the reservoir.
  • droplets of the cell lysate were then transported across the DNA capture beads in succession and the supernatant was pinched off while holding the beads.
  • droplets of wash buffer stored in separate on-chip reservoirs were then used to wash the beads to remove cell debris.
  • purified genomic DNA captured on the beads was then eluted and collected at the bead collection reservoir.
  • the collected DNA can then be amplified either on the chip as part of an integrated sample-to-answer chip or in a commercial thermocycler for further DNA processing or diagnostic applications.
  • the fluid includes a biological sample, such as whole blood, lymphatic fluid, serum, plasma, sweat, tear, saliva, sputum, cerebrospinal fluid, amniotic fluid, seminal fluid, vaginal excretion, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudates, exudates, cystic fluid, bile, urine, gastric fluid, intestinal fluid, fecal samples, fluidized tissues, fluidized organisms, biological swabs, biological washes, liquids with cells, tissues, multicellular organisms, single cellular organisms, protozoa, bacteria, fungal cells, viral particles, organelles.
  • 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, transu
  • the fluid includes a reagent, such as water, deionized water, saline solutions, acidic solutions, basic solutions, detergent solutions and/or buffers.
  • the fluid includes a reagent, such as a reagent for a biochemical protocol, such as a nucleic acid amplification protocol, an affinity-based assay protocol, a sequencing protocol, and/or a protocol for analyses of biological fluids.
  • the fluids may include one or more magnetically responsive and/or non-magnetically responsive beads.
  • droplet actuator techniques for immobilizing magnetically responsive beads and/or non-magnetically responsive beads are described in the foregoing international patent applications and in Sista, et al., U.S. Patent Application
  • Droplet actuator 1600 may include a path or array of droplet operations electrodes 1610 (e.g., electrowetting electrodes) and a wash reservoir 1612.
  • a magnet 1614 is arranged in close proximity to droplet operations electrodes 1610.
  • magnet 1614 is arranged such that a certain droplet operations electrode 1610 (e.g., droplet operations electrode 1610M) is within the magnetic field thereof.
  • Magnet 1614 may be a permanent magnet or an electromagnet.
  • Droplet actuator 1600 may contain a droplet 1618 that may be transported along droplet operations electrodes 1610 and upon which droplet operations may be performed.
  • Droplet 1618 may, for example, be a 3X droplet, meaning that its footprint is approximately 3 times the area of one droplet operations electrode 1610. Droplet 1618 may, for example, include 1 part magnetically responsive beads and 2 parts sample (e.g., an antigen to be evaluated).
  • Droplet 1618 may include one or more magnetically responsive beads 1622.
  • Magnetically responsive beads 1622 are coated with a primary antibody that has an affinity for a specific target antigen.
  • magnetically responsive beads 1622 are coated with a primary antibody that has an affinity for IL-6.
  • magnetically responsive beads 1622 are coated with a primary antibody that has an affinity for TNF- ⁇ .
  • An example of a process of cytokine detection on a droplet actuator may include, but is not limited to, the following steps:
  • Step A of Figure 16A shows a droplet 1618 that has magnetically responsive beads 1622 therein and is positioned at a certain droplet operations electrode 1610.
  • droplet 1618 includes 1 part beads 1622 and 2 parts sample.
  • Steps B and C of Figure 16A show an incubation process, in which droplet 1618 is repeatedly transported back and forth via droplet operations to adjacent electrodes 1610.
  • Droplet 1618 Repeated transporting of droplet 1618 is used during incubation of beads 1622 and sample in order to provide sufficient resuspension and mixing of magnetically responsive beads 1622 for optimal antibody and antigen binding.
  • two droplet operations electrodes 1610 may be used to transport a 3X droplet 1618, which takes on a slug shaped geometry. Elongation of droplet 1618 to a slug shape provides for sufficient flow of fluid within droplet 1618 to resuspend magnetically responsive beads 1622 therein.
  • droplet 1618 may be incubated for 6 minutes using 2 droplet operations electrodes 1610 and transporting droplet 1618 over a span of 8 electrodes at a switching speed of 5 Hertz (Hz).
  • Step D of Figure 16A shows droplet 1618 that has magnetically responsive beads 1622 therein transported to droplet operations electrode 1610M.
  • a supernatant droplet 1624 is split off using droplet operations. Because magnetically responsive beads 1622 are attracted to magnet 1614, they are retained at magnet 1614.
  • supernatant droplet 1624 is a IX droplet and droplet 1618 is now a 2X droplet. Supernatant droplet 1624 that includes unbound antigen (i.e., cytokine) is discarded (not shown).
  • Step E of Figure 16A shows a reagent droplet 1628 that includes secondary antibody transported via electrowetting to droplet operations electrode 1610M.
  • Reagent droplet 1628 is merged with droplet 1618 (i.e., a 2X droplet) using droplet operations to form, for example, a 3X droplet.
  • reagent droplet 1628 is a IX droplet that includes biotinylated secondary antibody that has an affinity to the target antigen.
  • the antigen target is captured by the primary antibody which is immobilized on the beads.
  • Merged droplet 1618 is incubated for 4 minutes using droplet operations, as described in steps B and C. Following the incubation period, droplet 1618 is transported via electrowetting to droplet operations electrode 1610M and a IX supernatant droplet is split off using droplet operations, as described in step D, in order to yield a 2X droplet 1618.
  • the supernatant droplet (not shown) that includes unbound secondary antibody is discarded.
  • the beads may in some embodiments be washed and then incubated with the streptavidin-peroxidase.
  • the entire complex thus consists of beads-primary antibody-antigen-secondary antibody- streptavidin-peroxidase.
  • Streptavidin-peroxidase may be substituted with streptavidin- alkaline phosphatase.
  • Step F of Figure 16B shows a bead washing step, in which a wash droplet 1630 is transported from wash reservoir 1612 along droplet operations electrodes 1610 and across droplet 1618, which is retained at droplet operations electrode 1610M.
  • droplet 1630 i.e., a 2X droplet.
  • wash droplet 1630 is a 2X droplet that has a slug geometry and the washing protocol is repeated 5 times.
  • a IX supernatant droplet is split off from droplet 1618, as described in step D of Figure 16A, in order to yield a IX droplet 1618.
  • the supernatant droplet (not shown) is discarded.
  • Step G of Figure 16B shows one or more reagent droplets 1632 (e.g., 1632a, 1632b) transported to droplet operations electrode 1610M.
  • reagent droplet 1632a that includes a blocking agent (e.g., Synblock) and reagent droplet 1632b that includes a streptavidin-enzyme conjugate are transported to droplet operations electrode 1610M and merged using droplet operations with droplet 1618.
  • Droplet 1618 is now a 3X droplet.
  • Merged droplet 1618 is incubated for 4 minutes using droplet operations, as described in steps B and C of Figure 16A. Following the incubation period, droplet 1618 is transported to droplet operations electrode 1610M and a supernatant droplet (i.e., a IX droplet) is split off using droplet operations, as described in step D of Figure 16A, in order to yield a 2X droplet 1618. The supernatant droplet (not shown) that includes unbound streptavidin-enzyme conjugate is discarded.
  • a supernatant droplet i.e., a IX droplet
  • Droplet 1618 is subsequently washed, for example 15 times, as described in step F of
  • FIG 16B Following bead washing, a IX supernatant droplet is split off droplet 1618, as described in step D of Figure 16A, in order to yield a IX droplet 1618. The supernatant droplet (not shown) is discarded. Droplet 1618 that includes antibody- antigen sandwich is now ready for detection.
  • Step H of Figure 16B shows droplet 1634 (IX droplet) that includes a detection substrate
  • wash buffer droplets may be transported across the detection window following each chemiluminescent droplet to clean up the detection window and the detection loop prior to the next detection.
  • the method of the invention is used to detect IL-6.
  • Figure 17 shows a plot of two 5-point standard curves for cytokine IL-6. Data was generated using the cytokine detection protocol described in Figures 16A and 16B. In this example, two 5- point standard curves (0, 0.05, 0.5, 5, and 25 ng/mL of IL-6) were obtained in 2 runs for IL-6 performed on 2 separate droplet actuators.
  • the method of the invention is used to detect TNF- ⁇ .
  • Figure 18 shows a plot of two 6-point standard curves for cytokine TNF- ⁇ . Data was generated using the cytokine detection protocol described in Figures 16A and 16B. In this example, two 6-point standard curves (0, 0.01, 0.1, 1, 10, and 100 ng/mL of TNF- ⁇ ) were obtained in 2 runs for TNF- ⁇ performed on 2 separate droplet actuators.

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Abstract

La présente invention concerne une incubation et un lavage de billes sur un actionneur à gouttelettes. Elle concerne également des procédés d’incubation de billes sensibles au magnétisme qui sont marquées avec un anticorps primaire, un échantillon (à savoir, un analyte) et des anticorps rapporteurs secondaires sur un aimant, sur un aimant et à distance de celui-ci, et entièrement éloignés d’un aimant. Elle concerne aussi des procédés de lavage de billes sensibles au magnétisme à l’aide d’une fusion de gouttelettes assistée par la forme. Des procédés de séparation induite par la forme, de transport et de distribution d’une gouttelette d’échantillon qui contient lesdites billes sont également décrits. Les appareils et les procédés de l’invention permettent d’obtenir des résultats rapides et une détection optimale d’un analyte dans un dosage immunologique.
PCT/US2009/059868 2006-04-18 2009-10-07 Incubation et lavage de billes sur un actionneur à gouttelettes Ceased WO2010042637A2 (fr)

Priority Applications (8)

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US13/081,927 US8637324B2 (en) 2006-04-18 2011-04-07 Bead incubation and washing on a droplet actuator
US14/081,376 US8846410B2 (en) 2006-04-18 2013-11-15 Bead incubation and washing on a droplet actuator
US14/466,193 US9081007B2 (en) 2006-04-18 2014-08-22 Bead incubation and washing on a droplet actuator
US14/731,740 US9395361B2 (en) 2006-04-18 2015-06-05 Bead incubation and washing on a droplet actuator
US15/210,634 US10078078B2 (en) 2006-04-18 2016-07-14 Bead incubation and washing on a droplet actuator
US16/132,175 US10585090B2 (en) 2006-04-18 2018-09-14 Bead incubation and washing on a droplet actuator
US16/811,789 US11525827B2 (en) 2006-04-18 2020-03-06 Bead incubation and washing on a droplet actuator
US18/063,874 US20230128722A1 (en) 2006-04-18 2022-12-09 Bead incubation and washing on a droplet actuator

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US10330208P 2008-10-07 2008-10-07
US61/103,302 2008-10-07
US12279108P 2008-12-16 2008-12-16
US61/122,791 2008-12-16

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US11/639,736 Continuation US7439014B2 (en) 2006-04-18 2006-12-15 Droplet-based surface modification and washing
US12/113,385 Continuation US8541176B2 (en) 2006-04-18 2008-05-01 Droplet-based surface modification and washing
US13/081,927 Continuation US8637324B2 (en) 2006-04-18 2011-04-07 Bead incubation and washing on a droplet actuator
US13/081,927 Continuation-In-Part US8637324B2 (en) 2006-04-18 2011-04-07 Bead incubation and washing on a droplet actuator

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Cited By (46)

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Publication number Priority date Publication date Assignee Title
WO2013041983A1 (fr) 2011-09-19 2013-03-28 Centre National De La Recherche Scientifique Système micro-fluidique
US8637324B2 (en) 2006-04-18 2014-01-28 Advanced Liquid Logic, Inc. Bead incubation and washing on a droplet actuator
US8637242B2 (en) 2011-11-07 2014-01-28 Illumina, Inc. Integrated sequencing apparatuses and methods of use
US8658111B2 (en) 2006-04-18 2014-02-25 Advanced Liquid Logic, Inc. Droplet actuators, modified fluids and methods
US8702938B2 (en) 2007-09-04 2014-04-22 Advanced Liquid Logic, Inc. Droplet actuator with improved top substrate
US8852952B2 (en) 2008-05-03 2014-10-07 Advanced Liquid Logic, Inc. Method of loading a droplet actuator
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US9140635B2 (en) 2011-05-10 2015-09-22 Advanced Liquid Logic, Inc. Assay for measuring enzymatic modification of a substrate by a glycoprotein having enzymatic activity
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US9377455B2 (en) 2006-04-18 2016-06-28 Advanced Liquid Logic, Inc Manipulation of beads in droplets and methods for manipulating droplets
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US12233390B2 (en) 2019-01-31 2025-02-25 Miroculus Inc. Nonfouling compositions and methods for manipulating and processing encapsulated droplets

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6911132B2 (en) * 2002-09-24 2005-06-28 Duke University Apparatus for manipulating droplets by electrowetting-based techniques
US8492168B2 (en) * 2006-04-18 2013-07-23 Advanced Liquid Logic Inc. Droplet-based affinity assays
US7439014B2 (en) * 2006-04-18 2008-10-21 Advanced Liquid Logic, Inc. Droplet-based surface modification and washing
EP2570812B1 (fr) * 2007-02-09 2018-07-18 Advanced Liquid Logic, Inc. Procédé d'assemblage d'un dispositif actionneurs de gouttelettes

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