Bead incubation and washing on a droplet actuator

• US 10,078,078 B2
• Filed: 07/14/2016
• Issued: 09/18/2018
• Est. Priority Date: 04/18/2006
• Status: Active Grant

• Current Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Pamula, Vamsee K., Pollack, Michael G., Sista, Ramakrishna, Sudarsan, Arjun
• Primary Examiner(s): Sines, Brian J
• Application Number:
  US15/210,634
• Publication Number:
  US 20160320378A1
• Time in Patent Office: 796 Days
• Field of Search: 422 50, 422 681, 422 81, 422 82, 422502, 422503, 422504, 436 43, 436174, 436177, 436179, 436180
• CPC Class Codes: B01F 31/65 the materials to be mixed being directly submitted to a pulsating movement, e.g. by means of an oscillating piston or air column
• CPC Class Codes: B01F 33/3021 the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets
• CPC Class Codes: B01F 33/3031 using electro-hydrodynamic [EHD] or electro-kinetic [EKI] phenomena to mix or move the fluids
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/089 Virtual walls for guiding liquids
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0424 Dielectrophoretic forces
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 2400/043 magnetic forces
• CPC Class Codes: B01L 2400/0436 acoustic forces, e.g. surface acoustic waves [SAW]
• CPC Class Codes: B01L 2400/0442 : thermal energy, e.g. vaporisation, bubble jet
• CPC Class Codes: B01L 3/502715 : characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502761 : specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/502784 : specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: B01L 3/502792 : for moving individual droplets on a plate, e.g. by locally altering surface tension
• CPC Class Codes: B01L 7/52 : with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B01L 7/525 : with physical movement of samples between temperature zones
• CPC Class Codes: B03C 1/02 : acting directly on the substance being separated
• CPC Class Codes: B03C 1/288 : disposed at the outer circumference of a recipient
• CPC Class Codes: B03C 1/30 : Combinations with other devices, not otherwise provided for
• CPC Class Codes: B03C 2201/18 : Magnetic separation whereby the particles are suspended in a liquid
• CPC Class Codes: B03C 2201/26 : for use in medical or biological applications
• CPC Class Codes: B03C 5/005 : Dielectrophoresis, i.e. dielectric particles migrating towards the region of highest field strength
• CPC Class Codes: C12N 15/1013 : by using magnetic beads
• CPC Class Codes: G01N 2035/1046 : Levitated, suspended drops
• CPC Class Codes: G01N 33/54333 : Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
• CPC Class Codes: G01N 33/561 : Immunoelectrophoresis
• CPC Class Codes: Y10T 436/11 : Automated chemical analysis
• CPC Class Codes: Y10T 436/25 : including sample preparation
• CPC Class Codes: Y10T 436/25625 : Dilution
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

The invention relates to bead incubating and washing on a droplet actuator. 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 are provided. Also provided are methods for washing magnetically responsive beads using shape-assisted merging of droplets. Also provided are methods for shape-mediated splitting, transporting, and dispensing of a sample droplet that contains magnetically responsive beads. The apparatuses and methods of the invention provide for rapid time to result and optimum detection of an analyte in an immunoassay.

Claims

1. A method of incubating droplets having magnetically responsive beads therein, the method comprising:

(a) providing a droplet actuator comprising;

(i) droplet operations electrodes arranged for conducting droplet operations on a droplet operations surface; and

(ii) 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;

(b) 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;

(c) 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;

(d) 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

(e) 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.

2. The method of claim 1, wherein in step (b) the concentration of magnetically responsive beads occurs at an edge of the droplet adjacent the magnet.

3. The method of claim 1, wherein the droplet transported has a footprint approximately three times the area of a single droplet operations electrode.

4. The method of claim 1, wherein the droplet comprises a sample comprising a target substance, and the magnetically responsive beads have an affinity for the target substance.

5. The method of claim 4, wherein the target substances are at least one of cells, protein, nucleic acid, and an antigen.

6. The method of claim 1, wherein the transporting, splitting, and merging operations are repeated a plurality of times.

7. The method of claim 1, wherein the droplet comprises one part magnetically responsive beads and one part sample.

8. The method of claim 1, wherein the droplet comprises one part magnetically responsive beads and one part secondary reporter antibodies.

9. The method of claim 1, wherein the incubation cycle is conducted substantially outside of the first region of the magnetic field.

10. The method of claim 1, wherein the droplet is a sample droplet having a target antigen as a sample therein.

11. The method of claim 1, wherein the droplet transported has a footprint approximately two times the area of a single droplet operations electrode.

12. The method of claim 4, wherein the target substance is a cytokine.