Bead incubation and washing on a droplet actuator

• US 10,585,090 B2
• Filed: 09/14/2018
• Issued: 03/10/2020
• 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:
  US16/132,175
• Publication Number:
  US 20190011443A1
• Time in Patent Office: 543 Days
• 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

Methods are provided for separating magnetically responsive beads from a droplet in a droplet actuator. Droplet operations electrodes and a magnet are arranged in a droplet actuator to manipulate a bead-containing droplet and position it relative to a magnetic field region that attracts the magnetically responsive beads. The droplet operations electrodes are operated to control the droplet shape and transport it away from the magnetic field region to form a concentration of beads in the droplet. The continued transport of the droplet away from the magnetic field causes the concentration of beads to break away from the droplet to yield a small, concentrated bead-containing droplet immobilized by the magnet.

Claims

1. A method of separating magnetically responsive beads from a droplet, 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 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;

(c) 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

(d) 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.

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

3. The method of claim 1, wherein in (b) the droplet is positioned at a droplet operations electrode within the first region of the magnetic field of the magnet.

4. The method of claim 1, wherein in (c) the droplet is caused to move from the droplet operations electrode within the first region of the magnetic field partially to an adjacent droplet operations electrode away from the magnet.

5. The method of claim 1, wherein the droplet comprises a sample comprising a target substance, and the magnetically responsive beads comprise beads having an affinity for the target sub stance.

6. The method of claim 5, wherein the target substance is at least one of a cell, a protein, a nucleic acid, and an antigen.

7. The method of claim 5, wherein the target substance is a cytokine.

8. The method of claim 1, wherein the droplet comprises, by volume, one part magnetically responsive beads to one part sample.

9. The method of claim 1, wherein the droplet comprises, by volume, one part sample plus magnetically responsive beads to one part secondary reporter antibodies.

10. The method of claim 1, wherein the magnet is positioned such that three droplet operations electrodes are within the magnetic field of the magnet.

11. The method of claim 1, wherein the magnet is one of a permanent magnet and an electromagnet.

12. The method of claim 1, wherein the droplet transport involves electrowetting.

13. The method of claim 1, wherein the droplet transported has a footprint approximately two times the area of any one of the droplet operations electrodes.

14. The method of claim 1, wherein the droplet transported has a footprint approximately three times the area of any one of the droplet operations electrodes.

15. The method of claim 1, wherein the magnetically responsive beads are coated with a primary antibody with an affinity for a specific target antigen.

16. A method of separating magnetically responsive beads from a droplet, 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 an elongated droplet containing magnetically responsive beads over at least three active droplet operations electrodes that include an intermediate droplet operation electrode, the at least three droplet operations electrodes positioned within the first region of the magnetic field of the magnet to cause the magnetically responsive beads to be attracted to the magnet;

(c) 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

(d) deactivating an intermediate droplet operations electrode to split the droplet into a supernatant droplet and a smaller droplet containing the beads held immobilized by the magnet.

17. The method of claim 16, wherein the droplet comprises a sample comprising a target substance, and the magnetically responsive beads comprise beads having an affinity for the target substance.

18. The method of claim 17, wherein the target substance is at least one of a cell, a protein, a nucleic acid, and an antigen.

19. The method of claim 17, wherein the target substance is a cytokine.

20. The method of claim 16, wherein in (c) the concentration of magnetically responsive beads occurs at an edge of the droplet adjacent to the magnet.