Bead Manipulation Techniques

• US 20110114490A1
• Filed: 01/06/2011
• Published: 05/19/2011
• Est. Priority Date: 04/18/2006
• Status: Active Grant

• Current Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Sudarsan, Arjun, Sista, Ramakrishna, Pamula, Vamsee K.
• Granted Patent: US 8,637,317 B2
• Time in Patent Office: Days
• US Class Current: 204/545
• 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 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 1/01 : by addition of magnetic adjuvants
• CPC Class Codes: B03C 1/288 : disposed at the outer circumference of a recipient
• CPC Class Codes: B03C 2201/18 : Magnetic separation whereby the particles are suspended in a liquid
• CPC Class Codes: G01N 2035/1046 : Levitated, suspended drops
• 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 provides a method of redistributing magnetically responsive beads in a droplet. The method may include providing a droplet including magnetically responsive beads. The droplet may be provided within a region of a magnetic field having sufficient strength to attract the magnetically responsive beads to an edge of the droplet or towards an edge of the droplet, or otherwise regionalize or aggregate beads within the droplet. The method may also include conducting on a droplet operations surface one or more droplet operations using the droplet without removing the magnetically responsive beads from the region of the magnetic field. The droplet operations may in some cases be electrode-mediated. The droplet operations may redistribute and/or circulate the magnetically responsive beads within the droplet. In some cases, the droplet may include a sample droplet may include a target analyte. The redistributing of the magnetically responsive beads may cause target analyte to bind to the magnetically responsive beads. In some cases, the droplet may include unbound substances in a wash buffer. The redistributing of the magnetically responsive beads causes unbound substances to be freed from interstices of an aggregated set or subset of the magnetically responsive beads.

Claims

1. A method of redistributing magnetically responsive beads in a droplet, the method comprising:

(a) providing a droplet comprising magnetically responsive beads within a region of a magnetic field having sufficient strength to attract the magnetically responsive beads to an edge of the droplet; and

(b) using electrodes to conduct on a droplet operations surface a droplet operation using the droplet without removing the magnetically responsive beads from the region of the magnetic field, thereby redistributing the magnetically responsive beads within the droplet.

2. The method of claim 1 wherein the droplet comprises a sample droplet comprising a target analyte.

3. The method of claim 1 wherein the redistributing of the magnetically responsive beads causes target analyte to bind to the magnetically responsive beads.

4. The method of claim 1 wherein the droplet comprises unbound substances in a wash buffer.

5. The method of claim 4 wherein the redistributing of the magnetically responsive beads causes unbound substances to be freed from interstices of an aggregated set or subset of the magnetically responsive beads.

6. The method of claim 1 wherein the droplet operation is selected to agitate contents of the droplet.

7. The method of claim 1 wherein the droplet operation comprises transporting the droplet.

8. The method of claim 1 wherein the droplet operation comprises elongating the droplet.

9. The method of claim 8 wherein elongating the droplet comprises flowing the droplet onto a region of the droplet operations surface atop two or more activated droplet electrodes causing the droplet to take on an elongated configuration.

10. The method of claim 1 wherein the droplet operation comprises merging the droplet with another droplet.

11. The method of claim 1 wherein the droplet operation comprises splitting the droplet to yield two or more daughter droplets.

12. The method of claim 11 wherein two or more of the daughter droplets each comprise a substantial subset of the magnetically responsive beads.

13. The method of claim 11 wherein the droplet operation comprises merging two or more of the daughter droplets.

14. The method of claim 1 further comprising removing the droplet or a sub-droplet thereof including at least a subset of the magnetically responsive beads from the magnetic field.

15. The method of claim 1 wherein one or more droplet operations is repeated in a series of two or more incubation cycles.

16. The method of claim 1 wherein the droplet operations surface is in a droplet operations gap of a droplet actuator.

17. The method of claim 1 wherein the droplet operations surface is coated by a liquid filler fluid.

18. The method of claim 1 wherein the droplet is surrounded by a liquid filler fluid.

19. A method of incubating magnetically responsive beads in a droplet, wherein:

(a) the droplet comprising magnetically responsive beads comprises one or more substances having affinity for one or more of the magnetically responsive beads;

(b) the method comprises redistributing the magnetically responsive beads in the droplet in accordance with the method of claim 1.

20. A method of washing magnetically responsive beads in a droplet, wherein:

(a) the droplet comprising aggregated magnetically responsive beads provided in step 1(a) comprises one or more unbound substances selected for removal;

(b) the method comprises;

(i) merging the droplet comprising the magnetically responsive beads with a wash droplet to yield a combined droplet;

(ii) redistributing the magnetically responsive beads in the droplet in accordance with the method of claim 1;

(iii) splitting the combined droplet to yield;

(1) a droplet comprising substantially all of the magnetically responsive beads and a reduced concentration of the unbound substances relative to the droplet provided in step 1(a); and

(2) a droplet substantially lacking magnetically responsive beads.

22. The method of claim 1 wherein the droplet operation is electrode-mediated.

23. The method of claim 1 wherein the droplet operation is electrowetting-mediated.

24. The method of claim 1 wherein the droplet operation is dielectrophoresis-mediated.

25. A method of washing magnetically responsive beads in a droplet, wherein:

(a) the droplet comprising magnetically responsive beads within a magnetic field comprises one or more unbound substances for removal;

(b) the method comprises;

(i) conducting one or more droplet operations using the droplet in the magnetic field to redistribute the magnetically responsive beads in the droplet in accordance with the method of claim 1;

(ii) merging the droplet comprising the redistributed magnetically responsive beads with a wash droplet to yield a combined droplet;

(iii) splitting the combined droplet to yield;

(1) a droplet comprising substantially all of the magnetically responsive beads and a reduced concentration of the unbound substances relative to the droplet provided in 1(a); and

(2) a droplet substantially lacking magnetically responsive beads.

21. A method of washing magnetically responsive beads in a droplet, wherein:

(a) providing a droplet comprising;

(i) magnetically responsive beads within a region of a magnetic field having sufficient strength to attract the magnetically responsive beads to an edge of the droplet;

(ii) one or more unbound substances selected for removal;

(b) the method comprises;

(i) merging the droplet comprising magnetically responsive beads with a wash droplet in the magnetic field to yield a combined droplet and to redistribute the magnetically responsive beads within the combined droplet;

(ii) splitting the combined droplet to yield;

(1) a droplet comprising substantially all of the magnetically responsive beads and a reduced concentration of the unbound substances relative to the droplet provided in (a); and

(2) a supernatant droplet substantially lacking magnetically responsive beads.

26. A method of redistributing magnetically responsive beads in a droplet, the method comprising:

(a) providing a droplet comprising magnetically responsive beads within a first region of a magnetic field having sufficient strength to attract the magnetically responsive beads to an edge of the droplet;

(b) using electrodes to transport droplet to a second region of a droplet operations surface in which the magnetic field is sufficiently reduced to permit the magnetically responsive beads to circulate in the droplet during the conduct of one or more droplet operations; and

(c) conducting the one or more droplet operations to cause the magnetically responsive beads to circulate in the droplet.

27. A method of incubating a droplet, the method comprising:

(a) providing a droplet comprising magnetically responsive beads within a region of a magnetic field in which the magnetically responsive beads are caused to become aggregated;

(b) using electrodes to conduct on a droplet operations surface droplet operations using the droplet wherein the droplet operations comprise;

(i) one or more droplet operations transporting the droplet away from the magnetic field to a locus of the droplet operations surface in which the magnetically responsive beads are resuspended in the droplet; and

(ii) one or more droplet operations effecting an incubation cycle in the locus in which the magnetically responsive beads are resuspended in the droplet.

28. A method of washing beads in a droplet, the method comprising:

(a) providing an elongated bead-containing droplet comprising one or more unbound substances;

(b) providing an elongated wash droplet;

(c) restraining movement of beads within the elongated bead-containing droplet;

(d) merging end-to-end the elongated bead-containing droplet with the elongated bead containing droplet to yield a combined droplet;

(e) splitting the combined droplet to form a first droplet comprising substantially all of the beads and a second droplet substantially lacking in beads.

29. A method of washing beads, the method comprising:

(a) providing the beads in a sample droplet comprising a target substance on a droplet operations substrate within a magnetic field;

(b) transporting the sample droplet away from the beads, causing the droplet to split, yielding a supernatant droplet and leaving behind a daughter droplet comprising substantially all of the magnetically responsive beads; and

(c) subjecting the daughter droplet to a merge-and-split bead washing protocol.