Method of reducing liquid volume surrounding beads

• US 8,927,296 B2
• Filed: 05/18/2009
• Issued: 01/06/2015
• 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: Sista, Ramakrishna, Sudarsan, Arjun, Srinivasan, Vijay, Thwar, Prasanna, Pamula, Vamsee
• Primary Examiner(s): HIXSON, CHRISTOPHER
• Application Number:
  US12/992,939
• Publication Number:
  US 20110091989A1
• Time in Patent Office: 2,059 Days
• Field of Search: 436/43, 436/174, 436/149, 422/50, 422/504, 422/68.1, 422/82.01, 422/82.02, 435/79, 435/7.4
• US Class Current: 436/174
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/089 Virtual walls for guiding liquids
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 2400/043 magnetic forces
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• 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 3/502792 for moving individual droplets on a plate, e.g. by locally altering surface tension
• CPC Class Codes: G01N 1/4077 by other techniques involving separation of suspended solids
• CPC Class Codes: G01N 2035/00564 Handling or washing solid phase elements, e.g. beads
• CPC Class Codes: G01N 35/0098 involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation magnetic particles used in immunoassays G01N33/54326; magnetic separation in general B03C
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

The invention provides droplet actuators and droplet actuator techniques. Among other things, the droplet actuators and methods are useful for manipulating beads on a droplet actuator, such as conducting droplet operations using bead-containing droplets on a droplet actuator. For example, beads may be manipulated on a droplet actuator in the context of executing a sample preparation protocol and/or an assay protocol. An output of the methods of the invention may be beads prepared for execution of an assay protocol. Another output of the methods of the invention may be results of an assay protocol executed using beads. Among the methods described herein are methods of concentrating beads in droplets, methods of washing beads, methods of suspending beads, methods of separating beads, methods of localizing beads within a droplet, methods of forming emulsions in which droplets include beads, methods of loading beads into a droplet operations gap of a droplet actuator, methods of organizing beads in a monolayer, and methods of capturing, trapping or restraining beads.

Claims

1. A method of reducing a liquid volume surrounding one or more magnetically responsive beads, the method comprising:

(a) providing a droplet comprising the one or more magnetically responsive beads;

(b) exposing the one or more magnetically responsive beads in the droplet to a first region of a magnetic field; and

(c) separating the droplet from the first region of the magnetic field via electrowetting, the one or more magnetically responsive beads remaining in a subdroplet atop an electrowetting electrode in the magnetic field.

2. The method of claim 1 further comprising heating the droplet to a temperature which permits the one or more magnetically responsive beads to break away from the droplet.

3. The method of claim 1 further comprising adjusting the droplet to a shape which permits the one or more magnetically responsive beads to break away from the droplet.

4. The method of claim 1 further comprising adjusting the droplet to an interfacial tension which permits the one or more magnetically responsive beads to break away from the droplet.

5. The method of claim 1 further comprising adding beads to the droplet until a mass of beads is accumulated which permits the one or more magnetically responsive beads to break away from the droplet.

6. The method of claim 1 further comprising adjusting the position and/or strength of a magnetic field in proximity to the droplet until one or more magnetically responsive beads breaks away from the droplet.

7. The method of claim 1 further comprising adjusting the position of the droplet in relation to a magnetic field until one or more magnetically responsive beads breaks away from the droplet.

8. The method of claim 1 wherein the droplet comprises a wash buffer.

9. The method of claim 1 wherein the droplet comprises one or more target substances for which at least a subset of the one or more beads has affinity.

10. The method of claim 1 wherein the one or more magnetically responsive beads remaining in the magnetic field is/are surrounded by liquid from the droplet.

11. The method of claim 1 wherein the one or more magnetically responsive beads is/are separated from the droplet during step 1(b).

12. The method of claim 1 wherein the one or more magnetically responsive beads is/are separated from the droplet during step 1(c).

14. The method of claim 1 wherein the one or more magnetically responsive beads is/are snapped out of the droplet as the droplet is transported away from the first region of the magnetic field.

15. The method of claim 1 wherein the one or more magnetically responsive beads is/are snapped out of the droplet while the droplet is in proximity to the first region of the magnetic field.

16. The method of claim 11 wherein during the separation, the droplet is restrained from following the one or more magnetically responsive beads by a surface property of an underlying substrate.

17. The method of claim 16 wherein during the separation, the surface property of the underlying substrate causes the droplet to remain in a substantially stationary position.

18. The method of claim 16 wherein during the separation, the surface property of the underlying substrate causes the droplet to move closer to the first region of the magnetic field.

19. The method of claim 16 wherein during the separation, the surface property of the underlying substrate causes the droplet to move away from the first region of the magnetic field.

20. The method of claim 16 wherein the surface property of the underlying substrate is mediated by an electrode.

21. The method of claim 20 wherein the electrode is underlying the substrate.

22. The method of claim 1 wherein during the separation, the droplet is heated.

23. The method of claim 1 wherein during the separation, the droplet is surrounded by a liquid filler fluid that is substantially immiscible with the droplet.

24. The method of claim 1 wherein following the separation, the one or more magnetically responsive beads remain(s) in the first region of the magnetic field.

25. The method of claim 1 wherein following the separation, the one or more magnetically responsive beads is/are caused by the magnetic field to relocate from the first region of the magnetic field to a second region of the magnetic field.

26. The method of claim 1 wherein exposing the one or more magnetically responsive beads in the droplet to a first region of a magnetic field comprises transporting the droplet into the first region of the magnetic field.

27. The method of claim 1 wherein exposing the one or more magnetically responsive beads in the droplet to a first region of a magnetic field comprises transporting the first region of the magnetic field into proximity with the one or more magnetically responsive beads.

28. The method of claim 1 wherein separating the droplet from the first region of the magnetic field comprises transporting the droplet away from the first region of the magnetic field.

29. The method of claim 1 wherein separating the droplet from the first region of the magnetic field comprises moving the first region of the magnetic field away from the droplet.

30. The method of claim 1 wherein separating the droplet from the first region of the magnetic field comprises eliminating or shielding the magnetic field.

31. The method of claim 1 further comprising concentrating a target substance on the one or more magnetically responsive beads by contacting the one or more magnetically responsive beads with a sample droplet.

32. The method of claim 31 further comprising separating the sample droplet from the first region of the magnetic field, the one or more magnetically responsive beads remaining in the magnetic field.

33. The method of claim 1 further comprising washing the one or more magnetically responsive beads by contacting the one or more magnetically responsive beads with a wash droplet.

34. The method of claim 33 further comprising separating the wash droplet from the first region of the magnetic field, the one or more magnetically responsive beads remaining in the magnetic field.

35. The method of claim 1 wherein providing a droplet comprising one or more magnetically responsive beads comprises providing the droplet in a droplet operations gap of a droplet actuator, wherein the droplet is subject to one or more droplet operations.

36. The method of claim 35 wherein exposing the one or more magnetically responsive beads in the droplet to a first region of a magnetic field comprises transporting the droplet using electrode-mediated droplet operations into the first region of the magnetic field.

37. The method of claim 35 wherein exposing the one or more magnetically responsive beads in the droplet to a first region of a magnetic field comprises transporting the first region of the magnetic field into proximity with the one or more magnetically responsive beads.

38. The method claim 35 wherein separating the droplet from the first region of the magnetic field comprises transporting the droplet using electrode-mediated droplet operations away from the first region of the magnetic field.

39. The method of claim 35 wherein separating the droplet from the first region of the magnetic field comprises transporting the first region of the magnetic field away from the one or more magnetically responsive beads.

40. The method of claim 35 wherein the one or more droplet operations is/are mediated by one or more electrodes.

41. The method of claim 1 wherein following separating the droplet from the first region of the magnetic field, the droplet is presented to a detector or detection window for detection of a property of and/or signal from the droplet.

42. The method of claim 1 wherein:

(a) the one or more magnetically responsive beads remaining in the magnetic field is positioned in a droplet atop an electrowetting electrode;

(b) the electrowetting electrode has a footprint; and

(c) the droplet has a footprint which is smaller than the footprint of the electrowetting electrode.

13. A method of reducing a liquid volume surrounding one or more magnetically responsive beads, the method comprising:

(a) providing a droplet comprising the one or more magnetically responsive beads;

(b) exposing the one or more magnetically responsive beads in the droplet to a first region of a magnetic field; and

(c) separating the droplet from the first region of the magnetic field via electrowetting, the one or more magnetically responsive beads remaining in a subdroplet atop an electrowetting electrode in the magnetic field, wherein separating the droplet from the first region of the magnetic field comprises magnetically attracting beads;

(i) from a droplet positioned at least partially in a droplet operations gap of a droplet actuator; and

(ii) through an opening in a substrate of the droplet actuator to a locus which is exterior to the droplet operations gap.