Bead manipulation techniques

• US 9,050,606 B2
• Filed: 09/19/2014
• Issued: 06/09/2015
• Est. Priority Date: 04/13/2006
• Status: Active Grant

• Current Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Pamula, Vamsee K., Sudarsan, Arjun, Sista, Ramakrishna
• Primary Examiner(s): Sines, Brian J
• Application Number:
  US14/491,231
• Publication Number:
  US 20150008128A1
• Time in Patent Office: 263 Days
• Field of Search: 422/50, 422/68.1, 422/82.01, 422/502, 422/503, 422/504, 436/43, 436/149, 436/150, 436/174, 436/179, 436/180
• US Class Current: 1/1
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2400/0424 Dielectrophoretic forces
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• 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: 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 2201/26 for use in medical applications
• CPC Class Codes: B03C 5/00 Separating dispersed particles from liquids by electrostatic effect flocculation or agglomeration of electric particles induced by electric field B01D21/0009; combined with centrifuges B04B5/10
• CPC Class Codes: B03C 5/005 Dielectrophoresis, i.e. dielectric particles migrating towards the region of highest field strength B03C5/02 takes precedence; electrophoresis B01D57/02
• CPC Class Codes: B03C 5/026 using open-gradient differential dielectric separation, i.e. using electrodes of special shapes for non-uniform field creation, e.g. Fluid Integrated Circuit [FIC]
• CPC Class Codes: B03C 7/026 using travelling or oscillating electric fields
• CPC Class Codes: G01N 33/54326 Magnetic particles

Abstract

The invention provides a method of redistributing magnetically responsive beads in a droplet. The method may 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 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, the droplet operations substrate comprising a path of electrowetting electrodes;

(b) retaining the beads using the magnetic field while transporting the sample droplet away from the beads by sequentially activating a series of the electrowetting electrodes selected to cause the sample droplet to be transported away from the beads, causing the beads to be snapped off from the sample droplet as the sample droplet is transported away from the beads, yielding a supernatant droplet and leaving behind a daughter droplet comprising substantially all of the magnetically responsive beads, wherein an electrowetting electrode underlying the beads remains deactivated while transporting the sample droplet away from the beads; and

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

2. The method of claim 1 wherein the supernatant droplet includes more than 50% of the unbound substances being removed.

3. The method of claim 1 wherein the supernatant droplet includes more than 75% of the unbound substances being removed.

4. The method of claim 1 further comprising:

(a) merging the daughter droplet comprising substantially all of the magnetically responsive beads with a wash droplet to form a merged droplet; and

(b) transporting the merged droplet away from the beads by sequentially activating the electrowetting electrodes, causing the beads to be snapped off as the droplet is transported away, yielding a second supernatant droplet and leaving behind a second daughter droplet comprising substantially all of the magnetically responsive beads.

5. The method of claim 1 further comprising

providing a droplet actuator comprising,

a first substrate, wherein the first substrate comprises the droplet operations substrate, wherein the path of electrowetting electrodes are configured for conducting one or more droplet operations on a surface of the droplet operations substrate.

6. The method of claim 5, further comprising a second substrate separated from the droplet operations substrate to form a gap.

7. The method of claim 6, wherein the gap comprises a filler fluid disposed therein.

8. The method of claim 7, wherein the filler fluid comprises an oil filler fluid.

9. The method of claim 8, wherein the oil filler fluid comprises a silicone oil.