Method of washing beads

• US 8,637,317 B2
• Filed: 01/06/2011
• Issued: 01/28/2014
• 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: Pamula, Vamsee K., Sudarsan, Arjun, Sista, Ramakrishna
• Primary Examiner(s): Sines, Brian J
• Application Number:
  US12/985,409
• Publication Number:
  US 20110114490A1
• Time in Patent Office: 1,118 Days
• Field of Search: 422/50, 422/68.1, 422/82.01, 422/82.02, 422/100, 436/43, 436/149, 436/150, 436/174, 436/179, 436/180
• US Class Current: 436/43
• 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 washing beads comprising:

(a) providing a droplet actuator comprising;

(i) two substrates arranged to provide a gap;

(ii) electrodes associated with one or both of the two substrates and arranged for conducting droplet operations in the gap;

(iii) a starting droplet situated in the gap comprising magnetically responsive beads within a region of a magnetic field and a starting concentration of the substance;

(b) conducting one or more electrowetting-mediated droplet operations to merge a wash droplet with the droplet provided in step (a)(iii) to yield a combined droplet;

(c) conducting one or more droplet operations while restraining the beads to divide the combined droplet, yielding a set of daughter droplets comprising;

(i) a first daughter droplet retaining at least about 99% of the beads and having a decreased concentration of the substance relative to the starting concentration;

(ii) a second daughter droplet which is separated from the at least one surface; and

(d) transporting the first daughter droplet away from the magnetic field.

2. The method of claim 1 comprising repeating steps (b) and (c) until a 99% reduction in the starting material is achieved relative to the starting concentration of the substance.

3. The method of claim 1 comprising repeating steps (b) and (c) until a 99.9% reduction in the starting material is achieved relative to the starting concentration of the substance.

4. The method of claim 1 comprising repeating steps (b) and (c) until a 99.99% reduction in the starting material is achieved relative to the starting concentration of the substance.

5. The method of claim 1 comprising repeating steps (b) and (c) until a 99.999% reduction in the starting material is achieved relative to the starting concentration of the substance.

6. The method of claim 1 comprising repeating steps (b) and (c) until a 99.999% reduction in the starting material is achieved relative to the starting concentration of the substance and substantially all of the beads are retained relative to the quantity of beads provided in the starting droplet.

7. The method of claim 1 comprising repeating steps (b) and (c) until a 99.999% reduction in the starting material is achieved relative to the starting concentration of the substance and substantially all of the beads are retained relative to the quantity of beads provided in the starting droplet.

8. The method of claim 1 comprising repeating steps (b) and (c) until a 99.999% reduction in the starting material is achieved relative to the starting concentration of the substance and substantially all of the beads are retained relative to the quantity of beads provided in the starting droplet.

9. The method of claim 1 comprising repeating steps (b) and (c) until a 99.999% reduction in the starting material is achieved relative to the starting concentration of the substance and substantially all of the beads are retained relative to the quantity of beads provided in the starting droplet.

10. The method of claim 1 wherein the restraining comprises aggregating the beads in a region of the droplet.

11. The method of claim 1 further comprising combining the first daughter droplet with a second droplet to yield a combined droplet and transporting the combined droplet away from the magnetic field.