Method of concentrating beads in a droplet

• US 9,675,972 B2
• Filed: 03/09/2009
• Issued: 06/13/2017
• Est. Priority Date: 05/09/2006
• Status: Active Grant

• Current Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Hua, Zhishan, Pamula, Vamsee K., Sudarsan, Arjun, Srinivasan, Vijay, Pollack, Michael G., Sista, Ramakrishna, Allen, Dwayne, Shenderov, Alexander, Thwar, Prasanna
• Primary Examiner(s): Brown, Melanie Y
• Application Number:
  US12/921,256
• Publication Number:
  US 20110104747A1
• Time in Patent Office: 3,018 Days
• Field of Search: 436526
• 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: B01F 33/452 using independent floating stirring elements
• CPC Class Codes: B01F 33/5011 portable during use, e.g. hand-held
• CPC Class Codes: B01F 33/50112 of the syringe or cartridge type
• CPC Class Codes: B01L 2200/0621 Control of the sequence of chambers filled or emptied
• CPC Class Codes: B01L 2200/0631 Purification arrangements, e.g. solid phase extraction [SPE]
• CPC Class Codes: B01L 2200/0652 Sorting or classification of particles or molecules
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2200/141 Preventing contamination, tampering
• CPC Class Codes: B01L 2300/0832 cylindrical, tube shaped
• 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 2400/043 magnetic forces
• CPC Class Codes: B01L 3/0282 mounted within a receptacle wash bottles B01L3/10
• CPC Class Codes: B01L 3/502753 characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation separation in general B01D; microapparatus for analysis using electrophoresis G01N27/44791; sample preparation G01N1/28
• CPC Class Codes: B03C 1/01 by addition of magnetic adjuvants
• CPC Class Codes: B03C 1/284 with associated cleaning means, e.g. retractable non-magnetic sleeve
• CPC Class Codes: B03C 1/286 disposed at the inner circumference of a recipient, e.g. magnetic drain bolt
• 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 2201/28 : Parts being designed to be removed for cleaning purposes
• CPC Class Codes: Y10T 436/25 : including sample preparation

Abstract

Methods of concentrating beads in a droplet and/or loading beads on a fluidic device are provided, including among other things, a method of concentrating beads in a droplet, the method comprising: (a) providing a droplet actuator comprising: (i) an interior droplet operations volume; and (ii) a reservoir exterior to the interior volume; (iii) a droplet established in a liquid path extending from the reservoir into the interior volume; (b) providing magnetically responsive beads in the portion of the droplet which is in the reservoir; (c) magnetically attracting the magnetically responsive beads through the liquid path into the portion of the droplet which is in the interior volume; and (d) forming a droplet comprising one or more of the magnetically responsive beads in the interior volume.

Claims

1. A method of concentrating beads in a droplet, the method comprising:

(a) providing a droplet actuator comprising;

(i) an interior droplet operations volume; and

(ii) a reservoir exterior to the interior volume;

(iii) a first substrate;

(iv) a second substrate separated from the first substrate to provide the interior volume between the first substrate and the second substrate, comprising the reservoir and a liquid path;

(v) electrodes associated with the first and/or second substrate and arranged for conducting one or more droplet operations in the interior volume; and

(vi) a magnet providing a magnetic field arranged to attract magnetically responsive beads from the liquid reservoir into the interior volume;

(b) establishing a droplet in the liquid path extending from the reservoir into the interior volume;

(c) providing a first concentration of magnetically responsive beads in the portion of the droplet which is in the reservoir;

(d) magnetically attracting the magnetically responsive beads through the liquid path into the portion of the droplet which is in the interior volume; and

(e) forming a droplet comprising a second concentration of magnetically responsive beads in the interior volume, wherein the second concentration of magnetically responsive beads in the droplet formed in the interior volume is greater than the first concentration of magnetically responsive beads provided in the portion of the droplet which is in the reservoir; and

(f) conducting one or more droplet operations on the droplet in the interior volume via electrowetting.

2. The method of claim 1 wherein:

(a) the beads are magnetically responsive; and

(b) step 1(d) comprises magnetically attracting the magnetically responsive beads to a terminus of the liquid path in the interior volume.

3. The method of claim 1 wherein step 1(e) comprises breaking the liquid path in a region lacking the magnetically responsive beads to yield a droplet comprising substantially all of the magnetically responsive beads attracted to the terminus of the liquid path in the interior volume.

4. The method of claim 1 wherein the droplet formed in step 1(e) comprises substantially all of the magnetically responsive beads provided in the reservoir.

5. The method of claim 1 wherein step 1(b) and step 1(c) comprise:

(a) providing a liquid comprising the magnetically responsive beads in the reservoir; and

(b) flowing a portion of the liquid comprising the magnetically responsive beads into the interior volume to establish the liquid path.

6. The method of claim 5 wherein step 5(b) is electrode-mediated.

7. The method of claim 5 wherein step 5(b) comprises changing an activation state of one or more electrodes to cause the liquid to flow onto a surface of the droplet actuator bounding the interior volume.

8. The method of claim 7 wherein step 1(e) comprises changing an activation state of one or more electrodes to cause the formation of a droplet comprising substantially all of the beads provided in the reservoir.

9. The method of claim 7 further comprising changing an activation state of one or more electrodes to cause the formation of one or more droplets substantially lacking the beads.

10. The method of claim 7 wherein step 1(d) comprises magnetically attracting the beads to a terminus of the flow of liquid.

11. The method of claim 10 wherein step 1(e) comprises changing an activation state of one or more electrodes to cause the formation of a droplet from the terminus of the flow, the droplet comprising substantially all of the beads provided in the reservoir.

12. The method of claim 7 wherein step 1(d) comprises magnetically attracting the beads to an intermediate locus of the flow.

13. The method of claim 12 further comprising changing an activation state of one or more electrodes to cause the formation of one or more droplets from the terminus of the flow, the one or more droplets substantially lacking the beads.

14. The method of claim 1 wherein magnetically attracting the magnetically responsive beads into the interior volume comprises magnetically attracting the beads towards a locus of the interior volume which is substantially opposite an entry point of the liquid path.

15. The method of claim 1 wherein the beads have affinity for a target substance in the liquid.

16. The method of claim 1 wherein:

(a) the liquid comprises a biological sample; and

(b) the beads have affinity for a target substance in the liquid.

17. The method of claim 1 wherein:

(a) the liquid comprises a lysis buffer; and

(b) the beads have an affinity for one or more target substances from cells lysed with the lysis buffer.

18. The method of claim 1, wherein the second concentration of magnetically responsive beads is at least 2×

the first concentration of magnetically responsive beads.

19. The method of claim 1, wherein the second concentration of magnetically responsive beads is at least 5×

the first concentration of magnetically responsive beads.

20. The method of claim 1, wherein the second concentration of magnetically responsive beads is at least 10×

the first concentration of magnetically responsive beads.

21. The method of claim 1, wherein the second concentration of magnetically responsive beads is at least 50×

the first concentration of magnetically responsive beads.

22. The method of claim 1, wherein the second concentration of magnetically responsive beads is at least 100×

the first concentration of magnetically responsive beads.