Droplet-based affinity assay device and system

• US 8,389,297 B2
• Filed: 12/15/2006
• Issued: 03/05/2013
• Est. Priority Date: 04/18/2006
• Status: Active Grant

• Current Assignee: Duke University
• Original Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Pamula, Vamsee K., Srinivasan, Vijay, Pollack, Michael G., Fair, Richard B.
• Primary Examiner(s): Brown, Melanie Y
• Application Number:
  US11/639,822
• Publication Number:
  US 20090280476A1
• Time in Patent Office: 2,272 Days
• Field of Search: 436/526, 436/518, 436/525, 436/174, 435/283.1, 435/287.1, 435/287.2, 435/287.3
• US Class Current: 436/526
• CPC Class Codes: B01L 2300/0645 Electrodes
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1894 Cooling means; Cryo cooling
• CPC Class Codes: B01L 2400/0424 Dielectrophoretic forces
• CPC Class Codes: B01L 2400/0427 Electrowetting
• 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: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

The present invention relates to a droplet-based affinity assay device and system. According to one embodiment, a droplet microactuator is provided and includes an antibody immobilized on a surface. According to another embodiment, a droplet microactuator is provided and includes a droplet on the droplet microactuator, the droplet comprising an antibody.

Claims

1. A droplet microactuator comprising:

(a) substrates spaced apart to form an intervening space and comprising electrodes for conducting electrowetting droplet operations, the droplet operations comprising merging and splitting operations, wherein the intervening space is substantially filled with a filler fluid comprising oil;

(b) reagent droplets for executing an immunoassay protocol, wherein the reagent droplets;

(i) comprise one or more wash droplets;

(ii) are situated in the intervening space and surrounded by the filler fluid;

(iii) are subject to the electrowetting droplet operations mediated by the electrodes; and

(iv) comprise an aqueous droplet surrounded by the filler fluid; and

(c) an antibody immobilized on a surface of a magnetically responsive bead comprised in the aqueous droplet.

4. The droplet microactuator of claim 1 comprising a sample for executing an immunoassay protocol.

5. The droplet microactuator of claim 4 wherein the volume of sample does not exceed about 1 mL.

6. The droplet microactuator of claim 4 wherein the volume of sample does not exceed about 100 μ

L.

7. The droplet microactuator of claim 4 wherein the volume of sample does not exceed about 50 μ

L.

8. The droplet microactuator of claim 1 comprising reagents and sample for executing an immunoassay protocol.

9. The droplet microactuator of claim 1 further comprising a means for producing a magnetic field in a droplet on the droplet microactuator, wherein the means is arranged to generally restrain the location of the bead during an electrowetting-mediated droplet splitting operation to yield a droplet with one or more magnetically responsive beads and a second droplet without a substantial amount of magnetically responsive beads.

10. The droplet microactuator of claim 9 wherein the means for producing the magnetic field comprises a magnet and a motorized assembly for moving the magnet into and out of proximity with the droplet microactuator.

11. The droplet microactuator of claim 9 wherein the means for producing a magnetic field comprises an electromagnet.

12. The droplet microactuator of claim 1 further comprising one or more heaters arranged to heat one or more droplets on the droplet microactuator.

13. The droplet microactuator of claim 12 comprising two or more heaters spaced to maintain distinct temperature zones on the droplet microactuator.

14. The droplet microactuator of claim 12 wherein one or more of the heaters comprises a block heater.

15. The droplet microactuator of claim 12 wherein one or more heaters maintains at least a portion of the droplet microactuator at a temperature suitable for an incubation step in an immunoassay.

16. The droplet microactuator of claim 1 wherein the filler fluid comprises silicone oil.

17. The droplet microactuator of claim 1 wherein the oil comprises a surfactant.

18. The droplet microactuator of claim 17 wherein the surfactant comprises a lipophilic surfactant.

19. The droplet microactuator of claim 17 wherein the surfactant comprises Triton X-15.

20. The droplet microactuator of claim 1 wherein the filler fluid comprises silicone oil comprising a surfactant.

21. The droplet microactuator of claim 20 wherein the surfactant comprises a lipophilic surfactant.

22. The droplet microactuator of claim 20 wherein the surfactant comprises Triton X-15.

23. The droplet microactuator of claim 1 comprising one or more reservoirs each comprising a sample processing reagent, an affinity-based assay reagent and/or a control reagent.

2. A droplet microactuator comprising:

(a) substrates separated to form an intervening space substantially filled with a filler fluid comprising oil, the substrates comprising electrodes for conducting electrowetting droplet operations comprising droplet transport, merging, dispensing and splitting operations; and

(b) droplets in the intervening space surrounded by the filler fluid, the droplets comprising reagents for executing an immunoassay protocol, one or more of the droplets comprising a magnetically responsive bead having antibodies immobilized thereon;

the droplets further comprising one or more wash droplets for washing the magnetically responsive antibody-capture bead and wherein each of the droplets is subject to one or more of the electrowetting droplet operations.

3. A droplet microactuator comprising:

(a) substrates separated to form an intervening space substantially filled with an oil filler fluid, the substrates comprising electrodes for conducting electrowetting droplet operations; and

(b) droplets in the intervening space comprising reagent droplets for executing an immunoassay protocol, including one or more droplets;

(i) surrounded by the oil filler fluid;

(ii) comprising a magnetically responsive antibody-capture bead; and

(iii) subject to the electrowetting droplet operations mediated by the electrodes.