Droplet-Based Surface Modification and Washing

• US 20100140093A1
• Filed: 11/10/2009
• Published: 06/10/2010
• Est. Priority Date: 04/18/2006
• Status: Active Grant

• Current Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Pamula, Vamsee K., Eckhardt, Allen E., Srinivasan, Vijay, Pollack, Michael G., Fair, Richard B.
• Granted Patent: US 8,313,895 B2
• Time in Patent Office: Days
• US Class Current: 204/547
• 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 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2200/0684 Venting, avoiding backpressure, avoid gas bubbles
• 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 2400/0424 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 2400/0448 : Marangoni flow; Thermocapillary effect
• CPC Class Codes: B01L 3/0268 : using pulse dispensing or spraying, eg. inkjet type, piezo actuated ejection of droplets from capillaries
• CPC Class Codes: B01L 3/502715 : characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502723 : characterised by venting arrangements
• 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: C12Q 1/686 : Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 : Methods for sequencing
• CPC Class Codes: G01N 2035/00158 : Elements containing microarrays, i.e. "biochip"
• CPC Class Codes: G01N 2035/00237 : Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: G01N 27/26 : by investigating electrochemical variables; by using electrolysis or electrophoresis
• CPC Class Codes: G01N 27/3271 : Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood amperometry per se G01N27/49; aspects concerning the enzyme reagent C12Q1/001
• CPC Class Codes: G01N 27/416 : Systems G01N27/27 takes precedence
• CPC Class Codes: G01N 27/447 : using electrophoresis
• CPC Class Codes: G01N 35/00029 : provided with flat sample substrates, e.g. slides G01N35/028 takes precedence

Abstract

The present invention relates to droplet-based surface modification and washing. According to one embodiment, a method of providing a bead-containing droplet with a reduced concentration of a substance is provided, the method including providing a droplet microactuator including a droplet having a starting volume and including one or more beads and a starting concentration and starting quantity of the substance. The method further includes conducting one or more droplet operations to merge a wash droplet with the droplet provided in the above step to yield a combined droplet and including one or more droplet operations to divide the combined droplet to yield a set of droplets including a droplet including substantially all of the one or more beads and having a decreased concentration of the substance relative to the starting concentration and a droplet which is substantially lacking in the beads.

Claims

1. A method of providing a bead-containing droplet with a reduced concentration of a substance, the method comprising:

(a) providing a droplet microactuator comprising a droplet having a starting volume and comprising;

(i) one or more beads; and

(ii) a starting concentration and starting quantity of the substance;

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

(c) conducting one or more droplet operations to divide the combined droplet to yield a set of droplets comprising;

(i) a droplet comprising substantially all of the one or more beads and having a decreased concentration of the substance relative to the starting concentration; and

(ii) a droplet which is substantially lacking in the beads.

2. The method of claim 1 wherein:

(a) the droplet microactuator comprises;

(i) a first substrate comprising electrodes configured for manipulating a droplet on a surface of the substrate; and

(ii) a second substrate arranged relative to the first substrate and spaced from the surface of the first substrate by a distance sufficient to define a space between the first substrate and second substrate; and

(b) the droplet is situated in the space between the first substrate and second substrate.

3. The method of claim 2 wherein the second substrate is arranged in a substantially parallel relationship to the first substrate.

4. The method of claim 1 wherein the starting droplet comprises a sample droplet having a volume which is less than about 100 μ

L.

5. The method of claim 1 wherein the starting droplet comprises a sample droplet having a volume which is less than about 50 μ

L.

6. The method of claim 1 wherein the starting droplet comprises a sample droplet having a volume which is less than about 25 μ

L.

7. The method of claim 1 wherein the one or more beads comprises beads having a diameter ranging from about 0.1 nm to about 25 mm.

8. The method of claim 1 wherein the immobilizing comprises attracting the beads prior to step 1(c).

9. The method of claim 1 wherein the immobilizing comprises immobilizing the beads prior to step 1(c).

10. The method of claim 1 wherein the immobilizing comprises retaining the beads during step 1(c).

11. The method of claim 1 further comprising releasing or resuspending one the beads following step 1(c).

12. The method of claim 11 further comprising agitating the beads following step 1(c).

13. The method of claim 11 further comprising agitating the beads following step 1(c) using a sonicator.

14. The method of claim 9 wherein the immobilizing step is achieved by using a physical object to block transport of one or more of the beads during a droplet operation.

15. The method of claim 9 wherein:

(a) one or more of the beads is substantially non-magnetically responsive; and

(b) the immobilizing step is achieved by using a physical object to block transport of the substantially non-magnetically responsive beads during a droplet operation.

16. The method of claim 9 wherein:

(a) the beads comprise magnetically responsive beads; and

(b) the immobilizing step is accomplished by exposing the magnetically responsive beads to a magnetic field.

17. The method of claim 16 wherein the immobilizing comprises using a magnetic field in of sufficient strength to generally restrain the location of the one or more magnetically responsive beads during the droplet operation.

18. The method of claim 16 comprising using a surfactant to prevent or reduce bead aggregation.

19. The method of claim 16 wherein step 16(b) comprises situating a droplet comprising the beads in proximity with a means for effecting a magnetic field in proximity to one or more of the electrodes, wherein the strength and proximity of the magnetic field is sufficient to immobilize magnetically responsive beads during execution of a washing protocol.

20. The method of claim 19 wherein the means for effecting a magnetic field comprises an electromagnet and a means for controlling power supply to the electromagnet.

21. The method of claim 19 wherein the means for effecting a magnetic field comprises a magnet and a means for moving the magnet into and out of proximity with the one or more of the electrodes.

22. The method of claim 1 wherein the droplet of 1(c)(i) comprises more than 99% of beads present in the combined droplet.

23. The method of claim 1 wherein the droplet of 1(c)(i) comprises more than 99.9% of beads present in the combined droplet.

24. The method of claim 1 wherein the droplet of 1(c)(i) comprises more than 99.999% of beads present in the combined droplet.

25. The method of claim 1 wherein step 1(c) is electrode-mediated.

26. The method of claim 1 wherein step 1(c) is electrowetting-mediated.

27. The method of claim 1 wherein step 1(c) is dielectrophoresis-mediated.

28. The method of claim 1 wherein the droplet operation comprises splitting, transporting, and/or dispensing.

29. The method of claim 1 wherein the substance comprises one or more contaminants.

30. The method of claim 1 wherein the substance comprises one or more analytes.

31. The method of claim 1 comprising repeating steps 1(b) and 1(c) until a predetermined reduction in the concentration and/or amount of the substance is achieved in the droplet of 1(c)(i).

32. The method of claim 1 comprising repeating steps 1(b) and 1(c) until a predetermined maximum quantity of the substance is met or exceeded in the droplet of 1(c)(i).

33. The method of claim 1 comprising repeating steps 1(b) and 1(c) until a substantial reduction the substance is achieved in the droplet of 1(c)(i).

34. The method of claim 1 comprising repeating steps 1(b) and 1(c) until the droplet of 1(c)(i) is substantially free of the substance.

35. The method of claim 1 comprising repeating steps 1(b) and 1(c) until the substance in the droplet of 1(c)(i) is reduced by 99 percent on a molar basis relative to the substance in the droplet provided in step 1(a).

36. The method of claim 1 comprising repeating steps 1(b) and 1(c) until the substance in the droplet of 1(c)(i) is reduced by 99.9 percent on a molar basis relative to the substance in the droplet provided in step 1(a).

37. The method of claim 1 comprising repeating steps 1(b) and 1(c) until the substance in the droplet of 1(c)(i) is reduced by 99.99 percent on a molar basis relative to the substance in the droplet provided in step 1(a).

38. The method of claim 1 comprising repeating steps 1(b) and 1(c) until the substance in the droplet of 1(c)(i) is reduced by 99.999 percent on a molar basis relative to the substance in the droplet provided in step 1(a).

39. The method of claim 1 comprising repeating steps 1(b) and 1(c) until the substance in the droplet of 1(c)(i) is reduced by 99.9999 percent on a molar basis relative to the substance in the droplet provided in step 1(a).

40. The method of claim 1 comprising repeating steps 1(b) and 1(c) until the substance in the droplet of 1(c)(i) is reduced by 99.99999 percent on a molar basis relative to the substance in the droplet provided in step 1(a).

41. The method of claim 1 comprising repeating steps 1(b) and 1(c) until the substance in the droplet of 1(c)(i) is reduced by 99.999999 percent on a molar basis relative to the substance in the droplet provided in step 1(a).

42. A method of providing a bead-containing droplet with a reduced concentration of a substance, the method comprising:

(a) providing an electrowetting device comprising;

(i) electrodes arranged to mediate droplet operations;

(ii) a bead-containing droplet having a starting volume and comprising a starting concentration and starting quantity of the substance;

(b) conducting droplet operations mediated by the electrodes to;

(i) merge a wash droplet with the bead-containing droplet provided in step 1(a) to yield a combined droplet; and

(ii) divide the combined droplet to yield a set of droplets comprising;

(1) a droplet comprising substantially all of the one or more beads and having a decreased concentration of the substance relative to the starting concentration; and

(2) a droplet which is substantially lacking in the beads.

43. The method of claim 42 wherein the droplet operations comprise electrowetting-mediated droplet operations.