Droplet-based affinity assays

• US 20070275415A1
• Filed: 12/15/2006
• Published: 11/29/2007
• Est. Priority Date: 04/18/2006
• Status: Active Grant

• Current Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Duke University
• Inventors: Eckhardt, Allen, Pamula, Vamsee, Srinivasan, Vijay, Pollack, Michael
• Granted Patent: US 8,492,168 B2
• Time in Patent Office: Days
• US Class Current: 435/7.400
• 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 2200/148 Specific details about calibrations
• 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 2300/0864 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 2400/0421 electrophoretic flow
• 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 3/502761 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 7/525 : with physical movement of samples between temperature zones
• CPC Class Codes: G01N 15/1484 : microstructural devices
• CPC Class Codes: G01N 2015/1006 : for cytology
• CPC Class Codes: G01N 33/54373 : involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Abstract

The present invention relates to droplet-based affinity assays. According to one embodiment, a method of detecting a target analyte in a sample is provided, wherein the method includes: (a) executing droplet operations to combine affinity-based assay reagents on a droplet microactuator with a sample potentially comprising the target analyte to generate a signal indicative of the presence, absence and/or quantity of analyte; and (b) detecting the signal, wherein the signal corresponds to the presence, absence and/or quantity of the analyte in the sample.

Claims

1. A method of detecting a target analyte in a sample, the method comprising:

(a) executing droplet operations to combine affinity-based assay reagents on a droplet microactuator with a sample potentially comprising the target analyte to generate a signal indicative of the presence, absence and/or quantity of analyte; and

(b) detecting the signal, wherein the signal corresponds to the presence, absence and/or quantity of the analyte in the sample.

2. The method of claim 1 wherein one or more of the droplet operations is effected on a droplet microactuator.

3. The method of claim 1 comprising an electric field-mediated droplet operation.

4. The method of claim 1 comprising an electrode-mediated droplet operation.

5. The method of claim 1 comprising an electrowetting-mediated droplet operation.

6. The method of claim 1 comprising an dielectrophoresis-mediated droplet operation.

7. The method of claim 1 wherein the affinity-based assay reagents comprise immunoassay reagents.

8. The method of claim 1 wherein the detecting step comprises detecting a signal from a droplet on a droplet microactuator.

9. The method of claim 1 wherein the detecting step comprises detecting a signal from a surface on a droplet microactuator.

10. The method of claim 9 wherein the surface comprises a surface of a bead.

11. The method of claim 10 wherein the bead is magnetically responsive.

12. The method of claim 10 wherein the bead is not substantially magnetically responsive.

13. The method of claim 9 wherein the surface comprises a surface of the droplet microactuator.

14. The method of claim 9 wherein:

(a) the surface comprises scintillant molecules and capture molecules; and

(b) the droplet operations comprise contacting a sample droplet comprising a radiolabeled molecule having affinity for a capture molecule with the surface, such that when a radiolabeled molecule is bound to a capture molecule, radioactive particles interact with the scintillant molecules to produce light.

15. The method of claim 14 wherein the surface comprises a surface of a bead or a surface of the droplet microactuator.

16. The method of claim 9 wherein:

(a) the surface comprises a roughened metal film comprising a capture antibody on a surface thereof; and

(b) the droplet microactuator comprises electrodes configured for transporting a droplet into contact with the roughened metal film.

17. The method of claim 16 wherein the roughened metal film comprises a silver or gold film.

18. The method of claim 9 wherein the surface is labeled with Raman reporter molecules.

19. The method of claim 9 wherein the signal comprises Raman spectra.

20. The method of claim 7 wherein the signal measured corresponds to unoccupied binding sites on an antibody.

21. The method of claim 7 wherein the signal measured corresponds to occupied binding sites on an antibody.

22. The method of claim 7 wherein the affinity-based assay reagents comprise an antibody immobilized on a surface.

23. The method of claim 22 wherein the surface comprises a solid phase within a droplet on the droplet microactuator.

24. The method of claim 22 wherein the surface comprises a surface of the droplet microactuator.

25. The method of claim 22 wherein the surface comprises a bead disposed in a droplet on the droplet microactuator.

26. The method of claim 25 wherein the bead is a magnetically responsive bead.

27. The method of claim 25 wherein the bead is not substantially magnetically responsive.

28. The method of claim 25 wherein the affinity-based assay reagents comprise a labeled analyte tracer.

29. The method of claim 28 wherein the labeled tracer generates a signal only when bound to the affinity reagent.

30. The method of claim 28 wherein the labeled tracer generates a signal only when not bound to the affinity reagent.

31. The method of claim 25 wherein the droplet operations effect a competitive affinity-based assay protocol.

32. The method of claim 7 wherein the affinity-based assay reagents comprise an antibody which is selective for the target temperature zone analyte.

33. The method of claim 7 wherein the affinity-based assay reagents comprise two or more antibodies, each selective for a specific epitope or region of the target analyte.

34. The method of claim 7 wherein the affinity-based assay reagents comprise two or more antibodies, including a first antibody selective for the target analyte and a second antibody selective for the first antibody.

35. The method of claim 25 wherein the affinity-based assay reagents include a labeled antibody.

36. The method of claim 35 wherein the labeled antibody comprises a radioactive isotope.

37. The method of claim 25 wherein the affinity-based assay reagents include an antibody conjugated to an enzyme capable of catalyzing a detectable reaction.

38. The method of claim 36 wherein the detectable reaction result in a colorimetric, fluorescent and/or luminescent property or signal.

39. The method of claim 35 wherein the droplet operations comprise a droplet-based washing protocol for separating labeled antibody which is bound to the analyte from labeled antibody which is not bound to the analyte.

40. The method of claim 39 wherein the labeled antibody which is bound to the analyte is also bound to a surface and the droplet-based washing protocol comprises:

(a) removing droplets from the surface which comprise labeled antibody which is not bound to the analyte; and

(b) adding droplets to the surface which do not include labeled antibody which is not bound to the analyte.

41. The method of claim 40 wherein the surface comprises a surface of a solid phase in contact with a droplet on a droplet microactuator.

42. The method of claim 40 wherein the surface comprises a surface of a droplet microactuator.

43. The method of claim 7 wherein the affinity-based assay reagents comprise reagents for conducting an assay type selected from the group consisting of sandwich assays, immunometric assays, immunometric assays, capture bridge assays, direct class capture assays, indirect class capture assays, assays for small molecules, noncompetitive assays for small molecules, ambient analyte assays and free analyte assays.

44. The method of claim 43 comprising conducting two or more of the assay types simultaneously on a droplet microactuator.

45. The method of claim 43 comprising conducting three or more of the assay types simultaneously on a droplet microactuator.

46. The method of claim 7 further comprising conducting droplet operations to develop a calibration curve.

47. The method of claim 7 wherein:

(a) the droplet operations bring a capture antibody and a labeled detecting antibody into contact with the sample to yield a capture antibody-analyte-detecting antibody complex in the presence of the analyte;

(b) droplet-based washing protocols are employed to separate the capture antibody-analyte-detecting antibody complex from unreacted components of the sample and/or unreacted excess antibody; and

(c) the detecting step comprises detecting a signal from the capture antibody-analyte-detecting antibody complex.

48. The method of claim 7 wherein the droplet operations employ a sample from a source selected from the group consisting of whole blood, lymphatic fluids, serum, plasma, sweat, tear, saliva, sputum, cerebrospinal fluids, amniotic fluids, seminal fluids, vaginal excretions, serous fluids, synovial fluids, pericardial fluids, peritoneal fluids, pleural fluids, transudates, exudates, cystic fluids, bile, urine, gastric fluids, intestinal fluids, fecal samples, fluidized tissues, fluidized organisms, biological swabs and biological washes.

49. The method of claim 7 wherein the affinity-based assay reagents comprise an antibody.

50. The method of claim 49 wherein the antibody is immobilized on a surface.

51. The method of claim 50 wherein the surface comprises a surface of a bead.

52. The method of claim 51 wherein the bead is magnetically responsive.

53. The method of claim 51 wherein the bead is not substantially magnetically responsive.

54. The method of claim 50 wherein the surface comprises a surface of the droplet microactuator.

55. The method of claim 50 wherein the droplet operations comprise immobilizing an antibody on the surface to provide an immobilized antibody.

56. The method of claim 55 wherein the droplet operations comprise contacting an antibody in a droplet with a surface of the droplet microactuator sufficient to result in immobilization of the antibody on the surface.

57. The method of claim 55 wherein the droplet operations comprise contacting an antibody in a droplet with a surface of a bead sufficient to result in immobilization of the antibody on the surface.

58. The method of claim 57 wherein the contact is achieved by droplet operations comprising combining the droplet comprising the antibody with a droplet comprising a bead.

59. The method of claim 55 wherein the droplet operations further comprise washing the surface to remove excess antibody.

60. The method of claim 55 wherein the droplet operations further comprise providing the sample as a sample droplet.

61. The method of claim 60 wherein the droplet operations further comprise exposing immobilized antibody on a surface to a sample droplet potentially including the target analyte.

62. The method of claim 61 wherein the droplet operations further comprise washing the surface thereby removing or reducing the quantity of unbound sample material.

63. The method of claim 50 wherein the droplet operations further comprise exposing the immobilized antibody to a droplet including a reporter antibody.

64. The method of claim 50 wherein the droplet operations further comprise washing away excess reporter antibody.

65. The method of claim 50 wherein the droplet operations further comprise performing additional steps to produce a measurable parameter or signal.

66. The method of claim 65 wherein the measurable parameter or signal comprises an electrochemical property of a droplet on a droplet microactuator.

67. The method of claim 66 wherein the electrical property comprises an amperometric, potentiometric and/or conductometric property of a droplet on the droplet microactuator.

68. The method of claim 65 wherein the measurable parameter or signal comprises an optical property of a droplet on a droplet microactuator.

69. The method of claim 68 wherein the optical property comprises absorbance, chemiluminescence and/or fluorescence.

70. The method of claim 68 wherein the optical property comprises Raman spectra.

71. The method of claim 65 wherein the measurable parameter or signal comprises a physical parameter of a droplet on a droplet microactuator.

72. The method of claim 71 wherein the physical parameter comprises temperature.

73. The method of claim 71 wherein the physical parameter comprises emission of radioactive particles.

74. The method of claim 49 wherein the droplet operations further comprise:

(a) exposing the immobilized antibody to a sample droplet potentially including the target analyte with binding affinity for the immobilized antibody;

(b) washing the immobilized antibody-target analyte complex thereby removing unbound components of the sample droplet;

(c) exposing the immobilized antibody to a droplet including a reporter antibody;

(d) washing away excess reporter antibody; and

(e) performing additional steps to produce a measurable parameter or signal.

75. The method of claim 74 further comprising providing an electronically produced output indicative of the signal.

76. The method of claim 49 wherein the droplet operations further comprise providing a sample droplet potentially including target analyte and including a reporter analyte.

77. The method of claim 76 wherein the droplet operations further comprise exposing the immobilized antibody to the sample droplet so that the reporter analyte competes with any target analyte for binding sites.

78. The method of claim 77 wherein the droplet operations further comprise washing the substrate to remove unbound analyte and reporter analyte.

79. The method of claim 78 further comprising performing additional droplet operations to yield a measurable parameter or signal.

80. The method of claim 78 further comprising quantifying bound reporter analyte, wherein the quantity of reporter analyte is inversely proportional to the quantity of target analyte.