Detection of Cardiac Markers on a Droplet Actuator

• US 20110076692A1
• Filed: 09/29/2009
• Published: 03/31/2011
• Est. Priority Date: 09/29/2009
• Status: Active Grant

• Current Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Sista, Ramakrishna, Pamula, Vamsee K., Sudarsan, Arjun
• Granted Patent: US 8,846,414 B2
• Time in Patent Office: Days
• US Class Current: 435/7.4
• CPC Class Codes: G01N 2333/4712 Muscle proteins, e.g. myosin, actin, protein
• CPC Class Codes: G01N 2333/58 Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Brain natriuretic peptide [BNP, proBNP]; Cardionatrin; Cardiodilatin
• CPC Class Codes: G01N 2800/32 Cardiovascular disorders
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/6893 related to diseases not provided for elsewhere
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

The present invention provides for the detection of cardiac markers on a droplet actuator. An aspect provides a method of assaying a cardiac marker in a biological sample from a subject, the method including providing a droplet actuator, loading the biological sample and assay reagents on the droplet actuator, executing droplet operations to create sample droplets from the sample and reagent droplets from the reagents on the droplet actuator, and executing droplet operations using the sample droplets and reagent droplets to produce a detectable signal indicative of the quantity of the cardiac marker in the biological sample. Still other aspects are provided.

Claims

1. A method of assaying a cardiac marker in a biological sample from a subject, the method comprising:

(a) providing a droplet actuator;

(b) loading the biological sample and assay reagents on the droplet actuator;

(c) executing droplet operations to create sample droplets from the sample and reagent droplets from the reagents on the droplet actuator; and

(d) executing droplet operations using the sample droplets and reagent droplets to produce a detectable signal indicative of the quantity of the cardiac marker in the biological sample.

2. The method of claim 1 comprising conducting assays for two or more cardiac markers in parallel.

3. The method of claim 1 comprising conducting assays for three or more cardiac markers in parallel.

4. The method of claim 1 comprising conducting assays for four or more cardiac markers in parallel.

5. The method of claim 1 wherein the cardiac marker is selected from the group consisting of cardiac troponin, creatine kinase, aspartate transaminase, lactate dehydrogenase, and Mb.

6. The method of claim 1 wherein the cardiac marker is selected from the group consisting of AST, ANP, BNP, proBNP, NT-proBNP, cTn, cTnI, cTnC, cTnT, cTn isoforms, CRP, CKMB, CNP, CSC, FABP, GPBB, IMA, LDH, MPO, Mb, PIGF, PAPPA, sCD40 ligand, and fragments, precursors, and isoforms of the foregoing.

7. The method of claim 1 wherein loading the biological sample to producing a detectable signal is accomplished in a time that is less than about 10 minutes.

8. The method of claim 1 wherein the biological sample loaded onto the droplet actuator has a volume ranging from about 0.001 to about 100 μ

L.

9. The method of claim 1 wherein the biological sample loaded onto the droplet actuator has a volume ranging from about 0.01 to about 50 μ

L.

10. The method of claim 1 wherein the biological sample loaded onto the droplet actuator has a volume ranging from about 0.1 to about 25 μ

L.

11. The method of claim 1 wherein the biological sample loaded onto the droplet actuator has a volume ranging from about 1 to about 20 μ

L.

12. The method of claim 1 further comprising providing an output indicative of the quantity of cardiac marker(s).

13. The method of claim 12 further comprising using the output to assess whether a subject has recently experienced or is contemporaneously experiencing an acute myocardial infarction.

14. The method of claim 12 further comprising using the output as a prognostic indicator.

15. The method of claim 1 further comprising, prior to loading the sample on the droplet actuator, binding cardiac markers of interest to capture beads, and loading the capture beads onto the droplet actuator.

16. The method of claim 15 wherein the beads are magnetically responsive.

17. The method of claim 15 wherein the beads are not substantially magnetically responsive.

18. The method of claim 15 further comprising subjecting the capture beads to a droplet-based wash protocol.

19. The method of claim 1 wherein step 1(d) comprises using one or more droplet operations in the execution of the following steps:

(a) combining a sample droplet with a reagent droplet comprising;

(i) beads comprising primary capture antibodies; and

(ii) reporter secondary antibodies;

to produce an assay droplet;

(b) incubating the assay droplet for a time sufficient to allow for the formation of capture antibody-antigen-reporter antibody complexes;

(c) executing a droplet-based wash protocol to remove unbound material from the beads to yield a washed-beads droplet;

(d) combining a chemiluminescence substrate droplet with the washed beads droplet; and

(e) detecting the quantity of the cardiac marker(s).

20. The method of claim 19 wherein step 19(c) is repeated until the unbound material is sufficiently depleted to permit accurate and precise detection of the cardiac marker.

21. The method of claim 19 wherein:

(a) step 19(c) is repeated less than about 10 times using droplets ranging from about 1X to about 5X the sample droplet; and

(b) upon completion of step 19(c), the unbound material is sufficiently depleted to permit accurate and precise detection of the cardiac marker.

22. The method of claim 19 wherein:

(a) step 19(c) is repeated less than about 5 times using droplets ranging from about 1X to about 5X the sample droplet; and

(b) upon completion of step 19(c), the unbound material is sufficiently depleted to permit accurate and precise detection of the cardiac marker.

23. The method of claim 19 wherein bead retention in step 19(c) is substantially complete.

24. The method of claim 19 wherein:

(a) the beads are magnetically responsive;

(b) step 19(c) comprises using a magnet to immobilize the beads as needed during droplet-based wash protocol; and

(c) following step 19(c), the washed-beads droplet is transported away from the magnet without substantial loss of beads.

25. The method claim 2 wherein the assays for each cardiac marker are run in multiplicate on a single droplet actuator.

26. The method claim 2 wherein each assay is run on a separate lane of the droplet actuator.

27. The method of claim 1 wherein the assay produces spike recovery data using physiological samples such as plasma and serum.

28. The method of claim 19 wherein the incubating step comprises serially incubating multiple sample droplets with a single bead population to pre-concentrate cardiac markers from multiple sample droplets onto a single bead population.

29. The method of claim 1 wherein droplets producing signals above a top-end of a standard curve are subjected to an auto-dilution sequence conducted using droplet operations to adjust the concentration of analytes to within a suitable range of detection.

30. The method of claim 1 wherein less than about 5 minutes elapses from loading the sample to producing a detectable signal.

31. The method of claim 1 wherein less than about 5 minutes elapses from loading the sample to producing a detectable signal for 2 or more cardiac markers.

32. The method of claim 1 wherein less than about 5 minutes elapses from loading the sample to producing a detectable signal for 3 or more cardiac markers.

33. The method of claim 19 wherein:

(a) less than about 600 seconds elapses from loading the sample to producing a detectable signal; and

(b) the assay droplet is incubated for a time that ranges from about 30 seconds to about 300 seconds.

34. The method of claim 19 wherein:

(a) less than about 600 seconds elapses from loading the sample to producing a detectable signal; and

(b) the assay droplet is incubated for a time that ranges from about 60 seconds to about 240 seconds.

35. The method of claim 19 wherein:

(a) less than about 600 seconds elapses from loading the sample to producing a detectable signal; and

(b) the assay droplet is incubated for a time that is less than about 240 seconds.

36. The method of claim 19 wherein:

(a) less than about 300 seconds elapses from loading the sample to producing a detectable signal; and

(b) the assay droplet is incubated for a time that ranges from about 30 seconds to about 180 seconds.

37. The method of claim 19 wherein:

(a) less than about 300 seconds elapses from loading the sample to producing a detectable signal; and

(b) the assay droplet is incubated for a time that ranges from about 60 to about 150 seconds.

38. The method of claim 19 wherein:

(a) less than about 300 seconds elapses from loading the sample to producing a detectable signal; and

(b) the assay droplet is incubated for a time that is less than about 120 seconds.

39. The method of claim 19 wherein:

(a) less than about 240 seconds elapses from loading the sample to producing a detectable signal; and

(b) the assay droplet is incubated for less than about 120 seconds.

40. The method of claim 19 wherein:

(a) less than about 600 seconds elapses from loading the sample to producing a detectable signal; and

(b) the droplet-based wash protocol is completed in a time that ranges from about 30 seconds to about 300 seconds.

41. The method of claim 19 wherein:

(a) less than about 600 seconds elapses from loading the sample to producing a detectable signal; and

(b) the droplet-based wash protocol is completed in a time that ranges from about 60 seconds to about 300 seconds.

42. The method of claim 19 wherein:

(a) less than about 600 seconds elapses from loading the sample to producing a detectable signal; and

(b) the droplet-based wash protocol is completed in a time that is less than about 300 seconds.

43. The method of claim 19 wherein:

(a) less than about 300 seconds elapses from loading the sample to producing a detectable signal; and

(b) the droplet-based wash protocol is completed in a time that ranges from about 30 seconds to about 240 seconds.

44. The method of claim 19 wherein:

(a) less than about 300 seconds elapses from loading the sample to producing a detectable signal; and

(b) the droplet-based wash protocol is completed in a time that ranges from about 30 seconds to about 180 seconds.

45. The method of claim 19 wherein:

(a) less than about 300 seconds elapses from loading the sample to producing a detectable signal; and

(b) the droplet-based wash protocol is completed in a time that is less than about 180 seconds.

46. The method of claim 19 wherein:

(a) less than about 600 seconds elapses from loading the sample to producing a detectable signal; and

(b) the detecting is completed in a time that ranges from about 30 seconds to about 180 seconds.

47. The method of claim 19 wherein:

(a) less than about 600 seconds elapses from loading the sample to producing a detectable signal; and

(b) the detecting is completed in a time that ranges from about 60 seconds to about 150 seconds.

48. The method of claim 19 wherein:

(a) less than about 600 seconds elapses from loading the sample to producing a detectable signal; and

(b) the detecting is completed in a time that is less than about 150 seconds.

49. The method of claim 19 wherein:

(a) less than about 300 seconds elapses from loading the sample to producing a detectable signal; and

(b) the droplet-based wash protocol is completed in a time that ranges from about 30 seconds to about 180 seconds.

50. The method of claim 19 wherein:

(a) less than about 300 seconds elapses from loading the sample to producing a detectable signal; and

(b) the detecting is completed in a time that ranges from about 60 seconds to about 180 seconds.

51. The method of claim 19 wherein:

(a) less than about 300 seconds elapses from loading the sample to producing a detectable signal; and

(b) the detecting is completed in a time that is less than about 180 seconds.

52. The method of claim 19 wherein:

(a) the assay droplet is incubated for a time that ranges from about 30 seconds to about 300 seconds;

(b) the droplet-based wash protocol is completed in a time that ranges from about 30 seconds to about 300 seconds; and

(c) the detecting is completed in a time that ranges from about 30 seconds to about 180 seconds.

53. The method of claim 19 wherein:

(a) the assay droplet is incubated for a time that ranges from about 60 seconds to about 240 seconds;

(b) the droplet-based wash protocol is completed in a time that ranges from about 60 seconds to about 300 seconds; and

(c) the detecting is completed in a time that ranges from about 60 seconds to about 150 seconds.

54. The method of claim 19 wherein:

(a) the assay droplet is incubated for a time that is less than about 240 seconds;

(b) the droplet-based wash protocol is completed in a time that is less than about 300 seconds; and

(c) the detecting is completed in a time that ranges from about 30 seconds to about 180 seconds.

55. The method of claim 19 wherein:

(a) the assay droplet is incubated for a time that ranges from about 30 seconds to about 180 seconds;

(b) the droplet-based wash protocol is completed in a time that ranges from about 30 seconds to about 240 seconds; and

(c) the detecting is completed in a time that ranges from about 60 seconds to about 180 seconds.

56. The method of claim 19 wherein:

(a) the assay droplet is incubated for a time that ranges from about 60 to about 150 seconds;

(b) the droplet-based wash protocol is completed in a time that ranges from about 30 seconds to about 180 seconds;

(c) the detecting is completed in a time that is less than about 180 seconds.