SPATIALLY ENCODED BIOLOGICAL ASSAYS USING A MICROFLUIDIC DEVICE

• US 20160145677A1
• Filed: 06/25/2014
• Published: 05/26/2016
• Est. Priority Date: 06/25/2013
• Status: Active Grant

• Current Assignee: Prognosys Biosciences Inc.
• Original Assignee: Prognosys Biosciences Inc.
• Inventors: CHEE, Mark S., ROUTENBERG, David A.
• Granted Patent: US 9,868,979 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2543/101 in situ amplification
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

The present disclosure provides methods and assay systems for use in spatially encoded biological assays, including assays to determine a spatial pattern of abundance, expression, and/or activity of one or more biological targets across multiple sites in a sample. In particular, the present disclosure provides methods and assay systems capable of high levels of multiplexing where reagents are provided to a biological sample in order to address tag the sites to which reagents are delivered; instrumentation capable of controlled delivery of reagents, in particular, microfluidic device based instrumentation; and a decoding scheme providing a readout that is digital in nature.

Claims

1. A method of determining a spatial pattern of abundance, expression and/or activity of a biological target in a sample, comprising:

(a) delivering a probe for a biological target to a sample, the probe comprising a binding moiety capable of binding to the biological target, and optionally, delivering an adaptor that specifically binds to the probe, wherein the probe and/or the adaptor comprises an identity tag that identifies the biological target or target-binding moiety;

(b) affixing the sample from step (a) to a first microfluidic device having multiple first addressing channels, wherein each first addressing channel identifies a first area in the sample;

(c) delivering a first address tag through each of the first addressing channels to each first area in the sample, wherein each first address tag is to be coupled to the probe and/or the adaptor;

(d) affixing the sample from step (c) to a second microfluidic device having multiple second addressing channels, wherein each second addressing channel identifies in the sample a second area that intersects with a first area identified by the first addressing channels at an angle greater than 0 degree;

(e) delivering a second address tag through each of the second addressing channels to each second area in the sample, wherein each second address tag is to be coupled to the probe and/or the adaptor, whereby the first address tag and the second address tag at an intersection between a first area and a second area determine the intersection'"'"'s address;

(f) analyzing the probe and/or the adaptor bound to the biological target, the analysis comprising;

(1) determining the abundance, expression and/or activity of the biological target by assessing the amount of the probe and/or the adaptor bound to the biological target; and

(2) determining the identities of the identity tag and the first and second address tags at each address; and

(g) determining a spatial pattern of the biological target abundance, expression and/or activity in the sample based on the analysis of step (f).

4. The method of any of claims 1-3, wherein the angle is about 90 degrees, about 80 degrees, about 70 degrees, about 60 degrees, about 50 degrees, about 40 degrees, about 30 degrees, about 20 degrees, or about 10 degrees.

5. The method of any of claims 1-4, wherein the multiple first addressing channels substantially parallel each other and the multiple second addressing channels substantially parallel each other.

6. The method of any of claims 1-5, wherein the sample is a biological sample selected from the group consisting of a freshly isolated sample, a fixed sample, a frozen sample, an embedded sample, a processed sample, or a combination thereof.

7. The method of any of claims 1-6, wherein the first addressing channels are disposed on the same device as the second addressing channels.

8. The method of any of claims 1-6, wherein the first addressing channels are disposed on a separate device from the second addressing channels.

9. The method of any of claims 1-8, wherein the first and/or second microfluidic device is manufactured by soft-lithographic techniques.

10. The method of any of claims 1-9, wherein the number of the first and/or second addressing channels is n, n being an integer between 100 and 150, between 150 and 200, between 200 and 250, between 250 and 300, between 300 and 350, between 350 and 400, between 400 and 450, between 450 and 500, between 500 and 550, between 550 and 600, between 600 and 650, between 650 and 700, between 700 and 750, between 750 and 800, between 800 and 850, between 850 and 900, between 900 and 950, between 950 and 1000, or greater than 1000.

11. The method of any of claims 1-10, wherein the width of the first and/or second addressing channels is about 5 μ

m, about 10 μ

m, about 50 μ

m, about 100 μ

m, about 150 μ

m, about 200 μ

m, about 250 μ

m, about 300 μ

m, about 350 μ

m, about 400 μ

m, about 450 μ

m, or about 500 μ

m.

12. The method of any of claims 1-11, wherein the depth of the first and/or second addressing channels is about 5 μ

m, about 10 μ

m, about 50 μ

m, about 100 μ

m, about 150 μ

m, about 200 μ

m, about 250 μ

m, about 300 μ

m, about 350 μ

m, about 400 μ

m, about 450 μ

m, or about 500 μ

m.

13. The method of any of claims 1-12, wherein the distance between each first addressing channel and/or between each second addressing channel is about 5 μ

m, about 10 μ

m, about 50 μ

m, about 100 μ

m, about 150 μ

m, about 200 μ

m, about 250 μ

m, about 300 μ

m, about 350 μ

m, about 400 μ

m, about 450 μ

m, about 500 μ

m, about 550 μ

m, about 600 μ

m, about 650 μ

m, about 700 μ

m, about 750 μ

m, about 800 μ

m, about 850 μ

m, about 900 μ

m, about 950 μ

m, about 1.0 mm, about 1.2 mm, about 1.3 mm, about 1.4 mm, about 1.5 mm, about 1.6 mm, about 1.7 mm, about 1.8 mm, about 1.9 mm, or about 2.0 mm.

14. The method of any of claims 1-13, wherein the first and/or second address tag comprises an oligonucleotide.

15. The method of any of claims 1-14, wherein the identity tag comprises an oligonucleotide.

16. The method of any of claims 1-15, wherein the biological target is a nucleic acid and the probe for the biological target comprises an oligonucleotide.

17. The method of any of claims 1-16, wherein the biological target is a nucleic acid and two probes for the nucleic acid target are used.

18. The method of any of claims 1-15, wherein the biological target is a protein, and the probe for the target protein comprises an oligonucleotide and a target-binding moiety, which is a protein.

19. The method of any of claims 1-15 and 18, wherein the biological target comprises an enzyme.

20. The method of any of claims 1-19, wherein the binding moiety of the probe for the biological target comprises an antibody.

21. The method of any of claims 1-20, wherein the binding moiety of the probe for the biological target comprises an aptamer.

22. The method of any of claims 1-17, wherein the binding moiety of the probe for the biological target is a small molecule.

23. The method of any of claims 1-22, wherein the analyzing step is performed by nucleic acid sequencing.

24. The method of any of claims 1-23, wherein the analyzing step is performed by high-throughput digital nucleic acid sequencing.

25. The method of any of claims 1-24, wherein spatial patterns of the abundance, expression and/or activity of multiple biological targets in the sample are determined in parallel.

26. The method of any of claims 1-25, wherein the product of the number of biological targets being assayed and the number of addresses being assayed in the sample is greater than 20.

27. The method of any of claims 1-26, wherein the product of the number of biological targets being assayed and the number of addresses being assayed in the sample is greater than 50.

28. The method of any of claims 1-27, wherein the product of the number of biological targets being assayed and the number of addresses being assayed in the sample is greater than 75.

29. The method of any of claims 1-28, wherein the product of the number of biological targets being assayed and the number of addresses being assayed in the sample is greater than 100.

30. The method of any of claims 1-29, wherein the product of the number of biological targets being assayed and the number of addresses being assayed in the sample is greater than 1,000.

31. The method of any of claims 1-30, wherein the product of the number of biological targets being assayed and the number of addresses being assayed in the sample is greater than 10,000.

32. The method of any of claims 1-31, wherein the product of the number of biological targets being assayed and the number of addresses being assayed in the sample is greater than 100,000.

33. The method of any of claims 1-32, wherein the product of the number of biological targets being assayed and the number of addresses being assayed in the sample is greater than 1,000,000.

34. The method of any of claims 1-33, wherein at least one hundred thousand probes bound to the biological target are analyzed in parallel.

35. The method of any of claims 1-34, wherein at least five hundred thousand probes bound to the biological target are analyzed in parallel.

36. The method of any of claims 1-35, wherein at least one million probes bound to the biological target are analyzed in parallel.

37. The method of any of claims 1-36, wherein software programmed hardware performs at least two steps of the delivering steps, the affixing steps, the analyzing step and the determining step.

38. The method of any of claims 1-37, wherein a known percentage of the probe for the biological target is an attenuator probe.

39. The method of claim 38, wherein the attenuator probe prevents production of an amplifiable product.

40. The method of claim 38 or 39, wherein the attenuator probe lacks a 5′

phosphate.

41. The method of any of claims 1, 3 and 4, wherein the address tag is coupled to the probe for the biological target by ligation, by extension, by extension followed by ligation, or any combination thereof.

2. A method of determining a spatial pattern of abundance, expression and/or activity of a biological target in a sample, comprising:

(a) affixing a sample to a first microfluidic device having multiple first addressing channels, wherein each first addressing channel identifies a first area in the sample;

(b) delivering a first probe for a biological target through each of the first addressing channels to each first area in the sample, the probe comprising;

(1) a first binding moiety capable of binding to the biological target;

(2) a first address tag that identifies an area in the sample to which the first probe is delivered; and

(3) a first identity tag that identifies the biological target or the first binding moiety;

(c) affixing the sample from step (b) to a second microfluidic device having multiple second addressing channels, wherein each second addressing channel identifies in the sample a second area that intersects with a first area identified by the first addressing channels at an angle greater than 0 degree;

(d) delivering a second probe for the biological target through each of the second addressing channels to each second area in the sample, the probe comprising;

(1) a second binding moiety capable of binding to the biological target;

(2) a second address tag that identifies an area in the sample to which the first probe is delivered; and

(3) a second identity tag that identifies the biological target or the second binding moiety, whereby the first address tag and the second address tag at an intersection between a first area and a second area determine the intersection'"'"'s address;

(e) analyzing the probes bound to the biological target, the analysis comprising;

(1) determining the abundance, expression and/or activity of the biological target by assessing the amount of the probes bound to the biological target; and

(2) determining the identities of the first and second identity tags and the first and second address tags at each address; and

(f) determining a spatial pattern of the biological target abundance, expression and/or activity in the sample based on the analysis of step (e).

3. A method of determining a spatial pattern of abundance, expression and/or activity of a biological target in a sample, comprising:

(a) affixing a sample to a first microfluidic device having multiple first addressing channels, wherein each first addressing channel identifies a first area in the sample;

(b) delivering a probe for a biological target through each of the first addressing channels to each first area in the sample, the probe comprising a binding moiety capable of binding to the biological target, and optionally, delivering an adaptor that specifically binds to the probe, wherein the probe and/or the adaptor comprises;

(1) a first address tag that identifies an area in the sample to which the probe and/or the adaptor is delivered; and

(2) an identity tag that identifies the biological target or the binding moiety;

(c) affixing the sample from step (b) to a second microfluidic device having multiple second addressing channels, wherein each second addressing channel identifies in the sample a second area that intersects with a first area identified by the first addressing channels at an angle greater than 0 degree;

(d) delivering a second address tag through each of the second addressing channels to each second area in the sample, wherein each second address tag is to be coupled to the probe and/or the adaptor, whereby the first address tag and the second address tag at an intersection between a first area and a second area determine the intersection'"'"'s address;

(e) analyzing the probe and/or the adaptor bound to the biological target, the analysis comprising;

(1) determining the abundance, expression and/or activity of the biological target by assessing the amount of the probe and/or the adaptor bound to the biological target; and

(2) determining the identities of the identity tag and the first and second address tags at each address; and

(f) determining a spatial pattern of the biological target abundance, expression and/or activity in the sample based on the analysis of step (e).

42. A method of address coding multiple sites in a sample, comprising:

(a) providing a sample affixed to a first microfluidic device having multiple first addressing channels, wherein each first addressing channel identifies a first area in the sample;

(b) delivering a first probe capable of binding to a target in the sample through each of the first addressing channels to each first area in the sample;

(c) affixing the sample from step (b) to a second microfluidic device having multiple second addressing channels, wherein each second addressing channel identifies in the sample a second area that intersects with a first area identified by the first addressing channels at an angle greater than 0 degree;

(d) delivering a second probe capable of binding to the target in the sample through each of the second addressing channels to each second area in the sample; and

(e) determining an address of an intersection between a first area and a second area in the sample based on the first probe and the second probe bound to the target at the intersection.

43. The method of claim 42, wherein the angle is about 90 degrees, about 80 degrees, about 70 degrees, about 60 degrees, about 50 degrees, about 40 degrees, about 30 degrees, about 20 degrees, or about 10 degrees.

44. The method of claim 42 or 43, wherein the first addressing channels substantially parallel each other and the second addressing channels substantially parallel each other.

45. The method of any of claims 42-44, wherein the sample is a biological sample selected from the group consisting of a freshly isolated sample, a fixed sample, a frozen sample, an embedded sample, a processed sample, or a combination thereof.

46. The method of any of claims 42-45, wherein the first addressing channels are disposed on the same device as the second addressing channels.

47. The method of any of claims 42-45, wherein the first addressing channels are disposed on a separate device from the second addressing channels.

48. The method of any of claims 42-47, wherein the first or second microfluidic device is manufactured by soft-lithographic techniques.

49. The method of any of claims 42-48, wherein the number of the first or second addressing channels is n, n being an integer between 100 and 150, between 150 and 200, between 200 and 250, between 250 and 300, between 300 and 350, between 350 and 400, between 400 and 450, between 450 and 500, between 500 and 550, between 550 and 600, between 600 and 650, between 650 and 700, between 700 and 750, between 750 and 800, between 800 and 850, between 850 and 900, between 900 and 950, between 950 and 1000, or greater than 1000.

50. The method of any of claims 42-49, wherein the width of the first or second addressing channels is about 5 μ

m, about 10 μ

m, about 50 μ

m, about 100 μ

m, about 150 μ

m, about 200 μ

m, about 250 μ

m, about 300 μ

m, about 350 μ

m, about 400 μ

m, about 450 μ

m, or about 500 μ

m.

51. The method of any of claims 42-50, wherein the depth of the first or second addressing channels is about 5 μ

m, about 10 μ

m, about 50 μ

m, about 100 μ

m, about 150 μ

m, about 200 μ

m, about 250 μ

m, about 300 μ

m, about 350 μ

m, about 400 μ

m, about 450 μ

m, or about 500 μ

m.

52. The method of any of claims 42-51, wherein the distance between each first addressing channel or between each second addressing channel is about 5 μ

m, about 10 μ

m, about 50 μ

m, about 100 μ

m, about 150 μ

m, about 200 μ

m, about 250 μ

m, about 300 μ

m, about 350 μ

m, about 400 μ

m, about 450 μ

m, about 500 μ

m, about 550 μ

m, about 600 μ

m, about 650 μ

m, about 700 μ

m, about 750 μ

m, about 800 μ

m, about 850 μ

m, about 900 μ

m, about 950 μ

m, about 1.0 mm, about 1.2 mm, about 1.3 mm, about 1.4 mm, about 1.5 mm, about 1.6 mm, about 1.7 mm, about 1.8 mm, about 1.9 mm, or about 2.0 mm.

53. The method of any of claims 42-52, wherein the first probes are different from each other, and the second probes are different from each other and different from the first probes.