Spatially encoded biological assays using a microfluidic device

Patent metadata

Field	Value
Publication number	US-9868979-B2
Application number	US-201414900602-A
Country	US
Kind code	B2
Filing date	Jun 25, 2014
Priority date	Jun 25, 2013
Publication date	Jan 16, 2018
Grant date	Jan 16, 2018

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.

First claim

We claim:

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 the biological target to the sample, the probe comprising a binding moiety capable of binding to the biological target, wherein the probe 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 to form multiple first addressing channels between the sample and the first microfluidic device, 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;
(d) affixing the sample from step (c) to a second microfluidic device to form multiple second addressing channels between the sample and the second microfluidic device, wherein each second addressing channel identifies in the sample a second area that intersects with the first area 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, whereby the first address tag and the second address tag at each intersection determine the intersection's address;
(f) analyzing the probe 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 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 the spatial pattern of the biological target abundance, expression and/or activity in the sample based on the analysis of step (f).

2. The method of claim 1 , 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.

3. The method of claim 1 , wherein the multiple first addressing channels substantially parallel each other and the multiple second addressing channels substantially parallel each other.

4. The method of claim 1 , 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.

5. The method of claim 1 , wherein the first addressing channels are disposed on the same device as the second addressing channels.

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

7. The method of claim 1 , wherein the first and/or second microfluidic device is manufactured by soft-lithographic techniques.

8. The method of claim 1 , 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.

9. The method of claim 1 , wherein the width of the first and/or second addressing channels is about 5 μm, about 10 μm, about 50 μm, about 100 μm, about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, or about 500 μm.

10. The method of claim 1 , wherein the depth of the first and/or second addressing channels is about 5 μm, about 10 μm, about 50 μm, about 100 μm, about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, or about 500 μm.

11. The method of claim 1 , wherein the distance between each first addressing channel and/or between each second addressing channel is about 5 μm, about 10 μm, about 50 μm, about 100 μm, about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about 400 μm, about 450 μm, about 500 μm, about 550 μm, about 600 μm, about 650 μm, about 700 μm, about 750 μm, about 800 μm, about 850 μm, about 900 μm, about 950 μm, 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.

12. The method of claim 1 , wherein the first and/or second address tag comprises an oligonucleotide.

13. The method of claim 1 , wherein the identity tag comprises an oligonucleotide.

14. The method of claim 1 , wherein the biological target is a nucleic acid and the probe for the biological target comprises an oligonucleotide.

15. The method of claim 1 , wherein the biological target is a nucleic acid and two probes for the nucleic acid target are used.

16. The method of claim 1 , 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.

17. The method of claim 1 , wherein the biological target comprises an enzyme.

18. The method of claim 1 , wherein the binding moiety of the probe for the biological target comprises an antibody, an aptamer, or a small molecule.

19. The method of claim 1 , wherein the analyzing step is performed by nucleic acid sequencing or high-throughput sequencing.

20. The method of claim 1 , wherein spatial patterns of the abundance, expression and/or activity of multiple biological targets in the sample are determined in parallel.

21. The method of claim 1 , wherein the number of biological targets being assayed is x and the number of the multiple sites being assayed in the sample is y, and the value of x×y is greater than 20, 50, 75, 100, 1,000, 10,000, 100,000, or 1,000,000.

22. The method of claim 1 , wherein at least one hundred thousand, at least five hundred thousand, or at least one million probes bound to the biological target are analyzed in parallel.

23. The method of claim 1 , wherein software programmed hardware performs at least two steps of the delivering steps, the affixing steps, the analyzing step and the determining step.

24. The method of claim 1 , wherein a known percentage of the probe for the biological target is an attenuator probe.

25. The method of claim 24 , wherein the attenuator probe prevents production of an amplifiable product.

26. The method of claim 24 , wherein the attenuator probe lacks a 5′ phosphate.

27. The method of claim 1 , 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.

28. The method of claim 1 , wherein:
step (a) comprises delivering to the sample an adaptor that specifically binds to the probe, and the adaptor comprises the identity tag that identifies the biological target or target-binding moiety,
step (c) comprises delivering the 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 adaptor,
step (e) comprises delivering the second address tag through each of the second addressing channels to eac

Assignees

• Prognosys Biosciences Inc

Inventors

• Chee Mark S
• Routenberg David A

Classifications

• C12Q1/6853 Primary

  using modified primers or templates · CPC title

• C12Q1/6841

  In situ hybridisation · CPC title

• B01L3/5027

  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 · CPC title

• C12Q1/6809 Primary

  Methods for determination or identification of nucleic acids involving differential detection · CPC title

• C12Q2565/629

  being a microfluidic device · CPC title