Spatially encoded biological assays

• US 10,787,701 B2
• Filed: 05/16/2019
• Issued: 09/29/2020
• Est. Priority Date: 04/05/2010
• Status: Active Grant

• Current Assignee: Prognosys Biosciences Inc.
• Original Assignee: Prognosys Biosciences Inc.
• Inventors: Chee, Mark S.
• Primary Examiner(s): Martinell, James
• Application Number:
  US16/414,213
• Publication Number:
  US 20190271031A1
• Time in Patent Office: 502 Days
• Field of Search: None
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2543/10 the purpose being "in situ" analysis
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/507 characterised by the density of the capture oligonucleotide
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/515 characterised by the interaction between or sequential use of two or more arrays
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C40B 30/04 by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• CPC Class Codes: C40B 60/04 : Integrated apparatus specially adapted for both screening libraries and identifying library members
• CPC Class Codes: G01N 2458/10 : Oligonucleotides as tagging agents for labelling antibodies
• CPC Class Codes: G01N 2458/40 : Rare earth chelates
• CPC Class Codes: G01N 33/5308 : for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/543 : with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/54366 : Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/6845 : Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: G01N 33/6848 : Methods of protein analysis involving mass spectrometry

Abstract

The present invention provides assays and assay systems for use in spatially encoded biological assays. The invention provides an assay system comprising an assay capable of high levels of multiplexing where reagents are provided to a biological sample in defined spatial patterns; instrumentation capable of controlled delivery of reagents according to the spatial patterns; and a decoding scheme providing a readout that is digital in nature.

Claims

1. A method of determining presence or abundance or both of a target biological molecule at a region of interest in a tissue sample, comprising:

(a) delivering a plurality of probes to a tissue sample, wherein a probe of the plurality of probes comprises a capture agent that specifically binds a target biological molecule of the tissue sample, wherein the capture agent is conjugated to an oligonucleotide having a sequence;

(b) separating the probe of the plurality of probes comprising the capture agent that is specifically bound to the target biological molecule at a region of interest in the tissue sample from a plurality of probes not specifically bound to the target biological molecule at the region of interest in the tissue sample;

(c) determining all or a portion of the sequence of the oligonucleotide of the probe of the plurality of probes comprising the capture agent that is specifically bound to the target biological molecule at the region of interest in the tissue sample via hybridization of a probe comprising (i) a sequence that is 100% complementary to a portion of the sequence of the oligonucleotide and (ii) a label; and

(d) using the determined sequence of the oligonucleotide of the probe of the plurality of probes comprising the capture agent that is specifically bound to the target biological molecule at the region of interest in the tissue sample to determine presence or abundance or both of the target biological molecule at the region of interest in the tissue sample.

2. The method of claim 1, wherein the method further comprises obtaining an image of the tissue sample.

3. The method of claim 1, wherein step (b) comprises washing the tissue sample to separate the probe of the plurality of probes comprising the capture agent that is specifically bound to the target biological molecule at the region of interest in the tissue sample from the plurality of probes not specifically bound to the target biological molecule at the region of interest in the tissue sample.

4. The method of claim 1, wherein step (c) comprises detecting the label.

5. The method of claim 1, wherein the label is a fluorophore.

6. The method of claim 5, wherein step (c) comprises detecting the fluorophore.

7. The method of claim 1, wherein the target biological molecule comprises a nucleic acid.

8. The method of claim 7, wherein the nucleic acid is DNA.

9. The method of claim 8, wherein the DNA is genomic DNA.

10. The method of claim 7, wherein the nucleic acid is mRNA.

11. The method of claim 7, wherein the nucleic acid comprises a single nucleotide polymorphism or a mutation.

12. The method of claim 1, wherein the target biological molecule comprises a protein.

13. The method of claim 12, wherein the capture agent is a small molecule or a substrate for an enzyme.

14. The method of claim 12, wherein the capture agent is an antibody.

15. The method of claim 1, wherein the target biological molecule is present in a subcellular target.

16. The method of claim 15, wherein the subcellular target comprises a mitochondrion or a nucleus.

17. The method of claim 1, wherein the determined sequence of the oligonucleotide of the probe of the plurality of probes comprising the capture agent that is specifically bound to the target biological molecule at the region of interest in the tissue sample is used to determine the presence of the target biological molecule at the region of interest in the tissue sample.

18. The method of claim 1, wherein the determined sequence of the oligonucleotide of the probe of the plurality of probes comprising the capture agent that is specifically bound to the target biological molecule at the region of interest in the tissue sample is used to determine the abundance of the target biological molecule at the region of interest in the tissue sample.

19. The method of claim 1, wherein the determined sequence of the oligonucleotide of the probe of the plurality of probes comprising the capture agent that is specifically bound to the target biological molecule at the region of interest in the tissue sample is used to determine associate the presence and abundance of the target biological molecule at the region of interest in the tissue sample.

20. The method of claim 1, wherein two or more probes of the plurality of probes specifically bind to unique target biological molecules of the tissue sample.

21. The method of claim 20, wherein step (c) comprises determining all or a portion of the sequence of the oligonucleotide of the two or more probes in parallel.

22. The method of claim 20, wherein the two or more probes is at least one thousand probes.

23. The method of claim 1, wherein the plurality of probes is delivered to multiple regions of interest in the tissue sample.

24. The method of claim 23, wherein the method further comprises detecting differential expression of the target biological molecule in two or more of the multiple regions of interest of the tissue sample.

25. The method of claim 23, wherein two or more of the multiple regions of interest are determined by histological features of the tissue sample.

26. The method of claim 1, wherein the method further comprises identifying a location of the target biological molecule in two or more serial sections of the tissue sample.

27. The method of claim 26, wherein the method further comprises generating a 3-dimensional map of the target biological molecule in the tissue sample.

28. The method of claim 1, wherein the tissue sample is a tissue section.

29. The method of claim 28, wherein the tissue section comprises a fresh-frozen or formalin-fixed paraffin embedded (FFPE) tissue sample.