Sequential analysis of biological samples

• US 9,201,063 B2
• Filed: 10/21/2009
• Issued: 12/01/2015
• Est. Priority Date: 11/16/2006
• Status: Active Grant

• Current Assignee: Leica Microsystems Gmbh (Danaher Corporation)
• Original Assignee: General Electric Company
• Inventors: Sood, Anup, Nelson, John Richard, Gerdes, Michael John
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): THOMAS, DAVID C
• Application Number:
  US12/582,729
• Publication Number:
  US 20100120043A1
• Time in Patent Office: 2,232 Days
• US Class Current: 1/1
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

Methods for detecting a plurality of targets in a biological sample are provided. The method comprises contacting the biological sample with a plurality of target-binding probes simultaneously to form a plurality of target-bound probes and observing the signals from the target-bound probes sequentially. An associated kit and device for detection of the plurality of targets are also provided.

Claims

1. A method for in situ detection of a plurality of targets in a biological sample, comprising:

(a) contacting the biological sample in situ with a plurality of target-binding probes to form a plurality of target-bound probes;

(b) detecting a first set of fluorescence signals from a first set of the plurality of target-bound probes;

(c) modifying the detected fluorescence signals using a chemical agent;

(d) generating a second set of fluorescence signals after step (c) from a second set of the plurality of target-bound probes;

(e) detecting the second set of fluorescence signals; and

(f) repeating steps (c) to (e) multiple times with a third, a fourth, or an n^th set of the plurality of target-bound probes to detect a third, a fourth, or an n^th set of fluorescence signals.

(f) repeating steps (c) to (e) multiple times with a third, a fourth, or an n^th set of the plurality of target-bound probes to detect a third, a fourth, or an n^th set of fluorescence signals.

2. The method of claim 1, further comprising correlating the detected fluorescence signals to the detection of the plurality of targets.

3. The method of claim 1, wherein the target-binding probe comprises a unit derived from a target-binding moiety and an independently detectable moiety.

4. The method of claim 3, wherein the independently detectable moiety of the first set of the plurality of target-bound probes comprises a fluorophore.

5. The method of claim 3, wherein the target binding moiety is coupled to the independently detectable moiety via a cleavable linker.

6. The method of claim 3, wherein the independently detectable moiety comprises a unique nucleic acid sequence, a hapten, an enzyme, or a combination thereof.

7. The method of claim 3, wherein the independently detectable moiety comprises a masked fluorophore.

8. The method of claim 7, wherein the fluorescence signals are generated from the plurality of target-bound probes by unmasking the masked fluorophore.

9. The method of claim 3, wherein the fluorescence signals are generated from the plurality of target-bound probes by associating a fluorophore with each of the target-bound probe via the independently detectable moiety.

10. The method of claim 1, further comprising steps, after the contacting step, for removing any target-binding probes that are not bound to the targets.

11. The method of claim 1, further comprising imaging the biological sample to obtain a background signal signature prior to contacting the biological sample with a plurality of target-binding probes.

12. The method of claim 1, wherein the detection of the plurality of targets comprises identifying the presence, absence, location, or amount of the plurality of targets.

13. The method of claim 1, wherein the plurality of targets is selected from the group consisting of an oligonucleotide, a nucleic acid, a peptide, a protein, a hormone, a receptor, a polysaccharide, a lipid, and a combination thereof.

14. The method of claim 1, wherein the plurality of targets consist essentially of a plurality of proteins.

15. A method for in situ detection of a plurality of targets in a biological sample, comprising:

(a) contacting the biological sample in situ with a plurality of target-binding probes to form a plurality of target-bound probes;

(b) generating a first set of fluorescence signals from a first set of the plurality of target-bound probes;

(c) detecting the first set of fluorescence signals;

(d) modifying the detected fluorescence signals after step (c) using a chemical agent;

(e) generating a second set of fluorescence signals after step (d) from a second set of the plurality of target-bound probes;

(f) detecting the second set of fluorescence signals; and

(g) repeating steps (d) to (f) multiple times with a third, a fourth, or an n^th set of the plurality of target-bound probes to detect a third, a fourth, or an n^th set of fluorescence signals.

16. The method of claim 15, wherein integer value of n ranges from 5 to 100.

17. The method of claim 15, further comprising correlating the detected fluorescence signals to the detection of the plurality of targets.

18. The method of claim 15, further comprising imaging the biological sample to obtain a background signal signature prior to generating the first set of fluorescence signals.

19. The method claim 18, further comprising subtracting the background fluorescence signal signature from the fluorescence signals observed in the subsequent signal observing steps prior to the correlating step.

20. A method for in situ detection of a plurality of targets in a tissue section, comprising:

(a) contacting the tissue section in situ with a plurality of target-binding probes to form a plurality of target-bound probes;

(b) contacting the plurality of target-bound probes with a first set of fluorescent probes that are capable of binding a first set of the plurality of target-bound probes, and are capable of generating a first set of fluorescence signals;

(c) detecting the first set of the plurality of targets via observing the first set of fluorescence signals;

(d) modifying the detected first set of fluorescence signals by applying a chemical agent; and

(e) repeating steps (b) to (d) multiple times after step (d) using a subsequent set of fluorescent probes to detect a subsequent set of the plurality of targets, wherein the subsequent set of fluorescent probes are capable of binding a subsequent set of the plurality of target-bound probes, and are capable of generating a subsequent set of fluorescence signals.

21. The method of claim 20, further comprising steps for removing the target-binding probes that are not bound to the plurality of targets, prior to contacting the plurality of target-bound probes with the first set of fluorescent probes.

22. The method of claim 21, further comprising steps for removing the first set of fluorescent probes that are not bound to the first set of the plurality of target-bound probes, prior to detecting the first set of the plurality of targets.

23. The method of claim 20, wherein the plurality of targets is selected from the group consisting of a deoxyribonucleic acid, a ribonucleic acid, a protein, and a combination thereof.