Sequential analysis of biological samples
View Patent ↗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.
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.