Sequential analysis of biological samples
View Patent ↗Methods for probing multiple targets in a biological sample are provided. The methods include the steps of providing a biological sample containing multiple targets adhered to a solid support, binding at least one fluorescent probe to one or more target present in the sample, and observing a signal from the fluorescent probe. The method further includes the steps of oxidizing the bound fluorescent probe with a solution containing an oxidizing agent that substantially inactivates the fluorescent probe, binding at least one fluorescent probe to one or more target present in the sample, and observing a signal from the fluorescent probe. The methods disclosed herein also provide for multiple iterations of binding, observing, and oxidizing for deriving information about multiple targets in a single sample. An associated kit is also provided.
1. A method of probing multiple targets in a biological sample comprising:
(a) providing a biological sample containing multiple targets adhered to a solid support;
(b) binding at least one fluorescent probe to one or more targets present in the sample;
(c) observing a signal from the fluorescent probe bound in step (b);
(d) oxidizing the bound fluorescent probe with a solution comprising an oxidizing agent that substantially inactivates the fluorescent probe;
(e) binding at least one fluorescent probe to one or more targets present in the sample of step (d); and
(f) observing a signal from the fluorescent probe bound in step (e).
2. The method of claim 1 , wherein the solution in step (d) is a basic solution.
3. The method of claim 2 , wherein the basic solution has a pH of about 10.
4. The method of claim 2 , wherein the basic solution does not contain a reducing agent or a surfactant.
5. The method of claim 1 , wherein the oxidation step is performed without stripping more than, or about, 20% of the fluorescent probe from the target adhered to the solid support.
6. The method of claim 1 , wherein the oxidizing agent is selected from hydrogen peroxide, potassium permanganate, sodium dichromate, aqueous bromine, iodine-potassium iodide, and t-butyl hydroperoxide.
7. The method of claim 1 , wherein the fluorescent probe comprises a binder and a fluorescent signal generator.
8. The method of claim 7 , wherein the fluorescent signal generator comprises a cyanine dye.
9. The method of claim 1 , wherein the sample comprises whole cells or tissue sections.
10. The method of claim 1 , wherein the sample comprises proteins or nucleic acids.
11. The method of claim 1 , wherein steps (d)-(f) are repeated one or more times.
12. The method of claim 1 , wherein steps (d)-(f) are repeated at least 5, at least 10, or at least 20 times.
13. The method of claim 1 , wherein the oxidation step (d) is performed for less than, or about, 30 minutes.
14. The method of claim 1 , wherein the oxidation step (d) is performed for about 30 seconds to about 15 minutes.
15. The method of claim 1 , wherein the oxidation step (d) is performed at room temperature.
16. The method of claim 1 , further comprising measuring one or more intensity values of the signal observed in observing step (c), step (f), or steps (c) and (f).
17. The method of claim 16 , further comprising correlating the intensity value with an amount of target present in the sample.
18. The method of claim 7 , wherein the fluorescent signal generator in step (b) is the same as the fluorescent signal generator in step (e).
19. The method of claim 7 , wherein the fluorescent signal generator in step (b) is different from the fluorescent signal generator in step (e).
20. The method of claim 1 , wherein the signals observed in step (c) and step (f) are both detectable in a single detection channel.
21. The method of claim 1 , wherein the signal observed in step (c) or step (f) is independently detectable in different detection channels.
22. A method of probing multiple targets in a biological sample comprising:
(a) providing a biological sample containing multiple targets adhered to a membrane;
(b) binding at least one fluorescent probe to one or more targets present in the sample;
(c) observing a signal from the fluorescent probe bound in step (b);
(d) oxidizing the bound fluorescent probe with a solution comprising an oxidizing agent that substantially inactivates the fluorescent probe;
(e) binding at least one fluorescent probe to one or more targets present in the sample of step (d);
(f) observing a signal from the fluorescent probe bound in step (e).
23. The method of claim 22 , wherein the membrane is selected from nylon, agarose, nitrocellulose, and polyvinylidene difluoride.
24. The method of claim 22 , further comprising separating the multiple targets by electrophoresis before step (a).