Sequential analysis of biological samples

• US 7,741,046 B2
• Filed: 09/28/2007
• Issued: 06/22/2010
• Est. Priority Date: 11/16/2006
• Status: Active Grant

• Current Assignee: Leica Microsystems Gmbh (Danaher Corporation)
• Original Assignee: General Electric Company
• Inventors: Larsen, Melinda, Ginty, Fiona, Gerdes, Michael J., Sood, Anup, Pang, Zhengyu, Montalto, Michael Christopher
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): Thomas; David C
• Application Number:
  US11/864,093
• Publication Number:
  US 20080118944A1
• Time in Patent Office: 998 Days
• Field of Search: None
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• 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
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

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.

Claims

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).