Photoactivated chemical bleaching of dyes

• US 8,568,991 B2
• Filed: 12/23/2011
• Issued: 10/29/2013
• Est. Priority Date: 12/23/2011
• Status: Active Grant

• Current Assignee: Leica Microsystems CMS GmbH (Danaher Corporation)
• Original Assignee: General Electric Company
• Inventors: Natarajan, Arunkumar, Sood, Anup, Chan, Kwok Pong, Kaanumalle, Lakshmi Sireesha
• Primary Examiner(s): Haq, Shafiqul
• Application Number:
  US13/336,409
• Publication Number:
  US 20130165330A1
• Time in Patent Office: 676 Days
• Field of Search: None
• US Class Current: 435/7.1
• CPC Class Codes: G01N 21/64 Fluorescence; Phosphorescence
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

Methods comprising the use of photoactivated chemical bleaching for detecting multiple targets in a biological sample are provided. The methods include the steps of providing a biological sample containing multiple targets, binding at least one probe to one or more target present in the sample, and observing a signal from the probe. The method further includes the steps of contacting the sample comprising the bound probe with an electron transfer reagent and irradiating the sample, thereby initiating a photoreaction that substantially inactivates the probe by photoactivated chemical bleaching. The method further includes the steps of binding at least one probe to one or more target present in the sample, and observing a signal from the probe. The process of binding, observing and bleaching may be iteratively repeated.

Claims

1. A method of probing multiple targets in a biological sample comprising:

(a) providing a biological sample containing multiple targets;

(b) binding at least one probe to the biological sample and wherein said probe comprises a binder and at least one of a optical signal generator or a fluorescent signal generator and wherein the binder is capable of binding specifically to one or more targets in the biological sample;

(c) observing a signal from the at least one probe bound in step (b);

(d) contacting the biological sample comprising the bound probe of step (b) with an electron transfer reagent, wherein the electron transfer reagent is a borate salt represented by the following structural formula;

2. The method of claim 1, wherein the probe in step (b) comprises an optical signal generator, and the signal observed in step (c) is an optical signal.

3. The method of claim 2, wherein the probe in step (b) comprises a fluorescent signal generator, and the signal observed in step (c) is a fluorescent signal.

4. The method of claim 1, wherein irradiating the sample in step (e) is carried out in the presence of a buffer at pH of 5-9.

5. The method of claim 1, wherein steps (d) and (e) are carried out at the temperature of 4-50°

C.

6. The method of claim 1, wherein irradiating the sample in step (e) is accomplished by exposing the sample to light of 350 nm-1.3 μ

M in wavelength.

7. The method of claim 6, wherein irradiating the sample in step (e) is accomplished by exposing the sample to light of 400-700 nm in wavelength.

8. The method of claim 1, wherein each R₁, R₂, and R₃ is an optionally substituted aryl and R₄ is an optionally substituted alkyl.

9. The method of claim 8, wherein each R₁, R₂, and R₃ is unsubstituted phenyl and R₄ is unsubstituted butyl, and the borate salt is triphenylbutyl borate salt.

10. The method of claim 1, wherein M⁺ is an inorganic cation selected from the group consisting of Li⁺, Na⁺ or K⁺.

11. The method of claim 3, wherein the fluorescent signal generator comprises a cyanine dye.

12. The method of claim 11, wherein the cyanine dye is Cy3 or Cy5.

13. The method of claim 1, wherein steps (d)-(g) are repeated one or more times.

14. The method of claim 1, wherein the steps (d) and (e) are performed for about 20 seconds to about 15 minutes.

15. The method of claim 1, further comprising measuring one or more intensity values of the signal observed in observing step (c), step (g), or steps (c) and (g).

16. The method of claim 15, further comprising correlating the intensity value with an amount of target present in the sample.

17. The method of claim 1, wherein the probe in step (b) and the probe in step (f) each comprise a signal generator, wherein the signal generator in step (b) is different from the signal generator in step (f).