Photoactivated chemical bleaching of dyes using borates
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 a cationic or zwitterionic borate compound 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.
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 one or more targets present in the sample;
(c) observing a signal from the at least one probe bound in step (b);
(d) contacting the sample comprising the bound probe of step (b) with a borate compound of Formula I;
R 1 R 2 R 3 R 4 B(X) n I
where each of R 1 , R 2 , R 3 and R 4 is, independently, an alkyl, an alkylaryl, an arylalkyl, or an aryl, with the proviso that at least one of R 1 , R 2 , R 3 and R 4 is an alkyl group and at least one of R 1 , R 2 , R 3 and R 4 is positively charged
X is an anion; and
n equals 0, 1, 2, or 3;
(e) irradiating the sample of step (d); and
(f) analyzing the sample for one or more targets.
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 3 , wherein the fluorescent signal generator is a xanthene dye.
5. The method of claim 4 , wherein the xanthene dye is eosin or fluorescein.
6. The method of claim 3 , wherein the fluorescent signal generator comprises a cyanine dye.
7. The method of claim 6 , wherein the cyanine dye is Cy3 or Cy5.
8. The method of claim 2 , wherein irradiation of sample in step (e) initiates a photoreaction that substantially inactivates the optical signal generator by photoactivated chemical bleaching.
9. 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.
10. The method of claim 1 , wherein steps (d) and (e) are carried out at the temperature of 4-50° C.
11. 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 μm in wavelength.
12. The method of claim 11 , wherein irradiating the sample in step (e) is accomplished by exposing the sample to light of 500-700 nm in wavelength.
13. The method of claim 1 , wherein the steps (d) and (e) are performed for about 20 seconds to about 15 minutes.
14. The method of claim 1 , wherein prior to step (e), the sample is rinsed with a buffer to remove excess borate.
15. The method of claim 1 , wherein analyzing the sample comprises:
binding at least one probe to one or more targets present in the sample of step (e); and
(ii) observing a signal from the probe bound in step (i).
16. The method of claim 15 , wherein steps (d)-(ii) are repeated one or more times.
17. The method of claim 15 , further comprising measuring one or more intensity values of the signal observed in observing step (c), step (ii), or steps (c) and (ii).
18. The method of claim 17 , further comprising correlating the intensity value with an amount of target present in the sample.
19. The method of claim 15 , wherein the probe in step (b) and the probe in step (i) each comprise a signal generator, wherein the signal generator in step (b) is different from the signal generator in step (i).
20. The method of claim 1 , wherein at least one of R 1 , R 2 , R 3 and R 4 is a 4-(trimethylamino)phenyl group.
21. The method of claim 1 , wherein at least one of R 1 , R 2 , R 3 and R 4 is a 3-(trimethylamino)propyl group.
22. The method of claim 1 , wherein at least two of R 1 , R 2 , R 3 and R 4 are positively charged.
23. The method of claim 1 , wherein Formula I is
24. The method of claim 1 , wherein more than one probe is bound to two or more targets.
25. The method of claim 1 , where n equals 1, 2, or 3.
26. The method of claim 25 , wherein X is selected from the group consisting of a halide, sulfonate, acetate, nitrate, phosphate ester and a carboxylate.
27. The method of claim 1 , wherein more than one probe is bound to one target.
28. The method of claim 1 , wherein the multiple targets are selected from the group consisting of peptides, proteins, nucleic acids, polysaccharides, lipids, enzymes, enzyme substrates, ligands, receptors, antigens, haptens and combinations thereof.
29. The method of claim 1 , wherein the biological sample is a tissue sample.