Application of reduced dyes in imaging
The present invention provides novel compounds and methods for hydrocyanines derived from near-infrared cyanine dyes, as reactive oxygen species probes in imaging. In certain embodiments, the present invention provides reduced dyes as substrates for ELISA and Western blots.
1. A compound, wherein the compound has the formula:
wherein R 1′ and R 1a′ are each independently hydrogen or deuterium;
M is an alkali metal cation; and
R 2 is CO 2 H or CO 2 M.
2. An assay method for measuring an analyte in a sample, the method comprising:
a) contacting said sample with a binding reagent that binds to an analyte to form an analyte complex;
b) contacting said sample with a compound of claim 1 , wherein said compound reacts with a radical, wherein said radical is generated from said analyte complex to form a detectable agent, which said detectable agent is a cyanine dye; and
c) upon excitation, detecting said detectable agent.
3. The method of claim 2 , wherein said analyte is a biological molecule.
4. The method of claim 2 , wherein said binding reagent is an antibody.
5. The method of claim 2 , wherein said analyte complex comprises an enzyme wherein a substrate of the enzyme is hydrogen peroxide.
6. The method of claim 2 , wherein said detectable agent is an oxidized form of a compound of claim 1 .
7. The method of claim 2 , wherein said assay method is an immunological detection method.
8. The method of claim 7 , wherein said immunological detection method is a member selected from the group consisting of an enzyme linked immunosorbent assay (ELISA), a Western blot analysis, and an immunohistochemical analysis.
9. A method for detecting a reactive oxygen species in a cell of a sample or a subject, said method comprising:
contacting a cell with one or more compounds of claim 1 , to form an oxidized form of said compound; and
exciting the oxidized form of said compound to emit light.
10. The method of claim 9 , wherein the oxidized form of said compound is detected by a method selected from the group consisting fluorescence spectroscopy or fluorescence microscopy.
11. A kit for conducting a fluorescent assay, said kit comprising a composition, said composition comprising:
a) a compound of claim 1 ; and
b) a buffer.
12. The kit of claim 11 , further comprising instructions for use of the composition in the fluorescent assay.