Photon reducing agents for use in fluorescence assays

• US 6,221,612 B1
• Filed: 07/23/1998
• Issued: 04/24/2001
• Est. Priority Date: 08/01/1997
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Aurora Biosciences Corporation (Vertex Pharmaceuticals Incorporated)
• Inventors: Negulescu, Paul, Rink, Tim, Zlokarnik, Gregor, Tsien, Roger Y., Knapp, Tom
• Primary Examiner(s): Le, Long V.
• Assistant Examiner(s): GABEL, GAILENE
• Application Number:
  US09/122,477
• Time in Patent Office: 1,006 Days
• Field of Search: 435/5, 435/6, 435/7.1, 435/7.2, 435/7.5, 435/7.21, 435/7.72, 435/7.92, 435/91.1, 435/230, 435/235, 436/501, 436/528, 436/529, 436/530, 436/531, 436/546, 436/800, 436/809, 427/102, 427/157, 427/213.34, 422/82.05, 422/99
• US Class Current: 435/7.1
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• 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: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/809 Multifield plates or multicontainer arrays
• CPC Class Codes: Y10T 436/13 Tracers or tags

Abstract

The present invention provides a method for reducing undesirable light emission from a sample using at least one photon producing agent and at least one photon reducing agent (e.g. dye-based photon reducing agents). The present invention further provides a method for reducing undesirable light emission from a sample (e.g., a biochemical or cellular sample) with at least one photon producing agent and at least one collisional quencher. The present invention also provides a method for reducing undesirable light emission from a sample (e.g., a biochemical or cellular sample) with at least one photon producing agent and at least one quencher, such as an electronic quencher. The present invention also provides a system and method of screening test chemicals in fluorescent assays using photon reducing agents. The present invention also provides compositions, pharmaceutical compositions, and kits for practicing these methods.

Claims

1. A method of identifying a chemical with a biological activity, comprising:

a) contacting a sample with a test chemical, said sample comprising a target,

b) contacting said sample with at least one photon reducing agent, wherein said sample comprises a membrane compartment in contact with a solid surface, wherein said membrane compartment includes at least one photon producing agent that directly or indirectly monitors the activity of said target and said at least one photon reducing agent is in an aqueous solution that contacts the outer surface of said membrane compartment, and wherein said at least one photon reducing agent has an absorption spectra that overlaps with the absorption, emission or excitation spectrum of said at least one photon producing agent or wherein said at least one photon producing agent exhibits fluorescence resonance energy transfer with said at least one photon reducing agent,

wherein said at least one photon reducing agent is substantially impermeant to said membrane compartment,

wherein said at least one photon reducing agent does not specifically bind to said membrane compartment, and

wherein said at least one photon reducing agent is present in said aqueous solution at a concentration sufficient to reduce light emitted from said aqueous solution by at least 10% compared to the light emitted of said aqueous solution in the absence of said at least one photon reducing agent,

c) detecting an optical signal from said at least one photon producing agent,

d) comparing the optical signal from said at least one photon producing agent to a background or control optical signal from a control sample.

2. The method of claim 1, wherein said membrane compartment comprises at least one living cell.

3. The method of claim 2, wherein said at least one living cell is part of a plurality of cells comprising at least two different photon producing agents.

4. The method of claim 2, wherein said at least one living cell is a mammalian cell.

5. The method of claim 4, wherein said at least one photon producing agent corresponds to an enzyme activity inside said mammalian cell.

6. The method of claim 5, wherein said at least one photon producing agent measures beta-lactamase activity.

7. The method of claim 6, wherein said mammalian cell is one of at least 100 living cells.

8. The method of claim 4, wherein said mammalian cell is a member of a clonal population of cultured cells.

9. The method of claim 4, wherein said at least one photon reducing agent is non-toxic to said living cell over the time frame of the assay.

10. The method of claim 4, wherein said at least one photon reducing agent is present in said aqueous solution at a concentration sufficient to reduce light emission from said aqueous solution by between 70 and 99% compared to light emission of said aqueous solution in the absence of said at least one photon reducing agent.

11. The method of claim 4, wherein said at least one photon reducing agent is present in said aqueous solution at a concentration of between 0.1 mM and 10 mM.

12. The method of claim 4, wherein said at least one photon reducing agent is present in said aqueous solution at a concentration of between 0.5 mM and 5 mM.

13. The method of claim 4, wherein said aqueous solution comprises at least two photon reducing agents.

14. The method of claim 4, wherein said aqueous solution comprises at least three photon reducing agents.

15. The method of claim 4, wherein said at least one photon reducing agent comprises Tartrazine.

16. The method of claim 4, wherein said at least one photon reducing agent comprises chromotrope 2R.

17. The method of claim 4, wherein said at least one photon reducing agent comprises Acid Fuchsin.

18. The method of claim 4, wherein said at least one photon reducing agent comprises Patent Blue.

19. The method of claim 4, wherein said at least one photon reducing agent comprises Acid Red 37.

20. The method of claim 4, wherein said at least one photon reducing agent comprises chromotrope F8.

21. The method of claim 13, wherein one of said at least two photon reducing agent comprises Tartrazine.

22. The method of claim 1, wherein said at least one photon reducing agent is selected from the group consisting of a collisional quencher, a particulate, an absorption quencher, a FRET quencher and a dark complex.

23. The method of claim 1, wherein said at least one photon reducing agent is a dye.

24. The method of claim 1, wherein said detecting comprises detecting a fluorescence signal.

25. The method of claim 24, wherein said at least one photon producing agent is produced from a precursor molecule that is a substrate for an esterase.

26. The method of claim 25, wherein said at least one photon producing agent detects the presence of an ion inside said membrane compartment.

27. The method of claim 25, wherein said at least one photon producing agent is a fluorescent protein.

28. The method of claim 27, wherein said fluorescent protein is a modified green fluorescent protein.

29. The method of claim 25, wherein said at least one photon producing agent detects voltage across a membrane of said membrane compartment.

30. A method of identifying a chemical with a toxicological activity, comprising:

contacting a sample with a test chemical,

wherein said sample comprises a membrane compartment in contact with a solid surface, and said sample comprises a target and at least one photon producing agent,

contacting said sample with at least one photon reducing agent,

wherein said at least one photon reducing agent is substantially impermeant to said membrane compartment,

wherein said at least one photon reducing agent does not specifically bind to said membrane compartment,

wherein said at least one photon reducing agent has an absorption spectrum that overlaps with the absorption, emission or excitation spectrum of said at least one photon producing agent, and

wherein said at least one photon reducing agent is present in said sample at a concentration sufficient to reduce light emitted from said sample by at least 10% compared to the light emitted of said sample in the absence of said at least one photon reducing agent, and

detecting an optical signal from said at least one photon producing agent, wherein said optical signal directly or indirectly monitors the toxicological activity of said test chemical.

31. A method of identifying a chemical having a biological activity, comprising:

a) contacting a sample with a test chemical, said sample comprising a target,

b) contacting said sample with at least one photon reducing agent, wherein said sample comprises a membrane compartment, wherein said membrane compartment includes at least one photon producing agent that directly or indirectly monitors the activity of said target and said at least one photon reducing agent is in an aqueous solution that contacts the outer surface of said membrane compartment, and

wherein said at least one photon reducing agent is substantially impermeant to said membrane compartment,

wherein said at least one photon reducing agent does not specifically bind to said membrane compartment,

wherein said at least one photon reducing agent has an absorption spectrum that overlaps with the absorption, emission or excitation spectrum of said at least one photon producing agent, and

wherein at least one photon reducing agent is present in said aqueous solution at a concentration sufficient to reduce light emitted from said aqueous solution by at least 10% compared to the light emitted of said aqueous solution in the absence of said at least one photon reducing agent,

c) detecting an optical signal from said at least one photon producing agent by fluorescence activated cell sorting (FACS),

d) comparing the optical signal from said at least one photon producing agent to a background or control optical signal from a control sample.