Photon reducing agents and compositions for fluorescence assays

• US 6,200,762 B1
• Filed: 07/17/1998
• Issued: 03/13/2001
• 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, Knapp, Tom, Rink, Tim, Zlokarnik, Gregor, Tsien, Roger Y.
• Primary Examiner(s): Le, Long V.
• Assistant Examiner(s): GABEL, GAILENE
• Application Number:
  US09/118,497
• Time in Patent Office: 970 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, 436/529, 436/531, 436/800, 436/528, 436/530, 436/546, 436/809, 427/102, 427/213.34, 427/157, 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: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/809 Multifield plates or multicontainer arrays

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 further provides a method of determining bound and free analyte in a sample using at least one photon reducing agent. The present invention also provides a method of screening test chemicals in fluorescent assays using photon reducing agents. The present invention also provides compositions and kits for practicing these methods.

Claims

1. A composition of matter, comprising:

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

b) an aqueous solution with at least one photon reducing agent, wherein said aqueous solution is in contact with an outer surface of said membrane compartment,

wherein said at least one photon reducing agent is substantially impermeant 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 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.

2. The composition of matter of claim 1, wherein said aqueous solution further comprises at least on photon producing agent.

3. The composition of matter of claim 2, wherein said at least one photon reducing agent is present in said aqueous solution at a concentration sufficient to reduce light emitted from said at least one photon producing agent present in said aqueous solution by at least 30% compared to the light emitted from said at least one photon producing agent in said aqueous solution in the absence of said at least one photon reducing agent.

4. The composition of matter of claim 2, wherein said aqueous solution further comprises a second photon producing agent that is a test chemical.

5. The composition of matter of claim 1, wherein said at least one photon reducing agent is present in said aqueous solution at a concentration that reduces undesired fluorescence from said aqueous solution by at least 40% compared to undesired fluorescence of said aqueous solution in the absence of said at least one photon reducing agent.

6. The composition of matter of claim 5, wherein said at least one photon reducing agent has solubility in water of about 100 mM or more.

7. The composition of matter of claim 1, wherein said at least one photon reducing agent has an absorption spectrum that overlaps with the emission or excitation spectrum of said at least one photon producing agent.

8. The composition of matter of claim 7, wherein said at least one photon reducing agent has an extinction coefficient of at least 10,000 M⁻

1cm<sup>−

1</sup>.

9. The composition of matter of claim 7, wherein said membrane compartment is a living cell.

10. The composition of matter of claim 9, wherein said at least one photon reducing agent is not acting as a pH indicator dye and said at least one photon reducing agent has an absorbance spectrum that overlaps with the emission or excitation spectrum of said at least one photon producing agent in said aqueous solution.

11. The composition of matter of claim 1, wherein said at least one photon producing agent in said aqueous solution exhibits transfer energy to said at least one photon reducing agent.

12. The composition of matter of claim 11, wherein said membrane compartment is a living cell and said at least one photon reducing agent is a dye.

13. The composition of matter of claim 12, wherein said living cell is a member of a plurality of living cells in a volume less than about 5 microliters.

14. The composition of matter of claim 12, wherein said composition further includes a microplate and said living cell is a member of a plurality of living cells in a well of said microplate.

15. The composition of matter of claim 14, wherein said microplate has 864 or greater wells.

16. The composition of matter of claim 1, wherein said at least one photon reducing agent has an extinction coefficient of at least 10,000 M⁻

1cm<sup>−

1</sup> and is present at a concentration of at least about 0.1 mM.

17. The composition of matter of claim 1, wherein said composition further comprises a system to launch light of a predetermined wavelength through the bottom of an assay surface, wherein said predetermined wavelength is an excitation wavelength for said at least one photon producing agent.

18. The composition of matter of claim 1, wherein said at least one photon reducing agent is present in said aqueous solution at a concentration that reduces fluorescence from said aqueous solution by at least 75% compared to fluorescence of said aqueous solution in the absence of said at least one photon reducing agent.

19. The composition of matter of claim 1, wherein said at least one photon reducing agent is non-toxic to said living cell over the time frame of the assay.

20. The composition of matter of claim 1, 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.

21. The composition of matter of claim 1, 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.

22. The composition of matter of claim 1, 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.

23. The composition of matter of claim 1, wherein said aqueous solution comprises at least two photon reducing agents.

24. The composition of matter of claim 23, wherein one of said at least two photon reducing agents includes Tartrazine.

25. The composition of matter of claim 1, wherein said aqueous solution comprises at least three photon reducing agents.

26. The composition of matter of claim 1, wherein said at least one photon reducing agents includes Tartrazine.

27. The composition of matter of claim 1, wherein said at least one photon reducing agents includes chromotrope 2R.

28. The composition of matter of claim 1, wherein said at least one photon reducing agents includes Acid Fuchsin.

29. The composition of matter of claim 1, wherein said at least one photon reducing agents includes Patent Blue.

30. The composition of matter of claim 1, wherein said at least one photon reducing agents includes Acid Red 37.

31. The composition of matter of claim 1, wherein said at least one photon reducing agents includes chromotrope F8.

32. The composition of claim 1, wherein said at least one photon reducing agent has an intracellular concentration of less than 30% of the concentration of said at least one photon reducing agent outside of said membrane compartment.

33. The composition of claim 1, wherein said at least one photon reducing agent has an intracellular concentration of less than 10% of the concentration of said at least one photon reducing agent outside of said membrane compartment.

34. A composition of matter, comprising:

a) at least one first photon producing agent internalized in a living cell in optical contact with a solid surface that can pass light of a predetermined wavelength that overlaps with the excitation spectrum of a second photon producing agent to excite said second photon producing agent, and

b) at least one photon reducing agent in an aqueous solution surrounding said living cell,

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

wherein said photon reducing agent is substantially impermeant 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.

35. The composition of matter of claim 34, wherein said at least one photon reducing agent is present in said aqueous solution at a concentration that reduces fluorescence from said aqueous solution by at least 75% compared to fluorescence of said aqueous solution in the absence of said at least one photon reducing agent.

36. The composition of matter of claim 34, 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.

37. The composition of matter of claim 34, 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.

38. The composition of matter of claim 34, wherein said aqueous solution comprises at least two photon reducing agents.

39. The composition of matter of claim 38, wherein one of said at least two photon reducing agents includes Tartrazine.

40. The composition of matter of claim 34, wherein said aqueous solution comprises at least three photon reducing agents.

41. The composition of matter of claim 34, wherein said at least one photon reducing agents includes Tartrazine.

42. The composition of matter of claim 34, wherein said at least one photon reducing agents includes chromotrope 2R.

43. The composition of matter of claim 34, wherein said at least one photon reducing agents includes Acid Fuchsin.

44. The composition of matter of claim 34, wherein said at least one photon reducing agents includes Patent Blue.

45. The composition of matter of claim 34, wherein said at least one photon reducing agents includes Acid Red 37.

46. The composition of matter of claim 34, wherein said at least one photon reducing agents includes chromotrope F8.

47. The composition of claim 34, wherein said at least one photon reducing agent has an intracellular concentration of less than 30% of the concentration of said at least one photon reducing agent outside of said membrane compartment.

48. The composition of claim 34, wherein said at least one photon reducing agent has an intracellular concentration of less than 10% of the concentration of said at least one photon reducing agent outside of said membrane compartment.