Photon reducing agents for fluorescence assays

• US 8,518,710 B2
• Filed: 04/02/2012
• Issued: 08/27/2013
• Est. Priority Date: 08/01/1997
• Status: Expired due to Term

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Knapp, Tom, Zlokarnik, Gregory, Negulescu, Paul, Tsien, Roger, Rink, Timothy
• Primary Examiner(s): Gabel, Gail R
• Application Number:
  US13/437,846
• Publication Number:
  US 20130004960A1
• Time in Patent Office: 512 Days
• Field of Search: 435/5, 435/6, 435/7.1, 435/7.2, 435/7.21, 435/29, 435/288.3, 435/288.4, 436/501, 436/523, 436/528, 436/544, 436/546, 436/63, 436/64, 436/164, 436/166, 436/172, 422/73, 422/82.05
• US Class Current: 436/166
• 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 reducing undesired light emission from a sample, comprising:

contacting a sample in need of reducing undesired light with an aqueous solution that comprises at least one photon reducing agent, wherein said sample comprises a living cell, wherein said cell comprises at least one photon producing agent and wherein said at least one photon reducing agent is substantially membrane impermeant, and

detecting an optical signal from said at least one photon producing agent.

2. The method of claim 1, 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.

3. The method of claim 2, wherein said at least one photon producing agent exhibits fluorescence resonance energy transfer with said at least one photon reducing agent.

4. The method of claim 1, wherein said sample comprises at least two photon reducing agents.

5. The method of claim 4, wherein at least one of said at least two photon reducing agents are dyes.

6. The method of claim 1, wherein said at least one living cell is a mammalian cell.

7. The method of claim 6, wherein said at least one photon producing agent corresponds to an enzyme activity inside said mammalian cell.

8. 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.

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

10. The method of claim 1, wherein said at least one living cell is part of a plurality of cells comprising at least two different photon producing agents.

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

12. The method of claim 11, wherein said at least one photon producing agent is produced from a precursor molecule that is a substrate for an esterase.

13. The method of claim 12, wherein said at least one photon producing agent detects the presence of an ion inside said membrane compartment.

14. The method of claim 12, wherein said at least one photon producing agent is a fluorescent protein.

15. The method of claim 12, wherein said at least one photon producing agent detects voltage across a membrane of said membrane compartment.

16. A composition of matter, comprising:

a) a living cell, wherein said cell comprises at least one photon producing agent, and

b) an aqueous solution with at least one photon reducing agent, wherein said at least one photon reducing agent is substantially membrane impermeant and does not specifically bind to said cell.

17. The composition of matter of claim 16, 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.

18. The composition of matter of claim 16, wherein said at least one photon reducing agent is a dye.

19. The composition of matter claim 16, 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.

20. 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 an aqueous solution that comprises at least one photon reducing agent, wherein said sample comprises a living cell, wherein said cell comprises 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 substantially membrane impermeant, and

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

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 can exhibit fluorescence resonance energy transfer with said at least one photon reducing agent.