Photon reducing agents for fluorescence assays

• US 7,785,536 B2
• Filed: 03/05/2009
• Issued: 08/31/2010
• 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: Rink, Timothy, Knapp, Tom, Zlokarnik, Gregory, Negulescu, Paul, Tsien, Roger
• Primary Examiner(s): GABEL, GAILENE
• Application Number:
  US12/398,744
• Publication Number:
  US 20090162926A1
• Time in Patent Office: 544 Days
• Field of Search: 422/82.05, 422/99, 422/73, 436/501, 436/528, 436/529, 436/530, 436/531, 436/546, 436/63, 436/84, 436/47, 436/56, 435/5, 435/6, 435/7.1, 435/7.2, 435/7.5, 435/7.21, 435/29, 435/40.5, 435/230, 435/235, 435/288.3, 435/288.4, 435/40.51
• US Class Current: 422/82.08
• 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 system for identifying a test chemical with a biological activity, the system comprising:

a) a plate containing at least 96 addressable wells, a well retriever, and having programmable selection and retrieval of said addressable wells, at least selected addressable wells of said 96 addressable wells containing an aqueous reagent mixture comprising a cell, a photon producing agent, and at least one photon reducing agent;

wherein said at least one photon reducing agent is substantially cell membrane impermeant and does not specifically bind to said cell membrane, and

wherein said at least one photon reducing agent is present in said aqueous reagent mixture at an amount sufficient to reduce light emitted from said aqueous reagent mixture by at least 10% compared to the light emitted from said aqueous reagent mixture in the absence of said photon reducing agent;

b) a distribution module for handling assay solutions comprising a liquid handler to aspirate or dispense assay solutions into selected addressable wells and programmable dispensation into selected addressable wells;

c) a transporter to transport said selected addressable wells to said distribution module and optionally having programmable control of transport of said selected addressable wells;

d) a reaction module comprising a reagent dispenser to dispense reagents into said selected addressable wells, wherein at least one said reagents includes said test chemical; and

e) a fluorescent detector to detect chemical reactions in said selected addressable wells,

wherein said plate, said distribution module, said reaction module, and said detector are integrated and programmably controllable; and

wherein said plate, said distribution module, said transporter, said reaction module, and said detector are operably linked to facilitate rapid processing of said addressable wells.

2. The system of claim 1, wherein the cell is a mammalian cell.

3. The system of claim 1, wherein the photon producing agent is selected from the group consisting of fluorescent reporter substrates, fluorescent labels, fluorescent membrane sensors, fluorescent proteins, and a member of a fluorescence resonance energy transfer (FRET) pair.

4. The system of claim 1, wherein the at least one photon reducing agent has an extinction coefficient of between about 10,000 M⁻

1cm<sup>−

1</sup> to about 200,000 M<sup>−

1</sup>cm<sup>−

1</sup> at an excitation or emission wavelength of said photon producing agent.

5. The system of claim 1, wherein the at least one photon reducing agent is present at a concentration between about 0.1 mM and about 10 mM.