Dye pair for flourescence resonance energy transfer (fret) measurements

• US 20040076979A1
• Filed: 10/24/2003
• Published: 04/22/2004
• Est. Priority Date: 11/16/2000
• Status: Active Grant

• Current Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Original Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Inventors: Josel, Hans-Peter, Hermann, Rupert, Mueller, Francis, Koenig, Bernard, Belik, Daniel
• Granted Patent: US 6,908,769 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention concerns new fluorescent dye systems especially for fluorescence resonance energy transfer determinations for example combined with the time-resolved measurement of the resulting fluorescence. The invention also concerns the use of these dyes to label biomolecules and for the homogeneous determination of interactions between biomolecules, for example for detecting an analyte.

Claims

1. Method for determining the interaction between biomolecules labelled with a donor or acceptor, respectively, based on the principle of fluorescence resonance energy transfer and measurement of the resulting fluorescence characterized in that metallic chelate complexes containing metal ions from the VIIth and VIIIth group of the transition elements are used as energy donors in combination with low molecular fluorophores having a molecular weight between 300 Da and 3000 Da as energy acceptors.

2. Method as claimed in claim 1, characterized in that the lower molecular fluorophores have an absorption maximum between 600 nm and 750 nm.

3. Method as claimed in claim 1 or 2, characterized in that the lower molecular fluorophores have a molecular weight of <

2000 Da.

4. Method as claimed in one of the claims 1 to 3, characterized in that the fluorophores are selected from the group comprising xanthenes, cyanins, rhodamines and oxazines.

5. Method as claimed in one of the claims 1 to 4, characterized in that a rhodamine of the following general formula I is used as the fluorophore

6. Method as claimed in claim 5, characterized in that the substituent R7 is an electron-attracting residue.

7. Method as claimed in one of the claims 1 to 4, characterized in that an oxazine of the following general formula II is used as the fluorophore

8. Method as claimed in one of the previous claims, characterized in that the metal chelate complex is a ruthenium complex.

9. Method as claimed in one of the claims 1 to 8, characterized in that a time-resolved measurement of fluorescence is carried out.

10. Method as claimed in one of the claims 1 to 8, characterized in that the fluorescence is measured by means of the phase modulation technique.

11. Use of metal chelate complexes containing metal ions from the VIIth and VIIIth group of the transition elements as fluorescence resonance energy donors in combination with low molecular fluorophores in a method for detecting an interaction of at least two biomolecules in a sample.

12. Use as claimed in claim 11, characterized in that the method is carried out according to the principle of a homogeneous binding test.

13. Reagent kit for detecting an analyte in a sample, characterized in that it contains at least one biomolecule labelled with a metal chelate complex containing metal ions from the VIIth and VIIIth group of the transition elements and at least one further biomolecule which is labelled with a low molecular fluorophore.

14. Reagent combination for determining the interaction between partners of a bioaffine binding pair, characterized in that one partner of the bioaffine binding pair is labelled with a metal chelate complex containing metal ions from the VIIth and VIIIth group of the transition elements and another partner of the bioaffine binding pair is labelled with a lower molecular fluorophore having a MW between 300 Da and 3000 Da.

15. Improved method for determining the interaction between biomolecules labelled with a donor or acceptor based on the principle of fluorescence resonance energy transfer and measurement of the resulting fluorescence characterized in that metallic chelate complexes containing metal ions from the VIIth and VIIIth group of the transition elements are used as energy donors in combination with low molecular fluorophores having a molecular weight between 300 Da and 3000 Da as energy acceptors.