Luminescent lanthanide chelates and methods of use

• US 5,639,615 A
• Filed: 12/09/1996
• Issued: 06/17/1997
• Est. Priority Date: 06/29/1994
• Status: Expired due to Term

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Selvin, Paul R., Hearst, John
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/762,598
• Time in Patent Office: 190 Days
• Field of Search: 435/5, 435/6, 435/7.1, 435/810, 436/501, 935/77, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: G01N 33/533 with fluorescent label
• 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 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10T 436/13 Tracers or tags

Abstract

The invention provides lanthanide chelates capable of intense luminescence. The celates comprise a lanthanide chelator covalently joined to a coumarin-like or quinolone-like sensitizer. Exemplary sensitzers include 2- or 4-quinolones, 2- or 4-coumarins, or derivatives thereof e.g. carbostyril 124 (7-amino-4-methyl-2-quinolone), coumarin 120 (7-amino-4-methyl-2-coumarin), coumarin 124 (7-amino-4-(trifluoromethyl)-2-coumarin), aminomethyltrimethylpsoralen, etc.

The chelates form high affinity complexes with lanthanides, such as terbium or europium, through chelator groups, such as DTPA. The chelates may be coupled to a wide variety of compounds to create specific labels, probes, diagnostic and/or therapeutic reagents, etc. The chelates find particular use in resonance energy transfer between chelate-lanthanide complexes and another luminescent agent, often a fluorescent non-metal based resonance energy acceptor. The methods provide useful information about the structure, conformation, relative location and/or interactions of macromolecules.

Claims

1. A method of detecting a portion of a sample by luminescence, said method comprising the steps of:

contacting a sample portion with a luminescent complex of a lanthanide chelate and a lanthanide capable of binding said chelate with an equilibrium constant of at least 10⁹ M^-1 ;

said lanthanide chelate comprising a lanthanide chelator covalently joined to a sensitizer. wherein a complex of said chelate and said lanthanide is capable of enhanced lanthanide luminescence, and said sensitizer comprises a polynuclear heterocyclic aromatic compound of the general formula;

##STR6## wherein X comprises an atom from periodic group 5 or 6, wherein a single position 2-8 carbon atom of said sensitizer is substituted with a linking up through which said sensitizer is covalently joined to said chelator;

exposing said sample portion to light at a first wavelength capable of inducing a first electronic transition in said chelate;

detecting a localized emission of light from said sample at a second wavelength, wherein said second wavelength is longer than said first wavelength and results from a second electronic transition in said chelate;

wherein the localized detection of said emission of light correlates with the localized presence of said sample portion.

2. A method according to claim 1, wherein said enhanced lanthanide luminescence is at least 50,000% greater intensity.

3. A method according to claim 1, wherein a first position 2-8 carbon atom of said sensitizer is substituted with an oxygen atom through a double covalent bond, and a second position 2-8 carbon atom of said sensitizer, different than said first position 2-8 carbon atom, is substituted with a linking group through which said sensitizer is covalently joined to said chelator.

4. A method according to claim 3, wherein said linking group consists essentially of an amine or carboxyl group.

5. A method according to claim 3, wherein said first position 2-8 carbon atom is the position 2 or 4 carbon atom and said second carbon atom is the position 7 carbon atom.

6. A method according to claim 3, wherein a third position 2-8 carbon atom of said sensitizer, different from said first and second position 2-8 carbon atoms, is substituted.

7. A method according to claim 5, wherein said third position 2-8 carbon atom is the position 4 carbon and is substituted with a hydrocarbon or halogen substituted hydrocarbon.

8. A method according to claim 1, wherein said sensitizer comprises a 2- or 4-or a 2- or 4- coumarin.

9. A method according to claim 1, wherein said sensitizer comprises carbostyril 124 (7-amino4-methyl-2-quinolone), coumarin 120 (7-amino4-methyl-2-coumarin), coumarin 124 (7-amino4-(trifluoromethyl)-2- coumarin), aminomethyltrimethylpsoralen.

10. A method according to claim 1, wherein a complex of said chelate and a lanthanide capable of binding said chelate with an equilibrium constant of at least 10⁹ M^-1 is capable of at least ten fold greater luminescence probability than is said lanthanide.

11. A method according to claim 1, wherein a first solution comprising complexes of said chelate and a lanthanide capable of binding said chelate with an equilibrium constant of at least 10⁹ M^-1 is capable of at least ten fold greater luminescence than is a second solution comprising said lanthanide, wherein said first and second solutions are identical except for the presence of said chelate in said first solution and absence of said chelate in said second solution.

12. A method according to claim 1, wherein said lanthanide is terbium or europium.

13. A method according to claim 1, wherein said chelator is capable of binding said lanthanide with an equilibrium constant of at least 10¹⁰ M^-1.

14. A method according to claim 1, wherein said chelator comprises a plurality of carboxylate or phosphonate groups.

15. A method according to claim 1, wherein said chelator comprises DTPA.

16. A method according to claim 1, wherein said chelate is covalently joined to a macromolecule.

17. A method according to claim 1, wherein said chelate is covalently joined to a reagent which selectively binds an analyte to form a molecular label.

18. A method according to claim 17, wherein said contacting step further comprises the step of incubating said sample portion under conditions wherein said reagent is capable of selectively binding said analyte;

wherein the detection of said emission of light correlates with the presence of said analyte sample portion.

19. A method according to claim 18, wherein said enhanced lanthanide luminescence is at least 50,000% greater intensity.

20. A method according to claim 18, wherein said chelator is capable of binding said lanthanide with an equilibrium constant of at least 10¹⁰ M^-1.