Method of amplifying the emission signal of a luminescent compound

• US 5,512,493 A
• Filed: 05/26/1993
• Issued: 04/30/1996
• Est. Priority Date: 07/13/1990
• Status: Expired due to Term

• Current Assignee: CIS Bio International (Institut National Radio-Elements et D'Util Pub)
• Original Assignee: CIS Bio International (Institut National Radio-Elements et D'Util Pub)
• Inventors: Dumont, Christophe, Jolu, Etienne J., Foyentin, Muriel, Nuti, Dominique, Mathis, Gerard, Aspe, Daniel
• Primary Examiner(s): Scheiner, Toni R.
• Assistant Examiner(s): Green, Lora M.
• Application Number:
  US08/068,843
• Time in Patent Office: 1,070 Days
• Field of Search: 435/7.1, 435/968, 436/536, 436/537, 436/546, 436/81, 436/82, 436/164, 436/800, 436/805, 436/172
• US Class Current: 436/537
• 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

Abstract

The invention relates to a method of amplifying the emission signal of a luminescent compound in a luminescent assay, and to its use in a luminescent method of detecting and/or determining an analyte in a medium in which it may be present.

Claims

1. A homogenous luminescent method of detecting and/or determining an analyte in a medium in which it may be present, said method comprising the steps of:

1) adding to said medium a first reagent consisting of at least one receptor which specifically binds to said analyte,

2) adding a second reagent selected from the analyte or at least one receptor which specifically binds to the analyte;

one of said first or second reagents being coupled with a luminescent donor compound consisting of a rare earth chelate or cryptate of terbium or europium, and the other reagent being coupled with a luminescent acceptor compound, wherein steps

     1) and

     2) may be reversed,

3) exciting the mixture with a light source at the excitation wavelength of the luminescent donor compound,

4) measuring the emission signal of the luminescent acceptor compound, wherein the rare earth chelate or cryptate of terbium or europium used as the donor compound possesses a low overall quantum yield and the yield of radiative deactivation of the emission level of the rare earth ion of terbium or europium in the chelate or cryptate is lower than the quantum yield of the acceptor and wherein the transfer efficiency between said luminescent donor compound and said luminescent acceptor compound times the quantum yield of the acceptor compound is greater than the yield of radiative deactivation of the emission level of the rare earth ion in the chelate or cryptate, whereby the emission signal of the rare earth chelate or cryptate of terbium or europium is amplified, and

5) correlating the measured emission signal of the luminescent acceptor compound to the presence and/or amount of analyte.

2. A method according to claim 1 wherein the first and second reagents are added simultaneously to the medium containing the analyte being tested.

3. A method according to claim 1 which comprises using a single receptor which specifically binds to the analyte.

4. A method according to claim 1 wherein the donor compound is a europium or terbium cryptate consisting of at least one europium or terbium salt complexed by a macrocyclic compound of the general formula ##STR8## in which Z is nitrogen, carbon, or phosphorous, R is present or absent, and when present is hydrogen, an hydroxyl group, an amino group or a hydrocarbon radical and the divalent radicals A, B, and C independently of one another are hydrocarbon chains which may or may not contain one or more heteroatoms and may or may not be interrupted by a heteromacrocycle, at least one of the radicals A, B, and C additionally containing at least one molecular unit or essentially consisting of a molecular unit, said molecular unit possessing a greater triplet energy than the emission level of the uncomplexed europium or terbium.

5. A method according to claim 1 wherein the europium or terbium cryptate compound is complexed by a macropolycyclic compound of the general formula ##STR9## in which A, B and C are independently selected from:

space="preserve" listing-type="equation">--C.sub.2 H.sub.4 --X.sub.1 --C.sub.6 H.sub.4 --X.sub.2 --C.sub.2 H.sub.4 -- or

space="preserve" listing-type="equation">--C.sub.2 H.sub.4 --X.sub.1 --CH.sub.2 --C.sub.6 H.sub.4 --CH.sub.2 --X.sub.2 --C.sub.2 H.sub.4 --;

wherein X₁ and X₂, are identical or different, and are selected from the group consisting of oxygen, nitrogen, sulfur, ##STR10## X being oxygen or hydrogen.

6. A method according to claim 1 wherein the donor compound is the terbium cryptate Tb trisbipyridine or the europium cryptate Eu trisbipyridine.

7. A method according to claim 3 wherein the luminescent donor compound is a europium or terbium cryptate consisting of at least one europium or terbium salt complexed by a macropolycyclic compound of formula I or II:

##STR11## in which the ring of the formula ##STR12## is one of the following rings ##STR13## wherein R'"'"' is hydrogen;

--COOR", in which R" is selected from a C₁ to C₁₀ alkyl group;

or --CO--NH--Y--Z;

Y is a group or spacer arm which consists of a divalent organic radical selected from linear or branched C₁ to C₂₀ alkylene groups which may or may not contain one or more double bonds and/or may or may not be interrupted by one or more heteroatoms selected from oxygen, nitrogen, sulfur or phosphorus;

C₅ -C₈ cycloalkylene groups;

or C₆ to C₁₄ arylene groups, said alkylene, cycloalkylene or arylene being unsubstituted or substituted by alkyl, aryl, or sulfonate groups;

Z is a functional group capable of bonding covalently with a biological substance; and

R is a methyl group or the group --Y--Z.

8. A method according to claim 1 wherein the fluorescent donor compound is am europium cryptate and wherein the fluorescent acceptor compound is allophycocyanine, allophycocyanine B, C phycocyanine or R phycocyanine.

9. A method according to claim 1 wherein the fluorescent donor compound is a terbium cryptate and wherein the fluorescent acceptor compound is selected from rhodamine, thionine, R phycocyanine, phycoerythrocyanine, C phycoerythrin, B phycoerythrin or R phycoerythrin.

10. The method according to claim 1 including the additional step of incubating said medium after each addition of reagents or after the addition of the two reagents, prior to exciting the resulting mixture.

11. The method according to claim 10 wherein said first reagent includes at least one receptor for said analyte, said first reagent having a receptor coupled with a luminescent donor compound consisting of a europium or terbium chelate or cryptate, and wherein said second reagent consists of one or more different receptors for said analyte, said second reagent being coupled with a luminescent acceptor compound.

12. The method according to claim 10 wherein said first reagent includes a receptor for said analyte, coupled with a luminescent donor compound consisting of a europium or terbium chelate or cryptate, and wherein said second reagent includes the analyte coupled with a luminescent acceptor compound.

13. The method according to claim 10 wherein said first reagent includes a receptor for said analyte, said receptor being coupled with a luminescent acceptor compound, and wherein said second reagent includes the analyte coupled with a luminescent donor compound.

14. A homogenous luminescent method of detecting and/or determining an analyte in a medium in which it may be present, said method comprising the steps of:

1) adding to said medium a first reagent consisting of at least one receptor which specifically binds to said analyte,

2) adding a second reagent selected from the analyte or at least one receptor which specifically binds to the analyte,

3) selecting and coupling a luminescent donor compound to one of the two reagents, said luminescent donor compound consisting of a europium cryptate, and said luminescent donor compound having a low overall quantum yield,

4) selecting and coupling a luminescent acceptor compound to the other reagent, said luminescent acceptor compound selected from the group consisting of allophycocyanine, allophycocyanine B, C phycocyanine, and R phycocyanine, and said luminescent acceptor compound having a quantum yield greater than the yield of radiative deactivation of the emission level of the europium ion of said luminescent donor compound,

5) exciting the mixture with a light source at the excitation wavelength of said luminescent donor compound such that the transfer efficiency between said luminescent donor compound and said luminescent acceptor compound times the quantum yield of said luminescent acceptor compound is greater than the quantum yield of radiative deactivation of the emission level of the europium ion of said luminescent donor compound, to amplify the emission signal of said luminescent donor compound,

6) measuring the emission signal of the luminescent acceptor compound, and

7) correlating the measured emission signal of the luminescent acceptor compound to the presence and/or amount of analyte.

15. A homogenous luminescent method of detecting and/or determining an analyte in a medium in which it may be present, said method comprising the steps of:

1) adding to said medium a first reagent consisting of at least one receptor which specifically binds said analyte,

2) adding a second reagent selected from the analyte or at least one receptor which specifically binds to the analyte,

3) selecting and coupling a luminescent donor compound to one of the two reagents, said luminescent donor compound consisting of a terbium cryptate, and said luminescent donor compound having a low overall quantum yield,

4) selecting and coupling a luminescent acceptor compound to the other reagent, said luminescent acceptor compound selected from the group consisting of rhodamine, thionine, R phycocyanine, phycoerythrocyanine, C phycoerythrin, B phycoerythrin, and R phycoerythrin, and said luminescent acceptor compound having a quantum yield greater than the yield of radiative deactivation of the emission level of the terbium ion of said luminescent donor compound,

5) exciting the mixture with a light source at the excitation wavelength of said luminescent donor compound such that the transfer efficiency between said luminescent donor compound and said luminescent acceptor compound times the quantum yield of said luminescent acceptor compound is greater than the quantum yield of radiative deactivation of the emission level of the terbium ion of said luminescent donor compound, to amplify the emission signal of said luminescent donor compound,

6) measuring the emission signal of the luminescent acceptor compound, and

7) correlating the measured emission signal of the luminescent acceptor compound to the presence and/or amount of analyte.