Luminescent metal chelate labels and means for detection

• US 5,310,687 A
• Filed: 11/04/1991
• Issued: 05/10/1994
• Est. Priority Date: 10/31/1984
• Status: Expired due to Term

• Current Assignee: BioVeris Corporation (Roche Holding AG)
• Original Assignee: IGEN, Inc. (Teligent Incorporated)
• Inventors: Whitesides, George M., Bard, Allen J.
• Primary Examiner(s): KIM, KAY KWANG AE
• Application Number:
  US07/789,418
• Time in Patent Office: 918 Days
• Field of Search: 435/4, 435/5, 435/6, 435/7.1, 435/7.2, 436/7.9, 436/7.92-7.95, 436/544, 436/518, 436/537, 436/546, 436/548, 436/519, 436/525, 436/536, 436/512, 436/84, 436/800, 436/904, 436/905, 546/2, 544/225, 540/145, 525/370, 525/281, 422/50, 422/52, 422/82.05, 422/82.08
• US Class Current: 436/518
• CPC Class Codes: C07F 15/0026 without a metal-carbon linkage
• CPC Class Codes: C07F 15/0053 without a metal-carbon linkage
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/806 Electrical property or magnetic property
• CPC Class Codes: Y10S 436/904 Oxidation - reduction indicators
• CPC Class Codes: Y10S 436/905 Photochemical activation of reactions

Abstract

A chemical moiety is disclosed which comprises a chemical, biochemical, or biological substance attached to one or more electrochemiluminescent organometallic compounds. In a preferred embodiment of the invention the substance is attached to one or more ruthenium-containing or osmium-containing luminescent organometallic compounds. Methods are disclosed for detecting low concentrations of the chemical moiety using chemiluminescent, electrochemiluminescent, and photoluminescent means. Compounds are disclosed which are useful for labeling substances of interest with ruthenium-containing and osmium-containing labels or other electrochemiluminescent labels. These labeled substances are useful in methods provided for detecting and quantifying analytes of interest in binding assays and competitive binding assays. The labeled substances are of particular use in homogeneous binding assays. These methods form the bases for systems designed to enable the rapid, efficient, and sensitive determination of a broad array of chemical, biochemical, and biological materials of interest.

Claims

1. A method of determining the presence of a chemical moiety, which comprises

(a) forming a reagent mixture containing the chemical moiety, or the chemical moiety and an agent which upon exposure of the reagent mixture to electrochemical energy forms either a reductant or an oxidant;

said chemical moiety having the formula ##STR4## wherein;

M is ruthenium or osmium;

P, L¹, and L², each is a bidentate aromatic heterocyclic nitrogen-containing ligand of M selected from the group consisting of bipyridyl, substituted bipyridyl, bipyrazyl, substituted bipyrazyl, phenanthrolyl and substituted phenanthrolyl, wherein each of said substituted groups is substituted by an alkyl, aryl, aralkyl, carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxycarbonyl, hydroxyamino, aminocarbonyl, amidine, guanidinium, ureide, sulfur-containing group, phosphorus-containing group, or the carboxylate ester of N-hydroxysuccinimide, each of said ligands being the same or not the same as each other ligand;

B is a biological substance, a synthetic substance which is capable of competing with a biological substance in a competitive binding reaction with a complementary material, or a non-biological polymer;

m is 1;

each of n and o is 1;

t is an integer equal to or greater than 1; and

u is 1;

said link being covalent bonding linking B with at least one of P, L¹ or L² ;

P, L¹, L² and B being of such composition and number that the chemical moiety is capable of being induced to electrochemiluminesce;

(b) exposing the reagent mixture to electrochemical energy the potential of which oscillates between a potential sufficiently positive to oxidize the chemical moiety and a potential sufficiently negative to reduce the chemical moiety, or to electrochemical energy such that said chemical moiety is oxidized and the agent forms a reductant, or such that said chemical moiety is reduced and the agent forms an oxidant, thereby to induce the chemical moiety to electrochemiluminesce; and

(c) detecting emitted luminescence thereby to determine the presence of the chemical moiety.

2. A method as defined in claim 1, wherein said link is one or more amide linkages, ester or thioester linkages, or ether or thioether linkages, each said linkage covalently bonding B with one of P, L¹ and L².

3. A method as defined in claim 2, wherein said link is one or more amide linkages, each said linkage covalently bonding B with one of P, L¹ and L².

4. A method of determining the presence of an analyte of interest which binds to a chemical moiety, which comprises

(a) forming a reagent mixture comprising the chemical moiety, or comprising the chemical moiety and an agent which upon exposure of the reagent mixture to electrochemical energy forms either a reductant or an oxidant, and the analyte of interest;

such that the chemical moiety and the analyte specifically bind to one another, said chemical moiety having the formula ##STR5## wherein;

M is ruthenium or osmium;

P, L¹, and L², each is a bidentate aromatic heterocyclic nitrogen-containing ligand of M selected from the group consisting of bipyridyl, substituted bipyridyl, bipyrazyl, substituted bipyrazyl, phenanthrolyl and substituted phenanthrolyl, wherein each of said substituted groups is substituted by an alkyl, aryl, aralkyl, carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxycarbonyl, hydroxyamino, aminocarbonyl, amidine, guanidinium, ureide, sulfur-containing group, phosphorus-containing group, or the carboxylate ester of N-hydroxysuccinimide, each of said ligands being the same or not the same as each other ligand;

B is a biological substance, or a synthetic substance which is capable of competing with a biological substance in a competitive binding reaction with a complementary material, said biological substance or synthetic substance being capable of specifically binding to the analyte of interest;

m is 1;

each of n and o is 1;

t is an integer equal to or greater than 1; and

u is 1;

said link being covalent bonding linking B with at least one of P, L¹ or L² ;

or

P, L¹, L² and B being of such composition and number that the chemical moiety is capable of being induced to electrochemiluminesce;

(b) exposing the reagent mixture to electrochemical energy the potential of which oscillates between a potential sufficiently positive to oxidize the chemical moiety and a potential sufficiently negative to reduce the chemical moiety, or to electrochemical energy such that said chemical moiety is oxidized and the agent forms a reductant, or such that said chemical moiety is reduced and the agent forms an oxidant, thereby to induce the chemical moiety to electrochemiluminesce; and

(c) detecting emitting luminescence thereby to determine the presence of the analyte of interest.

5. A method as defined in claim 4, wherein said link is one or more amide linkages, ester or thioester linkages, or ether or thioether linkages, each said linkage covalently bonding B with one of P, L¹ and L².

6. A method as defined in claim 5, wherein said link is one or more amide linkages, each said linkage covalently bonding B with one of P, L¹ and L².

7. A competitive binding method of determining the presence of an analyte of interest wherein the analyte and a chemical moiety bind competitively to a complementary material, which comprises

(a) forming a reagent mixture comprising the analyte of interest, the complementary material and the chemical moiety, or the chemical moiety and an agent which upon exposure of the reagent mixture to electrochemical energy forms either a reductant or an oxidant, such that the chemical moiety and the analyte of interest bind competitively to the complementary material;

said chemical moiety having the formula ##STR6## wherein;

M is ruthenium or osmium;

P, L¹, and L², each is a bidentate aromatic heterocyclic nitrogen-containing ligand of M selected from the group consisting of bipyridyl, substituted bypyridyl, bipyrazyl, substituted bipyrazyl, phenantholyl and substituted phenanthrolyl, wherein each of said substituted groups is substituted by an alkyl, aryl, aralkyl, carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxycarbonyl, hydroxyamino, aminocarbonyl, amide, guanidinium, ureide, sulfur-containing group, phosphorus-containing group, or the carboxylate ester of N-hydroxysuccinimide, each of said ligands being the same or not the same as each other ligand;

B is a biological substance, or a synthetic substance which is capable of competing with a biological substance in a competitive binding reaction with said complementary material, said biological substance or synthetic substance being capable of binding to the complementary material;

m is 1;

each of n an o is 1;

t is an integer equal to or greater than 1; and

u is 1;

said link being covalent bonding linking B with at least one of P, L¹ or L² ;

or

P, L¹ L² and B being of such composition and number that the chemical moiety is capable of being induced to electrochemiluminesce;

(b) exposing the reagent mixture to electrochemical energy the potential of which oscillates between a potential sufficiently positive to oxidize the chemical moiety and a potential sufficiently negative to reduce the chemical moiety, or to electrochemical energy such that said chemical moiety is oxidized and the agent forms a reductant, or such that said chemical moiety is reduced and the agent forms an oxidant, thereby to induce the chemical moiety to electrochemiluminesce; and

(c) detecting emitted luminescence thereby to determine the presence of the analyte of interest.

8. A competitive binding method as defined in claim 7, wherein said link is one or more amide linkages, ester or thioester linkages, or ether or thioester linkages, each said linkage covalently bonding B with one of P, L¹ and L².

9. A competitive binding method as defined in claim 8, wherein said link is one or more amide linkages, each said linkage covalently bonding B with one of P, L¹ and L².

10. A system for determining the presence of a chemical moiety, which system comprises

(a) a reagent mixture comprising the chemical moiety, or the chemical moiety and an agent which upon exposure of the reagent mixture to electrochemical energy forms either a reductant or an oxidant, said chemical moiety having the formula ##STR7## wherein:

M is ruthenium or osmium;

P, L¹, and L², each is a bidentate aromatic heterocyclic nitrogen-containing ligand of M selected from the group consisting of bipyridyl, substituted bipyridyl, bipyrazyl, substituted bipyrazyl, phenanthrolyl and substituted phenanthrolyl, wherein each of said substituted groups is substituted by an alkyl, aryl, aralkyl, carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxycarbonyl, hydroxyamino, aminocarbonyl, amidine, guanidinium, ureide, sulfur-containing group, phosphorus-containing group, or the carboxylate ester of N-hydroxysuccinimide, each of said ligands being the same or not the same as each other ligand;

B is a biological substance, a synthetic substance which is capable of competing with a biological substance in a competitive binding reaction with a complementary material, or a non-biological polymer;

m is 1;

each of n is o is 1;

t is an integer equal to or greater than 1; and

u is 1;

said link being covalent bonding linking B with at least one of P, L¹ or L² ;

or

P, L¹, L² and B being of such composition and number that the chemical moiety is capable of being induced to electrochemiluminesce;

(b) means for exposing the reagent mixture to electrochemical energy the potential of which oscillates between a potential sufficiently positive to oxidize the chemical moiety and a potential sufficiently negative to reduce the chemical moiety, or to electrochemical energy such that said chemical moiety is oxidized and the agent forms a reductant, or such that said chemical moiety is reduced and the agent forms an oxidant, thereby to induce the chemical moiety to electrochemiluminesce; and

(c) means for detecting emitted luminescence thereby to determine the presence of the chemical moiety.

11. A system as defined in claim 10, wherein said link is one or more amide linkages, ester or thioester linkages, or ether or thioether linkages, each said linkage covalently bonding B with one of P, L¹ and L².

12. A system as defined in claim 11, wherein said link is one or more amide linkages, each said linkage covalently bonding B with one of P, L¹ and L².

13. A system for determining the presence of an analyte of interest which binds to a chemical moiety, which system comprises

(a) a reagent mixture comprising the chemical moiety, or comprising the chemical moiety and an agent which upon exposure of the reagent mixture to electrochemical energy forms either a reductant or an oxidant, and the analyte of interest;

said chemical moiety having the formula ##STR8## wherein;

m is ruthenium or osmium;

P, L¹, and L², each is a bidentate aromatic heterocyclic nitrogen-containing ligand of M selected from the group consisting of bipyridyl, substituted bipyridyl, bipyrazyl, substituted bipyrazyl, phenanthrolyl and substituted phenanthrolyl, wherein each of said substituted groups is substituted by an alkyl, aryl, aralkyl, carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxycarbonyl, hydroxyamino, aminocarbonyl, amidine, guanidinium, ureide, sulfur-containing group, phosphorus-containing group, or the carboxylate ester of N-hydroxysuccinimide, each of said ligands being the same or not the same as each other ligand;

B is a biological substance, or a synthetic substance which is capable of competing with a biological substance in a competitive binding reaction with a complementary material, or synthetic substance being capable of binding to the analyte of interest;

m is 1;

each of n is o is 1;

t is an integer equal to 1; and

u is 1;

said link being covalent bonding linking B with one of P, L¹ or L² ;

or

P, L¹, L² and B being of such composition and number that the chemical moiety is capable of being induced to electrochemiluminesce;

(b) means for contacting the chemical moiety with the analyte of interest to form a reagent mixture;

(c) means for exposing the reagent mixture to electrochemical energy the potential of which oscillates between a potential sufficiently positive to oxidize the chemical moiety and a potential sufficiently negative to reduce the chemical moiety, or to electrochemical energy such that said chemical moiety is oxidized and the agent forms a reductant, or such that said chemical moiety is reduced and the agent forms an oxidant, thereby to induce the chemical moiety to electrochemiluminesce; and

(d) means for detecting emitted luminescence thereby to determine the presence of the chemical moiety.

14. A system as defined in claim 13, wherein said link is one or more amide linkages, ester or thioester linkages, or ether or thioether linkages, ester or thioester linkages, or ether or thioether linkages, each said linkage covalently bonding B with one of P, L¹ and L².

15. A system as defined in claim 14, wherein said link is one or more amide linkages, each said linkage covalently bonding B with one of P, L¹ and L².

16. A composition which comprises a chemical moiety having the formula ##STR9## wherein:

M is ruthenium or osmium;

P, L¹, and L², each is a bidentate aromatic heterocyclic nitrogen-containing ligand of M selected from the group consisting of bipyridyl, substituted bipyridyl, bipyrazyl, substituted bipyrazyl, phenanthrolyl and substituted phenanthrolyl, wherein each of said substituted groups is substituted by an alkyl, aryl, aralkyl, carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxycarbonyl, hydroxyamino, aminocarbonyl, amidine, guanidinium, ureide, sulfur-containing group, phosphorus-containing group, or the carboxylate ester of N-hydroxysuccinimide, each of said ligands being the same or not the same as each other ligand;

B is a biological substance, a synthetic substance which is capable of competing with a biological substance in a competitive binding reaction with a complementary material, or a non-biological polymer;

m is 1;

each of n and o is 1;

t is an integer equal to or greater than; and

u is 1;

said link being covalent bonding linking B with one of P, L¹ or L² ;

or

P, L¹, L² and B being of such composition and number that the chemical moiety is capable of being induced to electrochemiluminesce, and

one or more different chemical moieties each of which is capable of being included to luminesce at a wavelength different from that at which any other of the chemical moieties luminesces.

17. A composition as defined in claim 16, wherein said link is one or more amide linkages, ester to thioester linkages, or ether or thioether linkages, each said linkage covalently bonding B with one of P, L¹ and L².

18. A composition as defined in claim 17, wherein said link is one or more amide linkages, each said linkage covalently bonding B with one of P, L¹ or L².