Electrochemiluminescent label based on multimetallic assemblies

• US 6,613,583 B1
• Filed: 06/26/1998
• Issued: 09/02/2003
• Est. Priority Date: 06/27/1997
• Status: Expired due to Term

• Current Assignee: Administrators Of The Tulane Educational Fund (ADMINISTRATORS|DAVID H. COY), Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: IGEN International, Inc. (Roche Holding AG)
• Inventors: Richter, Mark M., Schmehl, Russel H., Bard, Allen J.
• Primary Examiner(s): Ceperley, Mary E.
• Application Number:
  US09/105,524
• Time in Patent Office: 1,894 Days
• Field of Search: 436/544, 436/500, 436/817, 436/815, 436/816, 436/519, 436/806, 530/391.3, 530/391.5, 530/402, 435/6
• US Class Current: 436/544
• CPC Class Codes: F16F 1/422 the stressing resulting in flexion of the spring
• CPC Class Codes: F16F 15/06 with metal springs with rubber springs also F16F15/08
• CPC Class Codes: Y10S 436/806 Electrical property or magnetic property
• CPC Class Codes: Y10S 436/815 Test for named compound or class of compounds
• CPC Class Codes: Y10S 436/816 Alkaloids, amphetamines, and barbiturates
• CPC Class Codes: Y10S 436/817 Steroids or hormones

Abstract

Electrochemiluminescent label compounds containing multimetallic centers separated by bridging ligands are described. An example of a multimetallic electrochemiluminescent label suitable for use in electrochemiluminescence (ECL) methods, [(bpy)₂Ru]₂(bphb)⁴⁺, demonstrates ECL efficiencies 2 to 3 times greater than those for Ru(bpy)₃²⁺ in acetonitrile and aqueous media. Such multimetallic ECL compounds may be especially useful in the design of new labels for bioanalytical applications, such as immunoassays and DNA probes.

Claims

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

(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:

 

wherein;

M′ and

M″

are independently selected from the group consisting of ruthenium and osmium;

L¹, L², L³, and L⁴ each is a bidentate aromatic heterocyclic nitrogen-containing ligand selected from the group consisting of bipyridyl, substituted bipyridyl, bipyrazyl, substituted bipyrazyl, terpyridyl, substituted terpyridyl, phenanthrolyl and substituted phenanthrolyl, wherein each of said substituted ligands 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;

P is a bridging ligand selected from the group consisting of dpp, bbpe, bphb, α

,ω

-(2,2′

-bipyrid-4-yl)-alkanes), 4,4′

-bipyridines, and 1,2-bis(diphenylphosphine)ethane;

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;

t is an integer equal to or greater than 1;

u is an integer equal to or greater than 1;

said link being covalent bonding linking B with at least one of L¹, L², L³, and L⁴;

L¹, L², L³, L⁴, P 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. The method of 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 L¹, L², L³, and L⁴.

3. The method of claim 2, wherein said link is one or more amide linkages, each of said linkages covalently bonding B with one of L¹, L², L³, and L⁴.

4. The method of claim 1, wherein both M′

and M″

are ruthenium.

5. The method of claim 1, wherein both M′

and M″

are osmium.

6. The method of claim 1, wherein M′

is osmium and M″

is ruthenium.

7. The method of claim 4, wherein P is bphb.

8. The method of claim 7, wherein L¹, L², L³, and L⁴ are independently selected from the group consisting of bipyridyl and substituted bipyridyl.

9. The method of claim 8, wherein at least one of L¹, L², L³, and L⁴ is substituted bipyridyl.

10. The method of claim 1, wherein B is a whole cell, subcellular particle, nucleic acid, polysaccharide, protein, lipoprotein, lipopolysaccharide, glycoprotein, polypeptide, amino acid, cellular metabolite, hormone, tranquilizer, barbiturate, alkaloid, steroid, vitamin, or non-biological polymer.

11. The method of claim 10, wherein B is a serum-derived antibody or a monoclonal antibody.

12. The method of claim 10, wherein B is a nucleotide or polynucleotide.

13. The method of claim 10, wherein B is T4 thyroid hormone.

14. A method of determining the presence of an analyte of interest which binds to a chemical moiety, said method comprising

(a) forming a reagent mixture compnsing 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

 

wherein;

M′ and

M″

are independently selected from the group consisting of ruthenium and osmium;

L¹, L², L³, and L⁴ each is a bidentate aromatic heterocyclic nitrogen-containing ligand selected from the group consisting of bipyridyl, substituted bipyridyl, bipyrazyl, substituted bipyrazyl, terpyridyl, substituted terpyridyl, phenanthrolyl and substituted phenanthrolyl, wherein each of said substituted ligands 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;

P is a bridging ligand selected from the group consisting of dpp, bbpe, bphb, α

,ω

-(2,2′

-bipyrid-4-yl)-alkanes), 4,4′

-bipyridines, and 1,2-bis(diphenylphosphine)ethane;

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;

t is an integer equal to or greater than 1;

u is an integer equal to or greater than 1;

said link being covalent bonding linking B with at least one of L¹, L², L³, and L⁴;

L¹, L², L³, L⁴, P 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.

15. The method of claim 14, 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 L¹, L², L³, and L⁴.

16. The method of claim 15, wherein said link is one or more amide linkages, each said linkage covalently bonding B with one of L¹, L², L³, and L⁴.

17. The method of claim 14, wherein both M′

and M″

are ruthenium.

18. The method of claim 14, wherein both M′

and M″

are osmium.

19. The method of claim 14, wherein M′

is osmium and M″

is ruthenium.

20. The method of claim 17, wherein P is bphb.

21. The method of claim 20, wherein L¹, L², L³, and L⁴ are independently selected from the group consisting of bipyridyl and substituted bipyridyl.

22. The method of claim 21, wherein at least one of L¹, L², L³, and L⁴ is substituted bipyridyl.

23. The method of claim 14, wherein B is a whole cell, subcellular particle, nucleic acid, polysaccharide, protein, lipoprotein, lipopolysaccharide, glycoprotein, polypeptide, amino acid, cellular metabolite, hormone, tranquilizer, barbiturate, alkaloid, steroid, vitamin, or non-biological polymer.

24. The method of claim 23, wherein B is a serum-derived antibody or a monoclonal antibody.

25. The method of claim 23, wherein B is a nucleotide or polynucleotide.

26. The method of claim 23, wherein B is T4 thyroid hormone.

27. The method of claim 14, wherein said analyte is a whole cell, subcellular particle, nucleic acid, polysaccharide, protein, lipoprotein, lipopolysaccharide, glycoprotein, polypeptide, amino acid, cellular metabolite, hormone, tranquilizer, barbiturate, alkaloid, steroid, vitamin, or non-biological polymer.

28. The method of claim 27, wherein said analyte is insulin, digoxin, digitoxin, or T4 thyroid hormone.

29. The method of claim 27, wherein said analyte is a fungus.

30. The method of claim 27, wherein said analyte is an antibody.

31. The method of claim 27, wherein said analyte is a pharmacological agent or sugar.

32. 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, said method comprising

(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

 

wherein;

M′ and

M″

are independently selected from the group consisting of ruthenium and osmium;

L¹, L², L³, and L⁴ each is a bidentate aromatic heterocyclic nitrogen-containing ligand selected from the group consisting of bipyridyl, substituted bipyridyl, bipyrazyl, substituted bipyrazyl, terpyridyl, substituted terpyridyl, phenanthrolyl and substituted phenanthrolyl, wherein each of said substituted ligands 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;

P is a bridging ligand selected from the group consisting of dpp, bbpe, bphb, α

,ω

-(2,2′

-bipyrid-4-yl)-alkanes), 4,4′

-bipyridines, and 1,2-bis(diphenylphosphine)ethane;

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;

t is an integer equal to or greater than 1;

u is an integer equal to or greater than 1;

said link being covalent bonding linking B with at least one of L¹, L², L³, and L⁴;

L¹, L², L³, L⁴, P 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.

33. The method of claim 32, 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 L¹, L², L³, and L⁴.

34. The method of claim 33, wherein said link is one or more amide linkages, each said linkage covalently bonding B with one of L¹, L², L³, and L⁴.

35. The method of claim 32, wherein both M′

and M″

are ruthenium.

36. The method of claim 32, wherein both M′

and M″

are osmium.

37. The method of claim 32, wherein M′

is osmium and M″

is ruthenium.

38. The method of claim 35, wherein P is bphb.

39. The method of claim 38, wherein L¹, L², L³, and L⁴ are independently selected from the group consisting of bipyridyl and substituted bipyridyl.

40. The method of claim 39, wherein at least one of L¹, L², L³, and L⁴ is substituted bipyridyl.

41. The method of claim 32, wherein B is a whole cell, subcellular particle, nucleic acid, polysaccharide, protein, lipoprotein, lipopolysaccharide, glycoprotein, polypeptide, amino acid, cellular metabolite, hormone, tranquilizer, barbiturate, alkaloid, steroid, vitamin, or non-biological polymer.

42. The method of claim 41, wherein B is a serum-derived antibody or a monoclonal antibody.

43. The method of claim 42, wherein B is a nucleotide or polynucleotide.

44. The method of claim 42, wherein B is T4 thyroid hormone.

45. The method of claim 32, wherein said analyte is a whole cell, subcellular particle, nucleic acid, polysaccharide, protein, lipoprotein, lipopolysaccharide, glycoprotein, polypeptide, amino acid, cellular metabolite, hormone, tranquilizer, barbiturate, alkaloid, steroid, vitamin, or non-biological polymer.

46. The method of claim 45, wherein said analyte is insulin, digoxin, digitoxin, or T4 thyroid hormone.

47. The method of claim 45, wherein said analyte is a fungus.

48. The method of claim 45, wherein said analyte is an antibody.

49. The method of claim 45, wherein said analyte is a pharmacological agent or sugar.

50. A system for determining the presence of a chemical moiety, said system comprising

(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

 

wherein;

M′ and

M″

are independently selected from the group consisting of ruthenium and osmium;

L¹, L², L³, and L⁴ each is a bidentate aromatic heterocyclic nitrogen-containing ligand selected from the group consisting of bipyridyl, substituted bipyridyl, bipyrazyl, substituted bipyrazyl, terpyridyl, substituted terpyridyl, phenanthrolyl and substituted phenanthrolyl, wherein each of said substituted ligands 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;

P is a bridging ligand selected from the group consisting of dpp, bbpe, bphb, α

,ω

-(2,2′

-bipyrid-4-yl)-alkanes), 4,4′

-bipyridines, and 1,2-bis(diphenylphosphine)ethane;

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;

t is an integer equal to or greater than 1;

u is an integer equal to or greater than 1;

said link being covalent bonding linking B with at least one of L¹, L², L³, and L⁴;

L¹, L², L³, L⁴, P 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.

51. The system of claim 50, 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 L¹, L², L³, and L⁴.

52. The system of claim 51, wherein said link is one or more amide linkages, each said linkage covalently bonding B with one of L¹, L², L³, and L⁴.

53. 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 electrochemiical energy forms either a reductant or an oxidant, and the analyte of interest;

said chemical moiety having the formula

 

wherein;

M′ and

M″

are independently selected from the group consisting of ruthenium and osmium;

L¹, L², L³, and L⁴ each is a bidentate aromatic heterocyclic nitrogen-containing ligand selected from the group consisting of bipyridyl, substituted bipyridyl, bipyrazyl, substituted bipyrazyl, terpyridyl, substituted terpyridyl, phenanthrolyl and substituted phenanthrolyl, wherein each of said substituted ligands 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;

P is a bridging ligand selected from the group consisting of dpp, bbpe, bphb, α

,ω

-(2,2′

-bipyrid-4-yl)-alkanes), 4,4′

-bipyridines, and 1,2-bis(diphenylphosphine)ethane;

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 a synthetic substance being capable of binding to the analyte of interest;

t is an integer equal to or greater than 1;

u is an integer equal to or greater than 1;

said link being covalent bonding linking B with at least one of L¹, L², L³, and L⁴;

L¹, L², L³, L⁴, P 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.

54. The system of claim 53, 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 L¹, L², L³, and L⁴.

55. The system of claim 54, wherein said link is one or more amide linkages, each said linkage covalently bonding B with one of L¹, L², L³, and L⁴.

56. A composition which comprises a chemical moiety having the formula

wherein:

M′ and

M″

are independently selected from the group consisting of ruthenium and osmium;

L¹, L², L³, and L⁴ each is a bidentate aromatic heterocyclic nitrogen-containing ligand selected from the group consisting of bipyridyl, substituted bipyridyl, bipyrazyl, substituted bipyrazyl, terpyridyl, substituted terpyridyl, phenanthrolyl and substituted phenanthrolyl, wherein each of said substituted ligands 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;

P is a bridging ligand selected from the group consisting of dpp, bbpe, bphb, α

,ω

-(2,2′

-bipyrid-4-yl)-alkanes), 4,4′

-bipyridines, and 1,2-bis(diphenylphosphine)ethane;

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;

t is an integer equal to or greater than 1;

u is an integer equal to or greater than 1;

said link being covalent bonding linking B with at least one of L¹, L², L³, and L⁴;

L¹, L², L³, L⁴, P 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 induced to luminesce at a wavelength different from that at which any other of the chemical moieties luminesces.

57. The composition of claim 56, 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 L¹, L², L³, and L⁴.

58. The composition of claim 57, wherein said link is one or more amide linkages, each said linkage covalently bonding B with one of L¹, L², L³, and L⁴.

59. The composition of claim 56, wherein both M′

and M″

are ruthenium.

60. The method of claim 56, wherein both M′

and M″

are osmium.

61. The method of claim 56, wherein M′

is osmium and M″

is ruthenium.

62. The composition of claim 59, wherein P is bphb.

63. The composition of claim 62, wherein L¹, L², L³, and L⁴ are independently selected from the group consisting of bipyridyl and substituted bipyridyl.

64. The composition of claim 63, wherein at least one of L¹, L², L³, and L⁴ is substituted bipyridyl.

65. The composition of claim 56, wherein B is a whole cell, subcellular particle, nucleic acid, polysaccharide, protein, lipoprotein, lipopolysaccharide, glycoprotein, polypeptide, amino acid, cellular metabolite, hormone, tranquilizer, barbiturate, alkaloid, steroid, vitamin, or non-biological polymer.

66. The composition of claim 65, wherein B is a serum-derived antibody or a monoclonal antibody.

67. The composition of claim 65, wherein B is a nucleotide or polynucleotide.

68. The composition of claim 65, wherein B is T4 thyroid hormone.

69. A method of determining the presence of a chemical moiety having the formula:

70. The method of claim 69, wherein B is further defined as a substance covalently bound to one or more of L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹, L¹⁰, L¹¹, or L¹² through one or more amide linkages.

71. The method of claim 69, wherein B is a whole cell, subcellular particle, nucleic acid, polysaccharide, protein, lipoprotein, lipopolysaccharide, glycoprotein, polypeptide, amino acid, cellular metabolite, hormone, tranquilizer, barbiturate, alkaloid, steroid, vitamin, or non-biological polymer.

72. The method of claim 71, wherein B is a serum-derived antibody or a monoclonal antibody.

73. The method of claim 71, wherein B is a nucleotide or polynucleotide.

74. The method of claim 71, wherein B is T4 thyroid hormone.

75. A method of determining the presence of an analyte of interest which binds to a chemical moiety, said moiety having the formula:

76. The method of claim 75, wherein B is further defined as a substance covalently bound to one or more of L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹, L¹⁰, L¹¹, or L¹² through one or more amide linkages.

77. The method of claim 75, wherein B is a whole cell, subcellular particle, nucleic acid, polysaccharide, protein, lipoprotein, lipopolysaccharide, glycoprotein, polypeptide, amino acid, cellular metabolite, hormone, tranquilizer, barbiturate, alkaloid, steroid, vitamin, or non-biological polymer.

78. The method of claim 77, wherein B is a serum-derived antibody or a monoclonal antibody.

79. The method of claim 77, wherein B is a nucleotide or polynucleotide.

80. The method of claim 77, wherein B is T4 thyroid hormone.

81. The method of claim 75, wherein said analyte is a whole cell, subcellular particle, nucleic acid, polysaccharide, protein, lipoprotein, lipopolysaccharide, glycoprotein, polypeptide, amino acid, cellular metabolite, hormone, tranquilizer, barbiturate, alkaloid, steroid, vitamin, or non-biological polymer.

82. The method of claim 81, wherein said analyte is insulin, digoxin, digitoxin, or T4 thyroid hormnone.

83. The method of claim 81, wherein said analyte is a fungus.

84. The method of claim 81, wherein said analyte is an antibody.

85. The method of claim 81, wherein said analyte is a pharmacological agent or sugar.

86. A competitive binding method of determining the presence of an analyte of interest wherein the analyte and a chemical moiety bind competitively to a chemical material, the chemical moiety having the formula:

87. The method of claim 86, wherein B is further defined as a substance covalently bound to one or more of L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹, L¹⁰, L¹¹, or L¹² through one or more amide linkages.

88. The method of claim 86, wherein B is the same substance as said analyte.

89. The method of claim 86, wherein said material is a whole cell, subcellular particle, nucleic acid, polysaccharide, protein, lipoprotein, lipopolysaccharide, glycoprotein, polypeptide, cellular metabolite, hormone, tranquilizer, barbiturate, alkaloid, steroid, vitamin, or amino acid.

90. The method of claim 89, wherein said chemical material is a serum-derived antibody or a monoclonal antibody.

91. The method of claim 89, wherein said chemical material is a DNA or RNA fragment.

92. The method of claim 86, further defined as a competitive binding method wherein the material is fixed to an insoluble matrix.

93. The method of claim 92, further defined as a heterogeneous method wherein the material is a monoclonal antibody and the insoluble matrix is the surface of an assay vessel.

94. The method of claim 92, further defined as a homogeneous method wherein the material is a monoclonal antibody and the insoluble matrix is the surface of an assay vessel.

95. The method of claim 86, wherein said material is a pharmacological agent or sugar.

96. The method of claim 86, wherein B is a whole cell, subcellular particle, nucleic acid, polysaccharide, protein, lipoprotein, lipopolysaccharide, glycoprotein, polypeptide, amino acid, cellular metabolite, hormone, tranquilizer, barbiturate, alkaloid, steroid, vitamin, or non-biological polymer.

97. The method of claim 96, wherein B is a serum-derived antibody or a monoclonal antibody.

98. The method of claim 96, wherein B is a nucleotide or polynucleotide.

99. The method of claim 96, wherein B is T4 thyroid hormone.

100. The method of claim 86, wherein said analyte is a whole cell, subcellular particle, nucleic acid, polysaccharide, protein, lipoprotein, lipopolysaccharide, glycoprotein, polypeptide, amino acid, cellular metabolite, hormone, tranquilizer, barbiturate, alkaloid, steroid, vitamin, or non-biological polymer.

101. The method of claim 100, wherein said analyte is insulin, digoxin, digitoxin, or T4 thyroid hormone.

102. The method of claim 100, wherein said analyte is a fungus.

103. The method of claim 100, wherein said analyte is an antibody.

104. The method of claim 100, wherein said analyte is a pharmacological agent or sugar.

105. A system for determining the presence of a chemical moiety having the formula:

106. The system of claim 105, wherein the reagent mixture also comprises one or more different chemical moieties each of which can be induced to luminesce at a different wavelength.

107. The system of claim 105, wherein the reagent mixture also comprises one or more different chemical moieties each of which can be induced to luminesce by exposure to energy of a different value or from a different source.

108. A system for determining the presence of an analyte of interest which binds to a chemical moiety, the moiety having the structural formula:

109. The system of claim 108, wherein the reagent mixture comprises one or more different chemical moieties each of which can be induced to luminesce at a different wavelength, each moiety being attached to a different analyte of interest.

110. The system of claim 108, wherein the reagent mixture comprises one or more different chemical moieties each of which can be induced to luminesce by exposure to energy of a different value or from a different source, each moiety being bound to a different analyte of interest.

111. A composition which comprises a chemical moiety having the formula:

112. The method of claim 111, wherein B is further defined as a substance covalently bound to one or more of L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹, L¹⁰, L¹¹, or L¹² through one or more amide linkages.

113. The composition of claim 111, wherein B is a whole cell, subcellular particle, nucleic acid, polysaccharide, protein, lipoprotein, lipopolysaccharide, glycoprotein, polypeptide, amino acid, cellular metabolite, hormone, tranquilizer, barbiturate, alkaloid, steroid, vitamin, or non-biological polymer.

114. The composition of claim 113, wherein B is a serum-derived antibody or a monoclonal antibody.

115. The composition of claim 113, wherein B is a nucleotide or polynucleotide.

116. The composition of claim 113 wherein B is T4 thyroid hormone.

117. A method of determining the presence of a chemical moiety, the method comprising

(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:

 

wherein;

M′ and

M″

are metal ions;

L¹, L², L³, and L⁴ each is a bidentate aromatic heterocyclic nitrogen-containing ligand selected from the group consisting of bipyridyl, substituted bipyridyl, bipyrazyl, substituted bipyrazyl, terpyridyl, substituted terpyridyl, phenanthrolyl and substituted phenanthrolyl, wherein each of said substituted ligands is substituted by an alkyl, aryl, aralkyl, carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxycarbonyl, hydroxyainino, aminocarbonyl, amidine, guanidinium, ureide, sulfur-containing group, phosphorus-containing group, or the carboxylate ester of N-hydroxysuccinimide, each of said ligands beings the same or not the same as each other ligand;

P is a bridging ligand selected from the group consisting of dpp, bbpe, bphb, α

,ω

-(2,2′

-bipyrid-4-yl)-alkanes), 4,4′

-bipyridines, and 1,2-bis(diphenylphosphine)ethane;

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;

t is an integer equal to or greater than 1;

u is an integer equal to or greater than 1;

said link being covalent bonding linking B with at least one of L¹, L², L³, and L⁴;

L¹, L², L³ , L⁴, P 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 electrochemilaminesce; and

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