Electrochemiluminescent reaction utilizing amine-derived reductant

• US 6,165,729 A
• Filed: 04/15/1994
• Issued: 12/26/2000
• Est. Priority Date: 04/30/1986
• Status: Expired due to Term

• Current Assignee: BioVeris Corporation (Roche Holding AG)
• Original Assignee: Hyperion Catalysis International Incorporated
• Inventors: Leland, Jonathan Kent, Powell, Michael Joseph
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): GABEL, GAILENE
• Application Number:
  US08/196,315
• Time in Patent Office: 2,447 Days
• Field of Search: 435/5, 435/6, 435/7.1, 435/8, 435/808, 435/968, 435/975, 422/52, 422/90, 422/98, 436/501, 436/546, 436/800, 436/805, 436/806, 436/807, 436/84, 436/172, 935/78
• US Class Current: 435/7.1
• CPC Class Codes: C07D 213/22 containing two or more pyridine rings directly linked together, e.g. bipyridyl
• CPC Class Codes: C07D 213/48 Aldehydo radicals
• CPC Class Codes: C07D 213/55 Acids; Esters
• CPC Class Codes: C07D 401/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 405/14 containing three or more hetero rings
• CPC Class Codes: C07D 473/08 with methyl radicals in positions 1 and 3, e.g. theophylline
• CPC Class Codes: C07F 13/00 Compounds containing elements of Groups 7 or 17 of the Periodic Table
• 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: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: G01N 2458/30 Electrochemically active labels
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/569 for microorganisms, e.g. protozoa, bacteria, viruses
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/76 Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
• CPC Class Codes: Y10S 435/808 Optical sensing apparatus
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 435/975 Kit
• 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 436/807 : Apparatus included in process claim, e.g. physical support structures

Abstract

Electrochemiluminescent assay methods for determining an analyte of interest, which methods include forming one or more compositions which include, in the aggregate, (a) a sample to be tested for the analyte of interest, (b) a component capable of specifically binding with the analyte, (c) a label reagent which, when oxidized, is capable of electrochemiluminescence, (d) an amine which, when oxidized, forms a reducing agent, and (e) an electrolyte solution; subjecting such a composition containing the sample to conditions sufficient for specific binding to occur between the analyte of interest, if present, and one or more of the other components in the composition; thereafter, a composition containing the label reagent and amine, and reflecting the outcome of applying the aforementioned binding conditions, is exposed to conditions, such that both the label reagent and the amine are oxidized, the amine forms a reducing agent which interacts with the label reagent, electrochemiluminescence occurs, and the luminescence emitted from the label reagent is used to determine the analyte of interest.

Claims

1. An electrochemiluminescent assay method of determining the presence or absence of or quantitating an analyte of interest, which consists essentially of the steps of:

(a) forming a composition consisting essentially of

(i) a sample to be tested for said analyte of interest;

(ii) a specific binding component capable of specifically binding with said analyte to form a binding product in the event said analyte is present in said sample;

(iii) a label reagent comprising a chelate of a transition metal or a rare earth metal which chelate, when oxidized, is capable of being converted to an excited state which electrochemiluminesces, and further comprising a species linked to said chelate which species is said analyte, an analog thereof, said specific binding component as recited in subparagraph (ii), a binding partner of said analyte, analog or specific binding component, or a binding partner capable of forming part of a combination of binding partners wherein at least one of the binding partners in such combination is capable of binding with said analyte, said analog, or said specific binding component;

(iv) an amine which, when oxidized, forms a reducing agent, said amine being selected from the group consisting of

(x) aliphatic amines, aromatic amines, diamines, polyamines, and heterocyclic amines,

(y) the amines defined in subparagraph (a)(iv)(x) substituted by one or more substituents selected from the group consisting of --OH, alkyl, chloro, fluoro, bromo, iodo, --SO₃, aryl, --SH, --C(O)--H, --C(O)--OH, ester groups, ether groups, alkenyl, alkynyl, --C(O)--, --N₂⁺, cyano, epoxide groups and heterocyclic groups, and

(z) protonated salts of the amines defined in subparagraph (a)(iv)(x) and protonated salts of the substituted amines defined in subparagraph (a)(iv)(y); and

(v) an electrolyte solution which functions as a medium in which said chelate and said amine can be oxidized;

(b) subjecting said composition to conditions sufficient for said specific binding to occur in the event said analyte is present in said sample;

(c) thereafter oxidizing the chelate and the amine such that an interaction between resulting oxidized products produces electrochemiluminescence; and

(d) detecting luminescence emitted as a result of the electrochemiluminescence of said label reagent to determine the presence or absence, or the amount, of said analyte.

2. The method as defined in claim 1, which comprises forming said composition from a kit for performing an electrochemiluminescent assay by combining a first kit component including a member of the group consisting of said label reagent moiety (i), said amine (ii), and said electrolyte solution (iii), and a second kit component including the remaining members of said group.

3. The method as defined in claim 1, wherein said pH is 6-9.

4. The method as defined in claim 1, wherein the amine is present in a stoichiometric excess in respect of the label reagent.

5. The method as defined in claim 1, wherein the metal of the metal chelate is selected from the group consisting of ruthenium, osmium, rhenium, iridium, rhodium, platinum, indium, palladium, molybdenum, technetium, copper, chromium and tungsten.

6. The method as defined in claim 5, wherein said metal is ruthenium or osmium.

7. The method as defined in claim 1, wherein the reducing agent formed by said amine has a redox potential, E_a, defined as follows:

##EQU3## wherein h is Planck'"'"'s constant, c is the speed of light, λ

is the wavelength characteristic of electrochemiluminescence emitted from the excited state of the metal chelate, K is the product of the absolute temperature in degrees Kelvin and the change in entropy, and E_m is the redox potential of the metal chelate.

8. The method as defined in claim 1, wherein said amine is tripropyl amine.

9. The method as defined in claim 1, wherein the metal chelate is a ruthenium-containing chelate, the amine is tripropyl amine and the electrolyte solution consists essentially of a phosphate buffer.

10. The method as defined in claim 1, wherein the composition is aqueous.

11. The method as defined in claim 1, wherein the composition is non-aqueous.

12. The method as defined in claim 1, wherein the electrolyte solution consists essentially of a phosphate buffer.

13. The method as defined in claim 1, wherein the electrolyte solution consists essentially of an organic liquid.

14. The method as defined in claim 1, wherein the electrolyte solution consists essentially of acetonitrile, dimethylsulfoxide, dimethylformamide, methanol, ethanol, or a mixture of two or more thereof.

15. The method of claim 1, wherein said chelate is incorporated in a moiety which also includes said specific binding component.

16. The method of claim 1, wherein said conditions sufficient for said specific binding to occur include immobilizing one of said analyte of interest and one of the species selected from the group consisting of said specific binding component as recited in subparagraph (a)(ii), said binding partner of said analyte, analog or specific binding component, and a binding partner capable of forming part of a combination of binding partners to a solid support, wherein said species binds to said analyte if said analyte is present, and contacting said species or analyte immobilized on the solid support with the other of said analyte of interest or said species.

17. The method as claimed in claim 1 wherein said species is said analyte, an analog thereof, or said specific binding component.

30. The method of any one of claims 1, 18 or 28, wherein said specific binding is immunological binding, hybridization, enzyme-substrate binding or ligand-receptor binding.

18. An electrochemiluminescent assay method of determining the presence or absence of or quantitating an analyte of interest, which consists essentially of the steps of:

(a) forming a first composition consisting essentially of a sample to be tested for said analyte of interest and a specific binding component capable of specifically binding with said analyte to form a binding product in the event said analyte is present in said sample;

(b) subjecting said first composition to conditions sufficient for said specific binding to occur in the event said analyte is present in said sample;

(c) forming a second composition consisting essentially of

(i) said specific binding component, or said product of said specific binding, or both;

(ii) a label reagent comprising a chelate of a transition metal or a rare earth metal which chelate, when oxidized, is capable of being converted to an excited state which electrochemiluminesces, and further comprising a species linked to said chelate which species is said analyte, an analog thereof, said specific binding component as recited in subparagraph (i), the product of said specific binding as recited in subparagraph (i), a binding partner of said analyte, analog, specific binding component, product of specific binding, or a binding partner which is capable of forming part of a combination of said binding partners wherein at least one of the binding partners in such combination is capable of binding with said analyte, said analog, said specific binding component, or said product of said specific binding;

(iii) an amine which, when oxidized, forms a reducing agent, said amine being selected from the group consisting of;

(x) aliphatic amines, aromatic amines, diamines, polyamines, and heterocyclic amines,

(y) the amines defined in subparagraph (c)(iii)(x) substituted by one or more substituents selected from the group consisting of --OH, alkyl, chloro, fluoro, bromo, iodo, --SO₃, aryl, --SH, --C(O), --C(O)--OH, ester groups, ether groups, alkenyl, alkynyl, --C(O)--, --N₂⁺, cyano, epoxide groups, and heterocyclic groups, and

(z) protonated salts of the amines defined in subparagraph (c)(iii)(x), and protonated salts of the substituted amines defined in subparagraph (c)(iii)(y); and

(iv) an electrolyte solution which functions as a medium in which said chelate and said amine can be oxidized;

(d) thereafter oxidizing the chelate and the amine such that an interaction between resulting oxidized products produces electrochemiluminescence; and

(e) detecting luminescence emitted as a result of the electrochemiluminescence of said label reagent to determine the presence or absence, or the amount, of said analyte.

19. The method as defined in claim 18, wherein said amine has a redox potential, E_a, defined as follows:

##EQU4## wherein h is Planck'"'"'s constant, c is the speed of light, λ

is the wavelength characteristic of electrochemiluminescence emitted from the excited state of the label reagent moiety, K is the product of the absolute temperature in degrees Kelvin and the change in entropy, and E_m is the redox potential of the electrochemiluminescent moiety.

20. The method as defined in claim 18, wherein said amine is tripropyl amine.

21. The method as defined in claim 18, wherein the metal is selected from the group consisting of ruthenium, osmium, rhenium, iridium, rhodium, platinum, indium, palladium, molybdenum, technetium, copper, chromium and tungsten.

22. The method as defined in claim 21, wherein the metal is ruthenium or osmium.

23. The method as defined in claim 18, wherein the composition is aqueous.

24. The method as defined in claim 18, wherein the chelate is a ruthenium-containing chelate, the amine is tripropyl amine and the electrolyte solution consists essentially of a phosphate buffer.

25. The method of claim 18, wherein said chelate is incorporated in a moiety which also includes said specific binding component.

26. The method of claim 18, wherein said conditions sufficient for said specific binding to occur include immobilizing one of said analyte of interest and one of the species selected from the group consisting of said specific binding component as recited in subparagraph (a)(ii), said binding partner of said analyte, analog or specific binding component, and a binding partner capable of forming part of a combination of binding partners to a solid support, wherein said species binds to said analyte if said analyte is present, and contacting said species or analyte immobilized on the solid support with the other of said analyte of interest or said species.

27. The method as claimed in claim 18 wherein said species is said analyte, an analog thereof, or said specific binding component.

28. A method of determining the presence or absence of or quantitating an analyte of interest by electrochemiluminescent assay, which consists essentially of:

(1) forming a reaction mixture consisting essentially of

(a) a sample to be tested for the analyte;

(b) at least one substance which is

(i) a predetermined amount of said analyte or an analog of the analyte, and

(ii) a binding partner of the analyte or said analog thereof, or a binding partner which is capable of forming part of a combination of binding partners wherein at least one of the binding partners in such combination is capable of binding with said analyte or said analog,

(c) a label reagent comprising a chelate of a transition metal or a rare earth metal, which reagent

(i) is capable of specifically binding with the analyte or at least one substance present from among (b)(i) and(b)(ii), and

(ii) when oxidized, is capable of being converted to an excited state which electrochemiluminesces;

(d) an amine which, when oxidized, forms reducing agent, said amine being selected from the group consisting off

(x) aliphatic amines, aromatic amines, diamines, polyamines, and heterocyclic amines, and

(y) the amines defined in subparagraph (1)(d)(x) substituted by one or more substituents selected from the group consisting of --OH, alkyl, chloro, fluoro, bromo, iodo, --SO₃, aryl, --SH, --C(O)--H, --C(O)--OH, ester groups, ether groups, alkenyl, alkynyl, --C(O)--, --N₂⁺, cyano, epoxide groups, and heterocyclic groups, and

(z) protonated salts of the amines defined in subparagraph (1)(d)(x), and protonated salts of the substituted amines defined in subparagraph (1)(d)(y); and

(e) an electrolyte solution which functions as a medium in which said chelate and said amine can be oxidized;

(2) subjecting said composition to conditions sufficient for said specific binding to occur in the event said analyte is present in said sample;

(3) thereafter oxidizing the chelate and the amine such that an interaction between resulting oxidized products produces electrochemiluminescence; and

(4) detecting luminescence emitted as a result of the electrochemiluminescence of said label reagent to determine the presence or absence of or the amount of said analyte.

29. The method as defined in claim 28 wherein the analyte of interest selected from the group consisting of a cell, subcellular particle, virus, prion, viroid, antibody, antigen, hapten, fatty acid, nucleic acid, lipoprotein, polysaccharide, lipopolysaccharide, glycoprotein, peptide, polypeptide, cellular metabolite, hormone, pharmacological agent, nonbiological polymer, synthetic organic molecule, organometallic molecule, tranquilizer, barbituate, alkaloid, steroid, vitamin, amino acid, sugar, lectin, biotin, avidin, streptavidin, and inorganic molecule.

31. The method as defined in claim 28, wherein said amine is tripropyl amine.

32. The method as defined in claim 28, wherein the electrolyte solution is aqueous.

33. The method as defined in claim 28, wherein the chelate is a ruthenium-containing chelate, the amine is tripropyl amine and the electrolyte solution consists essentially of a phosphate buffer.

34. The method as claimed in claim 28 wherein said species is said analyte, an analog thereof, or said specific binding component.

35. A system for electrochemiluminescent detection or quantitation of an analyte of interest in a sample, said system consisting essentially of

(a) at least one substance which is a predetermined amount of

(i) said analyte or an analog of the analyte, and

(ii) a binding partner of the analyte or said analog thereof, or a binding partner which is capable of forming part of a combination of binding partners wherein at least one of the binding partners in such combination is capable of binding with said analyte or said analog,

said at least one substance being linked to a chelate of a transition metal or a rare earth metal, which chelate is capable of being converted to an excited state which electrochemiluminesces;

(b) an amine which, when oxidized, forms a reducing agent, said amine being selected from the group consisting of

(x) aliphatic amines, aromatic amines, diamines, polyamines, and heterocyclic amines,

(y) the amines defined in subparagraph (b)(x) substituted by one or more substituents selected from the group consisting of --OH, alkyl, chloro, fluoro, bromo, iodo, SO₃, aryl, --SH, --C(O)--H, --C(O)--OH, ester groups, ether groups, alkenyl, alkynyl, --C(O)--, --N₂⁺, cyano, epoxide groups, and heterocyclic groups, and

(z) protonated salts of the amines defined in subparagraph (b)(x), and protonated salts of the substituted amines defined in subparagraph (b)(y); and

(c) an electrolyte;

(d) means for oxidizing both the chelate and the amine such that an interaction between resulting oxidized products produces electrochemiluminescence; and

(e) means for detecting emitted luminescence to determine the presence or absence or the amount of said analyte in the sample.

36. The method as defined in claim 35, wherein said composition is at a physiological pH during said oxidation step.

37. The system as claimed in claim 35 wherein said species is said analyte, an analog thereof, or said specific binding component.

38. An electochemiluminescent assay method for determining the presence of or quantitating an analyte of interest, said method comprising the steps of:

(a) forming a complex comprising;

(i) said analyte;

(ii) a first specific binding partner of said analyte; and

(iii) an electrochemiluminescent label comprising a chelate of a transition metal or a rare earth metal, which chelate, when oxidized, is capable of being converted to an excited state which electrochemiluminesces, wherein said chelate is linked to said first specific binding partner;

(b) forming a composition comprising;

(i) said complex;

(ii) an amine which forms a reducing agent upon oxidation, said amine being selected from the group consisting of;

(x) aliphatic amines, aromatic amines, diamines, polyamines, and heterocyclic amines,

(y) the amines defined in subparagraph (b)(ii)(x) substituted by one or more substituents selected from the group consisting of --OH, alkyl, chloro, fluoro, bromo, iodo, --SO₃, aryl, --SH, --C(O)--H, --C(O)--OH, ester groups, ether groups, alkenyl, alkynyl, --C(O)--, --N₂⁺, cyano, epoxide groups, and heterocyclic groups, and

(z) protonated salts of the amines defined in subparagraph (b))(ii)(x) and protonated salts of the substituted amines defined in subparagraph (b)(ii)(y); and

(iii) an electrolyte solution which functions as a medium in which said chelate and said amine can be oxidized;

(c) exposing said composition to electrochemical energy such that both the chelate and the amine are oxidized and an interaction between resulting oxidized products produces electrochemiluminescence, said electrochemiluminescence being related to the amount of said analyte;

(d) detecting said electrochemiluminescence and thereby determining the presence or amount of said analyte.

39. The method as claimed in claim 38, wherein said amine functions to ensure the generation of electrochemiluminescence.

40. The method as claimed in claim 38, wherein said composition does not include an additional reducing agent in an amount sufficient to ensure the generation of electrochemiluminescence.

41. The method as claimed in claim 38, wherein said linkage between said electrochemiluminescent label and said first specific binding partner is covalent.

42. The method as claimed in claim 38, wherein said chelate is covalently attached to a second specific binding partner bound through a specific binding interaction with said first specific binding partner, thereby providing a link between said chelate and said first specific binding partner.

43. The method of as claimed in claim 38, wherein said first specific binding partner and said analyte enter into a binding event selected from the group consisting of immunological binding, hybridization, enzyme-substrate binding, and ligand-receptor binding.

44. An electrochemiluminescent assay method for determining the presence of or quantitating an analyte of interest, said method comprising the steps of:

(a) forming a complex comprising;

(i) an analog of said analyte;

(ii) a first specific binding partner of said analyte and said analog; and

(iii) an electrochemiluminescent label comprising a chelate of a transition metal or a rare earth metal, which chelate, when oxidized, is capable of being converted to an excited state which electrochemiluminesces, wherein said chelate is linked to said analog and said analog competes for binding with said analyte for said first specific binding partner;

(b) forming a composition comprising;

(i) said complex;

(ii) said analyte;

(iii) an amine which forms a reducing agent upon oxidation, said amine being selected from the group consisting of;

(x) aliphatic amines, aromatic amines, diamines, polyamines, and heterocyclic amines,

(y) the amines defined in subparagraph (b)(iii)(x) substituted by one or more substituents selected from the group consisting of --OH, alkyl, chloro, fluoro, bromo, iodo, --SO₃, aryl, --SH, --C(O)--H, --C(O)--OH, ester groups, ether groups, alkenyl, alkynyl, --C(O)--, --N₂⁺, cyano, epoxide groups, and heterocyclic groups, and

(z) protonated salts of the amines defined in subparagraph (b)(iii)(x) and protonated salts of the substituted amines defined in subparagraph (b)(iii)(y); and

(iii) an electrolyte solution which functions as a medium in which said chelate and said amine can be oxidized;

(c) exposing the composition to electrochemical energy such that both the chelate and the amine are oxidized and an interaction between resulting oxidized products produces electrochemiluminescence, said electrochemiluminescence being related to the amount of said analyte present; and

(d) detecting said electrochemiluminescence and thereby determining the presence or amount of said analyte.

45. The method as claimed in claim 44, wherein said amine functions to ensure the generation of electrochemiluminescence.

46. The method as claimed in claim 44, wherein said composition does not include an additional reducing agent in an amount sufficient to ensure the generation of electrochemiluminescence.

47. The method as claimed in claim 44, wherein said linkage between said electrochemiluminescent label and said first specific binding partner is covalent.

48. The method as claimed in claim 44, wherein said chelate is covalently attached to a second specific binding partner bound through a specific binding interaction with said first specific binding partner, thereby providing a link between said chelate and said first specific binding partner.

49. The method as claimed in claim 44 wherein said first specific binding partner and said analyte and/or said analog enter into a binding event selected from the group consisting of immunological binding, hybridization, enzyme-substrate binding, and ligand-receptor binding.

50. An electrochemiluminescent assay method for determining the presence of or quantitating an analyte of interest, said method comprising the steps of:

(a) forming a first composition comprising;

(i) said analyte;

(ii) a first specific binding partner of said analyte; and

(iii) an electrochemiluminescent label comprising a chelate of a transition metal or a rare earth metal, which chelate, when oxidized, is capable of being converted to an excited state which electrochemiluminesces, wherein said chelate is linked to said first specific binding partner;

(b) forming a second composition comprising;

(i) said first composition;

(ii) an amine which forms a reducing agent upon oxidation, said amine being selected from the group consisting of;

(x) aliphatic amines, aromatic amines, diamines, polyamines, and heterocyclic amines,

(y) the amines defined in subparagraph (b)(ii)(x) substituted by one or more substituents selected from the group consisting of --OH, alkyl, chloro, fluoro, bromo, iodo, --SO₃, aryl, --SH, --C(O)--H, --C(O)--OH, ester groups, ether groups, alkenyl, alkynyl, --C(O)--, --N₂⁺, cyano, epoxide groups, and heterocyclic groups, and

(z) protonated salts of the amines defined in subparagraph (b)(ii)(x) and protonated salts of the substituted amines defined in subparagraph (b)(ii)(y); and

(iii) an electrolyte solution which functions as a medium in which said chelate and said amine can be oxidized;

(c) exposing said second composition to electrochemical energy such that both the chelate and the amine are oxidized and an interaction between resulting oxidized products produces electrochemiluminescence, said electrochemiluminescence being related to the amount of said analyte present;

(d) detecting said electrochemiluminescence and thereby determining the presence or amount of said analyte.

51. The method as claimed in claim 50, wherein said amine functions to ensure the generation of electrochemiluminescence.

52. The method as claimed in claim 50, wherein said second composition does not include an additional reducing agent in an amount sufficient to ensure the generation of electrochemiluminescence.

53. The method as claimed in claim 50, wherein said linkage between said chelate and said first specific binding partner is covalent.

54. The method as claimed in claim 50, wherein said chelate is covalently attached to a second specific binding partner bound through a specific binding interaction with said first specific binding partner, thereby providing a link between said chelate and said first specific binding partner.

55. The method as claimed in claim 50, wherein first specific binding partner and said analyte enter into a binding event selected from the group consisting of immunological binding, hybridization, enzyme-substrate binding, and ligand-receptor binding.

56. An electrochemiluminescent assay method for determining the presence or absence of or quantitating an analyte of interest, said method comprising the steps of:

(a) forming a first composition comprising;

(i) an analog of said analyte;

(ii) a first specific binding partner of said analyte and said analog; and

(iii) an electrochemiluminescent label comprising a chelate of a transition metal or a rare earth metal, which chelate, when oxidized, is capable of being converted to an excited state which electrochemiluminesces, wherein said chelate is linked to said analog and said analog and said first specific binding partner bind to one another if said analyte is present, such that said analyte competes for binding with said analog;

(b) forming a second composition comprising;

(i) said first composition;

(ii) said analyte;

(iii) an amine which forms a reducing agent upon oxidation, said amine being selected from the group consisting of;

(x) aliphatic amines, aromatic amines, diamines, polyamines, and heterocyclic amines,

(y) the amines defined in subparagraph (b)(iii)(x) substituted by one or more substituents selected from the group consisting of --OH, alkyl, chloro, fluoro, bromo, iodo, --SO₃, aryl, --SH, --C(O)--H, --C(O)--OH, ester groups, ether groups, alkenyl, alkynyl, --C(O)--, --N₂⁺, cyano, epoxide groups, and heterocyclic groups, and (z) protonated salts of the amines defined in subparagraph (b)(iii)(x) and protonated salts of the substituted amines defined in subparagraph (b)(iii)(y); and

(iv) an electrolyte solution which functions as a medium in which said chelate and said amine can be oxidized;

(c) exposing the second composition to electrochemical energy such that both the chelate and the amine are oxidized and an interaction between resulting oxidized products produces electochemiluminescence, said electrochemiluminescence being related to the amount of said analyte present; and

(d) detecting said electrochemiluminescence and thereby determining the presence or amount of said analyte.

57. The method as claimed in claim 56, wherein said amine functions to ensure the generation of electrochemiluminescence.

58. The method as claimed in claim 56, wherein said second composition does not include an additional reducing agent in an amount sufficient to ensure the generation of electrochemiluminescence.

59. The method as claimed in claim 56, wherein said linkage between said electrochemiluminescent label and said first specific binding partner is covalent.

60. The method as claimed in claim 56, wherein said chelate is covalently attached to a second specific binding partner bound through a specific binding interaction with said first specific binding partner, thereby providing a link between said chelate and said first specific binding partner.

61. The method as claimed in claim 56 wherein said first specific binding partner and said analyte enter into a binding event selected from the group consisting of immunological binding, hybridization, enzyme-substrate binding, and ligand-receptor binding.