Electrochemiluminescence of rare earth metal chelates

Abstract

Luminescent chemical reagents that include complexes of rare earth metals with ligands such as aromatic heterocyclic nitrogen-containing compounds and semi-aromatic oxygen-containing compounds are used to detect small quantities of complex substances such as pharmaceuticals, metabolites, and microorganisms in complex sample mixtures.

What is claimed is:  
     
         1 . A chemical moiety having the formula  
       [MPL 1 L 2 -(-link-)-] t B  wherein M is a lanthanide element;    P is a polydentate ligand of M;    L 1  and L 2  are ligands of M, each of which may be a substance covalently bound to one or more of P, L 1 , or L 2  through one or more covalent bond linkages, said linkages designated as (-link-) and being covalent bonds linking B with at least one of P, L 1 , or L 2 , and t is an integer equal to or greater than 1;    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;    P, L 1 , L 2 , and B are of such number that the total number of bonds to M provided by the ligands of M equals the coordination number of M; and    P, L 1 , L 2 , and B are of such composition that the chemical moiety can be induced to emit electromagnetic radiation.    
     
     
         2 . The chemical moiety of  claim 1 , wherein B is a member selected from the group consisting of whole cells, subcellular particles, polypeptides, proteins, nucleic acids, polysaccharides alkaloids, steroids, vitamins, amino acids, plant pathogens, serum-derived antibodies, monoclonal antibodies, and T4 thyroid hormones.  
     
     
         3 . The chemical moiety of  claim 1 , wherein (-link-) is the linkage formed between one of said ligands and a free amino group which is part of B.  
     
     
         4 . The chemical moiety of  claim 1 , wherein (-link-) is the linkage formed between a carboxyl group and a free amino group which is part of B.  
     
     
         5 . The chemical moiety of  claim 1 , wherein M is europium or terbium and (-link-) is an amide linkage that covalently bonds B with a substituted bipyridyl ligand, the amide linkage being formed by reaction of a carboxyl substituent on said substituted bipyridyl ligand with a free amino group which is part of B.  
     
     
         6 . The chemical moiety of  claim 1 , wherein M is europium and P, L 1 , and L 2  are each 6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedionato ligands.  
     
     
         7 . The chemical moiety of  claim 1 , wherein M is terbium and P, L 1 , and L 2  are each 2,2,6,6-tetramethyl-3,5-heptanedionato ligands.  
     
     
         8 . A method of determining the presence of a chemical moiety, said chemical moiety having the formula  
       [MPL 1 L 2 -(-link-)-] t B  wherein M is a lanthanide;    P is a polydentate ligand of M;    L 1  and L 2  are ligands of M, each of which may be a substance covalently bound to one or more of P, L 1 , or L 2  through one or more covalent bond linkages, said linkages designated as (-link-) and being covalent bonds linking B with at least one of P, L 1 , or L 2 ;    t is an integer equal to or greater than 1;    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;    P, L 1 , L 2 , and B are of such number that the total number of bonds to M provided by the ligands of M equals the coordination number of M; and    P, L 1 , L 2 , and B are of such composition that the chemical moiety can be induced to emit electromagnetic radiation;    which method 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;    (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 electrochemiluminescence; and    (c) detecting emitted luminescence thereby to determine the presence of the chemical moiety.    
     
     
         9 . The method of  claim 8 , wherein M is europium; 
 P, L 1 , and L 2 , each is a semi-aromatic oxygen-containing ligand, each of said ligands being the same or not the same as each other ligand; and    (-link-) is one or more amide linkages, ester or thioester linkages, or ether or thioether linkages, each said linkage covalently bonding B with at least one of P, L 1 , and L 2 .    
     
     
         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    [MPL 1 L 2 -(-link-)-] t B  wherein M is a lanthanide;    P is a polydentate ligand of M;    L 1  and L 2  are ligands of M, each of which may be a substance covalently bound to one or more of P, L 1 , or L 2  through one or more covalent bond linkages, said linkages designated as (-link-) and being covalent bonds linking B with at least one of P, L 1 , or L 2 ;    t is an integer equal to or greater than 1;    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 non-biological polymer;    P, L 1 , L 2 , and B are of such number that the total number of bonds to M provided by the ligands of M equals the coordination number of M; and    P, L 1 , L 2 , and B are of such composition that the chemical moiety can be induced to emit electromagnetic radiation;      (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 electrochemiluminescence; and    (c) means for detecting emitted luminescence thereby to determine the presence of the chemical moiety.    
     
     
         11 . 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    [MPL 1 L 2 -(-link-)-] t B  wherein M is a lanthanide;    P is a polydentate ligand of M;    L 1  and L 2  are ligands of M, each of which may be a substance covalently bound to one or more of P, L 1 , or L 2  through one or more covalent bond linkages, said linkages designated as (-link-) and being covalent bonds linking B with at least one of P, L 1 , or L 2 ;    t is an integer equal to or greater than 1;    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;    P, L 1 , L 2 , and B are of such number that the total number of bonds to M provided by the ligands of M equals the coordination number of M; and      P, L 1 , L 2 , and B are of such composition that the chemical moiety can be induced to emit electromagnetic radiation;    (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 electrochemiluminescence; and    (c) detecting emitted luminescence thereby to determine the presence of the analyte of interest.    
     
     
         12 . A composition which comprises 
 (i) a chemical moiety having the formula    [MPL 1 L 2 -(-link-)-] t B  wherein M is a lanthanide;    P is a polydentate ligand of M;    L 1  and L 2  are ligands of M, each of which may be a substance covalently bound to one or more of P, L 1 , or L 2  through one or more covalent bond linkages, said linkages designated as (-link-) and being covalent bonds linking B with at least one of P, L 1 , or L 2 , and    t is an integer equal to or greater than 1;    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;    P, L 1 , L 2 , and B are of such number that the total number of bonds to M provided by the ligands of M equals the coordination number of M; and    P, L 1 , L 2 , and B are of such composition that the chemical moiety can be induced to emit electromagnetic radiation;    said linkages being covalent bonding linking B with at least one of P, L 1 , or L 2 , and      (ii) 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.    
     
     
         13 . 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    [MPL 1 L 2 -(-link-)-] t B  wherein M is a lanthanide;    P is a polydentate ligand of M;    L 1  and L 2  are ligands of M, each of which may be a substance covalently bound to one or more of P, L 1 , or L 2  through one or more covalent bond linkages, said linkages designated as (-link-) and being covalent bonds linking B with at least one of P, L 1 , or L 2 ;    t is an integer equal to or greater than 1;    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 binding to the complementary material;    P, L 1 , L 2 , and B are of such number that the total number of bonds to M provided by the ligands of M equals the coordination number of M; and    P, L 1 , L 2 , and B are of such composition that the chemical moiety can be induced to emit electromagnetic radiation;      (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 electrochemiluminescence; and    (c) detecting emitted luminescence thereby to determine the presence of the analyte of interest.    
     
     
         14 . 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    [MPL 1 L 2 -(-link-)-] t B  wherein M is a lanthanide;    P is a polydentate ligand of M;    L 1  and L 2  are ligands of M, each of which may be a substance covalently bound to one or more of P, L 1 , or L 2  through one or more covalent bond linkages, said linkages designated as (-link-) and being covalent bonds linking B with at least one of P, L 1 , or L 2 ;    t is an integer equal to or greater than 1;    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;    P, L 1 , L 2 , and B are of such number that the total number of bonds to M provided by the ligands of M equals the coordination number of M; and    P, L 1 , L 2 , and B are of such composition that the chemical moiety can be induced to emit electromagnetic radiation;      (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, 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 electrochemiluminescence; and    (d) means for detecting emitted luminescence thereby to determine the presence of the chemical moiety.    
     
     
         15 . A compound having the formula  
       MPL 1 L 2 -(-link-)  wherein M is a lanthanide element;    P is a polydentate ligand of M;    L 1  and L 2  are ligands of M, each of which may be a substance covalently bound to one or more of P, L 1 , or L 2  through one or more covalent bond linkages, said linkages designated as (-link-) and being covalent bonds;    P, L 1 , and L 2  are of such number that the total number of bonds to M provided by the ligands of M equals the coordination number of M; and    P, L 1 , and L 2  are of such composition that the chemical moiety can be induced to emit electromagnetic radiation.    
     
     
         16 . The chemical moiety of  claim 15 , wherein M is europium and P, L 1 , and L 2  are each 6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedionato ligands.  
     
     
         17 . The chemical moiety of  claim 15 , wherein M is terbium and P, L 1 , and L 2  are each 2,2,6,6-tetramethyl-3,5-heptane-dionato ligands.  
     
     
         18 . The chemical moiety of  claim 15 , wherein (-link-) contains an N-hydroxysuccinimidyl moiety  
     
     
         19 . The chemical moiety of  claim 15 , wherein (-link-) contains an amino moiety  
     
     
         20 . The chemical moiety of  claim 15 , wherein (-link-) contains a thiol moiety