CA1339835C - Luminescent metal chelate labels and meand for detection - Google Patents

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 very small amounts of the chemical moiety using chemiluminescent, electrochemiluminescent, and photoluminescent means. Compounds are disclosed which are useful for labelling substances of interest with ruthenium-containing and osmium-containing labels or other electrochemiluminescent labels. These labelled substances are useful in methods provided for detecting and quantifying analytes of interest in binding assays and competitive binding assays. The labelled 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.

Description

1339.~35 LUMINESCENT ~lETAL r~T.I~TE r.~R~.T.~;
AND MEANS FOR DETECrION

8ackground of the Invention s There is a eontinuous ~nd ~Ypan~ing need for rapid, highly6pecific methods of detecting and quantifying chemical, biochemical, and biological ~ubstanees. Of partieular value ~re methods for measuring ~mall quantities of phar-maceuticals, metabolites, microorganisms and other ma-- terials of diagnostic value. Examples of ~uch materials include narcoties and poisons, drugs administered for therapeutic purposes, hormones, pathogenie mieroorganisms and viruses, antibodies, metabolites, enzymes and nucleic acids.

The presence of these materials can often be determined by binding methods which exploit the high degree of spe-~ificity whieh eharacterizes many bioehemical andbiological ~ystems. Frequently used methods are based on, for example, antigen-antibody ~ystems, nucleic acid hy-bridization techniques, and protein-ligand systems. In these methods, the existence of the eomplex of diagnostic value is typically indicated by the presence or absence of an observable ~label" which has been attached to one or more of the eomplexing materials.

The specific labelling method chosen often dictates the usefulness and versatility of a particular system for detecting a material of interest. A preferred label should ~

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-2- 133983~

be inexpensive, safe, and capable of being attached ef-ficiently to a wide variety of chemical, biochemical, and biological materials without changing the impcrtant bind-ing characteristics of those materials. The label should give a highly characteristic signal, and should be rarely, or preferably never found in nature. The label should be stable and detectable in aqueous systems over periods of time ranging up to months. Detection of the label should be rapid, sensitive, and reproducible without the need for expensive, specialized facilities or personnel. Quanti-fication of the label should be relatively independent of variables such as temperature and the composition of the mixture to be assayed. Most advantageous are labels which can be used in homogeneous systems, i.e. systems in which separation of the complexed and uncomplexed labelled ma-terial is not necessary. This is possible if the detect-ability of the label is modulated when the labelled ma-terial is incorporated into a specific complex.

A wide variety of labels have been developed, each with particular advantages and disadvantages. For example, radiocctive labels are quite versatile, and can be detected at very low concentrations. However, they are expensive, hazardous, and their use requires sophisticated equipment and trained personrel. Furthermore, the sensitivity of radioactive labels is limited by the fact that the detect-able event can, in its essential nature, occur only once per radioactive atom in the labelled material. Moreover, ra-dioactive labels cannot be used in homogeneous methods.

Thus, there is wide interest in non-radioactive labels.
These include molecules observable by spectrophotometric, spin resonance, and luminescence techniques, as well as enzymes which produce such molecules. Among the useful non-radioactive labelling materials are organometallic ~3~ 1~983~

compounds. Because of the rarity of some metals in bio-logical systems, methods which specifically assay the metal component of the organometallic compounds can be successfully exploited. For example, Cais, U.S. Patent No.
4,205,952 discloses the use of immunochemically active materials labelled with certain organometallic compounds for use in quantitating specific antigens. Any general method of detecting the chosen metals can be used with these labels, including emission, absorption and fluorescence spectrometry, atomic absorption, and neutron activation.
These methods often suffer from lack of sensitivity, can seldom be adapted to a homogenous system, and as with atomic absorption, sometimes entail destruction of the sample.

Of particular interest are labels which can be made to luminesce through photochemical, chemical, and elec-trochemical means. "Photoluminescence" is the process whereby a material is induced to luminesce when it absorbs electromagnetic radiation. Fluorescence and phosphorescence are types of photoluminescence. "Chemi-luminescent" processes entail the creation of the lumi-nescent species by a chemical transfer of energy.
"Electrochemiluminescence" entails the creation of the luminescent species electrochemically.

These luminescent systems are of increasing importance.
For example, Mandle, U.S. Patent No. 4,372,745 discloses the use of chemiluminescent labels in immur!ochemical ap-plications. In the disclosed systems, the labels are excited into a luminescent state by chemical means such as by reaction of the label with H2~2 and an oxalate. In these systems, H2~2 oxidatively converts the oxalate into a high energy derivative, which then excites the label. This system will, in Frinciple, work with any luminescent ma-terial that is stable in the oxidizing conditions of the , 13;~983~

assay and can be excited by the high energy oxalate de-rivative. Unfortunately, this very versatility is the source of a major limitation of the technique: typical biological fluids containing the analyte of interest also contain a large number of potentially luminescent sub-stances that can cause high background levels of luminescence.

Another example of the immunochemical use of chemilumin-escence which suffers from the same disadvantages is Ober-hardt et al., U.S. Patent No. 4,280,815, who disclose the in situ electrochemical generation of an oxidant (e.g., H2O2) in close proximity to an immunoreactant labelled with a chemiluminescent species. The electrogenerated oxidant diffuses to the chemiluminescent species and chemically oxidizes it, resulting in the net transfer of one or more electrons to the electrogenerated oxidant. Upon oxida-tion, the chemiluminescent species emits a photon. In contrast, the subject invention requires the direct trans-fer of electrons from a source of electrochemical energy toa chemiluminescent species which is capable of repeatedly emitting photons.

The present invention is concerned with electrochemilum-inescent labels. Suitable labels comprise electrochem-iluminescent compounds, including organic compounds and organometallic compounds. Electrochemiluminescent meth-ods of determining the presence of labelled materials are preferred over other methods for many reasons. They are highly diagnostic of the presence of a particular label, sensitive, nonhazardous, inexpensive, and can be used in a wide variety of applications. Organic compounds which are suitable electrochemical labels include, for example, ru-brene and 9,10-diphenyl anthracene. Many organometallic compounds are suitable electrochemical labels, but of 1~3983~

particular use are Ru-containing and Os-containing com-pounds.

The present invention is concerned with the use of Ru-containing and Os-containing labels which can be detected by a wide variety of methods. These labels are advantageous for many reasons that will be discussed herein.

Ru-containing and Os-containing organometallic compounds have been discussed in the literature. Cais discloses that any metal element or combination of metal elements, in-cluding noble metals from group VIII such as Ru, would be suitable components of organometallic labels detectable by atomic absorption methods. (Cais, column 11, line 20). However, ruthenium is not a preferred metal in Cais, osmium is not specifically mentioned, no data is presented on the efficiency of using Ru or Os in any of the methods disclosed, and the preferred method of detection, atomic absorption, entails destruction of the sample.

Weber, U.S. Patent No. 4,293,310, discloses the use of Ru-containing and Os-containing complexes as electrochem-ical labels for analytes in immunoassays. The disclosed complexes are linked to amino groups on the analytes through a thiourea linkage. Weber also suggests the pos-sibility of forming carboxylate esters between the labels and hydroxy groups on other analytes.

According to Weber, the presence of the labelled materials can be determined with an apparatus and method which comprises a quencher and an electrochemical flow cell with light means. The photoelectrochemically active label upon photoexcitation transfers an electron to a quencher molecule; the oxidized molecule is subsequently reduced with an electron from an electrode of the flow cell which ~33~83-~

is held at suitable potential. This electron is measured as photocurrent. The amount of free labelled analyte in the system is determined by the photocurrent signal. Note that this method is the reverse of electrochemiluminescent detection of luminescent materials.

In subsequent reports, Weber et al. (1983), Clinical Chemistry 29, pp. 1665-1672, Photoelectroanalytical Chem-istry: Possible Interferences in Serum and Selective De-tection of Tris(2,2'-bipyridine)ruthenium(II) in the Pre-sence of Interferents, have discussed the problems asso-ciated with the use of this method to detect Ru-containing labels. In Table 2 of Weber et al., the extrapolated detection limit for tris(bipyridyl)ru-thenium(I~) is l.lxlO-l~ moles/L under optimal condi-tions. In anticipating that the actual use of these labels would entail measurements in the presence of complex mix-tures, Weber et al. tested for potential interferents in their system. Table 3 of Weber et al. lists dimethylalkyl amines, EDTA, N-methylmorpholine, N,N'-dimethylpipera-zine, hydroxide, oxalate, ascorbate, uric acid, and serum as interferents which would presumably raise the practical detection limit substantially above l.lxlO-10 moles/L.
These studies were performed with a simple Ru-containing compound. No studies were reported in Weber or Weber et al. regarding the limits of detection of complex sub-stances labelled with Ru-containing labels, or on whether the thiourea linkage between the labelled material and label is stable under conditions of the assay.

The particular labels with which the present invention is concerned are electrochemiluminescent. They can often be excited to a luminescent state without their oxidation or -7- ~3~833 reduction by exposing the compounds to electromagnetic radiation or to a chemical energy source such as that created by typical oxalate-H2O2 systems. In addition, luminescence of these compounds can be induced by electro-chemical methods which do entail their oxidation and re-duction.

Extensive work has been reported on methods for detecting Ru(2,2'-bipyridine)32+ using photoluminescent, chemi-luminescent, and electrochemiluminescent means: Rubin-stein and Bard (1981), "Electrogenerated Chemilum-inescence. 37. Aqueous Ecl Systems based on Ru(2,2'-bipyridine)32+ and Oxalate or Organic Acids", J. Am. Chem.
Soc., 103, pp. 512-516; and White and Bard (1982), "Electrogenerated Chemiluminescence. 41. Electrogen-erated Chemiluminescence and Chemiluminescence of the Ru(2,2'-bpy)32+ - S20g2~ System in Acetonitrile-Water So-lutions", J. Am. Chem. Soc., 104, p. 6891. This work demonstrates that bright orange chemiluminescence can be based on the aqueous reaction of chemically generated or electrogenerated Ru(bpy)33+ (where "bpy" represents a bipyridyl ligand) with strong reductants produced as in-termediates in the oxidation of oxalate ions or other organic acids. Luminescence also can be achieved in organic solvent-H2O solutions by the reaction of elec-trogenerated, or chemically generated, Ru(bpy)31+ with strong oxidants generated during reduction of peroxy-disulfate. A third mechanism for production of elec-trochemiluminescence from Ru(bpy)32+ involves the oscil-lation of an electrode potential between a potential suf-ficiently negative to produce Ru(bpy)31+ and sufficiently positive to produce Ru(bpy)33+. These three methods are called, respectively, "oxidative-reduction," "reduc-tive-oxidation," and "the Ru(bpy)33+/+ regenerative sys-tem".

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13398~

The oxidative-reduction method can be performed in water, and produces an intense, efficient, stable luminescence, which is relatively insensitive to the presence of oxygen or impurities. This luminescence from Ru(bpy)32+ depends upon the presence of oxalate or other organic acids such as pyruvate, lactate, malonate, tartrate and citrate, and means of oxidatively producing Ru(bpy)33+ species. This oxidation can be performed chemically by such strong oxi-dants as PbO2 or a Ce(IV) salt. It can be performed elec-trochemically by a sufficiently positive potential appliedeither continuously or intermittently. Suitable elec-trodes for the electrochemical oxidation of Ru(bpy)32+
are, for example, Pt, pyrolytic graphite, and glassy car-bon. Although the oxalate or other organic acid is consumed during chemiluminescence, a strong, constant chemiluminescence for many hours can be achieved by the presence of an excess of the consumed material, or by a continuous supply of the consumed material to the reaction chamber.
The reductive-oxidation method can be performed in par-tially aqueous solutions containing an organic cosolvent such as, for example, acetonitrile. This luminescence depends upon the presence of peroxydisulfate and a means of reductively producing Ru(bpy)31+ species. The reduction can be performed chemically by strong reductants such as, for example, magnesium or other metals. It can be performed electrochemically by a sufficiently negative potential ap-plied either continuously or intermittently. A suitable electrode for the electrochemical reduction of Ru(bpy)32+
is, for example, a polished glassy-carbon electrode. As with the oxidative-reduction method, continuous, intense luminescence can be achieved for many hours by inclusion of excess reagents, or by continuous addition of the consumed reagents to the reaction mixture.

~339~3~
g Thè Ru(bpy)33+/+ regenerative system can be performed in organic solvents such as acetonitrile or in partially aque-ous systems, by pulsing an electrode potential between a potential sufficiently negative to reduce Ru(bpy)32+ and a potential sufficiently positive to oxidize Ru(bpy)32+. A
suitable electrode for such a regenerative system is, for example, a Pt electrode. This system does not consume chemical reagents and can proceed, in principle, for an unlimited duration.
These three methods of producing luminescent Ru-containing compounds have in common the repetitive oxida-tion-reduction or reduction-oxidation of the Ru-con-taining compound. The luminescence of solutions con-taining these compounds is therefore highly dependent onthe electric potential of the applied energy source, and is therefore highly diagnostic of the presence of a Ru-con-taining compound.

Mandle cites Curtis et al. (1977), "Chemiluminescence; A
New Method for Detecting Fluorescent Compounds Separated By Thin Layer Chromatography", J. Chromatography 134, pp. 343-350, as identifying Ru-tris(bipyridyl)(II) as a possible label in chemiluminescent applications. Curtis et al. reports only unpublished observations that Ru com-plexes can be induced to emit light when chemically excited by an oxalate/H2O2 system (Curtis et al. p. 350).

Neither Mandle nor Curtis recognized the exceptional util-ity of ruthenium and osmium complexes in chemiluminescent applications or the utility of electrochemilumiscent sys-tems. Sprintschnik, G. et al. (1977), "Preparation and Photochemical Reactivity of Surfactant Ruthenium (II) Com-ple:~es in Monolayer Assemblies and at Water-Solid In-terfaces", J. Am. Chem. Soc. 99, pp. 4947-4954, have 1 3 ~ 3 described complexes of tris(2,2'-bipyridine)ruthenium(II) esterified with octadecanol or dehydrocholesterol, and have created monolayer films of these surfactant com-plexes. The complexes were photoluminescent. But when the films were exposed to water, and then to light, the Ru-complexes failed to photoluminesce. This was attrib-uted to photohydrolysis of ester groups in the presence of light.

It has been discovered and is disclosed herein, that a wide variety of analytes of interest and chemical moieties that bind to analytes of interest may be conveniently attached to Ru-containing or Os-containing labels through amide or amine linkages. The labelled materials may then be de-termined by any of a wide variety of means, but by far themost efficient, reliable, and sensitive means are photo-luminescent, chemiluminescent, and electrochemilumines-cent means. It is also disclosed herein that electro-chemilumiscent labels, including Ru-containing and Os-containing labels and organic molecules such as rubreneand 9,10-diphenyl anthracene, are particularly versatile and advantageous. The great advantages of the use of these novel labelled materials, and of the methods of detecting them, are further discussed hereinbelow.

1~9~3~

Summary of the Invention According to the present invention, there is provided a chemical moiety having the formula [M(P)m(Ll)n(L2)o(L3)p(L4)q(L5)r(L6)s]t(B)u wherein M is ruthenium or osmium; P is a polydentate ligand of M; Ll, L2, L3, L4, L5 and L6 are ligands of M, each of which may be the same as, or different from, each other ligand; B is a substance covalently bound to one or more of P, Ll, L2, L3, L4, L5 or L6 through one or more amide or amine linkages; m is an integer equal to or greater than l; each of n, o, p, q, r and s is zero or an integer; t is an integer equal to or greater than 1; u is an integer equal to or greater than l; and P, Ll, L2, L3, L4, L5, L6 and B
are of such composition and number that the chemical moiety can be induced to emit electromagnetic radiation and the total number of bonds to M provided by the ligands of M
equals the coordination number of M.

The present invention provides compounds particularly suitable as intermediates for attaching a luminescent ru-thenium- or osmium-containing label to amino groups of chemical, biochemical and biological substances. These intermediates are thus particularly suitable for creating chemical moieties according to the present invention. The intermediates ~:I e the mono- and di-N-hydroxysuccini-mide esters of ruthenium or osmium bis(2,2'-bipyridine) (2,2'-bipyridine-4,4'-dicarboxylic acid) and their salts; and ruthenium or osmium bis (2,2'-bipyridine) (4,4'-di(chloromethyl)-2,2'-bipyridine). These compounds may be synthesized by means known in the art.

~33~3~

The present invention provides methods for determining the presence of the novel chemical moieties.

The present invention also provides methods of determining - 5 the presence of a chemical moiety having the formula [M(P)m(Ll)n(L2)o(L3)p(L4)q(L5)r(L6)s]t(B)u wherein M is ruthenium or osmium; P is a polydentate ligand of M; Ll, L2, L3, L4, L5 and L6 are ligands of M, each of which may be the same, or different from, each other ligand;
B is a substance which is a ligand of M or is attached to one or more of P, Ll, L2, L3, L4, L5 or L6; m is an integer equal to or greater than l; each of n, o, p, q, r and s is zero or an integer; t is an integer equal to or greater than l; and u is an integer equal to or greater than l; and P, Ll, L2, L3, L4, L5, L6 and B are of such composition and number that the chemical moiety can be induced to emit electromagnetic radiation and the total number of bonds to M provided by the ligands of M equals the coordination number of M.

The method comprises:

a) forming a reagent mixture under suitable con-ditions containing the chemical moiety;

b) inducing the moiety to emit electromagnetic ra-diation by exposing the reagent mixture to chemical energy or electrochemical energy; and c) detecting the emitted electromagnetic radiation and thereby determining the presence of the chemical moiety.

-13- 133983~

This invention further provides for the use of ruthenium-containing and osmium-containing labels in binding methods for determining the presence of substances of interest. These methods may be used to determine labelled moieties of interest, to employ labelled moieties to determine analytes of interest, or to use labelled analogues of analytes of interest to determine analytes of interest in competitive binding assays. These binding methods may be homogeneous or heterogeneous binding meth-ods.

Still further, the present invention provides systems fordetermining the presence of the ruthenium-containing or osmium-containing chemical moieties of this invention.
These systems comprise a means for inducing the chemical moiety to emit electromagnetic radiation and a means for detecting the emitted electomagnetic radiation.

The present invention also provides systems for employing the ruthenium-containing or osmium-containing chemical moieties in binding methods for the determination of ana-lytes of interest.

According to the present invention, there is provided a method of determining the presence of a chemical moiety having the formula (A)k (B)u wherein A is a compound which can be induced to repeatedly emit electromagnetic radiation by direct exposure to an electrochemical energy source; B is a substance which is attached to A; k is an integer equal to or greater than l;
and u is an integer equal to or greater than 1, comprising:
a) forming a reagent mixture under suitable conditions 1339'~3~

containing the chemical moiety; b) inducing the chemical moiety to repeatedly emit electromagnetic radiation by directly exposing the moiety to electrochemical energy and c) detecting the emitted electromagnetic radiation and thereby determining the presence of the chemical moiety.

The present invention also provides for use of electro-chemiluminescent labels in binding methods for determining the presence of substances of interest. These methods can be used to determine labelled moieties of interest, to employ labelled moieties to determine analytes of inter-est, or to use labelled analogues of analytes of interest to determine analytes of interest in competitive binding assays. These binding methods can be homogeneous or heterogeneous binding methods.

A specific embodiment of the invention provides for com-positions which contain two or more different chemical moieties. Each of the moieties may be chemical species which can be induced to emit electromagnetic radiation of a different wavelength. In another embodiment of the invention the chemical moieties may be chemical species each of which is induced to emit electromagnetic radiation by exposure to energy of a different value or from a different source. A different substance or analyte of interest may then be specifically attached to each of the different chemical moieties. By using these compositions and methods it is possible to determine two or more differ-ent substances or analytes of interest that may be present in the sample under examination.

-15- 133~8~3 Detailed Description of the Invention According to the present invention, there is provided a chemical moiety having the formula s [M(P)m(Ll)n(L2)o(L3)p(L4)q(L5)r(L6)s]t(B)u wherein M is ruthenium or osmium; P is a polydentate ligand of M; Ll, L2, L3, L4, L5 and L6 are ligands of M, each of which may be the same as, or different from, each other ligand; B
is a substance covalently bound to one or more of P, Ll, L2, L3, L4, L5 or L6 through one or more amide or amine linkages;
m is an integer equal to or greater than l; each of n, o, p, q, r and s is zero or an integer; t is an integer equal to or greater than 1; u is an integer equal to or greater than l; and P, Ll, L2, L3, L4, L5, L6 and B are of such composition and number that the chemical moiety can be induced to emit electromagnetic radiation and the total number of bonds to M provided by the ligands of Mequals the coordination number of M.

This chemical moiety must have at least one polydentate ligand of M. If the moiety has greater than one polydentate ligand, the polydentate ligands may be the same or dif-ferent. Polydentate ligands include aromatic and aliphaticligands. Suitable aromatic polydentate ligands include aromatic heterocyclic ligands. Preferred aromatic hetero-cyclic ligands are nitrogen-containing, such as, for ex-ample, bipyridyl,bipyrazyl, terpyridyl, and phenanthrolyl.

Suitable polydentate ligands may be unsubstituted, or sub-stituted by any of a large number of substituents known to the art. Suitable substituents include for example, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substi-133~3~

tuted aralkyl, carboxylate, carboxaldehyde, carbo-xamide, cyano, amino, hydroxy, imino, hydroxycarbonyl, aminocar-bonyl, amidine, guanidinium, ureide,-sulfur-containing groups, phosphorus containing groups, and the carboxylate ester of N-hydroxysuccinimide.

This chemical moiety may have one or more monodentate ligands, a wide variety of which are known to the art.
Suitable monodentate ligands include, for example, carbon monoxide, cyanides, isocyanides, halides, and aliphatic, aromatic and heterocyclic phosphines, amines, stibines, and arsines.

Particularly preferred embodiments of this chemical moiety comprise bis(2,2'-bipyridyl)ruthenium(II) and tris(2,2'-bipyridyl)ruthenium(II).

It is within the scope of this invention for one or more of the ligands of M to be attached to additional chemical labels, such as, for example, radioactive isotopes, fluo-rescent components, or additional luminescent ruthenium- or osmium-containing centers.

It is also within the scope of this invention for the labelled substance (B) to be labelled by greater than one, or many, electrochemilumescent centers.

Suitable substances (B) include many biological substances, for example, whole cells, viruses, subcellular particles, proteins, lipoproteins, glycoproteins, polypeptides, nu-cleic acids, polysaccharides, lipopolysaccharides, cell-ular metabolites, hormones, pharmacological agents, tran-quilizers, barbituates, alkaloids, steroids, vitamins, a-mino acids and sugars. Whole cells may be animal, plant, or bacterial, and maybe viable or dead. Examples include plant 133~83~

pathogens such as fungi and nematodes. The term "sub-cellular particles" is meant to encompass, for example, subcellular organelles, membrane particles as from dis-rupted cells, fragments of cell walls, ribosomes, multi-enzyme complexes, and other particles which can be derivedfrom living organisms. Nucleic acids include, for example, chromosomal DNA, plasmid DNA, viral DNA, and recombinant DNA
derived from multiple sources. Nucleic acids also include RNA's, for example messenger RNA's, ribosomal RNA's and transfer RNA's. Polypeptides include, for example, en-zymes, transport proteins, receptor proteins, and struc-tural proteins such as viral coat proteins. Preferred polypeptides are enzymes and antibodies. Particularly pre-ferred polypeptides are monoclonal antibodies. Hormones include, for example, insulin and T4 thyroid hormone.
Pharmacological agents include, for example, cardiac gly-cosides. It is of course within the scope of this invention to include synthetic substances which chemically resemble biological materials, such as synthetic polypeptides, syn-thetic nucleic acids, and synthetic membranes, vesicles andliposomes. The foregoing is not intended to be a com-prehensive list of the biological substances suitable for use in this invention, but is meant only to illustrate the wide scope of the invention.
It is within the scope of this invention to include labelled nonbiological substances, including polymeric materials.
These substances may be in the form of soluble polymeric molecules, or any of the large variety of known macroscopic forms such as, for example,beads, or containers suchas test tubes, bottles, assay wells or the like.

Biological and nonbiological substances (B) are covalently bound to a ligand of M through an amide or amine linkage. In the case of the amide linkage, the linkage may be oriented . .~.

-18- 1339~3~

so that the material (B) is bonded directly either to the carbonyl or to the nitrogen of the amide linkage. These chemical moieties may be ionized. If so, it is understood in the art that many different counterions will serve to neutralize the charge of preparations of the chemical mo-iety. Suitable cations include, for example, H+, NH4+, guanidinium, Ag+, Li+, Na+, K+, Mg2+, Mn2+, and Cd2+.
Suitable anions include, for example, halides, OH-, car-bonate, S042-, hexafluorophosphate and tetrafluoroborate.

The present invention also provides compounds particularly suitable as intermediates for attaching a luminescent ru-thenium-containing or osmium-containing label to amino groups of chemical, biochemical and biological substances.
These intermediates are thus particularly suitable for synthesizing chemical moieties according to the present invention. The inventive intermediates are the mono- and di-N-hydroxysuccinimide esters of ruthenium or osmium bis(2,2'-bipyridine)(2,2'-bipyridine-4,4'-dicarboxylic acid) and their salts; and ruthenium or osmium bis(2,2'-bipyridine) (4,4'-di(chloromethyl)-2,2'-bipyridine).

The chemical structures of these intermediates are as fol-lows. The mono-N-hydroxysuccinimide ester of ruthenium or osmium bis(2,2'-bipyridine)(2,2'-bipyridine-4,4'-dicarb-oxylic acid) includes ~ ~_ O ~

~ ,co~

.~

1~3g~3:~

wherein M is Ru or Os, n is the integer 1, 2, or 3, and salts and stereoisomers thereof. The di-N-hydroxysuccinimide esters of ruthenium- or osmium-bis(2,2'-bipyridine)(2,2'-bipyridine-4,4' dicarboxylic acid) includes 10 e~

wherein M is Ru or Os, n is the integer 1, 2, or 3, and salts and stereoisomers thereof. Ruthenium or osmium bis(2,2'-bipyridine) (4,4'-di(chloromethyl)-2,2'-bipyridine) in-cludes ~5 ~ c~
~1 c~2c/

wherein M is Ru or Os, n is the integer 1, 2, or 3, and salts and stereoisomers thereof. These compounds may be syn-thesized by means known to the art.

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A preferred method of synthesizing the ruthenium-containing N-hydroxysuccinimide esters is to first react ruthenium dichlorobis 2,2'-bipyridine) with 2,2'-bipyridine-4, 4'-dicarboxylic acid in a hot aqueous methanol solution of sodium bicarbonate. After acidification, an aqueous solu-tion of NaPF6 is added to the solution of carboxylated ruthenium compound. The isolated hexafluorophosphate salt of the ruthenium complex is then esterified by reaction with N-hydroxy-succinimide in the presence of dicyclohexylcar-bodiimide in dimethylformamide. Of course, many variationson the structure of the N-hydroxysuccinimide component are possible without substantially altering the usefulness of the inventive intermediates. These intermediates may be ionized. If so, it is understood in the art that many different counterions will serve to neutralize the charge of preparations of the intermediate. Suitable cations include for example H+, NH4+, guanidinium, Ag+, Li+, Na+, R+, Mg2+, Mn2+, and Cd2+. Suitable anions include, for example, halides, carbonate, S042-, hexafluorophosphate, and tetra-fluoroborate.
These intermediates are useful for labelling substancescontaining a free amino group capable of attacking the carboxylate ester, and thereby displacing N-hydroxysuc-cinimide, or of attacking the chloromethyl group, and thereby displacing chloride. Use of these intermediates to label analytes of interest is preferred over the isothio-cyanates of the prior art (e.g. Weber, U.S. Patent No.
4,293,310). Isothiocyanates are generally prepared by reaction of a primary amine with carbon disulfide or thio-phosgene, each of which is volatile and highly toxic. Carbondisulfide is also an acute fire and explosion hazard. The required precursor primary aromatic amines are more diffi-cult to obtain than the precursor aromatic carboxylic acids used in the present invention. Also, the intermediates of the present invention are less reactive and more easily -21- 133~3~

stored and handled than the isothiocyanate derivatives.

The present invention provides methods for determining the presence of chemical moieties according to this invention.
The metal-containing compositions may be detected by many means known to the art including, for example, emission, absorption, and fluorescence spectrometry, atomic absorp-tion, anodic stripping voltametry, neutron activation and electrochemical methods. Of particular interest are photo-luminescence, chemiluminescence and electrochemilum-inescence methods.

Ru(bpy)32+ may be determined at very low concentrations using luminescence techniques. Using the oxidative re-duction method, Ege et al. (1984) (Analytical Chemistry,in press) were able to detect Ru(bpy)32+ at concentrations of 5x10-8 M. In these experiments, sodium oxalate was lmM
in phosphate buffer pH 5.0, and the potential was pulsed at +1.0 to +1.4 volts versus a saturated sodium chloride reference electrode for 5 to 10 second intervals. These workers found the reductive oxidation method to be even more sensitive. Using 18mM Na2S2Og and 0.1 M tetra-n-butyl ammonium tetrafluoroborate in CH3CN: H2O (1:1 v/v), Ru(bpy)32+ concentrations as low as 10-13 M could be de-tected. Further refinements of the techniques promise evengreater sensitivity. These techniques also provide sensi-tive and accurate measurements of labelled substances, as demonstrated more fully in the Examples set out hereinbelow.

Our experience with Ru(bpy)32+-labelled substances indi-cates the advantages of using ruthenium-containing and osmium-containing compounds as chemical labels. They are stable for long periods and may be attached efficiently to a wide variety of chemical, biochemical and biological materials. The labels are safe and relatively inexpensive.

They give a highly characteristic signal and do not occur in nature. Measurements based on luminescence of the labels are sensitive, fast, reproducible and utilize simple in-strumentation. There is very little interference with detection based on luminescence of these labels by such components as phosphate buffered saline, Tween~ (a sur-factant), liver tissue extract or serum. Luminescence-based measurement of these labels does not destroy the sample or labelled materials and may be performed repeti-tively. The signal is generated repeatedly by each moleculeof label, thereby enhancing the sensitivity with which these labels may be detected. The presence of labelled materials may be determined qualitatively or quantitatively depending on the needs of the particular application. Note: the word "determined", as used in this patent application, refers to either qualitative or quantitative determinations of the labelled material.

Accordingly, this invention provides a method of deter-mining the presence of a chemical moiety having the formula:

[M(P)m(Ll)n(L2)o(L3)p(L4)q(L5)r(L6)s]t(B)u wherein M is ruthenium or osmium; P is a polydentate ligand of M; Ll, L2, L3, L4, L5 and L6 are ligands of M, each of which may be the same as, or different from each other ligand; B
is a substance which is a ligand of M or is attached to one or more of P, Ll, L2, L3, L4, L5 or L6; m is an integer equal to or greater than l; each of n, o, p, q, r and s is zero or an integer; t is an integer equal to or greater than 1; u is an integer equal to or greater than 1 and P, Ll, L2, L3, L4, L5, L6 and B are of such composition and number that the chemical moiety can be induced to emit electromagnetic radiation and the total number of bonds to M provided by the ligands of M equals the coordination number of M.

133983~

The method comprises:

a) forming a reagent mixture under suitable con-ditions containing the chemical moiety;

b) inducing the moiety to emit electromagnetic ra-diation by exposing the reagent mixture to chemical energy or electrochemical energy; and c) detecting the emitted electromagnetic radia-tion and thereby determining the presence of the ana-lyte of interest.

In the chemical moieties useful in these methods, biological and nonbiological substances (B) may be incorporated into the moieties by coordination directly to M or by attachment to a ligand of M. Attachment may be through covalent bonding, or by electrostatic or hydrogen bonding. Many diverse means of effecting covalent bonding of substances (B) to ligands of M are available. The attaching linkage may be, for example, an amide or amine bond, an ester or thioester linkage, an ether or thioether linkage or any of many other means known to the art. The type of linkage will be determined by the substituents of the ligand and the suit-able chemical groups available for binding with the ligandon the substance that is to be labelled. Suitable substances (B) include, for example, whole cells, subcellular part-icles, nucleic acids, polysaccharides, proteins, glyco-proteins, lipoproteins, lipopolysaccharides, polypep-tides, cellular metabolites, hormones, pharmacological a-gents, tranquilizers, barbituates, alkaloids, steroids, vitamins, amino acids, sugars, and non-biological polymers.
In a preferred embodiment of the invention the attaching linkage is an amide or amine bond. The amide or amine bond is formed between the substituent on the ligand and a free -24- ~3g~3~

amino group on the substance that is to bé labelled.

These methods include a method of determining the chemical moiety by formation of a specific complex with a com-plementary material. Of particular interest are antibody-antigen pairs of materials. This binding method may be used to determine the presence of labelled antigens, such as, for example, digoxin or digitoxin in complex mix-tures such as blood, urine, or synthetic reaction mixtures by first exposing the mixture to immobilized antibodies specific for the antigen of interest, and then measuring the amount of labelled material bound to the immobilized anti-bodies.

The phrase "inducing to emit electromagnetic radiation"
refers to creating an excited state of said moiety which luminesces at wavelengths between 200 nanometers and 900 nanometers at ambient temperatures. The present invention envisions osmium-containing moieties as well as ruthenium-containing moieties and encompasses the wide va-riety of luminescent moieties which can be made by varying the chemical structure of the ligands. Each of these variations in the metal and the ligands can change the precise value of the energy input required to create the luminescentexcited state. Similarly, the wavelength of the emitted electromagnetic radiation will be dependent upon the nature and environment of the ruthenium-containing or osmium-containing material. Generally, photoluminescence excitation and emission will occur with electromagnetic radiation of between about 200 nanometers and about 900 nanometers in wavelength. Chemiluminescent and elec-trochemiluminescent emission will generally occur with the emitted electromagnetic radiation being between about 200 nanometers and about 900 nanometers in wavelength. The potential at which the reduction or oxidation of the chem-1~39~3~j ical moiety will occur depends upon its exact chemical structure as well as factors such as the pH of the solution and the nature of the electrode used. Generally, it is well known in the art how to determine the optimal emission and excitation wavelengths in a photoluminescent system, and the optimal potential and emission wavelength of an elec-trochemiluminescent or chemiluminescent system.

It should be clear that there are many methods for quan-tifying the amount of luminescent species present. The rate of energy input into the system can provide a measure of the luminescent species. Suitable measurements include, for example, measurements of electric current when the lumi-nescent species is generated electrochemically, the rate of reductant or oxidant utilization when the luminescent spe-cies is generated chemically or the absorption of elec-tromagnetic energy in photoluminescent techniques. In addition, of course, the luminescent species can be detected by measuring the emitted electromagnetic radiation. All of these measurements can be made either as continuous, rate-based measurements, or as cumulative methods which accumulate the signal over a long period of time. An example of rate-based measurements is the use of photomultiplier tubes, photodiodes or phototransistors to produce electric currents proportional in magnitude to the incident light intensity. Examples of cumulative methods are the in-tegration of rate-based data, and the use of photographic film to provide cumulative data directly.

All of these luminescence-based methods entail repeated luminescence by the ruthenium-containing compound. The repetitive nature of the detectable event distinguishes these labels from radioactive isotopes or bound chemi-luminescent molecules such as luminol. The latter labels produce a detectable event only once per molecule (or atom) of label, thereby limiting their detectability.

-26- ~33983a This invention further provides for the use of ruthenium-containing and osmium-containing labels in bind-ing methods for determining the presence of analytes of interest. Many such binding methods are known to the art.
These methods often exploit the great specificity with which biochemical and biological agents bind together. Examples are methods based on nucleic acid hybridization techniques, antibody-antigen based techniques, and enzyme-ligand based techniques. These methods can employ labelled moieties to determine analytes of interest or to use labelled analogues of analytes of interest to determine analytes of interest in competitive binding assays.

The analyte of interest and the chemical moiety can be any pair of substances which are capable of binding together in a specific manner. Such substances include for example, whole cells, subcellular particles, nucleic acids, poly-saccharides, proteins, glycoproteins, lipoproteins, lipo-polysaccharides, polypeptides, cellular metabolites, hor-mones, pharmacological agents, tranquilizers, barbituates,alkaloids, steroids, vitamins, amino acids, sugars, and non-biological polymers. Of particular interest are anti-body-antigen pairs. For example, this method encompasses the use of labelled antibodies to determine the presence of cell surface antigens, or to label particular cells for detection by cell sorting methods. Antigens immobilized by, for example, attachment to immobilized, unlabelled anti-bodies can be detected by labelled antibodies in a method commonly known as a "sandwich" method.

In competitive binding assays, the analyte of interest and the labelled analogue of the analyte can be any substances capable of participating in formation of a specific complex with a complementary material, such as for example, whole cells, subcellular particles, nucleic acids, polysaccha-. .

-27- ~ 339~3~ï

rides, proteins, glycoproteins, lipoproteins, lipo-polysaccharides, polypeptides, cellular metabolites, hor-mones, pharmacological agents, tranquilizers, barbituates, alkaloids, steroids, vitamins, amino acids, sugars and non-biological polymers. Of particular interest are anti-body-antigen-based methods. These methods are analogous to the well known radioimmunoassay, wherein an analyte of interest is detected when it displaces a radioactive ana-logue of the analyte from an antibody. The many variations10 on radioimmunoassay known to the art can, in principle, be used to advantage by employing moieties labelled according to the present invention in place of radioactively labelled compounds.

It is further provided by the present invention to use labelled chemical moieties in either heterogeneous or homogeneous binding methods. In heterogeneous binding methods, the bound labelled substance must be physically separated from the unbound labelled substance before mea-surement of the presence of label. This is fre~uently accomplished in antibody-antigen systems by immobilizing one component, the antibody for example, by attachment to an insoluble matrix such as a filter or to the surface of beads or reaction vessels such as test tubes. The antigen-con-taining solution is poured through the filter or into the reaction vessel, and then washed away from the filter or sides of the reaction vessel. Only antigen specifically bound to antibody will remain to be determined.
In homogeneous methods, by contrast, the bound and unbound labelled material are present in the same reaction mixture when the presence of label is measured. This is possible when binding modifies the properties of the signal de-tectable from the label. There are many ways that !3 8 3 ~i luminescent labels can be used in homogeneous systems. For example, if a luminescence quencher were properly posi-tioned on an antibody, binding of a labelled antigen could result in suppression of the luminescence of the label by the luminescence quencher on the antibody. Many homogeneous methods for luminescent labels are known to the art, and some of them are reviewed in Boguslaski and Li (1982), "Homo-geneous Immunoassays," Applied Biochemistry and Biotech-nology, 7, pp. 401-414.

A particularly unique and useful class of homogeneous bind-ing assays is provided by the present invention. As de-scribed hereinbefore, these labels can be measured elec-trochemically by means of exposing a solution of the la-belled substance of interest to an electrode. Any labelledsubstance which is present in the solution but cannot gain access to the surface of the electrode will not be detected.
This can occur, for example, if the labelled substance is bound directly or indirectly to the surface of the reaction vessel into which the electrode is placed, or if the label is imbedded deep into the interior of the specific complex, such as within an antigen-antibody complex, or if the electrode itself were coated with a layer through which labelled material could pass but complexed labelled mat-erial could not pass. In addition, it should be possible tocoat the surface of an electrode with antibodies, so that only labelled antigen bound to the immobilized antibodies can obtain access to the electrode and thereby be deter-mined. This particular homogenous method may be most effective if the required electrode potential is applied in short pulses.

It is within the scope of the present invention to use a combination of means for determining the presence of la-belled compounds. For example, it may be desirable to , 1339~3~

measure the total amount of labelled substance by a means which does not distinguish between bound and unbound la-belled substance such as photoluminescence or chemilumi-nescence, and to determine the amount of bound labelled substance by a means which does distinguish between bound and unbound labelled substance, such as electrochemilumi-nescence, for example. Such a combination of methods could be performed on the same sample, and thus provide a richer source of information about the sample than could any method when used individually. It is also within the scope of~this invention to determine the presence of two or more dif-ferently labelled compounds within the same reaction mix-ture. This is possible either if the labels emit elec-tromagnetic radiation of differing wavelengths or if the labels can be induced to emit electromagnetic radiation by exposure to energy of different values or sources.

The present invention further provides-systems for de-termining the presence of the ruthenium-containing or os-mium-containing chemical moieties. The systems comprise reagentmixtures comprising the chemical moiety, a means for inducing the chemical moiety to emit electromagnetic radi-ation, and a means for detecting the emitted electromagnetic radiation.

The present invention further provides systems for em-ploying ruthenium-containing or osmium-containing labelled chemical moieties for the determination of analytes af interest.

The systems of the present invention are envisioned to be useful in the rapid, efficient, and versatile performance of the diverse methods disclosed and suggested by the instant invention disclosure.

~33!~83~

This invention also provides a method of determining the presence of a chemical moiety having the formula: ~A)k(B)U.
A is a compound which can be induced to repeatedly emit electromagnetic radiation by direct exposure to an electro-chemical energy source. These compounds can be inorganic,organometallic or organic compounds, e.g. rubrene, 9,10-diphenyl anthracene, or ruthenium-containing or os-mium-containing labels. B is a substance which is attached to A, k is an integer equal to or greater than one and n is an integer equal to or greater than 1.

The method comprises forming a reagent mixture under suit-able conditions containing the chemical moiety and inducing the chemical moiety to repeatedly emit electromagnetic radiation by directly exposing the moiety to electroc-hemical energy. The emitted electromagnetic radiation isthen detected by suitable methods thereby determining the presence of the chemical moiety.

Biological and nonbiological substances (B) may be incor-porated into the moieties by any form of attachment to A.
The attachment may be by coordination to a metal atom present in A or to a ligand of A. The attachment can be through covalent, electrostatic, or hydrogen bonding. The type of linkage will be determined by the suitable chemical groups available for binding on both A and B.

Suitable substances (B) include, for example, whole cells, subcellular particles, nucleic acids, polysaccha-rides, proteins, glycoproteins, lipoproteins, lipopolysac-charides, polypeptides, cellular metabolites, hormones, pharmacological agents, tranquilizers, barbituates, alka-loids, steroids, vitamins, amino acids and sugars. The substances are not limited to biological substances and may 3 1 1 ~ 3 ~ 8 be any suitable non-biological substance such as a polymer, organic or inorganic compound.

~ The chemical moiety is induced to emit electromagnetic radiation by creating an excited state of the moiety which - luminesces at wavelengths from about 200 nanometers to about 900 nanometers at ambient temperatures. In this embodiment of the invention the chemical moiety is excited by exposing the reagent mixture to electrochemical energy. The poten-tial at which the reduction or oxidation of the inventive chemical moiety will occur depends upon its exact chemical structure as well as factors such as the pH of the solution and the nature of the electrode used. It is well known to those of ordinary skill in the art how to determine the lS optimal potential and emission wavelength of an electro-chemiluminescent system. The electrochemiluminescent species may be measured by any suitable measurement such as the measurement of electric current or emitted electro-magnetic radiation.

The method of determining the presence of the moiety may also be performed when the moiety is capable of binding to another chemical agent. The chemical agent may be any substance capable of binding to the moiety in a specific manner.
Examples of such methods are nucleic acid hyridization techniques, antibody-antigen based techniques and enzyme-ligand techniques.

In another embodiment of the invention the electrochemilum-inescent moiety, (A)k(B)U may be used in methods to deter-mine the presence of an analyte of interest which binds to the moiety.

1339~3~

The analyte of interest can be any substance which is capable of binding to the electrochemiluminescent moiety, such as the binding of an antigen to a antibody labelled with an electrochemiluminescent moiety. The method involves con-tacting the analyte with the chemical moiety under suitableconditions so as to form a reagent mixture. The chemical moiety is then induced to repeatedly emit electromagnetic radiation by directly exposing the moiety to electrochemi-cal energy. The presence of the analyte is determined by detecting the electromagnetic radiation emitted by the chemical moiety bound to the analyte.

Competitive binding methods may also be used to determine the presence of an analyte of interest. The analyte and the chemical moiety, (A)k(B)U, bind competitively to a chemical material. The material is contacted with the chemical moiety and analyte under suitable conditions so as to form a reagent mixture. The chemical moiety is induced to repeatedly emit electromagnetic radiation by directly ex-posing the moiety to electrochemical energy. The presenceof the analyte of interest is determined by detecting the amount of emitted electromagnetic radiation.

This invention also concerns compositions which comprise the ruthenium-containing or osmium-containing chemical moieties of this invention and one or more different chem-ical moieties each of which can be induced to emit electro-magnetic radiation of a different distinct wavelength.
These compositions are useful in methods and systems of detecting two or more different substances or analytes of interest contained in a mixture of the same and other sub-stances.

The other different chemical moiety or moieties may be any suitable chemical moiety such as inorganic, organic and -33- 133983~

organometallic compounds which can be induced to emit elec-tromagnetic radiation, e.g. rubrene or 9,10-dipheny-lanthracene. These moieties may be such moieties that are induced to emit electromagnetic radiation when exposed to energy of different values or sources than the energy used to induce electromagnetic radiation from the ruthenium-containing or osmium-containing chemical moieties. In a specific embodiment of the invention, each other chemical moiety emits electromagnetic radiation of a different distinct wavelength when induced to emit electromagnetic radiation by energy of the same source and value that induces the ruthenium-containing or osmium-containing chemical mo-iety to emit electromagnetic radiation.

Methods for determining these chemical moieties comprise forming a reagent mixture under suitable conditions con-taining the chemical moieties and then inducing the chemicalmoieties to emit electromagnetic radiation by exposing the reagent mixture to chemical energy or electrochemical en-ergy. The presence of each of the moieties is determined bydetecting the electromagnetic radiation of different wave-lengths emitted by each of the moieties.
.

The invention also concerns a method of determining the presence of one or more analytes of interest which bind selectively to the different chemical moieties present in the same mixture. The method comprises contacting the analytes with the chemical moieties under suitable con-ditions so as to form a reagent mixture. The moieties are induced to emit electromagnetic radiation by exposing the reagent mixture to chemical energy or electrochemical ener-gy and the emitted electromagnetic radiation of different wavelengths is detected to determine the presence of each of the analytes of interest.

.

133~83i These methods in which the presence of two or more chemical moieties is determined in a mixture are applicable to all instances described previously for determining the ruthe-nium-containing and osmium-containing luminescent labels.
This embodiment, however, allows for the determination of two or more different substances present in the same sample simultaneously.

In another embodiment of the invention the different chemi-cal moieties are induced to emit electromagnetic radiation by exposure to energy of different values or different sources. The methods of determining these different chem-ical moieties are essentially the same as those for deter-mining the chemical moieties which emit different wave-lS lengths of electromagnetic radiation, except for the induc-tion step. These chemical moieties are induced to emitelectromagnetic radiation by energy of different values or sources. The sample containing the moieties is exposed to each of the different energy values or sources at a different time and the electromagnetic radiation emitted by the spe-cific moiety is detected, thus determining the presence of the moiety. This method is.also useful for determining the presence of analytes of interest which bind selectively to the different chemical moieties present in the sample.
Another embodiment of the invention involves methods and systems of determining one or more different electrochemi-luminescent moieties of the formula (A)k(B)U present in the same sample. These chemical moieties contain different compounds which emit electromagnetic radiation of different wavelengths when exposèd to an electrochemical energy source or can each be induced to emit electromagnetic radiation by exposure to distinct electrochemical energy sources. These different electrochemiluminescent moieties may be specifically attached to different substances or ana-1~39~3~

lytes of interest. Determination of the different moietiesinvolves the same procedures as discussed previously.

This invention is illustrated in the examples which follow.
The examples are set forth to aid in an understanding of the invention but are not intended to, and should not be con-strued to, limit in any way the invention as set forth in the claims which follow thereafter.

-36- 133~83~

EXAMPLE I

Preparation of Ruthenium bis (2,2'-bipyridine) (2,2'-bi-pyridine-4,4'-dicarboxylic acid) bis(hexafluorophosph-ate).

Sodium bicarbonate (0.40 g), ruthenium dichlorobis(2,2'-bipyridine)(0.40 g), and 2,2'-bipyridine-4,4'-dicarb-oxylic acid (0.30 g) were stirred in refluxing methanol (20 ml)-water (5 ml) for 9 hours. The resulting solution was cooled in an ice bath, treated with 5 drops concentrated H2SO4, and allowed to stand at ice temperature for 1.5 hours. A precipitate formed, which was separated by filtration and washed with MeOH (8 ml).
The combined filtrate and wash solution were treated with a solution of sodium hexafluorophosphate (5.0 g) in water (25 ml). The resulting solution was cooled in an ice bath for 3 hours, and the resulting precipitate of red-purple crystals was collected by filtration (0.40 g).

EXAMPLE II

Preparation of Active Ester of Ruthenium bis(2,2'-bipyrid-ine)(2,2'-bipyridine-4,4'-dicarboxylic acid).

Dicyclohexylcarbodiimide (DCC, 0.046 g) and N-hydroxysuc-cinimide (0.034 g) were dissolved in DMF (2 ml) with stir-ring, and cooled in an ice bath. A solution of ruthenium bis(2,2'-bipyridine) (2,2'-bipyridine-4,4'-dicarboxylic acid) (0.101 g, prepared as in ~.xample I) dissolved n DMF (1 ml) was added, and the mixture was stirred 5 hours at ice bath temperature. A precipitate formed and was separated by centrifugation. The supernatant containing the activated ruthenium complex was retained for labelling 133~3;~j of substrates.

EXAMPLE III

Labelling of Bovine Serum Albumin (BSA) with Activated Ruthenium Complex.

The DMF solution of activated ruthenium complex prepared in Example II (1 ml) was added to a stirred solution of BSA in aqueous Physiologic Buffered Saline (PBS, 5 ml; 25 mg/ml BSA). The mixture was stirred overnight, and precipitate was removed by centrifugation. The supernatant containing ruthenium-labelled BSA was analyzed by two methods.

METHOD 1: Dialysis Ruthenium-labelled BSA solution was dialyzed with PBS
solution. As a control, the unbound, activated ruthenium complex prepared in Example II was also dialyzed with PBS
solution. After 8 hours, the control showed no fluorescent species within the dialysis tube. The ruthenium labelled BSA solution, however, showed strong fluorescence, in-dicating that the ruthenium complex was bound to the high molecular weight BSA.
METHOD 2: Microfiltration Ruthenium-labelled BSA solution was placed in an Amicon microconcentrator and centrifuged at 8000 rpm. A small fraction of red-orange solution remained above the filter, and this colored fraction was diluted with wash PBS so-lution and centrifuged. This procedure was repeated sev-eral times. After the fourth wash, colorless solution passed through the filter, while highly red-orange colored material remained above the filter. This result indicates .. ~

-38- 13~9~3~

that the ruthenium complex was bound to the high molecular weight BSA.

EXAMPLE IV

Labelling of Human Immunoglobulin G (IgG) with Activated Ruthenium Complex.

The DMF solution of activated ruthenium complex prepared in Example II was added to a stirred solution of affinity purified human IgG in aqueous buffer. The ruthenium labelled IgG solution fluoresced brightly after extensive dialysis, indicating that the ruthenium complex was bound to the high molecular weight affinity purified human IgG.

EXAMPLE V

Labelling of Rabbit Anti-salmonella Antibody The DMF solution of activated ruthenium complex prepared in Example II (0.1 ml) was stirred with rabbit serum con-taining anti-Salmonella antibody (1 ml) at room temper-ature for 1 hour, and then quenched by addition of die-thanolamine (0.1 ml). Salmonella cells were treated with the resultant solution containing ruthenium labelled anti-Salmonella antibody. The cells were separated by cen-trifugation and resuspended in fresh buffer five times, in order to separate the cells from any unbound antibody (including ruthenium labelled unbound antibody) and from any free ruthenium complex. The Salmonella cells, treated with ruthenium labelled anti-Salmonella antibody, emitted bright red-orange light when viewed on a fluorescence optical microscope, indicating that the anti-Salmonella antibody was labelled with ruthenium complex, and that the ruthenium labelled antibody retained its ability to bind to ~....

:L~39835 Salmonella cells under conditions where the ruthenium complex fluoresced.

EXAMPLE VI

The procedure of Example V was repeated using normal mouse serum (i.e., lacking anti-Salmonella antibody) in place of rabbit serum containing anti-Salmonella antibody. The Salmonella cells, after treatment, did not emit red-orange light when viewed on a fluorescence optical microscope, indicating that non-specific binding of ruthenium labelled normal mouse serum components to Salmonella cells did not occur.

EXAMPLE VII

Labelling of Goat Anti-Rabbit Immunoglobulin (IgG) and Comparison with Rhodamine The DMF solution of activated ruthenium complex prepared in Example II was added to a stirred solution of affinity purified goat anti-rabbit IgG. After reaction, the mixture was dialyzed against buffer. Material remaining in the dialysis tube fluoresced under W light.
The ruthenium labelled IgG was tested for reactivity toward Salmonella coated with rabbit anti-Salmonella antibodies.
Rabbit anti-Salmonella antibodies were reacted Salmonella worthington that had been fixed to a glass microscope slide, and unreacted antibody was washed away with buffer.
The ruthenium labelled goat anti-rabbit IgG was then re-acted with the antibody treated S. worthington, and unre-acted material was washed away with buffer. The slide was examined under an optical microscope equipped with a 50 W
mercury lamp, and very bright orange-red fluorescence was .

133g8~j' observed on and around the bacterium.

A control experiment tested for non-specific binding of ruthenium-labelled antibody. S. worthington, fixed to a glass microscope slide, was reacted with normal mouse serum, and then with ruthenium labelied goat anti-rabbit IgG antiserum. The same washing proce-dures were followed. No orange-red fluorescence was observed.

For comparison purposes, a rhodamine isothiocyanate conjugated goat anti-rabbit IgG antiserum tat a pro-tein concentration equivalent to the ruthenium-con-jugated antibody) was reacted with S. worthington coated with rabbit anti-Salmonella antibodies. Red fluorescence was barely detectable and the intensity of fluorescence was significantly less than the ru-thenium conjugate.

EXAMPLE VIII

Electrochemiluminescent (ECL) Detection of Ruthenium Labelled Bovine Serum Albumin (BSA).

ECL measurements were carried out in a one compartment cell (30 ml) with an optically flat bottom. The working electrode was glassy carbon, the counter electrode was platinum gauze, and the pseudo-reference electrode was a silver wire. Light intensity measurements were made by applying a potential of -2.0v (versus the Ag wire), detecting the emitted light with a photo-multiplier tube (Hamamatsu 928), and inteyrating the resulting signal for 2 s with a Bascom-Turner Recorder.

--41- 1~9~3~

Acetonitrile-water (9 ml, 50:50 v/v), tetrabutylam-monium tetrafluoroborate (329 m~), and diammonium peroxydisulfate (42 mg) were mixed in the ECL cell, and background light intensity was recorded. Ruthenium labelled BSA solution (prepared in Example III) was diluted in acetonitrile-water (50:50 v/v) and the diluted BSA solution (1 ml) was added to the ECL cell.
The resulting solution was deaerated by bubbling solent-saturated nitrogen. Table I summarizes re-sults for different concentrations of ruthenium la-belled BSA.

TABLE I

Light Intensity (Ruthenium) (Arbitrary Units) _ M

5.2 blank 20.63 1 x lo-ll 33.25 1 x 10-1~
54.42 9 x 10-1~
150.2 8 x 10-9 .

-42- 1~3~3~

EXAMPLE IX

Preparation of 4,4'-Di(chloromethyl)-2,2'-bipyridine-bis (2,2'-bipyridine) ruthenium (II) 2+
s 4,4'-Diethoxycarbonyl-2,2'-bipyridine was prepared from 2,2-bipyridine-4,'dicarboxylic acid by the method of Sprin-tschink et al. (J. Amer. Chem. Soc. 99, 4947 (1977)). The diethyl ester (100 mg) was dissolved in anhydrous diethyl ether (35 ml). Lithium aluminum hydride (10~0 mg) was added, and following a 30 minute incubation diethyl ether (35 ml) and ice cold deionized water (100 ml) were added. The solution was mixed throughly and the ether layer collected.
The aqueous phase was extracted twice more with ether (70 ml). The combined ether extracts were dried over anhydrous sodium sulfate and filtered. The solvent was removed on a - rotary evaporator to give the desired product (43 mg). This 43 mg of 4,4-Di(hydroxymethyl)-2,2'-bipyridinè and 120 mg of cisdichloro bis (2,2'-bipyridine) ruthenium (II) dihy-drate were added to ethanol (25 ml), and the solution was refluxed for 14 hours. After the solution had cooled, 50 ul of solution of ammonium hexafluorophosphate (1 gm in 1 ml of water) was added, the resultant crystalline solid was col-lected, washed with a small volume of ethanol and dried to give 138 mg of the hexafluorophosphate salt of the 4,4-dihydroxymethyl complex. This complex (6 mg) was added to thionyl chloride (5 ml), and the solution was refluxed for 6 hours. The thionyl chloride was removed by distillation, and the solid residue was dissolved in a dioxane-water (1:1) mixture (500 ul). This solution containing 4,4'Di(chlorom-ethyl)-2,2'-bipyridine-bis-(2,2'-bipyridine) ruthenium (II) 2+ was used for labeling of antibody.

~43~ - 13~83;~

EXAMPLE X

Labeling of Rabbit and Sheep Anti-Mouse IgG with 4, 4'-Di(chloromethyl)-~,2'-bipyridine-bis-(2,2'-bipyridine) ruthenium (II) 2+

The rabbit and sheep antibodies were received as 2 mg/ml solutions in PBS. These solutions were dialyzed overnight against sodium bicarbonate (500 ml, 50 mM, pH9). The antibody (2 mg) was then diluted with sodium bicarbonate solution to give a final volume of 2 ml. This solution (1 ml) was added to sodium carbonate solution (1 ml, 50 mM) to adjust the pH to 10. The solution of activated complex in dioxane-water (100 ul) was added to the solution of antibody and allowed to react at 4~C for 16 hours. After this, BSA
(5 mg) was added to the reaction, and the solution was immediately dialyzed against carbonate buffer (500 ml, 25 mM, pH9). The dialysis buffer was changed three times at 24 hour intervals.

EXAMPLE IX

Demonstration of immunological Reactivity of Labeled Rabbit and Sheep Anti-Mouse Immunoglobulin by Fluorometric Anal-ysis A formalinized suspension of Legionel~a pneumophila 1 (Philadelphia 1 isolate) was adjusted to an optical density (at 425 nm) of 1.00 by dilution with PBS buffer. A 1.3 dilution (1 ml) of this suspension was added to a series of conical microcentrifuge tubes. The cells were centrifuged (10 minutes, 10,000 RPM), the supernatant was decanted, and the cells were resuspended in a 1:100 dilution in PBS (1 ml) of a mouse monoclonal IgG antibody ("lOC8", 0.85 mg/ml) or 3 j in PBS as a negative control. After incubation at room temperature for 1 hour, the cells were centrifuged as before, the supernatant was decanted, the cells were resu-spended in PBS buffer and centrifuged again. Following decantation of the supernatant, the cells were resuspended in a 1:5 dilution (in PBS) of the pooled ruthenium labeled rabbit and sheep anti-mouse IgG Antibody (1 ml, 10 mg ruthenium complex/ml), or in a 1:50 dilution (in PBS) of fluorescein labeled goat anti-mouse IgG antibody (1 ml, 0.5 mg IgG/ml) as a positive control, or in PBS as a negative control. After incubation at room temperature for 1 hour, the cells were centrifuged as before, the supernatant was decanted, and the cells resuspended in PBS and washed twice, with centrifugation, as before. Following the last wash the cells were resuspended in PBS (1 ml) and transferred to a polystyrene cuvette for fluorometric analysis.

Excitation and emission scans of the fluorescein labeled goat anti-mouse IgG antibody solution showed an excitation peak at 491 nm and an emission peak at 519 nm. These wavelengths were used for the fluorescence measurements of the fluorescein labeled cells. Excitation and emis~ion scans of the ruthenium labeled rabbit/sheep anti-mouse IgG
antibody solution showed an excitation peak at 455 nm and an emission peak at 623 nm. These wavelengths were used for the fluorescence measurements of the ruthenium labeled cells.
Cell suspensions which had been incubated with PBS instead of primary "lOC8" antibody and fluorescent conjugate served as negative controls and were used to blank the fluorometer.
The fluorescence of cell suspensions which had been incu-bated with both primary "lOC8" antibody and fluorescent conjugate was defined as 100, and all other fluorescence measurements were made in fluorescence units relative to these internal standards. Table I summarizes the results, which demonstrate that the ruthenium labeled anti-mouse IgG

133983~

conjugate fluoresces and exhibits specific immunological reactivity in the presence of primary "lOC8" antibody.

TABLE I

RELATIVE FLUORESCENCE UNITS

Fluorescein Ruthenium (at 519 nm) (at 623 nm) 1:3 cell 1:3 cell Dilution Dilution Fluorescence of conjugate with primary antibody -~100 _100 Fluorescence of conjugate without primary antibody 37 64 Background fluorescence 20 (with primary 6 48 antibody, without conjugate)

Claims (108)

1. A chemical moiety having the formula [M(P)m(L1)n(L2)o(L3)p(L4)q(L5)r(L6)s~t(B)u wherein:

M is ruthenium or osmium;

P is a polydentate ligand of M;

L1, L2, L3, L4, L5 and L6 are ligands of M, each of which may be the same as or different from each other ligand;
B is a biological, synthetic or non-biological substance, or variant thereof covalently bound to one or more of P, L1, L2, L3, L4, L5 or L6 through one or more amide or amine linkages;

m is an integer equal to or greater than 1;

each of n, o, p, q, r and s is zero or an integer;
t is an integer equal to or greater than 1; and u is an integer equal to or greater than 1, P L1, L2, L3, L4, L5, L6 and B being of such composition and number that the chemical moiety can be induced to emit electromagnetic radiation and the total number of bonds to M provided by the ligands of M equals the coordination number of M.

2. A chemical moiety according to claim 1, wherein M is ruthenium.

3. A chemical moiety according to claim 1, wherein M is osmium.

4. A chemical moiety according to claim 1, wherein P is an aromatic heterocyclic ligand.

5. A chemical moiety according to claim 4, wherein P
contains nitrogen.

6. A chemical moiety according to claim 5, wherein P
is bipyridyl, bipyrazyl, terpyridyl, phenanthrolyl, substituted bipyridyl, substituted bipyrazyl, substituted terpyridyl, or substituted phenanthrolyl;

wherein said substituted bipyridyl, substituted bipyrazyl, substituted terpyridyl, or substituted phenanthrolyl is substituted by an alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted 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 subgroup, or the carboxylate ester of N-hydroxysuccinimide substituent.

7. A chemical moiety according to claim 1, wherein at least one of L1, L2, L3, L4, L5 and L6 is a polydentate aromatic heterocyclic ligand.

8. A chemical moiety according to claim 7, wherein at least one of L1, L2, L3, L4, L5 and L6 contains nitrogen.

9. A chemical moiety according to claim 8, wherein at least one of L1, L2, L3, L4, L5 and L6 is bipyridyl, bipyrazyl, terpyridyl, phenanthrolyl, substituted bipyridyl, substituted bipyrazyl, substituted terpyridyl, or substituted phenanthrolyl.

10. A chemical moiety according to claim 9, wherein at least one of L1, L2, L3, L4, L5 and L6 is substituted with an alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted 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 substituent.

11. A chemical moiety according to claim 1 containing 2 bidentate ligands wherein each bidentate ligand is bi-pyridyl, bipyrazyl, terpyridyl, phenanthrolyl, substituted bipyridyl, substituted bipyrazyl, substituted terpyridyl, or substituted phenanthrolyl wherein said substituted bipyridyl, substituted bipyrazyl, substituted terpyridyl, or substituted phenanthrolyl is substituted by an alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted 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 subgroup, or the carboxylate ester of N-hydroxysuccinimide substituent.

12. A chemical moiety according to claim 1 containing 3 bidentate ligands, wherein each bidentate ligand is bi-pyridyl, bipyrazyl, terpyridyl, phenanthrolyl, substituted bipyridyl, substituted bipyrazyl, substituted terpyridyl, or substituted phenanthrolyl wherein said substituted bipyridyl, substituted bipyrazyl, substituted terpyridyl, or substituted phenanthrolyl is substituted by an alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, substituted 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 subgroup, or the carboxylate ester of N-hydroxysuccinimide substituent.

13. A chemical moiety according to claim 12 wherein M is ruthenium.

14. A chemical moiety according to claim 13 containing two bidentate bipyridyl ligands and one substituted bidentate bipyridyl ligand.

15. A chemical moiety according to claim 1, wherein B is a whole cell, subcellular particle, polypeptide, nucleic acid, polysaccharide, protein, glycoprotein, lipoprotein, lipopolysaccharide, polypeptide, cellular metabolite, hormone, pharmacological agent, tranquilizer, barbituate, alkaloid, steroid, vitamin, amino acid, or non-biological polymer.

16. A chemical moiety according to claim 15, wherein B is a plant pathogen.

17. A chemical moiety according to claim 15, wherein B is a serum-derived antibody or a monoclonal antibody.

18. A chemical moiety according to claim 15, wherein B is T4 thyroid hormone.

19. A chemical moiety according to claim 6, wherein the amide linkage is oriented so that the carbonyl group of the amide linkage is bonded directly to said substituted bi-pyridyl ligand.

20. A compound having the structure where n is the integer l, 2, or 3, and the salts thereof.

r~.

21. A compound having the structure wherein n is the integer 1,2 or 3, and the salts thereof.

22. A compound having the structure wherein n is the integer 1,2, or 3, and the salts thereof.

13398~5

23. A compound having the structure wherein n is the integer 1,2 or 3, and the salts thereof.

24. A compound having the structure wherein n is the integer 1, 2 or 3, and the salts thereof.

25. A compound having the structure wherein n is the integer l, 2 or 3, and the salts thereof.

26. A salt according to claim 20, 21, or 22 wherein the counterion is the cation NH4+, guanidinium, Li+, Na+, K+, Ca2+, Mg2+, Mn2+ or Cd2+, or the anion, OH-, SO42-, halide, carbonate, hexafluorophosphate or tetrafluoroborate.

27. A method for determining the presence of a chemical moiety as defined in claim 1 which comprises:
a) forming a reagent mixture containing the chemical moiety of claim 1; and b) detecting the presence of the moiety comprising, an atomic absorption, spectrophotometric, electrochemical, chemical, chemiluminescent, photoluminescent, or electrochemiluminescent method.

28. A method according to claim 27, wherein determining the presence of the moiety comprises inducing the chemical moiety to emit electromagnetic radiation and detecting the emitted electromagnetic radiation.

29. A method according to claim 28, wherein inducing the chemical moiety to emit electromagnetic radiation comprises exposing the reagent mixture to electromagnetic, chemical or electrochemical energy.

30. A method according to claim 29, wherein the reagent mixture is exposed to chemical or electrochemical energy.

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

[M(P)m(L1)n(L2)o(L3)p(L4)q(L5)r(L6)s]t(B)u wherein:

M is ruthenium or osmium;

P is a polydentate ligand of M;

L1, L2, L3, L4, L5 and L6 are ligands of M, each of which may be the same as or different from each other ligand;
B is a biological, synthetic or non-biological substance, or variant thereof substance which is a ligand of M or is attached to one or more of P, L1, L2, L3, L4, L5 and L6 .

m is an integer equal to or greater than 1;

each of n, o, p, q, r and s is zero or an integer;

t is an integer equal to or greater than 1; and u is an integer equal to or greater than 1, P, L1, L2, L3, L4, L5, L6 and B being of such composition and number that the chemical moiety can be induced to emit electromagnetic radiation and the total number of bonds to M provided by the ligands of M equals the coordination number of M;

the method comprising:

a) forming a reagent mixture containing the chemical moiety;

b) inducing the chemical moiety to emit electro-magnetic radiation by exposing the reagent mixture to chemical energy or electrochemical energy; and c) detecting the emitted electromagnetic radiation and thereby determining the presence of the chemical moiety.

32. A method according to claim 31, wherein said moiety is capable of binding to a chemical agent.

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

[M(P)m(L1)n(L2)o(L3)p(L4)q(L5)r(L6)s]t(B)u wherein:

M is ruthenium or osmium;

P is a polydentate ligand of M;

L1, L2, L3, L4, L5 and L6 are ligands of M, each of which may be the same as or different from each other ligand;
B is a biological, synthetic or non-biological substance, or variant thereof substance which is a ligand of M or is attached to one or more of P, L1, L2, L3, L4, L5 and L6 .

m is an integer equal to or greater than 1;

each of n, o, p, q, r and s is zero or an integer;

t is an integer equal to or greater than 1; and u is an integer equal to or greater than 1, P, L1, L2, L3, L4, L5, L6 and B being of such composition and number that the chemical moiety can be induced to emit electromagnetic radiation and the total number of bonds to M provided by the ligands of M equals the coordination number of M;

the method comprising:

a) contacting the analyte with the chemical moiety so as to form a reagent mixture;

b) inducing the chemical moiety to emit electro-magnetic radiation by exposing the reagent mixture to chemical energy or electrochemical energy; and c) detecting the emitted electromagnetic radiation and thereby determining the presence of the analyte of interest,

34. 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:

[M(P)m(L1)n(L2)o(L3)p(L4)q(L5)r(L6)s]t(B)u wherein:

M is ruthenium or osmium;

P is a polydentate ligand of M;

L1, L2, L3, L4, L5 and L6 are ligands of M, each of which may be the same as or different from each other ligand;
B is a biological, synthetic or non-biological substance, or variant thereof substance which is a ligand of M or is attached to one or more of P, L1, L2, L3, L4, L5 and L6.

m is an integer equal to or greater than 1;

each of n, o, p, q, r and s is zero or an integer;
t is an integer equal to or greater than 1; and u is an integer equal to or greater than 1, P, L1, L2, L3, L4, L5, L6 and B being of such composition and number that the chemical moiety can be induced to emit electromagnetic radiation and the total number of bonds to M provided by the ligands of M equals the coordination number of M;

said method comprising:

a) contacting the material, the chemical moiety and the analyte so as to form a reagent mixture;

b) inducing the chemical moiety to emit electro-magnetic radiation by exposing the reagent mixture to electrochemical or chemical energy; and c) detecting the emitted electromagnetic radiation and thereby determining the analyte of interest.

35. A method according to claim 31, 32, or 33 wherein B is a biological, synthetic or non-biological substance, or variant thereof substance covalently bound to one or more of P, L1, L2, L3, L4, L5 and L6 through one or more amide or amine linkages.

36. A method according to claim 30, wherein B is a whole cell, subcellular particle, nucleic acid, polysaccharide, protein, lipoprotein, lipopoly-saccharide, glycoprotein, polypeptide, cellular metabolite, hormone, pharmacological agent, tranquilizer, barbituate, alkaloid, steroid, vitamin, amino acid, sugar or non-biological polymer.

37. A method according to claim 36, wherein B is a nucleotide or polynucleotide.

38. A method according to claim 36, wherein B is a serum-derived antibody or a monoclonal antibody.

39. A method according to claim 32, wherein said chemical agent is a whole cell, subcellular particle, nucleic acid, polysaccharide, protein, lipoprotein, lipopolysaccharide, glycoprotein, polypeptide, cellular metabolite, hormone, pharmacological agent, tranquilizer, barbituate, alkaloid, steroid, vitamin, amino acid, sugar, or non-biological polymer.

40. A method according to claim 32, wherein said chemical agent is immobilized on the surface of an assay vessel.

41. A method according to claim 32, wherein said chemical agent is a serum-derived antibody or a monoclonal antibody.

42. A method according to claim 34, wherein B is the same substance as said analyte.

43. A method according to claim 33 or 34, wherein said analyte is a whole cell, subcellular particle, nucleic acid, polysaccharide, protein, lipoprotein, lipopolysaccharide, glycoprotein, polypeptide, cellular metabolite, hormone, pharmacological agent, tranquilizer, barbituate, alkaloid, steroid, vitamin, amino acid, sugar, or non-biological polymer.

44. A method according to claim 33 or 34, wherein said analyte is insulin, digoxin, digitoxin, or T4 thyroid hormone.

45. A method according to claim 33 or 34, wherein said analyte is a fungus or nematode.

46. A method according to claim 33 or 34, wherein said analyte is an antibody

47. A method according to claim 34, wherein said material is a whole cell, subcellular particle, nucleic acid, polysaccharide, protein, lipoprotein, lipopolysaccharide, glycoprotein, polypeptide, cellular metabolite, hormone, pharmacological agent, tranquilizer, barbituate, alkaloid, steroid, vitamin, amino acid, sugar, or non-biological polymer.

48. A method according to claim 33 or 34, wherein said chemical material is a serum-derived antibody or a monoclonal antibody.

49. A method according to claim 33 or 34, wherein said chemical material is a DNA or RNA fragment.

50. A method according to claim 32, 33, or 34,wherein the method is a homogeneous method and the conditions are such that bound chemical moiety and unbound chemical moiety are not separated before the reagent mixture is exposed to the energy source.

51. A method according to claim 32, 33, or 34, wherein the method is a heterogeneous method and the conditions comprise a separation of bound chemical moiety and unbound chemical moiety prior to exposure of the reagent mixture to the energy source.

52. A method according to claim 33, wherein the analyte is fixed to an insoluble matrix.

53. A method according to claim 52, the method being a sandwich method.

54. A method according to claim 32, wherein the chemical agent is fixed to an insoluble matrix, and the chemical moiety is a component of a biological fluid or synthetic reaction mixture.

55. A method according to claim 34, the method being a competitive binding method wherein the material is fixed to an insoluble matrix.

56. A heterogeneous method according to claim 55, wherein the material is a monoclonal antibody and the insoluble matrix is the surface of an assay vessel.

57. A homogeneous method according to claim 55, wherein the material is a monoclonal antibody and the insoluble matrix is the surface of an assay vessel.

58. A method according to claim 30, wherein the reagent mixture is exposed to electrochemical energy.

59. A method according to claim 30, wherein the reagent mixture is exposed to chemical energy.

60. A method according to claim 30, wherein the reagent mixture is exposed to a combination of electromagnetic radiation, chemical energy, and electrochemical energy.

61. A method according to claim 30, wherein the chemical moiety is oxidized by the exposure to the energy source.

62. A method according to claim 30, wherein the chemical moiety is reduced by the exposure to the energy source.

63. A method according to claim 29, wherein each chemical moiety which is induced to emit electromagnetic radiation is induced to emit electromagnetic radiation more than once.

64. A method according to claim 30, wherein the reagent mixture contains oxalate, pyruvate, lactate, malonate, citrate, tartrate or peroxydisulfate.

65. A method according to claim 62, wherein the energy source is a chemical reducing agent.

66. A method according to claim 65, wherein the chemical reducing agent is magnesium.

67. A method according to claim 61, wherein the energy source is a chemical oxidizing agent.

68. A method according to claim 67, wherein the oxidizing agent is a Ce (IV) salt or PbO2.

69. A method according to claim 62, wherein the reagent mixture contains peroxydisulfate.

70. A method according to claim 61, wherein the reagent mixture contains oxalate, pyruvate, lactate, malonate, citrate or tartrate.

71. A method according to claim 58, wherein the reagent mixture is continuously exposed to an electrode whose potential oscillates between a potential sufficient to effect the reduction of said chemical moiety, and a potential sufficient to effect the oxidation of the chemical moiety.

72. A method according to claim 61, wherein the energy source is an electrode whose potential oscillates above and below a potential sufficient to oxidize the chemical moiety and the reagent mixture contains oxalate, pyruvate, lactate, malonate, tartrate or citrate.

73. A method according to claim 61, wherein the energy source is an electrode whose potential is constant and sufficient to oxidize said chemical moiety, and the reagent mixture contains oxalate, pyruvate, lactate, malonate, tartrate or citrate.

74. A method according to claim 62, wherein the energy source is an electrode whose potential oscillates above and below a potential sufficient to reduce said chemical moiety and the reagent mixture contains peroxydisulfate.

75. A method according to claim 62, wherein the energy source is an electrode whose potential is constant and sufficient to reduce said chemical moiety, and the reagent mixture contains peroxydisulfate.

76. A method according to claim 74, wherein the reagent mixture contains an acetonitrile.

77. A method according to claim 75, wherein the reagent mixture contains acetonitrile.

78. A method according to claim 58 comprising continuous detection of the emitted electromagnetic radiation.

79. A method according to claim 78, wherein the detecting of the emitted electromagnetic radiation is performed visually.

80. A method according to claim 78, wherein the continuous detection of electromagnetic radiation comprises use of a photodiode.

81. A method according to claim 58 comprising cumulative detection of the emitted electromagnetic radiation.

82. A method according to claim 81, wherein detecting of the emitted electromagnetic radiation comprises use of photographic film.

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

[M(P)m(L1)n(L2)o(L3)p(L4)q(L5)r(L6)s]t(B)u wherein:

M is ruthenium or osmium;
P is a polydentate ligand of M;

L1, L2, L3, L4, L5 and L6 are ligands of M, each of which may be the same as or different from each other ligand;

B is a biological, synthetic or non-biological substance, or variant thereof substance covalently bound to one or more of P, L1, L2,L3, L4, L5 and L6 through one or more amide or amine linkages.
m is an integer equal to or greater than 1;

each of n, o, p, q, r and s is zero or an integer;

t is an integer equal to or greater than 1; and u is an integer equal to or greater than 1, P, L1, L2, L3, L4, L5, L6 and B being of such composition and number that the chemical moiety can be induced to emit electromagnetic radiation and the total number of bonds to M provided by the ligands of M equals the coordination number of M;

the system comprising:

a) a reagent mixture comprising the chemical moiety;

b) means for inducing the chemical moiety to emit electromagnetic radiation; and c) means for detecting the emitted electromagnetic radiation.

84. A system for determining the presence of an analyte of interest which binds to a chemical moiety, the moiety having the structural formula [M(P)m(L1)n(L2)o(L3)p(L4)q(L5)r(L6)s]t(B)u wherein:

M is ruthenium or osmium;
P is a polydentate ligand of M;

. L1, L2, L3, L4, L5 and L6 are ligands of M, each of which may be the same as or different from each other ligand;
B is a biological, synthetic or non-biological substance, or variant thereof substance covalently bound to one or more of P, L1, L2, L3, L4, L5 and L6 through one or more amide or amine linkages.

85. A method of determining the presence of a chemical moiety having the formula as defined in claim 1 comprising:
a) forming a reagent mixture containing the chemical moiety;

b) inducing the chemical moiety to repeatedly emit electromagnetic radiation by directly exposing the moiety to electrochemical energy;
and c) detecting the emitted electromagnetic radiation and thereby determining the presence of the chemical moiety.

86. A method according to claim 85, wherein the moiety is capable of binding to a chemical agent.

87. A method of determining the presence of an analyte of interest which binds to a chemical moiety having the formula as defined in claim 1, comprising:
a) contacting the analyte with the chemical moiety so as to form a reagent mixture;

b) inducing the chemical moiety to repeatedly emit electromagnetic radiation by directly exposing the moiety to electrochemical energy;
and c) detecting the emitted electromagnetic radiation and thereby determining the presence of the analyte of interest.

88. 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 as defined in claim 1 comprising:

a) contacting the material, the chemical moiety and the analyte so as to form a reagent mixture;

b) inducing the chemical moiety to repeatedly emit electromagnetic radiation by directly exposing the moiety to electrochemical energy;
and c) detecting the emitted electromagnetic radiation and thereby determining the presence of the analyte of interest.

89. A chemical moiety having the formula [M(P)m(L1)n(L2)o(L3)p(L4)q(L5)r(L6)s]t(B)u wherein:

M is ruthenium or osmium;

P is a polydentate ligand of M selected from the group consisting of aromatic and aliphatic ligands;

L1, L2, L3, L4, L5 and L6 are monodentate ligands of M selected from the group consisting of carbon monoxide, cyanides, isocyanides, halides, and aliphatic, aromatic and heterocyclic phosphines, amines, stibines, and arsines each of which may be the same or different from each other ligand;

B is a biological substance, or a non-biological analog thereof covalently bound to one or more of p, L1, L2, L3, L4, L5 or L6 through one or more amide or amine linkages;

m is an integer equal to or greater than 1;

each of n, o, p, q, r and s is zero or an integer;

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

P, L1, L2, L3, L4, L5, L6 and B being of such composition and number that the chemical moiety can be induced to emit electromagnetic radiation and the total number of bonds to M provided by the ligands of M equals the coordination number of M.

90. A chemical moiety according to claim 1, wherein P is an aromatic heterocyclic ligand selected from the group consisting of bipyridyl, bipyrazyl, terpyridyl and phenanthrolyl.

91. A chemical moiety according to claim 1, wherein at least one of L1, L2, L3, L4, L5 and L6 is a polydentate aromatic heterocyclic ligand selected from the group consisting of bipyridyl, bipyrazyl, terpyridyl and phenanthrolyl.

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

[M(P)m(L1)n(L2)o(L3)p(L4)q(L5)r(L6)s]t(B)u wherein:

M is ruthenium or osmium;

P is a polydentate of M selected from the group consisting of aromatic and aliphatic ligands;

L1, L2, L3, L4, L5 and L6 are monodentate ligands of M selected from the group consisting of carbon monoxide, cyanides, isocyanides, halides, and aliphatic, aromatic and heterocyclic phosphines, amines, stibines, and arsines, each of which may be the same as or different from each other ligand;

B is a biological, synthetic or non-biological substance, or variant thereof which is a monodentate ligand of M
selected from the group consisting of carbon monoxide, cyanides, isocyanides, halides and aromatic and heterocyclic phosphines, amines, stibines and arsines or is attached to one or more of P, L1, L2, L3, L4, L5 or L6;

m is an integer equal to or greater than 1;

each of n, o, p, q, r and s is zero or an integer;

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

P, L1, L2, L3, L4, L5, L6 and B being of such composition and number that the chemical moiety can be induced to emit electromagnetic radiation and the total number of bonds to M provided by the ligands of M equals the coordination number of M;

the method comprising:

a) forming a reagent mixture containing the chemical moiety;

b) inducing the chemical moiety to emit electromagnetic radiation by exposing the reagent mixture to chemical energy or electrochemical energy; and c) detecting the emitted electromagnetic radiation and thereby determining the presence of the chemical moiety.

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

[M(P)m(L1)n(L2)o(L3)p(L4)q(L5)r(L6)s]t(B)u wherein:

M is ruthenium or osmium;

P is a polydentate of M selected from the group consisting of aromatic and aliphatic ligands;

L1, L2, L3, L4, L5 and L6 are monodentate ligands of M selected from the group consisting of carbon monoxide, cyanides, isocyanides, halides, and aliphatic, aromatic and heterocyclic phosphines, amines, stibines, and arsines, each of which may be the same as or different from each other ligand;

B is a biological substances, or a non-biological analog thereof which is a monodentate ligand of M
selected from the group consisting of carbon monoxide, cyanides, isocyanides, halides and aromatic and heterocyclic phosphines, amines, stibines and arsines or is attached to one or more of P, L1 L2, L3, L4, L5 or L6;

m is an integer equal to or greater than 1;

each of n, o, p, q, r and s is zero or an integer;

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

P, L1, L2, L3, L4, L5, L6 and B being of such composition and number that the chemical moiety can be induced to emit electromagnetic radiation and the total number of bonds to M provided by the ligands of M equals the coordination number of M;

the method comprising:

a) contacting the analyte with the chemical moiety so as to form a reagent mixture;

b) inducing the chemical moiety to emit electromagnetic radiation by exposing the reagent mixture to chemical energy or electrochemical energy; and c) detecting the emitted electromagnetic radiation and thereby determining the presence of the analyte of interest.

94. 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:

[M(P)m(L1)n(L2)o(L3)p(L4)q(L5)r(L6)s]t(B)u of M equals the coordination number of M;

the method comprising:

a) contacting the analyte with the chemical moiety so as to form a reagent mixture;

b) inducing the chemical moiety to emit electromagnetic radiation by exposing the reagent mixture to chemical energy or electrochemical energy; and c) detecting the emitted electromagnetic radiation and thereby determining the presence of the analyte of interest.

95. A method according to claim 31, 32, or 33 wherein B
is a biological substance, or a non-biological analog thereof, covalently bound to one or more of P, L1, L2, L3, L4, L5, or L6 through one or more amide or amine linkages.

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

[M(P)m(L1)n(L2)o(L3)p(L4)q(L5)r(L6)s]t(B)u wherein:

M is ruthenium or osmium;

b) means for inducing the chemical moiety to emit electromagnetic radiation; and c) means for detecting the emitted electromagnetic radiation.

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

[M(P)m(L1)n(L2)o(L3)p(L4)q(L5)r(L6)s]t(B)u wherein:

M is ruthenium or osmium;

P is a polydentate ligand of M selected from the group consisting of aromatic and aliphatic ligands;

L1, L2, L3, L4, L5 and L6 are monodentate ligands of M selected from the group consisting of carbon monoxide, cyanides, isocyanides, halides, and aliphatic, aromatic and heterocyclic phosphines, amines, stibines, and arsines, each of which may be the same as or different from each other ligand;

B is a biological substance, or a non-biological analog thereof, covalently bound to one or more of p, L1, L2, L3, L4, L5 or L6 through one or more amide or amine linkages;

m is an integer equal to or greater than 1;

98. A method of determining the presence of a chemical moiety having the formula as defined in claim 1, wherein B is a biological substance, or a non-biological analogy thereof, comprising:
a) forming a reagent mixture containing the chemical moiety;

b) inducing the chemical moiety to repeatedly emit electromagnetic radiation by directly exposing the moiety to electrochemical energy;
and c) detecting the emitted electromagnetic radiation and thereby determining the presence of the chemical moiety.

99. A method of determining the presence of an analyte of interest which binds to a chemical moiety having the formula as defined in claim 1, wherein B is a biological substance, or a non-biological analogy thereof, comprising:
a) contacting the analyte with the chemical moiety so as to form a reagent mixture;

b) inducing the chemical moiety to repeatedly emit electromagnetic radiation by directly exposing the moiety to electrochemical energy;
and c) detecting the emitted electromagnetic radiation and thereby determining the presence of the analyte of interest.

100. 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 as defined in claim 1, wherein B is a biological substance, or a non-biological analogy thereof, comprising:
a) contacting the material, the chemical moiety and the analyte so as to form a reagent mixture;

b) inducing the chemical moiety to repeatedly emit electromagnetic radiation by directly exposing the moiety to electrochemical energy;
and c) detecting the emitted electromagnetic radiation and thereby determining the presence of the analyte of interest.

101. A chemical moiety according to claim 5, wherein P is bipyridyl, bipyrazyl, terpyridyl, or phenanthrolyl.

102. A chemical moiety according to claim 1, wherein P is 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 substituent.

103. A chemical moiety according to claim 8, wherein at least one of L1, L2, L3, L4, L5 and L6 is bipyridyl, bipyrazyl, terpyridyl, or phenanthrolyl.

104. A chemical moiety according to claim 1 containing 2 bidentate ligands wherein each bidentate ligand is bipyridyl, bipyrazyl, terpyridyl, or phenanthrolyl.

105. A chemical moiety according to claim 1 containing 3 bidentate ligands, wherein each bidentate ligand is bipyridyl, bipyrazyl, terpyridyl, or phenanthrolyl.

106. A chemical moiety according to claim 13 containing two bidentate bipyridyl ligands.

107. A chemical moiety according to claim 1 wherein P is lower alkyl, lower aryl, lower aralkyl, carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxycarbonyl, hydroxyamino, aminocarbonyl, amidine, guanidinium, ureide, sulfur-containing group, or the carboxylate ester of N-hydroxysuccinimide substituent.

108. A chemical moiety according to claim 9, wherein at least one of L1, L2, L3, L4, L5 and L6 are substituted with a lower alkyl, lower aryl, lower aralkyl, carboxylate, carboxaldehyde, carboxamide, cyano, amino, hydroxycarbonyl, hydroxyamino, aminocarbonyl, amidine, guanidinium, ureide, sulfur-containing group, or the carboxylate ester of N-hydroxysuccinimide substituent.