GB2233451A

GB2233451A - Chemiluminescent enhancement

Info

Publication number: GB2233451A
Application number: GB8914749A
Authority: GB
Inventors: John C Voyta; Edward Brooks; Irene Bronstein
Original assignee: Tropix Inc
Current assignee: Tropix Inc
Priority date: 1989-06-27
Filing date: 1989-06-27
Publication date: 1991-01-09
Anticipated expiration: 2009-06-27
Also published as: FR2649205B1; GB2233451B; GB8914749D0; DE3921609C2; FR2649205A1; DE3921609A1

Abstract

Water soluble enhancer substances, generally macromolecular in nature, for example globular proteins such as vobine serum albumin, and polymeric quaternary ammonium salts such as poly(vinylbenzyltrimethylammonium chloride), which have the ability to inhibit light-emitting fluorophores are disclosed as permitting the stabilization, and hence increasing the light intensity, of such light-emitting fluorophores in aqueous media as compared to the intensity of the light emitted by the same quantities of such fluorophores in aqueous media in the absence of such enhancer substances.

Description

CHEKIPUMINESCENCE EWHAN F1E1D OF THE INVERTION This invent-ion relates to

improvements in the detectability of electromagnetic, including optically detectable, energy released by the decomposition of chemiluminescent chemical compounds in aqueous media. More particularly, this invention relates to enhanced detection of electromagnetic energy released by the decomposition of chemiluminescent compounds used to determine the presence, concentration or structure of a substance in an aqueous sample, particularly when such chemiluminescent compounds are used to detect the presence or determine the concentration of chemical or biological substances by artrecognized im=unoassay techniques, chemical assays or nucleic acid probe assays, or when they are used as direct chemical/physical probes for studying the molecular structures or microstructures of various macromolecules: synthetic polymers, is proteins, nucleic acids and the like.

BACKGROURD OF THE INV M 10 The decomposition of chemiluminescent chemical compounds to release electromagnetic, and especially optically detectable, energy -- usually luminescence In the form of visible light -- is well known and understood. The incorporation of such light emitting reactants in art-recognized immunoassays, chemical assays, nucleic acid probe assays and chemical/physical probe techniques as the means by which the analyte, a substance whose I- 1 PATENT 183/68 presence, amount or structure is being determined, is actually identified or quantified has assumed increasing importance in recent years, particularly with the advent of enzymatically cleavable 1,2-dioxetanes; see, for example, copending Bronstein U.S. patent application Serial No. 889,523, 0Method of Detecting a Substance Using Enzymatically-Induced Decomposition of Dioxetanes", filed July 24, 1986; Bronstein et al U.S. patent application Serial No. 140,035, 0Dioxetanes for Use in Assays", filed December 31, 1987 and Edwards U.S. patent application Serial No. 140,197, Synthesis of 1,2-Dioxetanes and Intermediates Thereforll, filed December 31, 1987.

Reactions that produce chemiluminescence exemplify yet another instance in which the medium, although not the message.

can determine the intensity of the message transmitted.

Chemiluminescent compounds that, upon decomposition in substances such as moderately polar or polar aprotic organic solvents, e.g., n-butanol, acetonitrile, dinethylsulfoxide or dinethylformanide, produce fluorophores that in turn emit light of adequate intensity for easy detection and quantitation will produce light of considerably lessened intensity when decomposed In a polar protic environment, and especially in aqueous media. But since all biological systems are aqueous -- indeed, man himself is 97% water -- the need to enhance the Intensity of light produced by chemiluminescent labels or substrates in i=unoassays, nucleic acid probe assays, chemical/physical probe techniques and other bioassays is obvious. One way to provide such enhancement, of course, Is to use expensive optical or electronic equipment:

single photon counters, luminometers, scintillation counters, etc. The present invention provides a far less expensive yet 2 1 1 PATENT 183/68 equally effective way of providing the needed light enhancement in aqueous media, and in many cases provides enhanced light intensity to a degree which permits detection by simple, inexpensive means, such as with a caimera, instead of with complex detection instruments.

The present invention also permits the detection of lesser concentrations of analytes using the same quantities of chemiluminescent chemical compounds used in hitherto-practiced methods, and concomitantly permits the use of lessened amounts of chemiluminescent chemical compounds to detect the same concentrations of analytes as compared to those necessary in hitherto-practiced methods. By practicing this invention the intensity of light emitted by fluorophore decomposition products of chemiluninescent chemical compounds can be enhanced by a factor of at least 10%, but usually at least tenfold and oftentimes by factors of at least 100 to 1,000,000 times the intensities obtainable in aqueous media using the same chemiluminescent compounds in hitherto-practiced methods.

While we do not wish to be bound by any theory or mechanism advanced to explain the operation of this invention, we believe that our enhancer substances act in a polar protic environment, such as an aqueous medium, to bind fluorophore-containing fragments resulting from the decomposition of a chemiluminescent chemical compound and maintain such fragments in a stabilized conformation, possibly by hydrophobic or ionic interaction, or both, between the enhancer substance and the fluorophorecontaining fragment. This we believe in turn inhibits the fluorophore from releasing all or a substantial part of its excitational energy through non-light emitting pathways: 3 PATENT 183/68 vibrational relaxation in which energy is emitted as beat rather than light, intersystem crossing to other lower energy states, or other such mechanisms.

SUMMARY OF THE INVMIO

It has now been discovered that certain water soluble natural lyoccurring and synthetic substances, generally macromolecular in nature, for example water soluble globular proteins that include hydrophobic regions: mammalian serum albumins such as bovine serum albumin (BSA) and human serum albumin (HSA), or water soluble polymeric quaternary ammonium salts: poly(vinylbenzyltrinethyl-ammonium chloride) (TMQ) or poly[vinylbenzyl(benzyldinethyl-ammonium chloride)] (BDMQ), permit the stabilization, and hence increase the light intensity, of light-emitting fluorophores produced by the decomposition of chemiluminescent chemical compounds in aqueous media. Such chemiluminescent compounds include, but are not limited to, thermally, chemically, electrochenically and enzymatically cleavable 1,2-dioxetanes; acrid-inium esters; lucigenin, a diner of N-substituted acridines; luciferin, and the like, and mixtures of such chemiluminescent compounds with each other and with one or more auxiliary fluorophores, e.g., fluorescein-1, that accept energy from energy-emitting fluorophores produced by the decomposition of chemiluminescent compounds and in turn emit detectable energy. By virtue of the presence of effective amounts of an enhancer substance or substances the Intensity of the light emitted in aqueous medium by the thus-stabilized fluorophores is increased significantly as compared to the 4 PATENT 183/68 intensity of light emitted by the same quantities of fluorophores in the absence of such enhancers.

it is, therefore, an object of this invention to provide improvements in the detectability of electromagnetic, e.g., optically detectable, energy released by the decomposition of chemiluminescent chemical compounds in aqueous media.

Another object of this invention is to provide means for enhanced detection of electromagnetic, e.g., optically detectable, energy released by the decomposition of chemiluminescent chemical compounds used to detect the presence or determine the concentration or structure of a substance in an aqueous sample.

A further object of this invention is to provide improvements in the detectability of electromagnetic, e.g.,, is optically detectable, energy released by the decomposition of chemiluninescent chemical compounds used to detect the presence or determine the concentration of chemical or biological substances by art- recognized immunoassay, chemical assay or nucleic acid probe assay techniques.

A still further object of this invention is to provide improvements in the detectability of electromagnetic, e.g., optically detectable, energy released by the decomposition of chemiluminescent chemical compounds for studying molecular structures or microstructures.

Yet another object of this Invention is to provide aqueous compositions comprising water soluble naturally-occurring and synthetic enhancer substances which enable the stabilization, and hence increase the light intensity, of light-emitting fluorophores produced by the decomposition of chemiluminescent 5 PATENT 183/68 chemical compounds in aqueous media.

These and other objects, as well as the nature, scope and utilization of this invention, will become readily apparent to those skilled in the art from the following description, the drawings and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows plots of the luminescense intensity signals obtained in the TSH assays of Example 46 performed in the presence (Curve "All) and absence (Curve "B") of B5A.

FIG. 2 shows plots of the light emission resulting from the decomposition of the acridinium ester used to detect the AntiTSH-lgG of Example 47 in the absence ("Control) and presence CEnhanced") of a BDMQ/fluorescein disodium salt enhancer.

FIGS. 3-5 show the luminescence rate spectra of the emitting dioxetane fragment with and without enhancer: evaluated in Example 4 (0.1% BDMQ; FIG. 3) and Example 7 (1.0% BSA; FIG. 4), together with the luminescence rate spectrum of Example 30's buffer control (FIG. 5).

FIG. 6 shows a plot of Relative Turner Units (RTU) versus alkaline phosphatase concentration for the various systems of Example 48.

FIGS. 7 and 8 are reproductions of the photographs described in Example 49 taken of a substrate solution without (FIG. 7) and with (FIG. 8) B5A enhancer.

6 PATENT 183/68 DETAILED DESCRIPTION OF THE TNVENTION

The enhancer substances used in practicing this invention are water soluble naturally-occurring or synthetic materials which can provide a hydrophobic microenvironment of reduced polarity for fluorophore-containing fragments resulting from the decomposition of a chemiluminescent chemical compound contained In a polar medium, i.e., a medium consisting of water as a solvent or a mixture of water and other largely or entirely polar substances, such as methanol, acetonitrile, dimethylsulfoxide, dimethylformamide, or the like.

As noted above, included among such enhancer substances are macronolecular globular proteins, generally ones having molecular weights ranging from about 1000 to about 600,000 daltons, and preferably from about 40,000 to about 100,000 daltons, as is determined by SDS gel electrophoresis, that Include hydrophobic regions: mammalian serum albumins such as B5A, HSA, AFP and the like; globular proteins such as mammalian IgG, lgE, Protein A, avidins, and the like; serum lipoproteins, apolipoproteins, and the like.

Synthetic oligomeric or polymeric enhancer substances that can be used in practicing this invention include, first of all, water soluble poly(vinylaryl quaternary ammonium salts), such as the poly(vinylbenzyl quaternary ammonium salts) having the formula:

7 PATENT 183/68 (1) In this formula each of R,, R2 and R3 can be a straight or branched chain unsubstituted alkyl group having fro= 1 to 20 carbon atoms, inclusive, e. g., nethyl, ethyl, n-butyl, t-butYl, cetyl, or the like; a straight or branched chain alkyl group having from 1 to 20 carbon atoms, inclusive, substituted with one or more hydroxy, alkoxy, e.g., nethoxy, ethoxy, benzyloxy or polyoxethylethoxy, aryloxy, e- g., phenoxy, amino or substituted amino, e.g., rethylanino, a=ido, e.g., aceta=ido or cholesterylo>-ycarbonylazido, or fluoroalkane or fluoroaryl, e.g., heptaflucrobutyl, groups, an unsubstituted monocycloalkyl group having fro= 3 to 12 ring carbon atoms, inclusive, e.g., cyclohexyl or cyclooctyl, a substituted ronocycloalkyl group having fro= 3 to 12 ring carbon atoms, inclusive, substituted with one or =ore alkyl, alkoxy or fused benzo groups, e.g., nethoxycyclohexyl or 1,2,3,4-tetrahydronaphthyl, a polycycloalkyl group having 2 or =ore fused rings, each having from 5 to 12 carbon &tons, inclusive, unsubstituted or substituted with one or nore alkyl, alkoxy or aryl groups, e.g., 1-adanantyl or 3-phenyl- 1-adamantyl, an aryl, alkaryl or aralkyl group having at least one ring and from 6 to 20 carbon atoms jn toto, unsubstituted or substituted with one or =ore alkyl, aryl, or flucroalkane or fluoroaryl groups, e.g., phenyl, naphthyl, pentafluorophenyl, ethylphenyl, benzyl, hydroxybenzyl, phanylbenzyl or 8 is PATENT 183/68 dehydroabietyl; at least two of R,, R2 and R3, together with the quaternary nitrogen atom to which they are bonded, can form a saturated or unsaturated, unsubstituted or substituted nitrogencontaining, nitrogen and oxygen-containing or nitrogen and sulfur-containing ring having from 3 to 5 carbon atoms, inclusive, and 1 to 3 heteroatons, Inclusive, and which may be benzoanylated, e.g., 1-pyridyl,l-(3-alkyl or aralkyl)imidazolium, morpholino, piperidino or acylpiperidino, benzoxazole, benzthiazole or benzamidazole.

The symbol X- represents a counterion which can include, alone or in combination, moieties such as halide, i.e., fluoride, chloride, bromide or iodide, sulfate, alkylsulfonate, e.g., methylsulfonate, arylaulfonate, e.g., p-toluenesulfonate, substituted arylsulfonate, e.g., anilinonaphthylenesulfonate (various isomers), lucifer yellow CH and diphenylanthracenesulfonate, perchlorate, alkanoate, e.g., acetate, arylcarboxylate, e.g., fluorescein or fluorescein derivatives, Ibenzoheterocyclicarylcarboxylete, e.g., 7-diethylamino4cyanocoumarin-3-carboxylate, phosphate, or substituted monoaryloxyphosphate, e.g., a 3-(20-spiroadamantane)-4-nethoxy(3"phosphoryloxy)phenyl-1,2-dioxetane dianion or other dianions indicated in formula III, infra.

The symbol n represents a number such that the molecular weight of such poly(vinylbenzyl quaternary ammonium salts) will range from about 800 to about 200,000, and preferably from about 20,000 to about 70,000, as determined by intrinsic viscosity or LALLS techniques.

9 PATENT 183/68 illustrative of such water soluble poly(vinylbenzyl quaternary a-noniun salts) are TY&Q, EDYQ, and the like.

These vinylbenzyl quaternary ammonium salt polymers can be prepared by tree radical polymerization of the appropriate precursor monomers or by exhaustive alkylation of the corresponding tertiary amines with polyvinylbenzyl chloride. This same approach can be taken using other polymeric alkylating agents such as chloromethylated polyphenylene oxide or polyepichlorchydrin. The sane polymeric alkylating agents can be used as initiators of oxazoline ring-opening polymerization, which, after hydrolysis, yields polyethyleneimine graft copolymers. Such copolymers can then be quaternized, preferably with aralkyl groups, to give the final polymeric enhancer substance.

Water soluble acetals of a polyvinylalcohol and a forr.ylbenzyl quaternary annoniun salt, having the formula:

OL -,%W 4 (11) wherein each R4 is the same or a different aliphatic substituent and X, is an anion, as disclosed and claimed in Bronstein-Bonte et &I U.S. Patent No. 4,124,388, can also be used as enhancer substances in practicing this invention. And, the individual vinylbenzyl quaternary ammonium salt nonomers used to prepare the poly(vinylbenzyl quaternary ammonium salts) of formula 1 above can also be copolynerized with other vinylbenzyl quaternary PATENT 183/68 ammonium salt monomers whose polymers are depicted In formula 1, or with other ethylenically unsaturated monomers having no quaternary ammonium functionality, to give polymers such as those disclosed and claimed In land et al U.S. Patent No. 4,322, 489; Bronstein- Bonte - et al U.S. Patent No. 4,340,522, Land et al U.S. Patent No. 4,424, 326; Bronstein-Bonte et C U.S. Patent No. 4,503,138 and Bronstein-Bonte U. S. Patent No. 4,563,411, all of which polymers can also be used as enhancer substances in practicing this invention. Preferably these quaternized polymers will have molecular weights within the ranges given above for the poly(vinylbenzyl quaternary ammonium salts) of formula 1.

Other water soluble cligoneric, honopolymeric and copolyneric materials can be used as enhancer substances in addition to or instead of the foregoing polymers, including:

-poly-N-vinyl oxazolidinones; -polyvinyl carbanates (e.g., polyvinyl propylene carbamate); -polyhydroxyacrylates and nethacrylates [e.g., poly(phydroxyethyl)nethacrylate and polyethyleneglycol monomethacrylates); -amine-containing oligomers (e.g., Jeffamines) quaternized with alkylating or aralkylating agents; -synthetic polypeptides (e.g., polylysine W phenylalanine); -polyvinylalkylethers (e.g., polyvinyl methyl ether); -polyacids and salts thereof (e.g., polyacrylic acids, polymethacrylic acids, polyvinylbenzoic acid, polyethylenesulfonic acid, polyacrylemidonethylpropanesulfonic acid, polynaleic acid and poly(Nvinyl succinamidic acid)); 11 PATENT 183/68 -polyacrylamides and polymethecrylamides derived from ammonia or cyclic and acyclic primary or secondary amines; -polyvinyl alcohol and polyvinyl alcohol copolymers with vinyl acetate, ethylene and the like; -poly 2-, 3- or 4-vinylpyridinium salts where the haterocyclic nitrogen atom is bonded to a group as defined for R, R2 and R3 in formula 1 above; -polyvinylalkylpyrrolidinones (e.g., polyvinylmethyl pyrrolidinones); -polyvinylalkyloxazolidones (e.g., polyvinylmethyloxazolidones); -branched polyethyleneimines, acylated branched polyethyleneimines, or acylated branched polyethyleneinines further quaternized with alkyl or aralkyl groups; -poly N-vinylamines derived from ammonia or cyclic and acyclic primary or secondary amines, and salts thereof; -polyvinylpiperidine; -polyacryloyl, polymethacryloyl or 4-vinylbenzoyl aminimides where the three substituents on the positively charged nitrogen atom may be any of the R,, R2 and R3 groups defined in formula 1 above.

Here too, these oligomeric or pplymeric enhancer substances preferably will have molecular weights within the ranges given above for the poly(vinylbenzyl quaternary ammonium salts) of formula 1.

Water soluble monomeric quaternary soaps whose nitrogen atom has at least one benzyl substituent and in which the remaining nitrogen substituents correspond to the definitions given for R2, R3 and X in formula 1, supra. e.g., 12 PATENT 183/68 cetyldinethylbenzylamnonium chloride or cetyldibenzylmethyl ammonium bromide, can also be used.as enhancer substances when practicing this invention.

The amount of enhancer substance or mixture of enhancer substances employed when practicing this invention can vary within wide limits depending on the particular enhancer substance(s) chosen, the amount and type of chemiluminescent compound(s) present, etc. For example, certain enhancers, such as B5A, exhibit an optimum concentration, depending primarily on the cheniluninescent compound present, beyond which the addition of further amounts of the,enhancer produces no further increase in light intensity. Other enhancers, such as THQ, provide increased enhancement with increasing concentration. In general, however, amounts of enhancer substance ranging from about 0.01% is to about 25%, and preferably from about 0.1% to about 5%, based on the weight of enhancer divided by the weight of aqueous medium, will be employed.

Any chemiluminescent chemical compound that (1) can be induced to decompose to yield a moiety in an excited state, such moiety having a heteropolar character that makes it susceptible to environmental effects, and particularly to dampening or dininution'of luminescence in a polar protic environment, and that (2) is usable to determine the presence, concentration or structure of a substance In a polar protic environment, particularly a substance in an aqueous sample, can be used in practicing this invention.

1,2-Dioxetanes such as the enzymatically-cleavable dioxetanes disclosed and claimed In the aforementioned copending Bronstein, Bronstein et al and Edwards applications and their 13 PATENT 183/68 thermally, chemically and electrochenicallY cleavable analogs form one class of usable chemiluninescent chemical compounds. These 1,2-dioxetanes can be represented by the general formula:

0-0 L-1/XX (III) T C/_ Z In this formula T is an unsubsituted or substituted cycloalkyl, aryl, polyaryl or heteroaton group, e.g., an unsubstituted cycloalkyl group having from 6 to 12 ring carbon atoms, inclusive; a substituted cycloalkyl group having fro= 6 to 12 ring carbon atoms, inclusive, and having one or more substituents, 10 which can be an alkyl group having from 1 to 7 carbon atoms, inclusive, or a heteroaton group which can be an alkoxy group having fro= 1 to 12 carbon atoms, inclusive, such as nethoxy or ethoxy, a substituted or unsubstituted aryloxy group, such as phenoxy or carboxyphenoxy, or an alkoxyalkyloxy group, such as is net-hoxyethoxy or polyethyleneoxy, or a cycloalkylidene group bonded to the 3-carbon atom of the dioxetane ring through a spiro linkage and having from 6 to 12 carbon atoms, inclusive, or a fused polycycloalkylidene group bonded to the 3-carbon of the dioxetane ring through a spiro linkage and having two or more fused rings, each having from 5 to 12 carbon atoms, inclusive, e.g., an adamant-2-ylidene group.

The symbol Y represents a light-emitting fluorophore-forming fluorescent chromophore group capable of absorbing energy to form an excited energy state from which it emits optically detectable energy to return to its original energy state. 14 PATENT 183/68 The symbol X2 represents hydrogen or an alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl or cycloheteroalkyl group, e.g., a straight or branched chain alkyl group having from 1 to 7 carbon atoms, Inclusive; a straight or branched chain hydroxyalkyl group having from 1 to 7 carbon atoms, inclusive, or an -OR group in which R is a CI-C20 unbranched or branched, unsubstituted or substituted, saturated or unsaturated alkyl, cycloalkyl, cycloalkenyl, aryl, aralkyl or aralkenyl group, fused ring cycloalkyl, cycloalkenyl, aryl, aralkyl or aralkenyl group, or an N1 0 or S hereto atomcontaining group, or an enzyne-cleavable group containing a bond cleavable by an enzyme to yield an electron-rich moiety bonded to the dioxetane ring. Preferably X2 is a nethoxy group.

The symbol Z represents hydrogen (in which case the is dioxetane can be thermally cleaved via rupture of the oxygenoxygen bond), a chemically cleavable group such as a hydroxyl group, an alkanoyl or aroyl ester group, or an alkyl or aryl silyloxy group, or an enzyme- cleavable group containing a bond cleavable by an enzyme to yield an electron-rich moiety bonded to the dioxetane ring, e.g., a bond which, when cleaved, yields an oxygen anion, a sulfur anion or a nitrogen anion, and particularly an amido anion such as a sulfonamido anion.

One or more of the substituents T, X2 and Z can also include a substituent which enhances the water solubility of the 1,2dioxetane, such as a carboxylic acid, sulfonic acid or quaternary amino salt group, at least one of X2 and Z, and preferably Z, is an enzyme-cleavable group, and preferably an enzyme-cleavable phosphate ester group, and X2 and Y together can represent a fused fluorescent chronophore group bonded to the 4-carbon atom is is PATE9T 183/68 of the dioxetane ring through a spiro linkage, e.g., one having the general formula:

R7 Ra (1v) In this fOrmul& X3 is c 0 S - or -NM9, where each Of R7, RS and Rg, independently, is hydrogen, a branched or straight chain alkyl group having 1 to 20 carbon atoms, inclusive, e.g. , =ethyl, n-butyl or decyl, a branched or straight chain heteroalkyl group having 1 to 7 carbon atoms, inclusive, e.g., nethoxy, hydroxyethyl or hydroxypropyl; an aryl group having 1 or 2 rings, e.g., phenyl; a heteroaryl group having 1 or 2 rings, e.g., pyrrolyl or pyrazolyl; a cycloalkyl group having 3 to 7 carbon atoms, inclusive, in the ring, e.g., cyclohexyl; a heterocycloalkyl group having 3 to 6 carbon atoms, inclusive, in the ring, e.g., dioxane; an aralkyl group having 1 or 2 rings, e.g., benzyl; an alkaryl group having 1 or 2 rings, e.g., tolyl; or an enzyme-cleavable group as defined above; and each R6, independently, can be hydrogen; an electron-withdrawing group, such as a perfluoroalkyl group having between 1 and 7 carbon atoms, inclusive, e.g., trifluoromethyl; a halogen; C02H, Z1C02H, 503H, N02, ZIN02, C-N, or ZIC-N, where Z1 is a branched or straight chain alkyl group having 1 to 7 carbon atoms, inclusive, e.g., nethyl, or an aryl group having 1 or 2 rings, e.g., phenyl; an electron-dDnating group, e.g., a branched or 16 is PATENT 183/68 straight chain Cl-C7 alkoxy group, e.g., methoxy or ethoxy; an aralkoxy group having 1 or 2 rings, e.g., phenoxy; a branched or straight chain Cl-C7 hydroxyalkyl group, e.g., hydroxrymethyl or hydroxyethyl; a hydroxyaryl group having 1 or 2 rings, e.g., hydroxyphehyl; a branched or straight chain Cl-C7 alkyl ester group, e.g., acetate; or an aryl ester group having 1 or 2 rings, e.g., benzoate; a heteroaryl group having 1 or 2 rings, e.g., benzoxazole, benzthiazole, benzimidazole or benztriazole; or hydrogen or an enzyme-cleavable or chemically cleavable group Z as defined herein, with at least one of R6 being Z. Furthermore, all of the R6 groups together can form a ring which can be substituted or i=ubstituted, and one or more of the substituents T, X2 and Z can also Include a substituent which enhances the water solubility of the 1,2- dioxetane, such as a carboxylic acid, sulfonic acid or quaternary amino salt group.

Included among the substances whose residues can be present X in such 1,2dioxetanes as fluorescent chromophore (Y or < Y 11,1 Z groups are:

-anthracene and anthracene derivatives, e.g., 9,10- diphenylanthracene, 9-methylanthracene, 9-anthracene carboxaldehyde, anthrylalcohols and 9-phenylanthracene; -rhodanine and rhodamine derivatives, e.g., rhodols, tetranethyl rhodamine, tetraethyl rhodamine, diphenyldimethyl rhodamine, diphenyldiethyl rhodamine and dinaphthyl rhodamine; -fluorescein and fluorescein derivatives, e.g., 5- iodoacetamido fluorescein, 6-iodoacetamido fluorescein and fluorescein-5-maleimide; 17 PATENT 183/68 -counarin and coumarin derivatives, e.g., 7-dialkylamino-4inethylcoumarin, 4-bromonethyl-7-methoxycoumarin and 4bronomethyl-7-hydroxy coumarin; -erythrosin and erythrosin derivatives, e.g., hydroxy erythrosins, crythrosin-S-iodoacetamide and erythrosin-5- inaleimide; -aciridine and aciridine derivatives, e.g., hydroxy aciridines and 9- methyl aciridine; -pyrene and pyrene derivatives, e.g., N-(l-pyrene) iodoacetamide, hydroxy pyrenes and 1-pyrenemethyl iodoacetate; -stilbene and stilbene derivatives, e.g., 6,61dibromostilbene and hydroxy stilbenes; -naphthalene and naphthalene derivatives, e.g., 5dimethylamino naphthalene-l-sulfonic acid and hydroxy naphthalenes; -nitrobenzoxadiazoles and nitrobenzoxadiazole derivatives, e.g., hydroxy nitrobenzoxadiazoles, 4chloro-7-nitrobenz-2-oxa1,3-diazole, 2-(7nitrobenz-2-oxa-1,3-diazol-4-yl) nethylaminoacetaldehyde and 6-(7nitrobenz-2-oxa-1,3-diazol-4-y1 aminohexanoic acid; - -quinoline and quinoline derivatives, e.g., 6hydroxycluinoline and 6- aminoguinoline; -acridine and acridine derivatives, e.g., N-nethylacridine and Nphenylacridine; -acidoacridine and acidoacridine derivatives, e.g., 9nethylacidoacridine and hydroxy-g-nethylacidoacridine; -carbazole and carbazole derivatives, e.g., Nnethylcarbazole and hydroxyN-nethylcarbazole; is is PATENT 183/68 -fluorescent cyanines, e.g., DCM (a laser dye), hydroxy cyanines, 1,6- diphenyl-1,3,5-hexatriene, 1-(4-dimethyl aminophenyl)-6-phenylhexatriene and the corresponding 1,3butadienes; is -carbocyanines and carbocyanine derivatives, e.g.p phenylcarbocyanine and hydroxy carbocyanines; -pyridinium salts, e.g., 4(4-dialkyldianinostyryl)N-methyl pyridinium iodate and hydroxy-substituted pyridinium salts; -oxonols; and -resorofins and hydroxy resorofins.

When using an enzymatically-cleavable 1,2-dioxetane, cleavage can be accomplished using an enzyme such as an alkaline phosphatase that will cleave a bond in, for example, a Z substituent such as a phosphate ester group to produce a Y anion in a charge transfer state that will, in turn, destabilize the dioxetane and cleave its oxygen-oxygen bond. Cleavage can also be accomplished by using an enzyme such as an oxidoreductase enzyme that will cleave the oxygen-oxygen bond directly; see the aforementioned Bronstein and Bronstein et al pending U.S. patent applications. Besides a phosphate ester group, Z in formulas III and IV above can be an

enzyme-cleavable alkanoyloxy group, e.g., an acetate ester group, or an oxacarboxylate group, 1-phospho-2,3diacy1glyceride group, 1-thio-Dglucoside group, adenosine triphosphate analog group, adenosine diphosphate analog group, adenosine monophosphate analog group, adenosine analog group, aD-galactoside group, P-D-galactoside group, a-D-glucoside group, P-D-glucoside group, a-D-mannoside group, P-D-mannoside group, PDfructofuranoside group, P-D-glucosiduronate group. Pig PATENT 183/68 toluenesulfonyl-L-arginine 'dye ester group or p-toluenesulfonylL-arginine dye anide group.

Preferred enzynatically-cleavable 1,2-dioxetanes for use in practicing this invention are the 3-(21-spiroadanantane)-4z2ethoxy-4-(3-phosphoroyloxy)phenyl-1,2-dioxetane salts represented by the formula:

O-C) 0-p- 0- M"(V).0 M.4- (V) wherein K+ represents a cation such as alkali metal, e.g., sodium or potassium, annoniun, or a Cl-7 alkyl, aralkyl or aromatic quaternary c=oniun cation, N(M4 + in which each RS can be alkyl, e.g., =ethyl or ethyl, aralkyl, e.g., benzyl, or form part of a heterocyclic ring system, e.g., pyridiniun, and particularly the disodium salt.

Another class of cheniluninescent compounds usable when practicing this invention includes acridiniun esters or the phenanthradiniu= derivatives in which a phenoxycarbonyl group is present in the 9-position on an Nsubstituted acridine moiety, e.g., those having the general formula:

X X CW o, 1 0 (.11 (V1) 2D wherein RID is, for example, a Cl-CID straight or branched chain alkyl or alkoxy group; an aryl group, preferably one having a 2D Q 1 PATENT 183/68 single aryl ring, e.g., phenyl; an amino group; a hydroxyl group, a carbonyl-containing group; e.g., an acetylgroup, or a carboxylic acid or ester group; R,, can be any of RIO, hydrogen; a fused benzo ring, or a group suitable for conjugation to proteins or nucleic acids, e.g., an isothiocyanate group, an Nhydroxysuccinimide ester group, biotin or avidins, and X4 is a counterion which can include, alone or in combination, a halide, sulfate, alkylsulfonate, arylsulfonate, substituted aryloulfonate, perchlorate, alkanoate, arylcarboxylate, heteroaryl- carboxylate, phosphate or substituted monoaryloxyphosphate noiety, an anionic oligomer or an anionic homopolymer or copolymer.

These acridinium esters, in the presence of a peroxide such as hydrogen peroxide and an alkaline substance, e.g., sodium 2.5 hydroxide or potassium hydroxide, chemiluminesce from the excited state of the N-substituted acridone formed by cleavage of the phenoxy ester portion of the molecule.

one or more auxiliary fluorophores extraneous to the lightemitting fluorophores produced by the decomposition of the chemiluninescent compound(s) also present that will accept energy, especially light, from energy-emitting fluorophores produced by the decomposition of the chemiluminescent compound(s) and in turn emit detectable energy, again preferably light, can be used when practicing this invention. Among the auxiliary fluorophores that can be used, alone or in combination, are the substances listed above whose residues can be present in 1,2 dioxetanes as fluorescent chrorophore groups. Fluorescein and fluorescein derivatives, including derivatives capable of establishing a covalent bond with the enhancer substance, are 21 PATENT 183/68 especially preferred for use as the auxiliary fluorophore(s).

These auxiliary fluorophores can simply be admixed with the enhancer substance(s) and chemiluminescent compound(s) or bonded to either or both of these materials. Phycobiliproteins represent a naturally-occurring class of enhancer molecules in which a fluorophore is covalently bonded to a protein. When the auxiliary fluorophore is bonded to a chemiluninescent compound, it is preferably bonded to the portion of the chemiluminescent compound that, upon decomposition, forms a fragment containing the fluorophore portion of the chemiluminescent compound's molecule. In this way energy transfer is enhanced due to the two fluorophores being in close proximity to one another. Auxiliary fluorophores that are insoluble or partially insoluble in aqueous medium can be solubilized by first grafting them onto is solubilizing molecules, e.g., water soluble oligomer or polymer molecules.

When admixed with the enhancer substance(s) and chemiluminescent compounds(s), the amount of auxiliary fluorophore present used can range from about 1 ng (nanogram)/mL to about 10 =g/mL of enhancer-containing solution, and preferably from about 1 pg/=L to about 100 pg/mL of enhancer-containing solution.

When auxiliary fluorophores covalently attached to proteins are used, the molar ratio of fluorophore to protein can range from 1 to 60, and preferably (e.g., for BSA) from 1 to 6, per molecule of protein.

This invention"is particularly useful when conducting lmmunoassays, such as those employed to detect an enzyme or a member of a specific binding pair, e.g., an antigen-antibody pair 22 1 is PATENT 183/68 or a nucleic acid paired with a probe capable of binding to all or a portion of the nucleic acid. Such assays include immunoassays used to detect a hormone such as p-hunan chorionic gonadotropin (p-bCG), thyroid stimulating hormone (TSH), follicle stimulating hormone (FSH), luteinizing hormone (M) or the like, cell surface receptor assays, and nucleic acid assays used to detect viruses, e.g., RIV or ETW III and cytomegalovirus, or bacteria, e.g., E.Coli., and histocompatibility assays; for typical assay protocols see the working examples, infra, as well as the aforementioned Bronstein and Bronstein et al U.S. patent applications. The Invention can also be used in assays for a che=ical analyte, such as potassium or sodium ions, or in assays for substances such as cholesterol or glucose in which the analyte is caused to decompose, for example using an enzyme such as cholesterol oxidase or glucose oxidase, to form a substance, e.g., hydrogen peroxide, capable of causing the che=iluninescent compound to decompose.

In order that those skilled in the art can more fully understand this invention, the following examples are set forth. These examples are given solely for purposes of illustration, and should not be considered as expressing limitations unless so set forth in the appended claims.

EXAMPLES 1 - 45 The enhancer substances listed in Table I below, in which all parts and percentages are by weight, unless otherwise stated, were evaluated for their light intensity-enhancing properties by dissolving the appropriate amount of enhancer in 0.05 M carbonate 23 PATENT 183/68 ]buffer solution containing 1 nM magnesium chloride (pH - 9.5) to give the noted concentrations. To 1 =1 samples of each of the resulting solutions (including the control, Example 31, containing no enhancer) there was then added sufficient 3-(21 spiroadamantana-4-methoxy-4-(30-phosphoryloxy)phenyl-1,2- dioxetane disodium salt, dissolved in the same carbonate buffer solution used to dissolve the enhancer, to give a final concentration of this chemiluminescent compound of 0.125 W. The resulting solutions were then equilibrated to 301C, after which aqueous alkaline phosphatase solution was added to give a final enzyme concentration of 4.17 x 10 -10 M. The luminescence signal of each solution was then measured at 30C in a Turner 20 E Luminometer integrating over twenty minutes. Successful enhancers or amounts of enhancer were those that gave an is enhancement factor greater than 1 (i.e., greater than that of the sample, Example 30, containing the chemiluminescent compound but no enhancer).

24 Table 1

Example Enhancer Enhancement Concentration Factor 1 0.1% ADMO 0 fivoroactini 298.891832 2 0.1% AMAC2 4 ftworeactini 135.643835 3 1.0% ADMO 19.026097 4 0.1% ADMO 18.422605 0.25% TNO 7.194016 6 0.1% &SA 5.172073 7 1.0% &SA 4.790a37 a 0.1% 790 4.475993 9 0.1% BD9CAC 2.874500 1.0% PotyMAPTAC3 2.384163 11 1.0 PVP K-904 2.034744 12 0.01pq/CL ftuorercein stone 2.004920 13 1.0% PEDX5 1.454102 14 1.0% PYP K-306 1.419595 is 1.0% Potybrene7 1.391839 D.Olpilwi FLuoreacein added.

benzytdioethyLcety[aminonium chloride; TCI Potyinethecrylefdopropytenemethyt armonfum chloride; Polyscience.

Potyvinytpyrrottdont; CAF.

Potyethytoxazotlne; Dow Cheefcat Cc.

6 Potyvinytpyrrolidinont; CTAF.

7 1,5-Dfoethyt-1.5-dfbze-umdecamethylene polymethobrorlde; Aldrich.

Table 1 (contd).

Enhancer Enhancement Example Concentration Facto Protein As ftuoregcetn Pú1 10009 0.1% Potybrene 0.1% PVP K-30 13 14 1.391196 1.262907 1.167327 1.140866 1.139414 1.112314 1.091996.084700 - 077093.076141 1.045022 1.043893 1.025682 1.023954 16 17 It 19 20 21 22 23 24 25 26 27 28 29 a staphylococcus-derived; stole&. 9 poLyethytentiofne; Dow Chemicat Co. 10 Potyethytentoxide, Union Carbide Corp. 11 Atpinic acid (sodium aatt); Rerck. Sodium cettutose autfate; Werck. Potyethytemepropytene styeet; RASF Wyendotte. PotyOXAzoline; Dow Chericat C.

PvP K-90 Ketcolot Atoinil Ketco SCS XLIZ Pluronic F12713 PotVox W-1014 Kelco $CS XL PEDX PEI 1000 1 1 1 27 - Table 1 (contd).

Enhancer Enhancement Example Concentration Factor Buffer actution alone (no enhancer) 1.00000 31 1.0% Pturonic F127 0.959475 32 0.1% It-crown-6 etherls 0.953054 33 D. 1% Cue Ghatti16 0.946162 34 0.1% Protein A 0.940106 0.1% PVEP17 0.933433 36 0.1% Poty0x W-80 0.930573 37 0.1% Polyox W-10 0.907289 38 1.0% PEGI8 0.893920 39 1.0% It-crown-6 ether 0.571326 M% Ketcoact Atsin19 0.786052 ALdrich.

16 Cat. sic. G0378; Sign&.

17 Potyvfnythydrosenphthatete; Fastmen Kodak 5527.

Is Potyethytent glycol, Motetuter weight Mo, for biological use, P2139; úi9MA.

19 ALginate; teLeo Co.

TABLE 1 (contd).

Enhancer Enhancement Example Concentration Factor ú41 0.765806 42 PEG 0.751353 43 1.0% Gum Chatti 0.558062 44 1.0% POLY0X W-301 0.523886 1.0% pvbp 0.029226 Potyethytene oxide; Union Carbide.

PATENT 183/68 The light emission spectra of the samples of Examples 4, 7 and 30 are shown in FIGS. 3, 4 and 5, respectively, with light intensity being measured on the abscissa (X axis) and time on the ordinate (Y exi;) of each plot.

LXwPLE 46 A TSH assay was carried out as follows:

NATERIALS:

House monoclonal anti-TSH-P antibody was used to coat 1/8 inch beads for analyte capture. House monoclonal anti-TSH antibody was conjugated with alkaline phosphatase and used as a detection antibody.

TSH was obtained from Calbiochem, Catalog No. 609396, and B5A (type V fatty acid free) was obtained from Sigma, Catalog No. A6003.

The buffer solution used for the analyte and conjugate contained 0.1 M Tris-HCl, ImM M9C12, and 2% by weight B5A (pH=7.5) The substrate buffer solution contained 0.1 M Tris, 0.1 rM M9C12, 0.1% by weight B5A (pH-9.5), and 3-(29spiroadamantane)-4-nethoxy-4-(3u-phosphoryloxy)phenyl-1,2dioxetane disodium salt as the chemiluminescent compound. (50 pg/M1).

29 PATENT 183/68 PROTOCO:

15M1 of a TSH-containing analyte solution was mixed with 13591 of conjugate antibody solution. Two 1/8 inch beads coated as described above were added to the solution and incubated for 2 hours at 230C. The beads were then washed four times with 0.1 M Tris (pH - 7.5) and transferred to a reaction tube. 200M1 of the ame chemiluminescent compound used in the substrate buffer olution described above was added to the tube. Following an incubation period of 20 minutes, light emission was recorded as ten second counts using a Berthold Clinilumat Luminescence Analyzer.

An identical TSH essay was also performed with the same concentration of substrate in the same buffer without BSA for the sake of comparison. As shown in FIG. 1, a plot of the data in Table 11 below, the B5A-containing sample (Curve A) showed greater luminescence intensity for a given TSH concentration than the sample without B5A (Curve B).

Table 11

M Ca=ntration (Up/M1) 1 2 4 ilminesoent Signal with 0. 10% B5A 0.48 1.1 1.7 (C=Its/10 SW X 10-4) Withmit B5A 0.25 0.49 1.1 PA= 183/68 EXAKPLE 47 The enhancement of acridinium ester luminescence from an anti-TSH-IgGacridiniun ester (OAEII) conjugate (Ciba-Corning) was shown by means of the following procedure.

To 50 M1 portions of anti-TSH-lgG-AE conjugate containing 30 ng IgG/mI, with 2-3 labels in luminometer tubes there were added the amounts of an aqueous solution containing 0.1% BMQ and 0.01 =g/ral of fluorescein disodium salt indicated in Table 111 below. Each tube was placed in a Berthold Clinilumat Luminometer and a luminescent reaction initiated by injecting 200 M1 of 50 nM aqueous sodium hydroxide solution containing 0.05% hydrogen peroxide.

BDY,Q/fluorescein-enhanced light emission from the acridinium ester, at the 10 and 50 M1 levels, as compared to the control, is shown in Table III and illustrated by the plots of FIG. 2.

is Table 111

Enhancer Concentration. ul None (control) so 2.00 Light Intensity (10 second integral) 127,479 169,753 188,885 82,437 31 PATENT 183/68 EXAMPLE 48

An assay for alkaline phosphatase was conducted in the following mannet.

COMPONENTS:

Buffer: 0.05M carbonate, lmM M9C12 at PH 9.5 Substrate: 3-(21-opiroadamantane)-4-nethoxy-4-(319phosphoryloxy)phenyl-1, 2-dioxetane disodium salt at 0.4 nM concentration is Alkaline Phosphatase: stock solution at buffer Enhancing Systems Tested:

1. 2. 3.

1.168 gg/nl in the Buffer alone, control Buffer plus 0.1% BSA Buffer plus 0.1% BSA- fluorescein (BSA to fluorescein molar ratio of 1 to 3) 4. Buffer plus 0.1% BMQ 5. Buffer plus 0.1% BMQ and fluorescein (0.01 =g of fluorescein disodium salt added per =1 of BDMQ).

Serial dilutions of alkaline phosphatese stock solutions were made in tubes with final enzyme concentrations of:

32 PATENT 183/68 4.17 X 10-11M 8.34 X 10-12M 1.67 X 10-12M 3.34 X 10-13m 6.69 X 10-14M 1.34 X 10-14M 3.34 X 10-ISH PROCEDURE:

1.67 X 10-15M 8.34 X 10-16M 4.17 X 10-16M 2.09 X 10-16M 1.0 X 10-161q 5. 0 X 10-17M 2.5 X 10-17M Duplicate tubes at each of the above concentrations of alkaline phosphatase also containing 0.4 =Y 3-(21-spiroadamantane)-4nethoxy-4-(311-phosphoryloxy)phenyl-1,2-dioxetane disodiun salt in various enhancing systems were incubated at 30C. Systems 1, 4 and 5 were incubated for 20 minutes, while systems 2 and 3 were incubated for 80 minutes.

After incubation, 30 second light integrals were neasured in a Turner 20E Luminometer, and the effect of the enhancers tested on the limits of detection of alkaline phosphatase is shown in Table IV.

33 PATENT 183/68 Table

Concentration of Alkaline Phosphatase for 2X Addition --Background

None 1.0 X 10-14M 0.1% BSA 9.5 X 10-15M 0.1% BSA:Fluorescein 1.3 x 10-15M 0.1% WMQ 4.0 x 10-15M 0.1% BDMQ:Fluorescein3.4 X 10-15M Minimum Detectable Conc. of Alkaline Phosphatese 1.67 X 10-ISM (1.12) 8.34 X 10-16M (1.06) 4.17 X 10-16M (1.04) 1.00 X 10-16M (1.07) 2.09 X 10-16M (1.06) 1 Buffer: 0.05 M Carbonate, 1 MM H9C12, pH - 9.5.

Temperature: 30'C. Chemiluminescent compound concentration is 0.4 mM.

2 The number in parentheses is the multiple of background at the indicated concentration.

EXAMPLE 49

The ability to detect luninescense photographically by means of this invention was demonstrated in the following manner.

COKPOE=:

Buffer: 0.1M Tris, 1 =M MgC12 at pH 9.8 prepared with 0.1k BSA and without BSA, (BSA purchased from Sigma, Catalog number A7906).

34 1 PATENT 183/68 Enzyne: 0.05 Mg Alkaline Phosphatese (3.6 X 10-13 moles) purchased from Biozyme Corporation.

Membrane:

Substrate:

PROTOCO:

o.45 p pore-.zize nitrocellulose.

3-(20-sp.irioadamantane)-4-methoxy-4-(311phosphorylexy)phenyl-1,2dioxetane disodium salt at 0.08 mM concentration.

Enzyme solution was spotted on a dry nitrocellulose membrane. Subsequently, the membrane was blocked with 2.5% dry milk solids and l% fish gelatin in phosphate buffered saline at pH 7.3. The blocked membrane was washed with the substrate solution in buffers containing no B5A, and with B5A.

The membrane was placed in a camera luminometer and 10 minute exposures were Te=rded en Type 612 Polaroid Black and White is Instant Film. Reproductions of the photographs obtained appear as FIGS. 7 and 8.

The above discussion ofthis Invention Is directed primarily to preferred embodiments and practices thereof. It will be 35 PATENT 183/68 readily apparent to those skilled in the art that further changes and modifications in the actual Implementations of the concepts described herein can easily be made without departing from the spirit and scope of the invention as defined by the following claims.

36 X X

Claims

WE CLAIM:

1. In a process carried out in aqueous me,3ium in which electromagnetic energy released by the decomposition of a chemiluminescent chemical compound is detected to determine the presence, concentration or structure of an analyte, the improvement comprising carrying out the process in the presence of an amount of a water soluble substance sufficient to enhance the intensity of the released detectable energy as compared to the intensity of the detectable energy released in the absence of the enchancer substance.

2. The process of claim 1 in which the chemiluminescent chemical compound's decomposition results in the formation of a light-emitting fluorophore.

3. The process of claim 2 in which the enhancer substance has the ability to inhibit the fluorophore from releasing energy through non-light emitting pathways.

4. The process of claim 3 in which the enhancer substance comprises a naturally-occurring or synthetic macromolecular substance that can provide a hydrophobic microenvironment for the fluorophore.

5. The process of claim 4 in which the enhancer substance comprises a naturally-occurring macromolecular substance.

6. The process of claim 5 in which the macromolecular substance is a globular protein that includes hydrophobic regions.

7. The process of claim 6 in which the globular protein is a mammalian serum albumin.

8. The process of claim 4 in which the enhancer substance comprises a synthetic macromolecular substance.

9. The process of claim 8 in which the macromolecular substance comprises an oligomeric or polymeric quaternary ammonium salt.

10. The process of claim 9 in which the quaternary ammonium salt is a poly(vinylaryl quaternary ammonium salt).

11. The process of claim 10 in which the poly(vinylaryl quaternary ammonium salt) is poly(vinylbenzyltrimethylammonium chloride).

12. The process of claim 10 in which the poly(vinylaryl quaternary ammonium salt) is poly[vinylbenzyl-(benzyldimethylammonium chloride)!.

13. The process of any one of the preceding claims in which the chemiluminescent chemical compound is a thermally, chemically, electrochemically or enzymatically cleavable 1,2-dioxetane, acridinium ester, lucigenin or luciferin.

14. The process of claim 13 in which the chemiluminescent chemical compound comprises a 1,2-dioxetane.

15. The process of claim 14 in which the 1,2-dioxetane is enzymatically cleavable.

16. The process of claim 15 in which the 1,2-dioxetane is a 3-(21spiroadamantane)-4-methoxy(3n-phosphoryloxy)phenyl-1,2-dioxetane salt.

1

17. The process of any one of the preceeding claims carried out in the presence of an auxiliary fluorophore extraneous to the energy-emitting fluoroDhore produced by the decomposition of the chemiluminescent chemical compound and capable of accepting energy from said fluorophore to emit in turn detectable energy.

18. The process of claim 17 in which the auxiliary fluorophore is admixed with the chemiluminescent chemical compound and the enhancer substance.

19. The process of claim 17 in which the auxiliary fluorophore is bonded to the portion of the chemiluminescent compound that, upon decomposition, produces the energy-emitting fluorophore.

20. The process of any one of claims 17-19 in which the auxiliary fluorophore is a fluorescein.

21. The process of claim 17 in which the auxiliarv fluorophore is bonded to the enchancer substance.

22. The process of claim 21 in which the auxiliary fluorophore is a derivatized fluorescein capable of establishing a covalent bond with the enhancer substance.

23. The process of any one of the preceding claims in which the process carried out is a step in an immunoassay.

24. The process of any one of claims 1-22 in which the process carried out is a nucleic acid probe assay.

25. An aqueous composition comprising a chemiluminescent chemical compound as defined in any one of the preceding claims, capable of decomposing to release electromagnetic energy detectable to determine the presencet concentration or structure of an analyte and an amount of a water soluble substance as defined in any one of the preceding claims, sufficient to enhance the intensity of the released detectable energy as compared to the intensity of the detectable energy released in the absence of the enhancer substance.

26. The composition of claim 25 which also contains an auxiliary fluorophore as defined in any one of claims 17-22, extraneous to the energy-emitting fluorophore produced by the decomposition of the chemiluminescent chemical compound and capable of accepting energy from said fluorophore to in turn emit detectable energy.

Published 1991 at The Patent Office. State House. 66171 High Holborn. London WC1R4TP. Further copies maybe obtained from The Patent Office Sales Branch. St Mary Cray. Orpington. Kent BR5 3RD. Printed by Multiplex techniques lid. st mary Cray. Kent. Con 1187