CA2170873A1 - Fluorescent oxygen channeling immunoassays - Google Patents

Abstract

Methods are disclosed for determining an analyte in a medium suspected of containing the analyte. One method comprises treating a medium suspected of containing an analyte under conditions such that the analyte, if present, causes a photosensitizer and a photoactive indicator precursor molecule to come into close proximity. The photosensitizer generates singlet oxygen which activates the photoactive indicator precursor to generate a photoactive indicator molecule. Upon irradiation with light the photoactive indicator molecule produces light, which is measured. The amount of light produced by the photoactive indicator is related to the amount of analyte in the medium. Compositions, kits, and compounds are also disclosed.

Description

W O 9S/06877 2 ~ 7 3 PCTrUS94/09705 FL~ORESCENT O~YGEN ~NN~T-TNG rMM~NOASSAYS

R ~K~ O~ND OF THE lN V~ ~lON
Field of the Invention This invention relates to methods, compositions and kits for determi ni ng an analyte in a sample. In particular, this invention relates to specific hinfling assays which utilize a photoactive indicator p~ecursor which can react with singlet oxygen to form a fluorescent product.
The clinical diagnostic field has seen a broad expansion in recent years, both as to the variety of materials (analytes) that may be readily and accurately det~rmin~fl~ as well as the methods for the determinAtion.
Convenient, reliable and non-hazardous means for detecting the presence of low cnnC~ntrations of materials in liquids is desired. In clinical chemistry these materials may be present in body fluids in cnnc~ntrations below 10-l2 molar. The difficulty of detecting low cnnc~ntrations of these materials is ~nhAncefl by the relatively small sample sizes that can be utilized.
In developing an assay there are many considerations. One consideration is the signal response to changes in the concentration of an analyte. A second consideration is the ease with which the protocol for the assay may be carried out. A third consideration is the variation in interference from sample to fiample. Ease of preparation and purification of the reagents, availability of equipment, ease of Ant~ tion and interaction with material of interest are some of the additional considerations in developing a useful assay.
One broad category of techni~ues involves the use of a receptor which can specifically bind to a particular spAciAl and polar organization of a labeled ligand as a function of the presence of an analyte. The observed effect of hi nfl; ng by the receptor will depend upon the label. In some instances the hi nfli ng of the receptor merely provides for a differentiation in molecular weight between bound and unbound labeled ligand. In other instances the hi n~i ng of the receptor will facilitate separation of bound labeled ligand from free labeled ligand or it may affect the nature of the signal obtained from the label so that the signal varies with the amount of receptor bound to labeled ligand. A further variation is that the receptor is labeled and th~ ligand llnlAheled. AlternAtively, both the receptor and ligand are labeled or different receptors are labeled with two different labels, whereupon the labels interact when in close proximity and the amount of ligand present affects the degree to which the labels of the receptor may interact.
There is a cnnti nni ng need for new and accurate techniques that can be adapted for a wide spectrum of different ligands or be used in specific cases where other methods may not be readily adaptable.
~ g~n~oll~ i oA~says in which it is unnecessary to separate the bound and unbound label have previously been described for small molecules.
These a~says include SYVA's FRAT~ assay, EMIT~ assay, enzyme ~hAnneling W 095/06877 - PCTrUS94/09705 ; o~Rs~y, and fluorescence energy transfer ~ lnnA~say (FETI); enzyme inhibitor ~ lnn~s~ys (HofL.~ LaRoche and Abbott Laboratories):
fluorescence polarization i lnnARsay (Dandlicker), among others. All of these methods have limited sensitivity, and only a few including FETI and enzyme rh~nn~];ng, are suitable for large multiepitopic analytes.
Heterogenous ~ lnoAssays in which a separation step is required are generally useful for both small and large molecules. Various labels have been used including enzymes (ELISA), fluorescent labels (FIA), radiolabels (RIA), chemiluminescent labels (CLA), etc.
T~m;n~scent compounds, such as fluorescent compounds and chemiluminescent compounds, find wide application in the assay field because of their ability to emit light. For this reason, luminescers have been utilized as labels in assays such as nucleic acid assays and ~ o~Rsays. For example, a ~c} of a specific hin~;ng pair is conjugated to a luminescer and various protocols are employed. m e luminescer conjugate can be partitioned between a solid phase and a liquid phase in relation to the amount of analyte in a sample suspected of cnntA;n;ng the analyte. By measuring the luminescence of either of the phases, one can relate the level of luminescence observed to a cnnC~nt~ation of the analyte in the sample.
Particles, such as latex beads and liposomes, have also been utilized in assays. For example, in homogeneous assays an enzyme may be e..tLa~ped in the aqueous phase of a liposome labelled with an ~nt~ho~y or antigen.
m e liposomes are caused to release the enzyme in the presence of a sample Z5 and complement. ~nt~ho~y- or antigen-labelled liposomes, having water soluble fluorescent or non-fluorescent dyes encapsulated within an aqueous phase or lipid soluble dyes dissolved in the lipid bilayer of the lipid vesicle, have also been utilized to assay for analytes capable of entering into an immunochemical reaction with the surface bound antibody or antigen.
Detergents have been used to release the dyes from the aqueous phase of the liposomes. Particles have been dyed with fluorescent dyes and used as labels in ; ~no~Rsays. Undyed particles have also been used (e.g., latex aggl~lt;n~tion).

Related Art European Pnhl;~hed Patent Application No. 0 345 776 (McCapra) discloses specific h;n~;ng assays that utilize a sensitizer a~ a label.
m e sensitizers include any moiety which, when st;m~ te~ by excitation with radiation of one or more wavelengths or other chemical or physical stimuluB (e.g., electron transfer, electrolysis, electroluminescence or energy transfer) will achieve an excited state which (a) upon interaction with molecular oxygen will produce singlet molecular oxygen, or (b) upon interaction with a leuco dye will assume a reduced form that can be returned to its original unexcited state by interaction with molecular oxygen resulting in the production of 11YdLUYe~1 peroxide. Either interaction with the excited sensitizer will, with the addition of reagents, produce a detectible signal.
Buropean Published Patent Application No. 0 476 556 (Motsenbocker) discloses a method for det~rm;nAtion of a light sensitive substance wherein irradiation of lumigenic substance-light sensitive substance solution with mo~ Ate~ light is used to generate short wavelength light proportionally to the c~nCPntration of the light sensitive substance.
Tnm;n~scent labels for ; oA~says are described in McCapra et al., Journal of Bioluminescence and C&emiluminescence (1989), Vol. 4, pp. 51-58.
European Published Patent Application No. 0 515 194 (Ullman et al.) discloæes methods for det~- ;n;ng an analyte in a medium suspected of c~ntA;n;ng the analyte. One such disclosed method compriseæ treating a medium suspected of contA;n;ng an analyte under conditions such that the analyte, if present, causes a photosensitizer and a chemiluminescent compound to come into close proximity. The photosensitizer generates singlet oxygen and activates the chemiluminescent c~mrolln~ when it is in close proximity. The activated chemiluminescent subsequently produces light upon activation by singlet oxygen. The amount of light produced is related to the amount of analyte in the medium.
In this method, each singlet oxygen that is generated can react with no more than one chemiluminescent compound, which, in turn, can emit not more than one photon of light. The sensitivity of the method is therefore limited by the chemiluminescence ~uantum efficiency of the chemiluminescent cGm~ound, and, more importantly, by the ability to detect the limited '-I of photonæ that will be emitted upon reaction with singlet oxygen.

SUMMARY OF THE lN V~ .lON
The present invention is directed to methodæ for determ;n;ng an analyte, kitæ for conducting aææays for an analyte, and compounds useful in the methods and assays.
One aspect of the invention is a method for det~m;n;ng an analyte which is a specific hin~;ng pair (sbp) member. In one : 'o~; - t of this aspect the method compriæeæ a first step of providing in c 'inAtion a medium suspected of cont~; n; ng an analyte; a photoæenæitizer capable in itæ
excited state of generating singlet oxygen, wherein the photosPn~itizer is aæsociated with an sbp 1 ~-; and a photoactive indicator precursor capable of forming a photoactive indicator upon reaction with singlet oxygen, wherein the photoactive indicator precursor is associated with an sbp member; then a second step of exciting the photosensitizer by irradiation with light; and a final step of measuring the fluorescence of the photoactive indicator. At least one of the sbp members is capable of h;n~;ng directly or indirectly to the analyte or to an sbp member compl - Ary to the analyte. The fluorescence measured is related to the amount of the analyte in the medium.
In another P~ho~; t, the method comprises the firæt ætep of W O 95/06877 2 1 7 0 ~ 7 3 PCTrUS94/09705 - ' `n;ng in an aqueous medium a sample suspected of cont~in;ng an analyte;
a first 6uspendible particle comprised of a photosensitizer capable in its excited state of generating singlet oxygen, wherein the particle has a specific b;n~;~g pair (sbp) member bound thereto; and a second suspendible particle comprised of a photoactive indicator precursor capable of forming a photoactive indicator upon reaction with singlet oxygen, wherein the particle has an sbp member bound thereto; a second step of irrAA;At;ng the medium to excite the photosensitizer to generate singlet oxygen; and a final step of measuring the fluorescence of the photoactive indicator.
Each sbp member is capable of h; n~;n~ directly or indirectly with the analyte or to an sbp member compl: ~ry to the analyte. The fluorescence measured is related to the amount of the analyte in the medium.
In another ; _'; t, the method comprises a first step of providing in c b;n~tion a medium suspected of cnnt~;n;ng an analyte; a photosensitizer capable in its excited state of generating singlet oxygen, wherein the photos~n~itizer is associated with an sbp member; and a suspendible particle having bound thereto an sbp member, wherein the suspendible particle comprises a photoactive indicator precursor capable of forming a photoactive indicator upon reaction with singlet oxygen; a second step of irrA~i~t;ng the ~ ';n~tinn with light to excite the photosensitizer; and a final step of measuring the fluorescence of the photoactive indicator. Each sbp member is capable of h; n~; ng directly or indirectly to the analyte or to an sbp member co-m~pl~m~nt~ry to the analyte.
The fluorescence measured is related to the amount of the analyte in the medium.
Another aspect of the invention i8 a method for dete- 'n;ng an analyte. The method comprises a first step of providing in _ ;n~tion a medium suspected of cnntA;n;ng an analyte; a photosensitizer capable in its excited state of generating singlet oxygen, wherein the photosensitizer is associated with a first specific b;n~;ng pair (sbp) member; and a photoactive indicator precursor capable of forming a photoactive indicator upon reaction with singlet oxygen, wherein the photoactive indicator precursor is associated with a second sbp member; a second step of irr~;Ating the crmhin~t;on with light to excite the photosensitizer; and a final step of measuring the fluorescence of the photoactive indicator.
Each sbp member is capable of h; n~;n~ directly or indirectly to the analyte or to an sbp member compl: - Ary to the analyte. The fluorescence measured is related to the amount of the analyte in the medium.
Another aspect of this invention is a method for dete n;ng a polynucleotide analyte. The method comprises a first step of cnmh;n;ng in an aqueous medium the analyte; one or more polynucleotide probes (wherein each probe c~ntA;n~ a nucleotide sequence compl. Ary to a nucleotide sequence of the analyte and wherein at least one probe is associated with a specific hin~;n~ pair (sbp) member that is differenct from said compl - ~ry nucleotide sequence); a photo8ensitizer cAr~hle in its W O 9S106877 ~ 1 7 0 ~ 7 3 PCT~US94/0970S

excited state of generating singlet oxygen (wherein said photosensitizer is associated with a polynucleotide having a sequence compl.- - tAny to a nucleotide sequence of said probe); and a photoactive indicator precursor capable of forming a photoactive indicator upon reaction with singlet oxygen, wherein the photoactive indicator precursor is associated with an sbp member complementary to the sbp ~ ..~e associated with the probe; a second step of irrA~;At;ng the medium with light to excite the photosensitizer to generate singlet oxygen; and a third step of measuring the fluorescence of the photoactive indicator. The fluorescence is related to the amount of the analyte in the medium.
A method for det~ -n;ng an analyte, which method comprises (A) if the analyte is a polynucleotide (a) - ;n;ng in an aqueous medium (1) said analyte; (2) one or more polynucleotide probes, wherein each probe cnntA;n~ a nucleotide sequence complementary to a nucleotide sequence of said analyte and wherein at least one probe is bound to a specific bin~;ng pair (sbp) member, or is bound to or incorporated in a particle having said sbp member inco,~o-~ted therein or bound thereto, said sbp member being different from said compl~mentAry nucleotide sequence; (3) a photosensitizer cArAhle in its excited state of generating singlet oxygen, wherein said photosensitizer is bound to, or is bound to or incorporated in a particle having incoL~o~ated therein or bound thereto, a nucleotide sequence compl. - Ary to a nucleotide sequence of said probe; and (4) a photoactive indicator precursor capable of forming a photoactive indicator upon reaction with singlet oxygen, wherein said photoactive indicator precursor is bound to, or is bound to or incorporated in a particle having inco ~oL~ted therein or bound thereto, an sbp member complementary to said sbp member associated with said probe; (b) irrA~;At-;ng said medium with light to excite said photosensitizer to generate singlet oxygen; and (c) measuring the fluorescence of said photoactive indicator; wherein said fluorescence is related to the amount of said analyte in said medium; or (B) if the analyte is other than a polynucleotide (a) providing in c~m~;nAtion: (1) a medium suspected of c~ntA;ning an analyte; (2) a photosensitizer capable in its excited state of generating singlet oxygen, wherein said photosensitizer is bound to, or is bound to or inco ~o~ated in a particle having incG ~ol~ted therein or bound thereto, a first specific h;n~;ng pair (sbp) member; and (3) a photoactive indicator precursor capable of forming a photoactive indicator upon reaction with singlet oxygen, wherein said photoactive indicator precursor is bound to, or is bound to or incoL~or~ted in a particle having inco,~u~ted therein or bound thereto, a second sbp member; (b) irrA~;At;ng said . inAt;on with light to excite said photosensitizer; and (c) measuring the fluorescence of said photoactive indicator; wherein each sbp member is capable of h;n~;ng directly or indirectly to said analyte or to an sbp member compl: ~ry to said analyte, and wherein said fluorescence is related to the amount of said analyte in said medium, and wherein said W 095/06877 ~ ~ 7~ g ~ PCTrUS94/09705 photosensitizer is optionally part of a suspendible particle to which said first sbp member is bound, and wherein said photoactive indicator precursor is optionally part of a suspendible particle to which said second sbp member is bound.
Another aspect of this invention is a ~ _~osition comprising suspendible particles of average diameter of 20 to 4000 nanometers having associated therewith a photoactive indicator precursor, wherein the photoactive indicator precursor c~ntA;nR a selenium or tellurium atom.
Another aspect of this invention is a kit for conducting an assay for analyte. The kit comprises, in p~k~ed c~mh;n~t;on, sl~Rp~nA;hle particles comprising a photoactive indicator precursor, wherein said photoactive indicator precursor cnntA;n~ a selenium or a tellurium atom and wherein the particles have bound thereto an sbp member; and a photosensitizer which is assor;Ate~ with an sbp member and is capable in its excited state of activating oxygen to its singlet state, wherein at least one of the sbp ~ i8 capable of hinA;ng to the analyte or to an sbp member complementary to the analyte.
In another : '_ ; t of this aspect, the kit comprises, in pArk~geA
_ n~t;on, a co-,~osition, which comprises a first suspendible particle comprising a photoactive indicator precursor cnntA;n;ng a selenium or tellurium atom, wherein the first particle has bound thereto an sbp member;
and a second suspendible particle comprising a photosensitizer, wherein the second particle has bound thereto an sbp member. At least one o~ the sbp members is capable of h; nA;ng to the analyte or to an sbp member compl ~Ary to the analyte.
In another : '~A; t of this aspect, the kit comprises, in packaged ~ -nAt;on, a photoactive indicator precursor c~nt~;n;ng a selenium or tellurium atom, wherein the photoactive indicator precursor is associated with a ~irst sbp member; and a photosensitizer capable in its excited state of activating oxygen to its singlet state associated with a second sbp member. m e sbp members are capable of h; nA;ng to the analyte or to an sbp 'o- c~pAh]e of h;n~;ng the analyte.
Another aspect of this invention is a hinA;ng assay for an analyte that is an sbp member. The assay comprises the first step of , ;n;ng a medium suspected of cnnt~;n;ng the analyte with an sbp member capable of b;n~;n~ directly or indirectly to the analyte or to an sbp - '-I
complementary to the analyte; a second step of detecting the h; nA; ng of the sbp member to the analyte or the complementary sbp member, wherein the detection comprises exposing a photoactive indicator precursor in the medium to singlet oxygen to produce a photoactive indicator; and a final step of measuring the fluorescence of the photoactive indicator.
Another aspect of this invention are compounds useful as photoactive indicator precursors which cnntA;n the following structure:

W O 95/06877 2 1 7 ~ ~ ~ 3 PCTrUS94/09705 H XR

wherein H is cis to the XR group; X is a selenium or tellurium; R is an organic or organometallic group bound to X through an unsaturated carbon atom, a silicon atom, or a tin atom; and A, when taken with the carbon-carbon group, forms an alicyclic ring (optionally fused to one or more aromatic rings) or a heterocyclic ring; where upon reaction of the compound with singlet oxygen, the H and the XR group are replaced by a carbon-carbon double bond to yield a fluorescent molecule having an extinction coefficient of at least 10,000 M~cm~l at its absorption and a fluorescence emission quantum yield of at least 0.1.
Another aspect of this invention is a method for preparing a photoactive indicator molecule. The method comprises reacting a compound of the invention (as described above) with singlet oxygen to yield a photoactive indicator having an extinction coefficient of at least 10,000 M~cm~l at its absorption - ~ and a fluorescence emission ~lAntl yield of at least 0.1.
One of the advantages of the present invention is the ability of the fluore~cent photoactive indicator (which is produced from the reaction of the photoactive indicator precursor with singlet oxygen) to generate at least 105 times as many photons as the chemiluminescent compound used in the method described above in European pllhli~h~ Patent Application No. O
515 194. This is because a single fluorescent photoactive indicator molecule can often be excited up to 105 times before it is destroyed.
Thus, the fluorescent photoactive indicator molecule that is formed in the present invention can produce tens of thousands of photons on irradiation.
Detection of this fluorescence can therefore provide a more sensitive assay. Moreover, measu~ of the fluorescence of the photoactive indicator molecule in the present invention permits the use of a standard fluorometer whereas detection of the chemiluminescence produced on activation of the chemiluminescent compound in the previously described assay requires more specialized spectrometers.

BRIEF D~S~TPTION OF THE DRAWINGS
Figure 1 is a graphic depiction of the results of DNA detectionassays. The results of each assay are depicted by a different symbol.
DETAILED D~Cr~llON OF THE lNv~nllON
Definit~on~
As used in this specification and appended claims, unless specified to the Co-lL~a y, the following terms have the --n;ng indicated:
"Alkyl" refers to a monovalent brAnrh~ or unbrAn~h~ radical derived from an ~lirhAt;c hydrocarbon by removal of one hydLU~ell atom; includes W 095/06877 ~ 1 7 ~ ~ 7 3 PCT~US94109705 both lower alkyl and upper alkyl.
"Lower alkyl" refers to an alkyl radical contA;ni~g from 1 to 5 carbon atoms, e.g., methyl, ethyl, propyl, butyl, isopropyl, isobutyl, pentyl, isopentyl, and the like.
"Upper alkyl" refers to an alkyl radical contAin;ng more than 6 carbon atoms, usually 6 to 20 carbon atoms, e.g., hexyl, heptyl, octyl, and the like.
I'Alkylidene'' refers to a divalent organic radical derived from an alkyl radical in which two hYdLOge~ atoms are taken from the same carbon atom, e.g., ethylidene, and the like.
"Alkylene" refers to a divalent organic radical derived from an alkyl radical in which two hyd~e~- atoms are taken from different carbon atoms.
"Alicyclic ring" refers to a cyclic hydrocarbon radical of 5 to 7 cArb~n~ in length which may be l~n~tnrated or partially saturated.
"Aryl" refers to an organic radical derived from an aromatic hydrocarbon by the removal of one atom and contAining one or more aromatic rings, usually one to ~our aromatic rings, e.g., phenyl (from benzene), n~trhthyl (from naphthalene), and the like.
"Aralkyl" refers to an organic radical having an alkyl group to which is attached an aryl group, e.g., benzyl, phenethyl, 3-phenylpropyl, 1-n~phthylethyl, and the like.
"Alkoxy" refers to a radical of the formula -0~ where ~ is an alkyl group, e.g., methoxy, ethoxy, and the like.
"Aryloxy~ refers to a radical of the formula -0~ where R~ is an aryl group, e.g., phenoxy, nAphtho~y, and the like.
"Aralkoxy" refers to a radical of the formula -0~ where ~ is an aralkyl radical, e.g., benzyloxy, 1-~phthylethoxy, and the like.
"Alkylthio" refers to a radical of the formula -S~ where ~ is an alkyl group, e.g., methylthio, ethylthio, and the like.
"Arylthio" refers to a radical of the formula -S~ where Rb is an aryl group, e.g., phenylthio, nAphthylthio, and the like.
"Heterocyclic ring" refers to a stable mono-, bi- or tricyclic ring system which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur and which is either saturated or nn~Atllrated, wherein the nitrogen, carbon or sulfur atoms may optionally be oxidized, and the nitrogen atom may optionally be ~l~t~rni~ed, and includes any ring system in which any of the above-defined heterocyclic ring systems is fused to a benzene ring. The heterocyclic ring system may be substituted at any heteroatom or carbon atom which results in the creation of a stable structure. Examples of such heterocyclic ring system6 include, but are not limited to, piperidine, piperazine, 2-oxopiperazine, 2-oxopiperidine, 2-o~y~olidine, 2-oxoazepine, azepine, pyrrole, 4-piperidone, pyrrolidine, pyrazole, pyrazolidine, imidazole, imidazoline, imidazolidine, pyridine, pyrazine, pyrimidine, pyridazine, oxazole, oxazolidine, indane, isoxazole, 2 t 7087~
W O 95/06877 PCTfUS94/09705 isoxazolidine, morphol;ne, thi~701e, thiazolidine, isothiazole, quinuclidine, isothiazolidine, indole, isoindole, indoline, isoindoline, octahydroindole, octahydroisoindole, quinoline, iso~uinoline, decahydroisoqu;noline, benzimidazole, th;A~;~7ole, dihydrobenzofuran, benzofuran, benzopyran, 1,4-benzopyrone, 1,2-benzopyrone, benzothiazole, benzoxazole, furan, tetral-ydLuf~ran, pyran, tetral.ydlu~y ~, thiophene, benzothiophene, thi Amorpholine, th; A~orpholine sulfoxide, thi Amnrpholine sulfone, and nY~ ole. Preferred heterocyclic rings for the purposes of this invention are benzopyrones.
"Substituted" refers to the condition wherein a hyd u~-- atom of a molecule has been replaced by another atom, which may be a single atom such as a halogen, etc., or part of a group of atoms, such as an organic group.
"Electron-~nnAt;ng group" refers to a substituent which, when bound to a molecule, is capable of polarizing the molecule such that the electron-~nnAt;ng group becomes electron poor and positively charged relative to another portion of the molecule, i.e., has re~uce~ electron density. Such groups may be, by way of illustration and not limitation, amines, ethers, thioethers, phosph;n~s, hy~u~y, oxyanions, mercaptans and their anions, sulfides, etc.
"Organic group" refers to a substituent having from 1 to 50 atoms other than the requisite number of l-yd-ogell atoms necessary to satisfy the valencies of the atoms in the radical. Generally, the pre~om;nAnt atom in such a group is carbon tC) but may also be oxygen (0), nitrogen (N), sulfur (S), phn8rh9~U8 (P), wherein, if present, the 0, N, S, or P atom may be bound to carbon or to one or more of each other or to hydlo~e.l or to a metal atom to form various functional groups, such as carboxylic acids, alcohols, thiols, cA--~ des, c~hA~-tes, carboxylic acid esters, sulfonic acids, sulfonic acid esters, rh~sphoric acids, phosphoric acid esters, ureas, c~~'~ tes, rhosFhoramides, sulfonamides, ethers, sulfides, thioethers, olefins, acetylenes, amines, ketones, aldehydes, nitriles, and the like. Illustrative of such organic groups, by way of illustration and not limitation, are alkyl, alkylidine, aryl, aralkyl, and heterocyclyl, wherein the alkyl, alkylidine, aryl, aralkyl or heterocyclyl group may be substituted with one or more of the afo~. t; oned functional groups.
llOl~_ -tallic group" refers to a radical cnntA;ning an organic group (as defined above) 1 ink~ to a metal atom.
"Analyte" refers to the c o~-d or composition to be detected. The analyte can be comprised of a member of a specific bin~ing pair (sbp) and may be a ligand, which is monovalent ( oepitopic) or polyvalent (polyepitopic), usually antigenic or haptenic, and is a single compound or plurality of compounds which share at least one common epitopic or detr- inAnt site. The analyte can be a part of a cell such as bacteria or a cell bearing a blood group antigen such as A, B, D, etc., or an HLA
antigen or a miu~oul~J- i~m, e.g., bacterium, fungus, protozoan, or virus.
The polyvalent ligand analytes will nr 1 ~y be poly(amino acids), W O9~/06877 ~ 1 7 0 8 ~ 3 PCT~US94/09705 i.e., polypeptides and proteins, polys~crh~nides, nucleic acids, and ~ n~t;on6 thereof. Such ,_ 'in~tions include c ~on~nt~ of bacteria, viruses, ~ -somes, genes, mitochondria, nuclei, cell ...~ &les and the like.
For the most part, the polyepitopic ligand analytes to which the subject invention can be applied will have a molecular weight of at least about 5,000, more usually at least about 10,000. In the poly(amino acid) category, the poly(amino acids) of interest will generally be from about 5,000 to 5,000,000 molecular weight, more usually from about 20,000 to 1,000,000 molecular weight; among the hr ~S of interest, the molecular weights will usually range from about 5,000 to 60,000 molecular weight.
A wide variety of proteins may be considered as to the family of proteins having similar structural features, proteins having particular biological functions, proteins related to specific microorganisme, particularly disease causing miu UULJ~ P, etc. Such proteins include, for example, immunoglobulins, cytokines~ enzymes, h~ ?S, cancer antigens, nutritional markers, tissue specific antigens, etc.
The types of proteins, blood clotting factors, protein h~: ?S, antigenic polys~c~h~rides, mic oo~J~ F and other pathogens of interest in the present invention are specifically disclosed in U.S. Patent ~o.
4,650,770, the disclosure of which is incorporated by reference herein in its entirety.
The monoepitopic ligand analytes will generally be from about 100 to 2,000 molecular weight, more usually from 125 to 1,000 molecular weight.
The analytes include drugs, metabolites, pesticides, pollutants, and the like. Included among drugs of interest are the alkaloids. Among the ~lk~loids are morphine alkaloids, which includes morphine, co~ein~, heroin, dexLr~...ethorphan, their derivatives and metabolites; co~in~ alkaloids, which include coc~i ne and benzyl ecgonine, their derivatives and metabolites; ergot alkaloids, which include the diethylamide of lysergic acid; steroid alkaloids; iminazoyl alkaloids; quinazoline alkaloids;
iso~uinoline alkaloids; qllinnlin~ Alk~1oids, which include quinine and quinidine; diterpene alkaloids, their derivatives and metabolites.
The next group of drugs includes steroids, which includes the esL~uy~s~ androgens, adrenocortical steroids, bile acids, cardiotonic glycosides and aglycones, which includes digoxin and digoxigenin, saponins and sapogenins, their derivatives and metabolites. Also included are the steroid mimetic substances, such as diethylstilbestrol.
The next group of drugs is lactams having from 5 to 6 ~nnnl ~r ~_~, which include the barbiturates, e.~., ph~nnh~rhital and se~nh~rhital, diphenylhy~ntoin~ primidone, ethosn~ , and their metabolites.
The next group of drugs is ~inn~lkylbenzenes, with alkyl of from 2 to 3 carbon atoms, which includes the amphet~ in~s; cate~hol~mines, which includes ephedrine, L-dopa, epinephrine; narceine; papaverine; and W 095/06877 2 1 7 0 8 7 3 PCTrUS94/09705 metabolites of the above.
The next group of drugs is bPn~h~terocyclics which include oxazepam, chlo~, 7ine, tegretol, their derivatives and metabolites, the - heterocyclic rings being azepines, diazepines and phenothiazines.
The next group of drugs i8 purines, which includes theophylline, caffeine, their metabolites and derivatives.
The next group of drugs includes those derived from marijuana, which includes cAnnAhinol and tetrahydrocAnnAh;nol.
The next group of drugs is the hf ?8 such as thyroxine, cortisol, triiodothyronine, testosterone, estradiol, estrone, progesterone, polypeptides such as angiotensin, LHRH, and ; ~nosuppressants such as cyclosporin, FK506, mycoph~nolic acid, and 80 forth.
The next group of drugs includes the vitamins such as A, B (e.g., B~2), C, D, E and K, folic acid, th;; 'n~
The next group of drugs is prostaglAn~;nR, which differ by the degree and sites of hydroxylation and nn~At-lration.
The next group of drugs is the tricyclic antidepressants, which include imi~l 'n~, dismethylimi~l -n~, amitriptyline, nortriptyline, protriptyline, trimi~l n~, chlomi~l n~, doxepine, and desmethyldoxepin, The next group of drugs are the anti-neoplastics, which include methotrexate.
The next group of drugs is antibiotics, which include penicillin, chl-L~-,y-cetin, Actinn~ycetin, tetracycline, te~ y~in, the metabolites and derivatives.
l'he next group of drugs is the nucleosides and nucleotides, which include ATP, NAD, FMN, adenosine, guanosine, thymidine, and cytidine with their ~ u~Liate sugar and phosphAte substituents.
The next group of drugs is miscell~neoll~ individual drugs which include methA~nn~ te, serotonin, meperidine, lidocaine, proc~;n; ;de, acetylprocA;n; de, ~o~l~-olol~ griseofulvin, valproic acid, butyro~h~nnn~s, antihistA~;n~s, chlo- h~n;col, anti~hol;n~rgic drugs, such a~ atropine, their metabolites and derivatives.
~[etabolites related to diseased states include spermine, galactose, phenylpyruvic acid, and ~o.~hylin Type 1.
I'he next group of drugs is aminoglycosides, such as gentamicin, kAn; ycin, tobl ycin, and ; 'kAr;n Among pesticides of interest are polyhalogenated h;rh~nyls, phosrhAte esters, thiophosphAtes~ rA-- ~ tes, polyhalogenated sulfenamides, their metabolites and derivatives.
For receptor analytes, the molecular weights will generally range from 10,000 to 2x108, more usually from 10,000 to 106, For i oglnh~lll;n~
IgA, IgG, IgB and IgM, the molecular weights will generally vary from about 160,000 to about 106. Bnzymes will normally range from about 10,000 to 1,000,000 in molecular weight. Natural receptors vary widely, generally being at least about 25,000 molecular weight and may be 106 or higher W 095/06877 2 1 7 0 ~ 7 3 PCTrUS94/0970~

molecular weight, including such materials as avidin, streptavidin, DNA, RNA, thyroxine hin~;ng globulin, thyroxine hin~ng prealbumin, transcortin, etc.
m e term analyte further includes polynucleotide analytes such as those polynucleotides defined below. m ese include m-RNA, r-RNA, t-RNA, DNA, DNA-RNA duplexes, etc . me term analyte also includes receptors that are polynucleotide hin~;ng agents, such as, for example, restriction enzymes, activators, repressors, nllcleA~es, polymerases, histones, repair enzymes, chemotherapeutic agents, and the like.
The analyte may be a molecule found directly in a sample such as a body fluid from a host. The sample can be , ;ne~ directly or may be pretreated to render the analyte more readily detectible. Furthermore~ the analyte of interest may be determin~ by detecting an agent probative of the analyte of interest such as a specific h;n~;ng pair member compl: - t~ry to the analyte of interest, whose presence will be detected only when the analyte of interest is present in a sample. m us, the agent probative of the analyte becomes the analyte that is detected in an assay.
m e body fluid can be, for example, urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, and the like.
"Specific h~n~;nq pair (sbp) ~ ~ refers to one of two different molecules, having an area on the surface or in a cavity which specifically binds to and is thereby defined as complementary with a particular sp~t;~l and polar org~n;~t;on of the other molecule. m e members of the specific h;n~;ng pair are referred to as ligand and receptor (antiligand). m ese will usually be ~ 7 ~ of an ; ological pair such as antigen-~nt;ho~y, although other specific h;n~;ng pairs such as biotin-avidin, hl_ ?B-hr_ e receptorg, nucleic acid duplexes, IgG-protein ~, polynucleotide pairs such as DNA-DNA, DNA-R~A, and the like are not ; lnological pairs but are included in the invention and the definition of sbp member.
"Polynucleotide" refers to a compound or composition which is a polymeric nucleotide having in the natural state about 6 to 500,000 or more nucleotides and having in the iso1~te~ state about 6 to 50,000 or more nucleotides, usually about 6 to 20,000 nucleotides, more frequently 6 to 10,000 nucleotides. m e term "polynucleotide" also includes oligonucleotides and nucleic acids from any source in purified or unpurified form, naturally occurring or synthetically produced, including DNA (dsDNA and ssD~A) and RNA, usually D~A, and may be t-RNA, m-RNA, r-RNA, mitochondrial DNA and RNA, chloroplast DNA and RNA, DNA-RNA hybrids, or mixtures thereof, genes, ~h.~ ~somes, plasmids, the genomes of biological material such as mi~.ooly~isms, e.g., bacteria, yeasts, viruses, viroids, molds, fungi, plants, ~n; ls, humans, and fr~_ ts thereof, and the like.
"Polynucleotide probe" refers to single-stranded nucleic acid molecules having base sequences compl: ~ry to that of the target polynucleotide analyte. Probes will generally consist of chemically or ~ W 09S/06877 2 ~ 7 0 ~ ~ 3 PCTAUS94/09705 synthesized D~A or ~NA polynucleotides from 6 to 200 base pair in length and must be capable of forming a stable hybridization complex with the target polynucleotide analyte.
"Ligand" refers to any organic compound for which a receptor naturally exists or can be prepared. The term ligand also includes ligand analogs, which are modified ligand5, usually an organic radical or analyte analog, usually of a molecular weight greater than 100, which can compete with the analogous ligand for a receptor, the modification providing means to join the ligand analog to another molecule. The ligand analog will usually differ from the ligand by more than replS~_ t of a hydr ogen with a bond which links the ligand analog to a hub or label, but need not. The ligand analog can bind to the receptor in a manner similar to the ligand.
The analog could be, for example, an antibody directed against the idiotype of an Ant;hoAy to the ligand.
"Receptor" or "antiligand" refers to any compound or composition capable of recognizing a particular spAt;S~l and polar organization of a molecule, e.g., epitopic or det~rn~;nAnt site. Illustrative receptors include naturally occurring receptors, e.g., thyroxine h;nA;ng globulin, Ant;ho~';es, enzymes, Fab frAs ~, lectins, nucleic acids, avidin, protein A, barstar, complement component Clq, and the like. Avidin is ;nt~nA~A to include egg white avidin and biotin hinA;ng proteins from other sources, such as streptavidin.
"Specific b;nA;ng'l refers to the specific recognition of one of two different molecules for the other compared to substAnti~lly less recognition o~ other molecules. Generally, the molecules have areas on their surfaces or in cavities giving rise to specific recognition between the two molecules. Exemplary of specific hinA;ng are antibody-antigen interactions, enzyme-substrate interaction~, polynucleotide interactions, and 80 forth.
"Non-specific h;nA;ng" refers to the non-covalent binA;ng between molecules that is relatively ;nA~p~nA~nt of specific surface structures.
Non-specific h;nA;ng may result from several factors including hydrophobic interactions between molecules.
"AntihoAy" refers to an ; ~nnglobulin which specifically binds to and is thereby defined as compl: - A~y with a particular ~pAt; Al and polar orgS~n;~;~tion of another molecule. The antibody can be monoclonal or polyclonal and can be prepared by techniques that are well known in the art such as ; ln;7Sltion of a host and collection of sera (polyclonal) or by preparing cnnt;nuoll~ hybrid cell lines _nd collecting the secreted protein (monoclonal), or by cloning and expressing nucleotide sequences or mutagenized versions thereof coding at least for the amino acid sequences required for specific hinA;ng of natural ~nt;hoA;es. Antibodies may include a complete ; lnnglobulin or fragment thereof, which ; ln~glnhlll;n~ include the various classes and isotypes, such as IgA, IgD, IgE, IgGl, IgG2a, IgG2b and IgG3, IgM, etc. ErA~m~nt8 thereof may include W 095/06877 2 1 7 ~ 8 7 3 PCTrUS94/09705 . ' Fab, Fv and F(ab')2, Fab', and the like. In addition, aggregates, polymers, and conjugates of ; ~noglobulins or their fragments can be used where ~-u~ ate so long a~ hin~;n~ affinity for a particular molecule is int~in "Linking group" refers to the covalent linkage between molecules.
The linking group will vary depending upon the nature of the molecules, such as a photosensitizer, a photoactive indicator precursor, an sbp member or the molecule as~oc;~te~ with or part of a particle, being linked.
Functional groups that are n~ lly present or are intro~lce~ on a photosensitizer or a photoactive indicator precursor will be employed for linking these molecules to an sbp 1 b~ or to a particle such as a lipoph; lic cv.l.~onent of a 1;~OB' - or oil droplet, latex particle, silicon particle, metal 801, or dye crystallite.
For the most part, carbonyl functionalities are useful as linking groups, such as oxocarbonyl groups ~uch as aldehydes, acetyl and carboxy groups; and non-oxocarbonyl groups (including nitrogen and sulfur analogs) such as amidine, amidate, thiocarboxy and thionocarboxy. Altprn~t;ve functionalities of oxo are also useful as linking groups, such as halogen, diazo, mercapto, olefin (particularly activated olefin), amino, rhosph~oro and the like. A good description of link;n~ groups may be found in U.S.
Patent No. 3,817,837, which disclosure is inco~u~ated herein by reference.
m e link;ng groups may vary from a bond to a chain of from 1 to 100 atoms, usually from about 1 to 70 atoms" preferably 1 to 50 atoms, more preferably 1 to 20 atoms, each indepPn~Pntly selected from the group norr-lly consisting of carbon, oxygen, sulfur, nitrogen and phosphorous.
m e number of heteroatoms in the link;ng groups will norr~lly range from about 0 to 20, usually from about 1 to 15, more preferably 2 to 6. The atoms in the chain may be substituted with atoms other than hyd-oye~l in a manner similar to that de6cribed for organic groups. As a general rule, the length of a particular l;nk;ng group can be selected arbitrarily to provide for convenience of synthesis, minimize interference of hin~ng sbp ~Q'~, and permit the incu,~u ~tion of any desired group such as a fluorescent energy acceptor, or a catalyst of intersystem crossing such as a heavy atom, and the like. m e linking groups may be Al ;rh~t; c or aromatic, although with diazo groups, aromatic groups will usually be involved.
When heteroatoms are present, oxygen will n~ lly be present a~ oxo or oxy, hon~e~ to carbon, sulfur, nitrogen or phnsFhnrousi nitrogen will n- lly be present as nitro, nitroso or amino, normally bonded to carbon, oxygen, sulfur or rhosphorous; sulfur would be analogous to oxygen; while phosphorous will be hon~P~ to carbon, sulfur, oxygen or nitrogen, usually as phosphnn~te and phosph~te mono- or die8ter.
Common functionalities in forming a covalent bond between the l~nk;ng group and the molecule to be conjugated are alkylamine, amidine, thioamide, ether, urea, thiourea, guanidine, azo, thioether and carboxylate, W095/06877 2 1 7 0 ~ ~ 3 PCTtUS94tO970S

sulfonate, and phosphate esters, amides and thioesters.
For the most part, where the photosensitizer and the photoactive indicator precursor of the present invention are linked to a particle, surface or sbp member, they will have a non-oxocarbonyl group (including nitrogen and sulfur analogs), a rhosrhAte group, an amino group, an alkylating agent (e.g., such as halo or tosylalkyl), an ether group (including hydh~y), a thioether group (including mercapto), an oxocarbonyl group (e.g., aldehyde or ketone), or an active olefin such as a vinyl sulfone or an ~,~-un~aturated ester or amide. These functionalities will be linked to a particle, surface or an sbp member having functionalities such as amine groups, carboxyl groups, active olefins, or alkylating agents, e.g., bromoacetyl. Where an amine and carboxylic acid or its nitrogen derivative or phosphoric acid are l;nke~, amides, _midines and phngrhnramideg will be formed. Where mercaptan and activated olefin are linked, thioethers will be formed. Where a mercaptan and an alkylating agent are linked, thioethers will be formed. Where aldehyde and an amine are linked under re~r; ng conditions, an alkylamine will be formed. Where a carboxylic acid or pho~rhAte acid and an alcohol are linked, esters will be formed.
"A group or functionality imparting hydrophilicity or water solubility" refers to a hydrophilic functionality, which increases wettability of solids with water and the solubility in water of compounds to which it is bound. Such a functional group or functionality can be an organic group and can include a sulfonate, sulfate, rhosrhAte, amidine, rh~sph~nAte, carboxylate, hyd~v~yl particularly polyol~, amine, ether, amide, and the like. Illustrative functional groups are carboxyalkyl, sulfonoxyalkyl, CONHOCH2COOH, CO-(glucosamine), sugars, dextran, cyclodextrin, SO~ ~2COOH, SO3H, C~N~2CH2SO3H, PO3H2, OPO3H2, 1.YdL~Y1 carboxyl, ketone, and ~ 'inAt;ons thereof. Most of the above functionalities can also be utilized as attArhing groups, which permit attArh~nt of the photosensitizer or photoactive indicator precursor to an sbp member or a support.
"A group or functionality imparting lipophilicity or lipid solubility" is a lipophilic functionality, which decreases the wettability of surfaces by water and the solubility in water of compounds to which it is bound. Such a functional group or functionality can cnntAin 1 to 50 or more atoms, usually carbon atoms substituted with hydlo~e.~ or halogen and can include alkyl, alkylidene, aryl and aralkyl. The lipophilic group or functionality will _ lly have one to six straight or brAnrhe~ chain Al iphAt; C groups of at least 6 carbon atoms, more usually at least 10 carbon atoms, and preferably at least 12 carbon atoms, usually not more than 30 carbon atoms. The aliphatic group may be bonded to rings of from 5 to 6 members, which may be alicyclic, heterocyclic, or aromatic.
"Photosensitizer" refers to a molecule which, for the purposes of this invention, can be excited to a metastable state, usually a triplet W O 95/06877 2 1 7 0 8 7 3 PCTfUS~ 3705 state, which in the proximity of molecular oxygen can directly or indirectly transfer its energy to the oxygen with rnnrnm;tant excitation of the oxygen to a highly reactive excited state of oxygen often referred to as singlet oxygen or lo2 (l~,). m e photosensitizer will usually be excited by the absorption of light or by an energy transfer from an excited state of a suitable donor but may also be excited by chemiexcitation, electrochemical activation or by other means. Usually excitation of the photosensitizer will be caused by irradiation with light from an ~Yt~r source. m e photosensitizers of this invention will usually have an absorption Y~ in the wavelength range of 2S0 to 1100 nm, preferably 300 to 1000 nm, and more preferably 450 to 950 nm, with an extinction coefficient at its absorh~nce greater than 500 ~cm~l, preferably at least 5000 ~Icm~l, more preferably at least 50,000 ~Icm~l. The lifetime of the excited state, usually a triplet ætate, produced following absorption of light by the photosensitizer will usually be at least 100 nsec, preferably at least 1 ~sec in the absence of oxygen. In general, the lifetime must be sufficiently long to permit the energy transfer to oxygen, which will nr- lly be present at cnnC~ntrations in the range of 10-5 to 10-~M (~pen~;ng on the -~i ). m e excited state of the photosensitizer will usually have a different spin ~Ant- number (S) than its ground state and will usually be in a triplet (S=1) state when, as is usually the case, the ground state is a singlet (S=0). Preferably, the photosensitizer will have a high intersystem crossing yield. m at is, excitation o~ a photosensitizer will produce the long lived state (usually triplet) with an efficiency of at least 10~, desirably at least 40~, preferably greater than 80~. m e photos~n~itizer will usually be at most weakly fluore~cent under the assay conditions (quantum yield usually less than 0.5, preferably less that 0.1).
Photosensitizers of the instant invention are relatively photostable and will not react efficiently with the singlet molecular oxygen BO
generated. Several structural features are present in most useful photos~n~itizers. ~ost photosensitizers have at least one and frequently three or more conjugated double or triple bonds held in a rigid, frequently aromatic structure. They will fre~ntly cnnt~n at least one group that accelerates intersystem crossing such as a carbonyl or imine group or a heavy atom selected from rows 3 through 6 of the periodic table, especially iodine or L-~ 'n~, or they will frequently have polyaromatic structures.
Typical photosensitizers include ketones suCh as acetone, h~n~h~nnne and 9-thinY~nthone; Y~nthpne~ such a8 eosin and rose bengal; polyaromatic compounds such as hllr~ n~terfullerene and 9,10-dib-~ - h~acene;
porphyrins including metallo-porphyrins such as h toporphyrin and chlorophylls; oY~z;nes; cyanines; squarate dyes; phthalocyanines;
merocyanines; thiazines such as methylene blue, etc ., and deri~atives of these compounds substituted by an organic group for enh~nC~ng intersystem crossing and rendering such compounds more lipophilic or more hydrophilic ~ W O 95/06877 2 1 7 0 B 7 3 PCTrUS94/09705 and/or as attArh;ng groups for att~ t, for example, to an sbp member.
Examples of other photosensitizers that may be utilized in the present invention are tho~e that have the above properties and are enumerated in N.J. Turro, "Molecular Photochemistry", page 132, W.A. Benjamin Inc., N.Y.
1965.
The photosensitizers of the instant invention are preferably relatively non-polar to assure dissolution into a lipophilic member when the photosensitizer is inco-~oL~ted into a suspendible particle such as an oil droplet, liposome, latex particle, and the like.
"Photoactive indicator precursor" refers to those molecules which react with singlet oxygen to form photoactive indicators or to form a compound that can react with an auxiliary compound that i8 thereupon converted to a photoactive indicator. There are several types of reactions of singlet oxygen that can give compounds that will lead to a photoactive indicator ~ d. The type of reaction that is employed and the choice of the photoactive indicator that is desired provides a guide to the structures of the photoactive indicator precursors and any auxiliary compounds used in the present invention.
The photoactive indicator precursor will preferably undergo a reaction with singlet oxygen that is very rapid, usually at least 104 to 106 sec~~, preferably at least 106 to 108 sec-~, more preferably ~108 sec~l. When the initial product of the reaction is an ;nt~ te that reacts to give the photoactive precursor, the ;nt~ te will preferably have a lifetime that i8 short relative to the desired time between forming singlet oxygen and detecting the fluorescence emitted from the photoactive indicator upon exposure to light. For simultaneous singlet oxygen generation and fluorescence detection the lifetime will usually be 10-3 to 10 sec, preferably 10-3 sec. When generation of singlet oxygen and fluorescence detection are se~l~nti~l the lifetime may vary from 10-3 sec to 30 minutes or more, preferably ~1 sec to 60 sec.
Higher rates of reaction of singlet oxygen are achieved by providing singlet oxygen reactive groups in the photoactive indicator precursor that are electron rich. These groups will usually be an olefin or acetylene, hydrazine and hydru~ylamine derivatives, selenides and tellurides but are not limited to these groups. For example, tellurides have been found to be particularly useful because they react rapidly with singlet oxygen to produce an olefin. The reaction rate depends on the electron availability (oxidation potential) of the tellurium. For example, electron donating groups on an aryl ring substituent on the tellurium atom can increase the rate. Changing from tellurium to selenium (the next lower chalcogenide) will decrease the rate, but increase the oxidation stability of the molecule.
When the photoactive indicator precursor cnntA;n~ a hydrazine or hydrazide, reaction with singlet oxygen can produce a double bond. For example, singlet oxygen can convert hydrazides directly into fluorescent W 095/06877 2 1 7 0 8 7 ~ PCTrUS94/09705 photoactive indicators, as illustrated in the following reaction:

O O

~ N-~ , ~ ,N

The oxidation pot~ntiA1 of a hydrazine is an important factor in providing a high rate of reaction. Electron withdrawing groups such as an acyl group (e.g., as in a hydrazide) slow the reaction although acyl hydrazides and diacyl hydrazides can still be used as photoactive indicator precursors in the present invention. When the reaction is insufficiently rapid it can often be accelerated in the presence of a base. Por example, 3-~ 'n~phthaloyl hydrazide forms an anion in the presence of strong base that is electron rich and can react rapidly with singlet oxygen to form 3-: 'nnphth~l~te ag the photoactive indicator. However, the l-ydlu~l ion cannot be used as a base when the suspendible particles cnnt~ i n the photoactive indicator precursor within a hydrophobic matrix. Hydrophilic particles such as agarose can be used instead to permit access to the l-yd.~yl ion. Usually the photoactive indicator precursor will be covalently bound to the suspendible particle when the particle is hydrophilic.
Still another example of a u~eful singlet oxygen reaction is the 2S reaction with electron rich olefins such as those described in European Published Patent Application No. O S15 194. Two fnn~' tal types of reactions are described. One of these i8 the "ene" reaction which is exemplified by the following transformation:
~ 1 2 /~

H OOH

Thi6 reaction shifts the positiûn of a double bond and produces a hydroperoxide. The double bond shift can cause two ~n~n~hromic groups in the photoactive indicator precursor to come into conjugation and thus produce a fluorescent photoactive indicator.
Other photoactive indicator precursors react with singlet oxygen to form hydroperoxides which can react ~ntern~11y with an oxidizable group to give a fluorescent photoactive indicator. An example of such a precursor and the subsequent reaction and product include the following:

WO 95/06877 2 1 7 ~ 8 7 3 PCT/US94/09705 C H9 Cl ~ 3 C H9 \~5 e~ 9 G H 3 \~9 eJ~) !q~t~ C H 3 H O - o/l~l/ C H 9 CH3 CH~

C~9 \~f~0 Alternatively, a hydroperoxide formed by reaction of æinglet oxygen with a photoactive indicator precursor, such as 1,3-diphenylpropene, can serve to oxidize the leuco form of a dye which is present as an auxiliary S campound 80 a~ to form a fluorescent photoactive indicator. The hydLu~elu~ide can also oxygenate a group V element in an auxiliary compound to cause it to act a6 an electron ~nn~t;ng q~l~n~h~r of an associated fluore~cent group. For example, the ~ y compound:

~N I~ J C H 3 CH9~CH3 ~C~O)O

which is poorly fluorescent, can be oxygenated by a hydroperoxide to give the more highly fluorescent ~ ,,u~ld:

CH9; ~ `CH3 9 CH~ ~C l O ) O~

The auxiliary compound could alternatively have a selenium or tellurium atom that could react with a hydroperoxide to produce an int~rm~;Ate that could undergo sub6equent el;m;n~tion to form a W 095/06877 2 1 7 0 ~ 7 3 PCTrUS94/09705 fluorescent photoactive indicator.
Altern~tlvely, the photoactive indicator precursor will react 810wly or not at all with singlet oxygen but will react with a hydroperoxide reaction product of singlet oxygen and an ~n~;~iAry molecule. For example, in the following reaction, the auxiliary compound i5 reacted with singlet oxygen to form a hydroperoxide, which is then reacted with the photoactive indicator precursor of formula (Ik) to form a fluorescent photoactive indicator:
OOH

1 . ~/0 C H9 1 o z ~ C ~ a ~n) ~b) ~7C~ ~ ~ ~ ~b) C27 ~

In each of these examples the auxiliary compound and the photoactive indicator precursor may be covalently linked. In such an occurrence, the resulting molecule is referred to as a photoactive indicator precursor.
The second typical reaction of olefins with ~inglet oxygen is 2 + 2 addition to form a dioxetane. This reaction can lead to bond breaking and therefore can separate a ~l~nching group from a fl~n~m~n~lly fluorescent molecule. Alt~rn~tively the bond breaking step can lead to a ketone, aldehyde or ester which could be fluorescent or which could undergo subseguent reactions that could lead to a fluorescent molecule.
In all of the above olefin reactions the rate will be faster if the olefin is substituted with electron ~nn~tin~ groups such as ethers, thioethers, amines, and the like.
Still another type of reaction of singlet oxygen i8 4 + 2 cycloadditions with dienes. Such reactions lead initially to en~np~roxides. In some cases endoperoxides can rearrange to active esters or anhydrides that are capable of reaction with a suitably placed group to provide a lactone or lactam that can be fluorescent. For example, the endoperoxide formed in the following reaction scheme can rearrange to form a fluorescent lactone:

~ W O95/06877 -21- PCTrUS94/09705 ~3 ~ ~ N

o N
XO~

~ ~ o Altern~tively, the endoperoxides may oxidize a photoactive indicator precur60r much as described above for hydroperoxides.
Additional examples of photoactive indicator precursors~ reaction 40 with si~glet oxygen to produce fluorescent photoactive indicator molecules are illustrated below:

WO 95/06877 2 1 7 0 ~ 7 3 PCT/US94/(19705 3 ~ ~ TeC~IH~; loZ ~, 9 \ ~

H3 C )<~ N ~19 C >~C N
CN CN

and X9C- ~ / H9 2 ~9C - ~ ~ ~C~
~ CH9 CH~

The structure of the photoactive indicator precursor will therefore depend on the particular singlet oxygen reaction that i8 to be employed and it will usually be designed to assure that any subsequent reactions initiated by reaction with singlet oxygen that are required to produce a photoactive indicator will proceed relatively rapidly. Additionally the structure of the photoactive indicator precursor will lead to the ~ormation of a photoactive indicator that has the desired absorption and emission wavelengths, and has relatively high fluorescent quantum yields, preferably ~0.1, more preferably greater than 0.4, and a high extinction coefficient at the desired excitation wavelength, preferably ~1000 ~' cm~~, more preferably ~10,000 ~ cm~~.
Preferred photoactive indicator precursors of the present invention include compol~n~ c~nt~;n;ng the following structure (I):
El~ X R
~ \1~

wherein H is cis to the XR group; X is a selenium or tellurium atom; R is an organic or organometallic group bound to X through an nn~t~ated carbon atom, a silicon atom, or a tin atom; and A, when taken with the carbon-carbon group, forms an alicyclic ring (optionally fused to one or more aromatic rings) or a heterocyclic ring; where, upon reaction of the compound with singlet oxygen, the H and the XR groups are replaced by a carbon-carbon double bond to yield a fluorescent molecule having an extinction coefficient of at least 10,000 M~cm~~ at its absorption m~;m~m and a fluorescence quantum efficiency of at least 10~.
Particularly preferred within these compounds are those compounds wherein X is tellurium. Most preferred is the compound of the following formula:

W 095/06877 2 1 7 ~ ~ 7 3 PCT~US94/09705 ' ~ ~ YR
H

wherein R i5 an organic or organometallic group bound to X
through an nn~At-lrated carbon atom, a silicon atom, or a tin atom; a~d Rl is hydLUy~ll or alkyl; and wherein up to four of the L~ -; n; ng hydLogell atoms may be replaced by alkyl or alkylene substituents which may be taken together to form one or more alicyclic or aromatic rings.
m e compounds disclosed herein c~nt~;n~ng structure (I) are designated herein as derivatives of the structure, e.g., compound of formula (Ia), compound of formula (If) or compound of formula (Ik).
Example~ of such compounds where X is tellurium and the fluorescent photoactive indicator molecule formed upon the compounds' reaction with singlet oxygen are given below:

WO 95/068772 1 7 ~ ~ 7 . ~

~)<CN 2 ~ ~CII

(1~

10 C8H5~~eC~X5 102 ~ C~H5~ C10)0CH3 ( Ib) NaC~ ~ 2 N C~N\~ /C;L5 ( IC) ~T e C r H > ~C ( O ) O C H o C({~)O~H3 ( I d ) ~ WO 95/06871 2 1 7 0 8 7 3 PCT/US94/09705 CH9 Cl H3 H3~ ~ 21~3C~ ~ C~H sf ' ~le~

1~9 CH S IcHS
EaC~ \~e~ 1 ~
~lf ) T e C ~ ~ 6 o3~C H N C~O>~g olc (Ig, Presently, preferred photosensitive indicator precursors of the invention are the compounds of formula (Ie) and (If) above.
The phenyltelluridyl radical (-TeC~H5) in these compounds can be repl~ce~ with other tellurium derivatives, such as TeSiC(CH3)3 and TeSn((CH2)3CH3)3, or the phenyl group can be substituted, preferably with electron ~nn~t~ng groups such a~ -N(CH3)2 and -OCH3. When X i8 selenium it is preferable that the selenium is substituted by a strong electron donor group or atom, such as tin.
Other classes of photoactive indicator precursors can also be used in the present invention. For example, compounds that chemiluminesce on reaction with singlet oxygen are frequently converted to fluorescent products which can serve as photoactive indicators of the present invention. Examples of such photoactive indicator precursors and the photoactive indicators pro~nce~ upon reaction with singlet oxygen include the following:

W O9S/06877 2 1 7 Q ~ ~ ~ PCT~US94/0970~
.

~N~ 0 ~N

N-U U~ ~ c(0)~

~nd ~ o ~ 10 CH9 ~

"Photoactive indicator" refers to a molecule which, following absorption of light of wavelengths of 250 to 1100 nm, preferably 300 to 950 nm, emits light by fluorescence or rhnsrhnrescence, preferably by fluorescence, or transfers it excitation energy to an acceptor molecule which thereupon emits light by fluorescence or pho~rhorescence. Preferably the emission ~l -t yield will be high, usually at least 0.1, preferably at least 0.4, more preferably greater than 0.7 and the extinction coefficient of the absorption - will usually be greater than 5000 ~Icm~'.
Photoactive indicators of this invention are typically fluorescent compounds, such as fluorescent brightPnPrs~ which typically absorb light between 300 and 400 nanometers and emit between 400 and 500 nanometers;
Y~nthPn~Ps such as rho~Am~nP and fluoresceini bimanes; coumarins such as nmbell;ferone; aromatic amines such as dansyl; squarate dyes; benzofurans;
cyanines, merocyanines, rare earth chelates, and the like. Photoactive indicators that are phosphorescent include porphyrins, phthalocyanines, polyaromatic compounds such as pyrene, anthracene and ACPn~phthPnP
Photoactive indicators also include ~l.L~ es. Photoactive indicators that can transfer energy to an acceptor molecule will usually absorb at 250 to 550 nm. Such acceptor molecules are luminescent and can include any of the above mentioned fluorescent and phosphorescent photoactive indicators.
"Measuring the fluorescence" refers to the detection and calculation of the amount of light emitted from a photoactive indicator of the invention upon excitation by irradiation with light. While the fluorescence of the photoactive indicator will usually be measured by exciting the photoactive indicator by irradiation with light and simult~nPoll~ly detecting the light that is emitted therefrom (i.e., the fluorescence), other methods of detecting the fluorescence are contemplated W 095/06877 2 1 7 a ~ 7 3 PCTrUS94/09705 by this invention. The measu~. ~ t of fluorescence i8 intended to include detection of light emitted by the photoactive indicator simultaneous with or ~mm~ tely following irradiation with light regardless of whether the - light iB absorbed directly or indirectly or whether the emission is from an excited singlet state or state of higher multiplicity. Measurement of fluorescence is also ~ntPn~ to include the measu-l ~ t of light emitted from the photoactive indicator following transfer of energy from a donor that is excited through chemiexcitation other than chemiexcitation initiated by absorption of light by the photosensitizer. For example, measu-~ - t of fluorescence of the photoactive indicator includes activation of a chemiluminescent molecule, for example, by addition of 1~Y~LO~n peroxide and peroxidase to luminol, and measuL~ ~ t of the light emitted from the photoactive indicator as a result of the energy transfer from the luminol reaction product to the photoactive indicator.
"Su~o~L" or "surface" refers to a surface comprised of a porous or non-porous water insoluble material. The surface can have any one of a number of shapes, such as strip, rod, particle, including bead, and the like. The surface can be hydrophilic or capable of being rendered hydrophilic and includes inorganic powders such as silica, magnesium sulfate, and alumina; natural polymeric materials, particularly cellulosic materials and materials derived from cellulose, such as fiber cont~;n;ng papers, e.g., filter paper, chromatographic paper, etc.; synthetic or modified naturally occurring polymers, such as nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide, cross linked dexL ~-, agarose, polyacrylate, polyethylene, poly~-~ylene, poly(4-methylbutene), poly~Lyrene, polymethacrylate, poly(ethylene terephth~l~te)~ nylon, poly(vinyl butyrate), etc.; either used by themselves or in conjunction with other materials; glass available as Bioglass, ceramics, metals, and the like. Natural or synthetic assemblies ~uch as liposomes, lipid vesicles, and cell~ can also be employed.
R; n~ng of sbp members to the support or surface may be accomplished by well known techniques, _~ ly available in the literature. See, for example, "Immobilized Enzymes," Ichiro Chibata, Halsted Press, New York (1978) and Cuatrecasas, J. Biol. Chem., 245:3059 (1970).
The surface will usually be polyfunctional or be capable of being polyfunctionalized or be capable of h;n~ng a polynucleotide, an sbp member, a photos~n~itizer, and/or a photoactive chemiluminescent compound through specific or non-specific covalent or non-covalent interactions. A
wide variety of functional groups are available or can be incorporated.
Functional groups include carboxylic acids, aldehydes, amino groups, cyano groups, ethylene groups, l~yd-O~yl groups, --c~to groups and the like.
The manner of linking a wide variety of compounds to surfaces is well known and is ~mply illustrated in the literature. See for example Cautrecasas, J. Biol. Chem. 245,3059 (1970). The length of a linking group to the oligonucleotide or sbp member may vary widely, depending upon the nature of W 095/06877 2 ~ 7 ~ PCTrUS94/09705 the compound being linked, the effect of the distance between the compound being linked and the surface on the specific hin~; ng properties and the like.
"Suspendible particles" refers to particles capable of being able to be suspended in water which are at least about 20 nm and not more than about 20 microns, usually at least about 40 nm and less than about 10 microns, preferably from about 0.10 to 2.0 microns in diameter, and which normally have a volume of less than about 4 picoliters. The suspendible particles may be organic or inorganic, swellable or non-swellable, porous or non-porous, preferably of a density a~ ; t;ng water (generally from about 0.7 to about 1.5g/ml), and composed of material that can be transparent, partially transparent, or opaque. The suspendible particles will usually be charged, preferably negatively charged. The suspen~;hle particles may be solid (e.g., polymer, metal, gla~s, organic and inorganic such as minerals, salts and ~;~t~m~), oil droplets (e.g., hydrocarbon, fluorocarbon, silicon fluid), or vesicles (e.g., synthetic such as phospholipid or other lipids such as dialkyl phosph~tes or natural such as cells and organelles). The suspendible particles may be latex particles or other particles comprised of organic or inorganic polymers; lipid vesicles, e.g., liposomes; rhssphslipid vesicles; oil droplets; silicon particles;
metal 8018; cells; and dye crystallites.
If organic, the suspendible particles may be polymers, either addition or con~n~t;on polymers, which are readily suspendible in the assay medium. The organic suspendible particles will also be adsorptive or functi~n~liz~hle 80 as to bind at their surface an sbp member (either directly or indirectly) and to bind at their surface or incorporate within their volume a photosensitizer or a photoactive indicator precursor.
The suspendible particles can be derived from naturally occurring materials, naturally occurring materials which are synthetically modified and synthetic materials. Natural or synthetic ~8~ ~lies such as lipid bilayers, e.g., liposomes and non-phospholipid vesicles, are preferred.
Among organic polymers of particular interest are polysaccharides, particularly cross-linked polysAc~h~ides, such as agarose, which is available as Sepharose, dextran, available as S~ph~e~ and Sephacryl, cellulose, starch, and the like; addition polymers, such as pol~Lyr~l~e~
polyacrylamide, homopolymers and copolymers of derivatives of acrylate and methacrylate, particularly esters and amides having free hydLu~yl functionalities including hyd-ug~ls~ and the like. Inorganic polymers include silicones, glasses, available as Bioglas, and the like. Sols include gold, selenium, and other metals. Suspendible particles may also be dispersed water insoluble dyes such as porphyrins, phthalocyanines, etc., which may also act as photosensitizers. Suspendible particles may also include ~;Atl ~, cells, viral particles, oil droplets, fat particles such as alkyl triglycerides, magnetosomes, cell nuclei and the like.
Where non-polymeric particles are used, the particle size may be -W O 95/06877 PCT~US94/09705 ~ 217~87~

varied by breaking larger particles into smaller particles by mechanical means, such as grinding, sonication, agitation, etc.
Like the surface or support defined above, the suspendible particles - will usually be polyfunctional or be capable of being polyfunctionalized or be capable of being bound to an sbp member, photosensitizer, or photoactive indicator precursor through specific or non-specific covalent or non-covalent interactions. A wide variety of functional groups are available or can be incorporated. Exemplary functional groups include carboxylic acids, aldehydes, amino groups, cyano groups, ethylene groups, hyd-~yl groups, mercapto groups and the like. When covalent att~' t of an sbp member, chemiluminescent compound or photosensitizer to the particle is employed, the manner of 1 inkin~ is well known and is amply illustrated in the literature. See for example Cautrecasas, J. Biol. Chem., 245:3059 (1970). The length of a 1 ink;ng group may vary widely, depending upon the nature of the compound being l~nke~, the nature of the particle, the effect of the distance between the compound being linked and the particle on the bin~i ng of sbp members and the analyte and the like.
The photosensitizer and/or photoactive indicator precursor can be chosen to dissolve in, or covalently bind to suspendible particles, provided, however, that the phot~sensitizer and the photoactive indicator precursor are not associated with the same particle. When noncovalently bound, the compounds and the particles will all usually be hydrophobic to reduce the ability of the compounds to dissociate from the particles and to associate with the same particle. The problem of having both the photosensitizer and the photoactive indicator associated with the same particle may be ;n; ' ~ed by covalently h;n~;ng either one or both of the compounds to a particle, thereby allowing each ~_ _o~n~ to be either hydrophilic or hydrophobic.
The number of photosensitizer or photoactive indicator precursor molecules associated with each particle will be at lea6t one and may be sufficiently high enough 80 that the particle consists entirely of photosensitizer or photoactive indicator precursor molecules. The preferred number of molecules will be selected empirically to provide the highest signal to bachy~u~-d in the assay (where the signal is det~rm;ne~
under conditions where the particles are bound to each other and the background is dete ;ned where the particles are unassociated). Normally, the cnnc~nt~ation of photosensitizer and photoactive indicator precursor in the particles will range from 10~ to 5M, usually from 10-5 to 10-lM, preferably from 10-3 to 10-IM.
"Oil droplets" refers to fluid or waxy particles comprised of a lipophilic compound coated and stabilized with an emulsifier that is an hiph;liC molecule such as, for example, phospholipids, sphingomyelin, hl-m;n and the like.
The phospholipids are based upon ~liphatic carboxylic acid esters of aliphatic polyols, where at least one hyd ~ylic group is substituted with W 095/06877 ~ ~ 7 o ~ ~ 3 PCTrUS94/09705 a carboxylic acid ester of from about 8 to 36, more usually of from about 10 to 20 carbon atoms, which may have from O to 3 sites, more usually from O to 1 site of ethylenic unsaturation and at lea~t 1, normally only 1, hyd o~yl group substituted with phosphate to form a phosphate ester. The rh~sph~te group may be further substituted with small ~l;ph~tic compounds which are of di or higher functionality, generally having hydL~l or amino groups.
The oil droplets can be made in accordance with conventional procedures by c ~in;ng the ~ o~.iate lipophilic c ,,o~ds with a surfactant, anionic, cationic or nonionic, where the surfactant is present in from about 0.1 to 40, more usually from about 0.1 to 20 weight percent of the mixture and subjecting the mixture in an aqueous medium to agitation, such as sonication or vortexing. Illustrative lipophilic compounds include hydrocarbon oils, halorArhon~ including fluorocarbons, alkyl phthAlAtes, trialkyl phosph~tes, triglycerides, etc.
An sbp member will usually be A~orhe~ to the surface of the oil droplet or bonded directly or indirectly to a surface component of the oil droplet. The sbp member may be incol~oL~ted into the liquid particles either during or after the preparation of the liquid particles. The sbp '-~ will n~ -lly be present in from about 0.5 to 100, more usually 1 to 90, frequently from about 5 to 80 and preferably from about 50 to 100 mole percent of the molecules present on the surface of the particle.
The following is a list, by way of illustration and not limitation, of ; ,h;ph;l-c compounds, which may be utilized for stabilizing oil droplets: phosphAtidyl ethAnol~ n~, phosphAtidyl rhol;n~ phosph~t;dyl serine, dimyristoylrhosph~;dyl ~hol~ne, egg rhosphAtidyl choline, ~;~r~l 'toylphosphatidyl choline, phosphatidic acid, cardiolipin, lecithin, galactocerebroside, sphingomyelin, dicetylphosphate, phosphatidyl ino~itol, 2-tr;h~Y~cyl; ;umethylamine, 1,3-bis(octadecyl phosphate)-propanol, stearoyloxyethylene phnsrhAte~ phospholipids, dialkylphosph~t~s, sodium dodecyl sulfate, cationic detergents, anionic detergents, proteins such as ~lhl n, non-ionic detergents, etc.
Stabilization of oil droplets can also be achieved by co~t;ng with a polymer such as polycyanoacrylates, ~trAn, polymerized proteins such as A~ ' n, l~yd~yL~Lyl methacrylate, polyacrylamide and the like.
Other compound5 may also be used which have l;p~ph;lic groups and which have been described previously. For the most part, these compounds will be alkylben~en~s, having alkyl groups of from 6 to 20 carbon atoms, usually mixtures of alkyl groups, which may be straight or branched chain, and having a carboxyl group, an hyd-uxylic group, a polyoxy alkylene group (alkylene of from 2 to 3 carbon atoms), carboxylic group, sulfonic acid group, or amino group. Al;phAtic fatty acids may be used which will normally be of from about 10 to 36, more usually of from about 12 to 20 carbon atoms. Also, fatty alcohols having the carbon limits indicated for the fatty acids, fatty amines of similar carbon limitations and various W 095/06877 PCTrUS94109705 steroids may also find use.
The oil droplets can comprise a fluorocarbon oil or a silicone oil (silicon particle). Such droplets are described by Giaever in U.S. Patents - Nos. 4,634,681 and 4,619,904 (the disclosures of which are incorporated herein in their entirety). These droplets are formed by dispersing a fluorocarbon oil or silicone oil in an aqueous phase. The droplets are prepared by placing a small amount of the selected oil (generally, such oils are commercially available) in a container with a larger amount of the aqueous phase. The liquid system is subjected to agitation to bring about emulsification and then centrifuged. The h~ ~e.-eous phase is removed and the residual droplets are resuspended in an aqueous buffered medium. The above centrifugation and derAntAt;on steps can be repeated one or more times before the droplets are utilized.
Sbp members can be bound to the droplets in a number of ways. As described by Giaever, the particular sbp member, particularly a protein~ceQus sbp member, can be coated on the droplets by introducing an excess of the sbp member into the aqueous medium prior to or after the emulsification step. W~h;ng steps are desirable to remove excess sbp ' - L . Functionalization of the oil introduces functionalities described above for linking to sbp members. Such functionalities can also be employed to link the droplets to a photosensitizer or a photoactive indicator precursor. On the other hand, the photosensitizer or photoactive indicator precursor will frequently be chosen to be soluble in the oil phase of the oil droplet and will not be covalently bound. When the oil is a fluorocarbon, a fluorinated photosensitizer or photoactive indicator precursor will often be more soluble than the corresponding unfluorinated derivation.
"Liposomes" refers to microvesicles of apprn~i~-tely spherical shape and are one of the preferred materials for use in the present invention.
The liposomes have a diameter that is at least about 20 nm and not more than about 20 microns, usually at least about 40 nm and less than about 10 microns. Preferably, the diameter of the liposomes will be less than about two microns 80 as to limit settling or floatation.
The outer shell of a liposome consists of an amphiphilic bilayer that encloses a volume of water or an A~Oll~ solution. Liposomes with more than one bilayer are referred to as multil: -llA~ vesicles. Liposomes with only one bilayer are called unil; ~ vesicles. Multil; 11~ vesicles are preferred in the present invention when using a lipophilic photosensitizer or photoactive indicator precursor because of their ability to inco-~o.ate larger quantities of these materialg than llni 1; ~ 11 vesicles. The Amrh;ph;lic bilayer is frequently comprised of phosph~lipids. Phospholipids employed in preparing particles utilizable in the present invention can be any phospholipid or phospholipid mixture found in natural ~es including lecithin, or synthetic glyceryl phosphate diesters of saturated or -n~At~ated 12-carbon or 24-carbon linear fatty W 095/06877 ~ ~ 7 0 8 ~ 3 pcTrus94m97o5 acids wherein the rhosph~te can be present as a monoester, or as an ester of a polar alcohol such as ethAnol; n~, rh~linP, inositol, serine, glycerol and the like. Particularly preferred phospholipids include L-~-palmitoyl oleoyl-phosphatidylcholine (POPC), palmitoyl oleoylphss~h~tidyl-glycerol (POPG), L-~-dioleoylphosphatidylglycerol, L-~-(dioleoyl)-phnsphAt;dyl ethanolamine (DOPE) and L-~-(dioleoyl)-osrhAt;dyl ~-(4-(N-maleimidomethyl)-cy-cloh~Y~ne-l-caLbu~y~ido)ethan (DOP~-MCC).
The phosphslipids in the bilayer may be supplemented with cholesterol and may be replaced with other A~rh;philic compounds that have a polar head group, usually charged, and a hydLuphobic portion usually comprised of two linear hydrocarbon chains. Examples of such substituents include dialkylphosphate, ~;~Alk~l~y~Lu~ylphosphates wherein the alkyl groups have linear chains of 12 to 20 carbon atoms, N-(2,3-di-(9-(Z)-octa-decenyloxy))-prop-1-yl-N,N,N-trimethyl-ammonium chloride (DOTMA), sphingomyelin, cardiolipin, and the like.
Liposome~ utilized in the pre~ent invention preferably have a high negative charge density to stabilize the suspension and to ~L~v~
gpnnt~n~oug aggregation.
Liposomes may be produced by a variety of methods including hydration and mechanical dispersion of dried rhssph~lipid or phospholipid substitute in an aqueous solution. Tiposr -.~ prepared in this manner have a variety of ~ ions, _ ,ssitions and behaviors. One method of re~nc;ng the heterogeneity and inrnn~i~tenCy of behavior of mechanically dispersed liposomes is by sonication. Such a method decreases the average liposome size. Alt~rn~tively, extrusion is usable as a final step during the production of the liposomes. U.S. Patent 4,529,561 discloses a method of extruding liposomes under pressure through a uniform pore-size ~-e to i ,_uv~ gize uniformity.
For use in the present invention the liposomes should be capable of hi n~ing to an sbp member and be capable of having a photosensitizer or photoactive indicator precursor associated with either the aqueous or the nonaqueous phase. The liposomes utilized in the present invention will usually have sbp '-?~ bound to the outer surface of the lipid vesicle.
Preparation of liposomes contA;ning a hydrophobic or ; phirhilic photosensiti~er or a photoactive indicator precursor dissolved in the lipid bilayer can be carried out in a variety of methods, including a method described by Olsen, et al., Biochemica et Biophysica Acta, ~57(9), 1979.
Briefly, a mixture of lipids cnntAining the a~Lu~Liate compound in an organic solvent such as chloroform is dried to a thin film on the walls of a glass vessel. The lipid film is hydrated in an a~Lu~Liate buffer by ~h~king or vortexing. Thereafter, the lipid suspension i8 extruded through a series of polyc~hsn~te filter ~ '~ ~les having successively smaller pore sizes, for example, 2.0, 1.0, 0.8, 0.6, 0.4, and 0.2 microns. Repeated filtration through any of the filters, and in particular through the W O 95/06877 PCTrUS94/09705 217()873 smallest filter, is desirable. The liposomes can be purified by, for example, gel filtration, such as through a column of Sephacryl S-1000. The column can be eluted with buffer and the liposomes collected. Storage in - the cold prolongs shelf-life of the liposomes produced by this method.
Alternatively the photosensitizer or photoactive indicator precursor can be added to the liquid suspension following preparation of the liposomes.
Labeling of droplets and liposomes will often involve, for example, inclusion of thiol or maleimide or biotin groups on the molecules comprising the lipid bilayer. Photosensitizers, photoactive indicator precursor molecules or sbp members may then be bound to the surface by reaction of the particles with one of these materials that is bound to a sulfhydryl reactive reagent, a sulfhydryl group, or avidin, respectively.
Sulfhydryl reactive groups include alkylating reagents such as b-~ cetamide and maleimide.
Sbp members can be attracted to the surface of the liposome particles by weak hydrophobic interactions, however such interactions are not generally sufficient to withstand the shear force encountered during incubation and washing. It is preferable to covalently bond sbp members to a liposome particle that has been functionalized, for example by use of DOPB-NCC, as shown above, by c~mh;n;ng said liposome with the selected sbp member functionalized with a mercaptan group. For example, if the sbp member is an ~nt;ho~y, it may be reacted with S-acetyl-mercaptosuccinic anhydride (SAMSA) and hydrolyzed to provide a sulfhydryl modified antibody.
"Latex particles" refers to a particulate water-suspendible water-insoluble polymeric material usually having particle ~ ;ons of 20 nm to 20 mm, more preferably 100 to 1000 nm in diameter. The latex is freg~l~ntly a substituted polyethylene such as the following:
poly~ty-e,le-butadiene, polyacrylamide poly~Ly~ e, polystyrene with amino groups, poly-acrylic acid, polymethacrylic acid, acrylonitrile-butadiene, styrene copolymers, polyvinyl acetate-acrylate, polyvinyl pyridine, vinyl-chloride acrylate copolymers, and the like. Non-crosslinked polymers of styrene and carboxylated styrene or styrene functionalized with other active groups such as amino, hyd u~yl, halo and the like are preferred.
Fre~-ently, copolymers of substituted styrenes with dienes such as butadiene will be used.
The association of the photosensitizer or photoactive indicator precursor with latex particles utilized in the present invention may involve incorporation during formation of the particles by polymerization but will usually involve incorporation into preformed particles, usually by noncovalent dissolution into the particles. Usually a solution of the photoactive indicator precursor or photosensitizer will be employed.
Solvents that may be utilized include alcohols (including ethanol), ethylene glycol and benzyl alcohol; amides such as dimethyl formamide, formamide, acetamide and tetramethyl urea and the like; sulfoxides such as dimethyl sulfoxide and sulfolane; and ethers such as carbitol, ethyl W 095/06877 2 ~ 7 ~ PCTrUS94/09705 carbitol, dimethoxy ethane and the like, and water. The use of solvents having high boiling points in which the particles are insoluble permits the use of elevated temperatures to facilitate di6solution of the compounds into the particles and are particularly suitable. The solvents may be used singly or in cnmh~nAtion. Particularly preferred solvents for incorporating a photosensitizer are those that will not quench the triplet excited state of the photosensitizer either because of their intrinsic properties or because they can subsequently be removed from the particles by virtue of their ability to be dissolved in a solvent such as water that is insoluble in the particles. Aromatic solvents are preferred, and generally solvents that are soluble in the particle. For incorporating photoactive indicator precursors in particles a solvent should be selected that does not interfere with the fluorescence of the photoactive indicator so formed because of their intrinsic properties or ability to be removed from the particles. Frequently, aromatic solvents will also be preferred.
Typical aromatic solvents include dibutylphth~l~te, benzonitrile, n~rhth~n;trile, dioctylterephth~l~te, dichlorobenzene, diphenylether, dimethoxybenzene, etc.
Except when the photosensitizer or photoactive indicator precursor i5 to be covalently bound to the particles, it will usually be preferable to use electronically neutral photosensitizers or photoactive indicator precursors. It is preferable that the liquid medium ~elected does not soften the polymer beads to the point of stickiness. A preferred technique comprises suspending the selected latex particles in a liquid medium in which the photosensitizer or photoactive indicator precursor ha~ at least limited solubility. Preferably, the cnnc~n~ation~ of the photosensitizer and photoactive indicator precursor in the liquid media will be selected to provide particles that have the highest efficiency of singlet oxygen formation and highest ~ntl yield of emission from the photoactive indicator so formed in the media but less concentrated solutions will sometimes be preferred. Distortion or dissolution of the particles in the solvent can be prevented by adding a miscible cosolvent in which the particles are insoluble.
Generally, the temperature employed during the procedure will be chosen to ~ ze the singlet oxygen formation ability of the photosensitizer-labeled particles and the quantum yield of the photoactive indicator 80 formed from the photoactive indicator precursor-labelled particles with the proviso that the particles ~hould not become aggregated at the selected temperature. Elevated temperatures are normally employed.
The temperatures for the procedure will generally range from 20C to 200C, more usually from 50C to 170C. It has been observed that ~ome compounds that are nearly insoluble in water at room temperature, are soluble in, for example, low molecular weight alcohols, such as ethanol and ethylene glycol and the like, at elevated temperatures. Carboxylated modified latex particles have been shown to tolerate low molecular weight alcohols at such ~ W 095/06877 2 1 7 0 8 7 3 PCT~US94/09705 temperatures.
An sbp member may be physically ad80rbed on the surface of the latex particle or may be covalently hon~ to the particle. In cases wherein the sbp ~-- is only weakly bound to the surface of the latex particle, the h;n~;ng may in certain cases be unable to endure particle-to-particle shear forces enco~lntered during incubation and washings. Therefore, it is usually preferable to covalently bond sbp members to the latex particles under conditions that will n; ;ze adsorption. m is may be accomplished by chemically activating the surface of the latex. For example, the N-hyd~u~y~Llcc;n; de ester of surface carboxyl groups can be formed and are then contacted with a linker having amino groups that will react with the ester groups or directly with an sbp -~ that has an amino group. m e linker will usually be selected to reduce nonspecific hin~;ng of assay components to the particle surface and will preferably provide suitable functionality for both attAI ~ t to the latex particle and at~Achm~nt of the sbp member. Suitable materials include maleimidated : no~yt~an (MAD), polylysine, ~m;nosAc~hArides, and the like. M~D can be prepared as described by Hubert, et al., Proc. Natl. Acad. Sci., 75(7), 3143, 1978.
In one method, MAD is first attached to rA~hoYyl-cont~in;ng latex particles using a water soluble carbodiimide, for example, 1-(3-dimethyl~m;no~Lu~yl)-3-ethyl r~ho~;i de. m e coated particles are then eguilibrated in reagents to ~,~v~ nonspecific h;n~;ng Such reagents include proteins such as bovine gamma globulin (BGG), and detergent, such as Tween 20, TRITON X-100 and the like. An sbp member having a sulfhydryl group, or suitably modified to introduce a sulfhydryl group, is then added to a su~p~n~ion of the particles, whereupon a covalent bond is formed between the sbp member and the M~D on the particles. Any excess unreacted sbp member can then be removed by washing.
"~etal 8018" refers to those suspendible particles comprised of a heavy metal, i.e., a metal of atomic number greater than 20 such as a Group IB metal, e.g., gold or silver.
Metal 801 particles are described, for example, by Leuvering in U.S.
Patent ~o. 4,313,734, the disclosure of which is inco.~ol~ted herein by reference in its entirety. Such 8018 include colloidal agueous dispersion of a metal, metal compound, or polymer nuclei coated with a metal or metal compound.
The metal 8018 may be of metals or metal compounds, such as metal oxides, metal hyd~ides and metal salts of polymer nuclei coated with metals or metal compounds. Examples of such metals are plAt;n~ , gold, 6ilver, mercury, lead, pAll~;um, and copper, and of such metal compounds are sil~er iodide, silver bromide, copper hydrous oxide, iron oxide, iron hydL~ide or hydrous oxide, aluminum hydlo~ide or hydrous oxide, chromium hyd~ide or hydrous oxide, vanadium oxide, arsenic sulphide, ~ng~nese hyd~u~ide, lead sulphide, mercury sulphide, barium ~ulrh~te and titanium dioxide. In general, the metals or metal compounds useful may be readily W O 95/06877 PCTrUS94109705 2 ~ ~Q8~3 demonstrated by means of known techniques.
It i8 sometimes advantageous to use s018 comprised of dispersed particles consisting of polymer nuclei coated with the above mentioned metals or metal compounds. These particles have similar properties as the dispersed phase of pure metals or metal compounds, but size, density and metal contact can be optimally c~hine~.
The metal sol particles may be prepared in a large number of ways which are in themselves known. For example, for the preparation of a gold sol Leuvering refers to an article by G Frens in Nature Physical Science 241, 20 (1973).
The metal sol particles can be modi~ied to cnnt~; n various functional groups as described above for l;nk;ng to an sbp member or a photosensitizer or a photoactive indicator precursor. For example, polymeric hon~; ng agents can be used to coat the particles such as polymers contA;n;ng thiol groups that bond strongly to many heavy metals or silylating agents that can bond and form polymeric coatings as, for example, by reaction of metal particles with trialkoxy ~m; n~l kylsilanes as described in European Published Patent Application 84400952.2 by Advanced Magnetics for coAt;ng magnetic particles.
"Dye crystallites" refers to microcrystals of pure or mixed solid water insoluble dyes, preferably dyes that can serve as the photosensitizers described above. The dye crystallites useful in the present invention have a size range of 20 nm to 20 ~m.
One method for preparing dye crystallites is described in U.S. Patent ~o. 4,373,932 (Gribnau, et al.), the disclosure of which is incorporated herein by reference in its entirety. Gribnau describes colloidal dye particles and aqueous dispersions of a hydLo~hObic dye or pigment, which may have an immunochemically reactive component directly or indirectly attached. The dye particles are prepared in general by dispersing a dye in water and then centrifuging. A dye pellet is obtained and resuspended in water, to which glass beads are added. This suspension is rolled for several days at room temperature. The liquid is decanted and centrifuged, and the dye particles are obtained after aspiration of the liquid.
Another method for preparing dye crystallites is by slow addition of a solution of the dye in a water miscible solvent to water. Another method is by sonication of a suspension of the solid dye in water.
R; n~; ng of sbp members to the dye particles can be achieved by direct or indirect adsorption or covalent chemical att~ . The latter is governed by the presence of suitable functional groups in any co~t;ng material and in the dye. For example, functional groups can be introduced onto the surface of a dye crystallite by coupling a compound cont~;n;ng a diazotized aromatic amino group and the desired functional group to a phenol;c or anilino group of the dye.
Where the dye has a carboxyl group, the dye crystallite can be activated by a carbodiimide and coupled to a primary amino component.

~ W O 95/06X77 2 1 7 0 8 7 3 PCTrUS94/09705 Aliphatic primary amino group6 and hydroxyl group6 can be activated, for example, by cyanogen bromide or halogen-sub6tituted di- or tri-azines, after which att~l - t with a primary amino component or, for example, with a component containing a -SH or -OH group can take place. U6e can also be made of bifunctional reactive compounds. For example, glutaraldehyde can be used for the mutual coupling of primary amino component6 of the dye and an 6bp member, and, for example, a hetero-bifunctional reagent 6uch a6 N-6~ccin; ;dyl 3-(2-pyridyldithio) propionate can be employed for the coupling of a primary amino component to a component cont~;n;ng a thiol group.
"Wholly or partially 6ecrl~nt; Al ly~ refer6 to the condition when the component6 of the method6 of the present invention are - ;n~ other than c~nr ;tantly (simult~neoll~ly), one or more may be c ine~ with one or more of the L~ in;ng agents to form a 6~lh_c ~;n~tion. Each 8ubcnm~in~Ation can then be 6ubjected to one or more step6 of the pre6ent method. Thu6, each of the 6~lh_ ;nAtion6 can be ;ncllhAte~ under conditions to achieve one or more of the de6ired re8ult6.
Variou6 ancillary materials will frequently be employed in the a66ay in accold~ce with the present invention. For example, buffers will n- lly be pregent in the assay medium, as well as stabilizer6 for the assay medium and the assay components. Frequently, in addition to the6e additive6, proteins may be included, such as ~lh ;n~, organic solvents such a6 formamide, quaternary ammonium salt6, polycation8 such as dextran sulfate, surfactant6, particularly non-ionic gurfactant6, hjnd;ng ~nh~ncers, e.g., polyalkylene glycols, or the like.

Description o~ the Spec~fic Embod~ments In general, the present invention i6 directed to methods for determ;n;ng an analyte in a selected medium. The methods compri6e treating a medium su6pected of contA;n;ng an analyte under condition6 such that the analyte, if present, affects the amount of a photosensitizer and a photoactive indicator precursor that can come into close proximity wherein the short-lived singlet oxygen generated by the photosensitizer can react with the photoactive indicator precursor prior to the spnnt~n~o~ decay of the singlet oxygen in order to form a photoactive indicator. The method further comprises exposing the photoactive indicator to light which may be of the same or a different wavelength than the light used to excite the photosensitizer in order to excite the photoactive indicator 80 formed and then measuring the intensity of fluorescence emitted from the photoactive indicator upon excitation. The intensity of fluorescence pro~nce~ is related to the amount of analyte in the medium. The photoactive indicator is formed upon reaction of the photoactive indicator precursor with the singlet oxygen generated by the photosensitizer. The photosensitizer catalyze6 the generation of singlet oxygen u6ually in response to photoexcitation followed by energy transfer to molecular oxygen. Often one W 095/06877 2 ~ 7 ~ ~ 7. ~ PCTrUS94/0970~

or both of the photosensitizer and the photoactive indicator precursor will be associated with surfaces, wherein, in h~ -~el.eous a~says, the surface will preferably be the surface of suspendible particles.
For homogeneous assays the invention is predicated on an analyte causing or inhibiting molecules of the photosensitizer and the photoactive indicator precursor to be closer to each other than their average distance in the bulk solution of the assay medium. The amount of this partitioning will depend upon the amount of analyte present in the sample to be analyzed. The photosensitizer molecules that do not become associated with the photoactive indicator precursor produce singlet oxygen that is unable to reach the photoactive indicator precursor before undergoing decay in the aqueous medium. However, when the photosensitizer and the photoactive indicator precursor come in close proximity with each other in response to the presence of the analyte, the singlet oxygen produced upon irradiation o$ the photosensitizer can react with the photoactive indicator precursor to form a photoactive indicator before undergoing decay. Because numerous photoactive indicator precursor molecules and/or photosensitizer molecules can be associated with a surface or can be incol~o~ted into the material comprising the surface, the presence of a surface in conjunction with the photost~nRitizer and photoactive indicator precursor can increase the efficiency of, or action of, singlet oxygen with the photoactive indicator precursor prior to decay. It is therefore preferred to bring one member of the photoactive indicator precursor and photosensitizer pair into the proximity of a surface that inco-~oLates the other '-_ as a function of the presence of an analyte. The subject assay provides for a convenient method for detecting and measuring a wide variety of analytes in a simple, efficient, .u~ c~hle manner, which can employ simple e~ for measuring the amount of light produced during the reaction.
The amount of photosensitizer that comes in close proximity to the photoactive indicator precursor is affected by the presence of analyte by virtue of the photosensitizer and photoactive indicator precursor each being aRsociated with an sbp member. This may be accomplished in a number of ways and the term "associated with~ defined thereby. The photosensitizer and photoactive indicator precursor may contain functionalities for covalent att~t ~ to sbp members and the sbp members may contA~n functionalities for attAching to the photosensitizer and/or photoactive indicator precursor. The attA~' t may be accomplished by a direct bond between the two molecules or through a 1; nk; ng group which can be employed between an sbp member and the photosensitizer or photoactive indicator precursor. In another ~mhot~; t either or both of the photosensitizer and photoactive indicator precursor can be bound to surfaces or incu~u~ted in particles, to which are also attached sbp members. In both cases each of the sbp members ig capable of hi n~i ng directly or indirectly to the analyte or an assay component whose ct~nC~n~ation is affected by the presence of the analyte. Either or both ~ W O 95/06877 2 1 7 0 ~ 7 3 PCT/US94/09705 of the photosensitizer and photoactive indicator precursor can be incorporated into particles by virtue of being dissolved in at least one phase of the particles, in which case the solute will be at much higher concentration within the particle than in the bulk assay medium.
AlternAtively, either or both of the photosensitizer and photoactive indicator precursor may be covalently bound to particles, either by providing linking functional groups on the components to be bound or by incoL~oL~ting the photosensitizer or photoactive indicator precursor into a polymer that comprises the particles. Por particles that are oil droplets or liposomes the photosensitizer and photoactive indicator precursor can be attached to one or more long hydrocarbon chains, each generally having at least 10 to 30 carbon atoms. If the particles are droplets of a fluorocarbon, the photosensitizer or photoactive indicator precursor incol~o.ated into these particles may be fluorinated to ~nhAnce solubility and reduce ~hAnge into other particles bound with the other label, and the hydrocarbon chain used for linking will preferably be replaced with a fluorocarbon chain. For silicon fluid particles the photosensitizer and photoactive indicator precursor can be bound to a polysiloxane. In order to maximize the number of photosensitizer or photoactive indicator precursor molecules per particle, it will usually be desirable to minimize the charge and polarity of the photosensitizer or photoactive indicator precursor so that it resides within the non-aqueous portion of the particle. When the particle is a liposome and it is desired to retain the photosensitizer or photoactive indicator precursor in the aqueous phase of the liposome, it will be preferred to use photosensitizers or photoactive indicator precursors that are highly polar or charged.
~o matter how the photosensitizer and the photoactive indicator precursor are associated with their respective sbp member, it is critical that neither compound is capable of dissoci~ting from its sbp member and becoming associated with the sbp member associAte~ with the other member of the photosensitizer and photoactive indicator precursor pair during the course of the a~say. Thus, dissociation of these compounds from their respective sbp members must be slow relative to the time required for the assay.
The photoactive indicator precursor may be bound to an sbp -I
that is capable of h; n~i ng directly or indirectly to the analyte or to an assay e _ -nt whose cnnc~nt~ation is affected by the presence of the analyte. The term "capable of hin~in~ directly or indirectly" means that the designated entity can bind specifically to the entity (directly) or can bind specifically to a specific hin~;n~ pair member or to a complex of two or more sbp members which is capable of bin~i ng the other entity (indirectly).
The surface generally has an sbp member bound to it Preferably, the photoactive indicator precursor is associated with the surface, usually within a sll~p~n~ihle particle. This sbp member is generally capable of W O 95l06877 PCTrUS94/0970S
2 ~ 7 3 ~

h; n~;ng directly or indirectly to the analyte or a receptor for the analyte. When the sbp members associated with the photosensitizer and the photoactive indicator precursor are both capable of h;n~;ng to the analyte, a sandwich assay protocol can be used. When one of the sbp members associated with the photosensitizer or photoactive indicator precursor can bind both the analyte and an analyte analog, a competitive assay protocol can be used. m e att~ t to a surface or incorporation in a particle of the photoactive indicator precursor i8 governed generally by the same principles described above for the att~' ~ t to, or the incoL~o-Qtion into, a particle of the photosensitizer.
m e photosensitizer is usually caused to activate the photoactive indicator precursor by irradiating the medium cont~;n;ng these reactants.
Since it will fre~l~ntly be undesirable to excite the photoactive indicator precursor directly with light, the wavelength of light used to activate the photosensitizer will usually be longer than the longest wavelengths absorbed subst~nt;~lly by the photoactive indicator precursor. However, the medium must be irradiated with a short enough wavelength of light that has sufficient energy to convert the photosensitizer to an excited state and thereby render it capable of activating molecular oxygen to singlet oxygen. m e excited state for the photosensitizer capable of exciting molecular oxygen is generally a triplet state which is more than about 20, usually at least 23 Kcal/mol more energetic than the photosensitizer ground state. Preferably, the medium is irr~;Ate~ with light having a wavelength of about 450 to 950 nm although shorter wavelengths can be used, for example, 230 to 950 nm, and longer wavelengths of up to 2000 nm can be used by providing sufficiently intense light to provide for biphotonic excitation.
Although it will usually be preferable to excite the photosensitizer by irradiation with light of a wavelength that is efficiently absorbed by the photosensitizer, other means of excitation may be used, for example, by energy transfer from an excited state of an energy donor. When an energy donor is used, wavelengths of light can be used which are inefficiently ~hsorhe~ by the photosensitizer but are efficiently absorbed by the energy donor. m e energy donor may be bound to an assay component that is assoc;~ted, or becomes associated, with the photosensitizer, for example, bound to a surface or incorporated in the particle having the photosensitizer. When an energy donor is employed its lowest energy singlet and/or triplet state will usually be of higher energy than the lowest energy singlet and/or triplet state, respectively, of the photosensitizer.
m e singlet oxygen so formed reacts with the photoactive indicator precursor to form a photoactive indicator which is fluorescent.
Fluorescence of the photoactive indicator that is formed can be detected following electronic excitation of the photoactive indicator. ~ormally electL~ -gn~tic radiation, preferably light, will be used to excite the ~ W 095/06877 2 1 7 0 8 7 3 PCTrUS94109705 photoactive indicator, but energy transfer from molecules that have been excited by other means such as chemiexcitation can also be used when the chemiexcitation i8 separate from the above-mentioned singlet oxygen reaction. m e wavelength of light used to excite the photoactive indicator can be the same or different from the wavelength of light used to excite the photos~n~;tizer. Usually it will be preferable for the light emitted by the photoactive indicator to be shorter wavelength than any fluorescence of the photosensitizer. Preferably, therefore, when the photosensitizer is fluore~cent, the light used to excite the photoactive indicator will be shorter wavelength than that used to activate the photosensitizer, usually at least 50 nm shorter, preferably at least 200 nm shorter. m e fluorescence emitted from the excited photoactive indicator may be measured in any convenient manner such as photographically, visually or photometrically, to dete ~ne the amount thereof, which is related to the amount of analyte in the medium.
Irradiation of the photosensitizer and the excitation of the photoactive indicator may be carried out simultaneously but will preferably be carried out sequentially so that the light used to excite the photosensitizer does not interfere with the fluorescence meas-.~ t. m e photoactive indicator precursor must not 8Ub8tAnt~ Al ly absorb light at the wavelength used to generate the singlet oxygen and will therefore usually absorb at shorter wavelengths than the photosensitizer. In addition, the photoactive indicator precursor will preferably not absorb significantly at the wavelength required to excite the photoactive indicator and therefore will usually absorb at shorter wavelengths than the photoactive indicator.
The method and compositions of the invention may be adapted to most assays involving sbp members such as ligand-receptor; e.g., antigen-~nt~ho~y reactions; polynucleotide h;n~ng assays, and 50 forth.
m e assays may be hl -J~neous or heterogeneous, competitive or nnncnmretitive. m e assay components, photoactive indicator precursor and photosensitizer, can be associated in a number of ways to a receptor, to a ligand, or, when employed, to a surface. m e association may involve covalent or non-covalent bonds. m ose skilled in the art will be able to chO08e I~LU~ ' ate associations A~p~n~;ng on the particular assay desired in view of the foregoing and the following illustrative discussion.
The sample may be pretreated if necessary to remove unwanted materials. m e reaction for a noncnmretitive sandwich type assay can involve an sbp '-1, (e.g., an antibody, polynucleotide probe, receptor or ligand) compl: - Ary to the analyte and associated with a photoactive indicator precursor; a photosensitizer assor~Ate~ with an sbp member, (e.g., antibody, polynucleotide probe, receptor or ligand) that is also compl Ary to the analyte; the sample of interest; and any ancillary reagents required. In a competitive protocol one sbp member can be a derivative of the analyte and the other sbp member can be complementary to the analyte, e.g., an antibody. In either protocol the components may be W O 95/06877 PCTrUS94/09705 21 7087:~ ~

combined either simultAnPol-Rly or wholly or partially seq~lPnti~lly. The ability of singlet oxygen produced by an activated photosensitizer to react with the photoactive indicator precursor to form a photoactive indicator is governed by the h;nAing of an sbp member to the analyte. Hence, the presence or amount of analyte can be determ;n~A by measuring the amount of light emitted upon activation of the photoactive indicator 80 formed by irrAA;~tinn. Both the h;nA;n~ reaction and detection of the extent thereof can be carried out in a homogeneous solution without separation, wherein, preferably, one or both of the photosensitizer and the photoactive indicator precursor are inco- ~G~ ated in particles to which the sbp members are attached. This i8 an advantage of the present invention over prior art methods utilizing chemiluminescence.
In a heterogeneous assay approach, one of the sbp members will frequently be bound to a support or another means provided to separate it lS from the assay medium. The support may be either a non-dispersible surface or a particle. In one GmboA; t, the support or particle will have a~soci~ted with it one '-_ of a group consisting of the photoactive indicator precursor and the photosensitizer. Another sbp member will have the other member of the group associated with it wherein the sbp members can inAPpPnAPntly~ either directly or indirectly, bind the analyte or a receptor for the analyte. These components are generally _ linPA either simultAnPoll~ly or wholly or partially sequPnti~lly. The surface or particles are then separated from the liquid phase and either are subjected to conditions for activating the photosensitizer and the photoactive indicator so formed, usually by irr~A;~t;ng the separated phase, and measuring the amount of fluorescence emitted.
Altern~tively, a heterogenous assay of this invention may be carried out by providing means such as a surface to separate a first sbp member from the liquid assay medium and providing a second sbp member that is associated with a photosensitizer and that binds to the first sbp member as a function of the amount of analyte in the medium. The sample suspected of cnnt~;n;ng the analyte is then _ ''nP~ with the first and second sbp members either simultaneously or wholly or partially seq~pnti~lly and the first sbp member is separated from the medium. A third sbp member associated with a photoactive indicator precursor is then cnmh; nPd with the separated first sbp '~1 where the third sbp member is capable of hinA;ng directly or indirectly to the second sbp member. The c ~;n~t;nn is then irr~A;~teA to activate the photosensitizer and the fluorescence of the photoactive indicator 80 formed is measured.
The h;nA;ng reactions in an assay for the analyte will nf lly be carried out in an aqueous medium at a moderate pH, generally that which provides optimum assay sensitivity. Preferably, the activation of the photosensitizer will also be carried out in an aqueous medium. However, when a separation step is employed, non-aqueous media such a~, e.g., g5 acetonitrile, acetone, toluene, hPn7nn;trile~ etc. and aqueous media with ~ W 095/06877 2 1 7 0 8 7 3 PCTrUS94/09705 pH values that are very high, i.e., greater than 10.0, or very low, i.e., less than 4.0, preferably with pH values that are very high, can be used.
As explained above, the assay can be performed either without æeparation - (ht -,~..eoug) or with separation (heterogeneous) of any of the assay c~,.. Auonents or products.
The aqueous medium may be solely water or may include from 0.01 to 80 volume percent of a cosolvent but will usually include less than 40~ of a cosolvent when an sbp member is used that is a protein. The pH for the medium of the h;n~;n~ reaction will usually be in the range of about 4 to 11, more usually in the range of about 5 to 10, and preferably in the range of about 6.5 to 9.5. The pH will usually be a compromise between optimum hin~ing of the h;n~;n~ and the pH optimum for the production of signal and the stability of other reagents of the assay. Usually no change in pH will be required for signal production, although if desired, a step involving the addition of _n acid or a basic reagent can be inserted between the h;n~;ng reaction and generation of singlet oxygen and/or signal production. Usually in h~ ~;eA~ous assays the final pH will be in the range of 5 to 13. For heterogenous assays non-aqueous solvents may also be used as mentioned above, the main consideration being that the solvent not react efficiently with singlet oxygen.
Various buffers may be used to achieve the desired pH and maintain the pH during an assay. Illustrative buffers include borate, phosphAte~
cArhonAte, tris, barbital and the like. The particular buffer employed is not critical to this invention, but in an individual assay one or another buffer may be preferred.
Moderate temperatures are normally employed for carrying out the bin~ing reactions of proteinAceous ligands and receptors in the assay and usually con~tant temperature, preferably, 25 to 40, during the period of the measu~ ~ t. Incubation temperatures for the h;n~;ng reaction will normally range from about 5 to 45C, usually from about 15 to 40C, more usually 25 to 40C. Where hin~ing of nucleic acids occur in the assay, higher temperatures will frequently be used, usually 20 to 90, more usually 35 to 75C. ~ _c}~tures during measuLF ~ ts, that is, generation of singlet oxygen and light detection, will generally range from about 20 to 100, more usually from about 25 to 50C, more usually 25 to 40C.
The concentration of analyte which may be assayed will generally vary from about 10~ to below 10l6 M, more usually from about 10-6 to 10-l4 M.
Considerations, such as whether the assay is qualitative, semiquantitative or quantitative, the particular detection technique the concentration of the analyte of interest, and the desired incubation times will normally dete in~A the concentrations of the various reagents.
In competitive assays, while the cnncentrations of the various reagents in the assay medium will gerAerally be deter~in~ by the conc~ntration range of interest of the analyte, the final cnnc~ntration of each of the reagents will normally be dete ;n~ empirically to optimize W O95l06877 PCTrUS9410970~

the sensitivity of the assay over the range. That i5, a variation in concPntration of the analyte which is of 6ignificance should provide an accurately measurable signal difference.
The conc~ntration of the shp members will depend on the analyte concentration, the desired rate of h; n~ing, and the degree that the sbp members bind nonspecifically. Usually, the sbp members will be present in at least the lowest expected analyte cnnC~ntration~ preferably at least the highest analyte cnnC~ntration expected, and for nnnl __titive assays the cnnc~ntrations may be 10 to 106 times the highest analyte concentration but usually less than 10~ N, preferably less than 10~ M, frequently between 10-and 10-7 M. The amount of photosensitizer or photoactive indicator precursor asEoriAte~ with a sbp member will usually be at least one molecule per sbp member and may be as high as 105, usually at least 10 to 10~ when the photosensitizer or photoactive indicator precursor molecule is incorporated in a particle.
While the order of addition may be varied widely, there will be certain preferences depending on the nature of the assay. The simplest order of addition is to add all the materials simultaneously.
Alt~rn~tively, the reagents can be cnmhine~ wholly or partially sequenti~lly~ When the assay is competitive, it will often be desirable to add the analyte analog after c -;ning the sample and an sbp member capable of hin~ing the analyte. Optionally, an incubation step may be involved after the reagents are combined, generally ranging from about 30 seconds to 6 hours, more usually from about 2 mi n~tes to 1 hour before the photosensitizer is caused to generate singlet oxygen and the photoactive indicator is caused to fluoresce.
In a particularly preferred order of addition, a first set of sbp memhers that are compl: - t~ry to and/or homologous with the analyte are ~;n~ with the analyte followed by the addition of specific bin~;ng pair '- ~ compl ~ry to the first specific bin~in~ pair members, each asso~i~te~ with a different '- of the group consisting of a photosensitizer and a photoactive indicator precursor. The assay mixture, or a separated component thereof, is irr~ te~ first to produce singlet oxygen and then later to produce measurable fluorescence.
In a h~ neous assay after all of the reagents have been cnmhin~, they can be incubated, if desired. Then, the _ 'in~tion is irradiated (at the necessary wavelengths of light) and the resulting fluorescence emitted is measured. The emitted fluorescence is related to the amount of the analyte in the sample tested. The ; lnts of the reagents of the invention employed in a homogeneous assay depend on the nature of the analyte.
Generally, the h~ neous assay of the present invention exhibits an increased sensitivity over known assays such as the BMIT~ assay. This advantage results primarily because of the improved signal to noise ratio obtained in the present method.
The following assays are provided by way of illustration and not ~ W 095/06$77 2 1 7 0 8 7 3 PCTAUS94/09705 limitation to enable one skilled in the art to appreciate the scope of the present invention and to practice the invention without undue experimentation. It will be appreciated that the choice of analytes, photosensitizers, photoactive indicator precursors, surfaces, particles and reaction conditions will be suggested to those skilled in the art in view of the disclosure herein in the examples that follow.
In one embo~i ~rt of the invention a photoactive indicator precursor of the following formula (Im):
lo ~ fH3 ~ N ~ e ~ (Im) is covalently linked to an antibody for human chorionic gonadotropin (HCG) to provide Reagent l. The photoactive indicator precursor, functionalized with a N-Hydlu~y~l~C;n; dyl ester of the carboxyl group, reacts with the amino groups of the Ant;ho~y The l;nk;ng group is a rA~ -;de. The photosensitizer utilized is rose bengal, which is covalently bound to latex particle8 having an average ~i ~ion of 0.5 micron. The latex particles and rose bengal are covalently bound to each other by means of chloromethyl groups on the latex to provide an ester 1 in~i ng group as described in ~.
Am. Chem. Soc., ~7: 3741 (1975). The latex particle is further link~ to a second ~"tiho~y for HCG by means of N-llyd ~y~l~c~;nimidyl ester groups on the latex to provide ~eAgent 2. Both of the antibodies employed are monoclonal ~ntiho~;es prepared by standard hybrid cell line technology.
One Antiho~y recognizes the a-subunit of HCG and the other recognizes the ~-suhunit of HCG. In conducting the assay a serum sample suspected of contA;ning HCG is obtained from a patient. Fifty microliters of the sample is ~- 'ine~ in 500 microliters of aqueous medium, buffered with Tris buffer at pH 8.0, with Reagent 1 and ~gent 2 above. The amounts of Reagent 1 and Reagent 2 are sufficient to provide concentrations of each ~Antiho~y of about 1 P molar. The reaction mixture is then ;ncnhAte~ for a period of one hour at 25C and then irr~;Ate~ for 30 ~;nl~te~ with 560 nm light. The fluorescence of the solution is then measured by irradiating at 350 nm and detecting at 440 nm and is c~--~ared with values obtained in a similar procedure with samples having known conc~ntrations of HCG to det~ n~ the concentration of HCG in the unknown.
In an altern~t;ve approach based on the above, Reagent 2 is rose bengal covalently linked to the second antibody and no latex particle is employed. In still another alternative approach based on the above, Reagent 2 is rose bengal covalently linked to the second Ant; ho~y and Reagent 1 is the photoactive indicator precursor covalently bound to latex particles, to which the first antibody is covalently attached. In still W 095/06877 2 1 7 ~ 8 7 3 PCT~US94/0970S

another alt~rn~tive approach based on the above, Reagent 1 iæ as described ~ tely above, Reagent 2 i5 ro6e bengal covalently linked to latex particles, to which avidin iæ covalently attached, and a third reagent (Reagent 2A) that is the second ~ntiho~y covalently linked to biotin i8 employed. Reagent 1 and the third reagent are combined with sample and incubated. Then, an excess of Reagent 2 is added and the ~ F -;n; ng procedure is as described above.
In another ~ t in acco~ ce with the present invention, a first set of oil droplets (Reagent 3) is prepared from a solution of the photosensitizer and chlorophyll in mineral oil in accordance with Giaever, supra. The oil droplets, which range from 0.1 to 2 microns in diameter, are coated with a functionalized surfactant that is linked to a monoclonal ~nt;ho~y for C-reactive protein (CRP). The chlorophyll is lipophilic and i8 therefore diæsolved in the lipoph;lic oil droplet. A second set of oil droplets (Reagent 4) i8 prepared in a similar manner. In this set of droplets the oil droplet is similarly coated with a second monoclonal antibody for CRP, which recognizes a different portion of the CRP molecule than that recognized by the first monoclonal Antihs~y referred to above.
9-Benzal-10-methyl acridan iæ irreversibly dissolved in the lipophilic oil droplet by including a N,N-didodecylc~rhnY~mi~ group bound to one of the phenyl groups of the acridan. The monoclonal antibodies are prepared by standard hybrid cell line technology. A serum sample suspected of cnnt~;n;ng CRP (50 microliters) is combined with excess quantities of Reagent 3 and Reagent 4 in an aqueous buffered medium (500 ~L) at pH 7.5.
The medium is ;n~nh~te~ at 25C for a period of 20 minutes. The medium, without separation, is irr~ te~ at 633 nm using a He/Ne laser for a period of twenty m~n~ltes and the fluorescence of the solution i8 measured by irradiation at 360 nm and detection at 440 nm of the light emitted. The intensity of fluorescence is compared with that ~rom samples cnnt~;n;ng known : ln~ of CRP and the amount of CRP in the unknown sample is det~rm;ne~ by comparing the values. In thiæ way a convenient and sensitive h, ~neous ; ~no~say for CRP iæ cnn~llcte~.
In an altP~nAtive approach based on the above, Reagent 3 haæ an antibody for fluorescein in place of the antibody for CRP and an additional reagent (Reagent 3A) has the CRP ~nt;ho~y covalently linked to fluorescein.
Reagent 4 has avidin in place of the second CRP ~ntiho~y and a fourth reagent (Reagent 4A) has the second antibody covalently linked to biotin.
In the assay Reagent 4A and Reagent 3A are c~mhin~ with sample and ;n~llh~te~. Thereafter, Reagents 3 and 4 are added and inCllh~te~ The r~ in~r of the assay procedure as described above is then carried out.
In another embo~i ~rt of the present invention, one set of liposomes (Reagent 5) (0.2 micron in diameter) is formed by high pressure extrusion of a phospholipid suspension in pH 7.4 buffer through a 0.2 micron pore ...c..~.~.e using a c~ -rcially available instrument designed for such purpose. A thyroxine analog is covalently linked to the liposome by first ~ W 095/06877 2 1 7 0 ~ 7 3 PCTrUS94/09705 forming mercaptoacetamide groups on the liposome by reaction of phn5ph~tidyleth~nnll ;ne in the liposome with an N-hydlu~y-6uccinimide ester of methyl calbu~ylllethyl disulfide followed by reaction with dithioerythritol. Bromoacetyl thyroxine is then allowed to react with the sulfhydrylated liposomes. A metallo-porphyrin dye is dissolved in the lipophilic portion of the liposome. Another set of liposomes (Reagent 6) is utilized to attach a monoclonal ~nt;hofly for thyroxine. The ~nt;ho~y is attached covalently by means 8; ] ~ to the att~. - t of thyroxine. A
photoactive indicator precursor of the following formula:

H~C~ ~ N ~

is covalently linked by means of a carboxamide linking group to the surface of the liposome. Reagent 5 and Reagent 6 are ,_ ; ne~ in an aqueous buffered assay medium (500 ~L) of pH 8.0 together with a serum sample suspected of cnnt~;ning thyroxine that cont~;n~ a thyroxine releasing agent of the following formula:

S~H o HN ~ ~

to displace thyroxine from bin~;ng proteins (50 micro-liters). The assay medium is then ;ncnh~te~ at room temperature for 1 hour. The medium is irr~;Ate~ at 650 nm for a period of 1 nnte and the fluorescence is measured as in the preceding examples. The value obtained is compared with values obtained by conducting a similar assay with known amounts of thyroxine. In this way the amount of thyroxine in the serum sample is quantitated.
In an altern~t;ve approach based on the above, Reagent 6 has avidin in place of ~nt;ho~y for thyroxine. An additional reagent (Reagent 6A) has antibody for thyroxine covalently linked to biotin. Reagent 5 has antibody for fluorescein in place of thyroxine and an additional reagent (Reagent 5A) ha~ thyroxine l;nke~ covalently to fluorescein. In the assay Reagents 5A and 6A are cnmh; ne~ with sample and ;nc~h~tefl. Then, Reagent 5 and 6 are added, the mixture is ;nc~h~te~, and the r~m~;n~r of the assay procedure is followed.
In another '_~; t 2-hyd~u~y~thyl-9~lO-dibromo-anthracene is formed into a dye crystallite in a manner similar to that described by W 095/06877 2 1 7 0 8 7 3 PCTrUS94/09705 Gribnau. A 25mer oligonucleotide probe that recognizes a sequence of hepatitis B RNA is covalently attached to the dye crystallite by means of a rArh; te l;nk;ng group. A second 25mer oligonucleotide probe for hepatitis B RNA is covalently linked to 9-(benzal-9H-xanthene) by means of an amide linking group. The dye crystallite has a particle size 2 microns on the average. The oligonucleotides are prepared by standard automated synthesis technology. A sample (50 ~L) from a patient suspected of having hepatitis B is c~mh;n~ in an aqueous assay medium (500 ~L) at pH 7.0 with an excess of the dye crystallite and the second probe described above. The assay medium is then ;ncllhAte~ at 50C for a period of 30 ;n~ltes and the fluorescence is then measured by irradiation at 330 nm and detection at 390 nm. The presence of hepatitis B RNA in the sample causes the dye crystallite and the 9-(benzal-9H-yAnth~ne) to come into close proximity by virtue of the h; n~; n~ of the respective oligonucleotides with the RNA.
Upon irradiation of the medium the 9,10-diL~ nthracene is excited and converts ground state oxygen to singlet oxygen. The singlet oxygen reacts with the xanthene to give a xanthone, which is fluorescent. The fluorescence is measured photometrically and the amount of light over a certain threshold level indicates the presence of hepatitis B RNA in the sample. Irradiation of the medium is conducted at room temperature and the assay is conducted in a h~ eous manner to yield an assay for hepatitis B RNA.
In another '~ t the assay is for the det~rm;n~t;on of a particular blood group antigen on the surface of a red blood cell, namely, an A group antigen. Latex particles prepared as described above having a particle size of 150 to 500 nm are utilized. The latex particles have an ~nt;ho~y for the A group antigen covalently linked to the latex particle.
The particles also have the photoactive indicator precursor of formula (If):
l~ 3 ~ ,0~

C~a which is dissolved in the latex. This latex particle reagent is combined in the aqueous medium (500 ~1) with whole blood (100 ~1) and lX10~ M of a photosensitizer, which is a hydrophobic dye of the following formula:

XaC~DCE~CN~C ~ CE~CE~CE~CE~

E~SCX2C~2cE~5~c cX2c~2cH2cH9 o - OH

~ W O 95/06877 2 1 7 0 ~ 7 3 PCTrUS94/09705 The hydrophobic dye is incorporated into the red cells present in the sample. The medium is incubated at 25C for a period of 10 minutes and then irrA~;~te~ at >650 nm with a tungsten source for a period of 30 seconds. The fluorescence of the solution is then dete i n~ by irradiation at 360 nm and detection at 440 nm. The light emitted from the medium is measured and compared to the amount of light obtained in samples known to be free of A group antigen red blood cell6. Thus, the amount of light over a thre6hold level indicate6 the pre6ence of the A blood group antigen.
The pre6ent invention further encrmrA~ses compositions comprising a su6pendible particle of 25 to 4000 nAnt -ter average diameter comprising a photoactive indicator precur60r. The photoactive indicator precursor may be covalently bound to the particle matrix or may be di6solved in the matrix or di6solved in a 601vent that is di6solved in the matrix. The particles will preferably be polymeric or be oil droplets or vesicles such as liposomes. Where the particle is a liposome, the photoactive indicator precursor will be associated with the lipid bilayer or dissolved in the aqueous interior of the liposome. The particle will have an sbp member bound to it. Also t~n _-~sed are compositions comprised of two complementary sbp members bound to each other wherein one is associated with a photosensitizer and one is associated with a photoactive indicator precursor.
Another aspect of the present invention relates to kits useful for conveniently performing an assay method of the invention for determ;n;ntJ
the pre~ence or amount o~ an analyte in a sample su~pected o~ containing the analyte. To t~nhAnte the versatility of the subject invention, the reagents can be provided in packaged ~ ';nAtion, in the same or separate ct~ntA;n~s, 80 that the ratio of the reagents provides for substAnti~l optimization of the method and assay. The reagents may each be in separate contA;ners or various reagents can be _ n~tl in one or more contA;n~rs depending on the cross-reactivity and stability of the reagents. The kit comprises (1) a composition wherein the composition comprises a suspendible particle comprising a photoactive indicator precursor, the particle having an sbp member bound to it, and (2) a photosensitizer. The photosensitizer can be attached to an 6bp member or it can be associated with a particle, to which an sbp member is bound. The kit can further include other separately p~t~k~ged reagents for conducting an assay including An~; l 1A~Y
reagents, and so forth.
Another t~mho~ of a kit in acco d~ce with the present invention comprises in packaged - ;n~tion a photoactive indicator precursor associated with a first sbp member and a photosensitizer capable in its excited state of activating oxygen to its singlet state associated with a second sbp member.
EXAMP~ES
The invention is demonstrated further by the following illustrative W 095/06877 PCT~US94/09705 2~ 70873 examples. Parts and percentages used herein are by weight unles6 otherwise specified. Temperature~ are in degrees centigrade (C). The ~ollowing abbreviations are used in the Examples:
"Amino-GATTAG" - a modified 42mer oligonucleotide having the se~l~nce shown below:
5'-GATTAG-GATTAG-GATTAG-GATTAG-GATTAG-GATTAG-GATTAG-3' (SEQ ID NO:1) with the nucleotide (Clontech Laboratories, ~5202-1) at the 5'-end substituted as illustrated below:

H2N~_~0--P-- 5 B~se 0~~
o "Biotin-30mer" - a modified 30mer oligonucleotide having the se~-~nce shown below:
5'-CAA-TAC-AGG-TTG-TTG-CCT-TCA-CGC-TCG-AAA-3' (SEQ ID NO:2);
with biotin attached to a modi~ied cytosine (5-methylcytosine) at the 5'-end through a linking group as shown below:
O

~-N ~ N-CN~
N
N

"CTAATC-30mer" - a modified tailed 30mer oligonucleotide having the sequence shown below:
5'-CTG-CCG-GTG-CGC-CAT-GCT-CGC-CCG-CTT-CAC-CTA-ATC-CTA-ATC-CTA-ATC-CTA-ATC-CTA-ATC-CTA-ATC-3' (SEQ ID NO:3).
"DMF" - dimethyl f~
"EDAC" - 1-ethyl-3-(3-dimethyl; ;~v~ u~yl) -c~ho~;; de hydrochloride.
"EDTA" - ethyl~n~; ;n~tetraacetic acid.
"GATTAG-SH" - a modified 42mer oligonucleotide having the se~uence shown below:
5'-GATTAG-GATTAG-GATTAG-GATTAG-GATTAG--~ W 095/06877 2 1 7 0 8 7 3 PCTrUS94/09705 GATTAG-GATTAG-3' (SEQ ID NO:4) with the 5'-end nucleotide substituted as illustrated below:

H
O
HS ~ N ,^~"~ ~ ~ o-P-0 B~s~
IJ o~ 1~
o "MES" - 2-(N-morpholino)ethane sulfonic acid.
"SPDP" - N-sl-c~n;m~dyl 3-(2-pyridylthio)-propionate.
"Sulfo-SMCC" - 4-(~-maleimidomethyl)cycl~h~Y~ne-1-carboxylate.
"TCEP" - tris-car~oxyethyl phosphine.
"THF" - tetral-yd~uL~ran.

Example 1 Preparation of a Photoactive Indicator Precursor A.

c~u~n~r in-l 2 A solution of coumarin-l (11.0 g, 47.5 mmol) in ethyl acetate (150 mL) was treated with 10~ Pd/C (100 mg) in a parr bottle. The suspension was then hyd uy~nated at 80 psi and 80C for 6 hours. The suspension was filtered through a bed of celite to remove the Pd/C, and the celite bed washed with warm ethyl acetate (100 mL) The filtrate was cnnc~nt~ated and dried under vacuo to yield 11.0 g (100~) of the 3,4-dihydrocoumarin (2) as an oil;
H-NMR ~CDC13,250 MHz): ~ 7.02 (d,J=8.5Hz,lH); 6.42 (dd,J=8.5Hz,1.7Hz,lH); 6.35 (d,J=1.7Hz,lH); 4.07 (q,J=7.OHz,4H); 3.06 (m,lH); 2.80 (dd,J~=15.6Hz, J~c=5.4Hz,lH); 2.51 (dd,J~=15.6Hz,J~c=7.7Hz, lH); 1.28 (d,J=7.0Hz,3H);
1.15 (t,J=7.OHz,6H);
MS(EI) calculAte~ for Cl4HI~02, 233: found 233 (M+,40~); 218 (M+-CH3, 100~).

W 095/06877 2 1 7 ~ ~ 7 3 -52- PCTrUS94109705 S ~ ~

A solution of 3,4-dihydrocoumarin 2 (1.68 g, 7.20 mmol) in anhydrous THF (20 mL) was cooled to -78C under argon. Lithium diisopropylamide in THP (8.0 mL of 1.0 M, 8.0 mmol) was added to the stirred solution and the re~ultant yellow colored ~olution was further stirred for 1 hour. Phenyl selenyl chloride (1.50 g, 7.8 mmol) dissolved in THF (10 mL) wa~
15 subsequently added into the enolate solution. The orange color of the mixture quickly faded to yield a yellow solution. The solution was stirred for 3 hours and ql~nrh~d with aqueous NH4Cl (10 mL of 1~). Ater about 10 'nutes, dichloromethane (100 mL) was added and the organic phase separated. The aqueous portion wa~ further extracted with CH2Clz (2x20 mL) 20 and the organic portions com~ined. The . i n~fl organic portions were washed with brine (20 mL), dried over anhydrous NazSO4 (25 g) and c~ncPnt~ated to yield 2.10 g of an yellow oil. The oil was purified by chromatography on silica gel with hexane in dichloromethane gradient to yield 1.80 g (67~) of the coumarin-3-phenyl selenide 3, as a white powder.
25 CrystA~ ion from hot hexane afforded 1.30 g of 3 as white n~eflles, m.p.
99-101C;
H-~MR (C6D6,250MHz) ~ 7.65 (m,2H); 6.92 (m,3H); 6.74 (d,~=8.0Hz,lH); 6.38 (d,J=1.5Hz,lH); 6.25 (dd,J=8.0Hz,1.5Hz,lH); 3.88 (d,~=2Hz,lH); 3.02 (m,lH); 2.84 (q,J=7.OHz,4H); 0.96 (d,J=7.OHz, 3H);
0.80 (t,J=7.OHz,6H);
MS (EI) calcd. for C ~"~O,~e 389; found 389 (M~,100~);
232 (M~-C6H5Se 70~); 218(60~); 202(45~);
UV-Vis (toluene) 300 nm (4600); 310 nm (4600); 330 nm (2100).

E~ample a Preparation of a Photoacti~e Indicator Precursor Li Te~r ~. ~ ~ ~ ~

To a stirred suspension of tellurium powder (100 mesh, 13.0 g, 0.10 mol) in dry THF (150 mL) was added a solution of phenyl lithium (60 mL of W 0 9S/06877 ~ 1 7 0 8 7 3 PCTAUS94/09705 1.8M, 0.10 mol) in ether-h~n~s. The ~uspenæion was stirred at room temperature for 2 hours and then refluxed for 1 hour. The suspension was allowed to cool and water (100 mL) was added followed by overnight - stirring. Oxygen gas was bubbled through the suspension for 3 hours.
Methylene chloride (200 mL) was added and the organic phase separated. The aqueous phase was further extracted with CH2Cl2 (2xlOO mL) and the combined portions washed with brine (100 mL) and dried over anhydrous Na2SO4. The dried ~olution was passed through a plug of silica (300 g); the filtrate thus obtained was concentrated and crystallized from hot ethanol to yield 13.2 g of the diphenyl ditelluride 4, as orange red needles, m.p. 63-65~C
(lit 63.5-65C);
MS (EI) calcd for C~2HIOTe2 414; found 414 (25~); 412 (45~);
410 (50~); 408 (40~); 207 (40~).
The diphenyl ditelluride 4 (1.0 g, 2.5 mmol) was dissolved in THF
lS (10.0 mL) and cooled to 0C. Bromine (125 ~L, 2.5 mmol) in THF (5.0 mL) was added and the solution stirred at 0C for 1 hour and allowed to attain room temperature. The reaction mixture was stirred at room temperature until no more starting material was detectable by analytical thin layer chromatography to yield compound 5.

2 ~ [ ~

A solution of the 3,4-dihydrocoumarin (240 mg, 1.0 mmol) in anhydrous THF (10 mL) was cooled to -78C under argon. Lithium diiso~ ~yl amide (1.1 mL of 1.0 M, 1.1 mmol) in THF was added and the solution stirred at -78C for 1 hour. A solution of 5 (3 mmol, prepared as described above) in THF waE r~nn~ te~ into the ester ~nol~te and the mixture stirred for 2 hours at -78C and then allowed to attain room temperature. The reaction mixture was qn~nrh~ with aqueous NH~Cl (1~, 5 mL) and stirred for another 5 'n~ltes. The reaction mixture was then extracted with CH2Cl2 (3.25 mL) - and the _ ~in~ organic portions washed with brine (20 mL) and dried over anhydrous Na2SO4 (20 g). Cnnc~ntration followed by flash chromatography (under 8nh~ lighting) on silica with CH2Cl2 gave 190 mg (43~) of an yellow oil. Cryst~ At;on from cyclnh~Y~ne afforded 165 mg of the coumarin telluride 6 as a light yellow colored solid;
H-NMR (CDCl3,250MHz) ~ 7.82 (dd,J=7.OHz,1.2 Hz,2H); 7.31 (m,lH); 7.26 (m,2H); 6.91 (d,J=8.5Hz,lH); 6.38 (dd,J=8.5Hz,2.5Hz,lH);
6.25 (d,J=2.5Hz,lH); 4.05 (d,2.OHz,lH); 3.33 (q,J=7.OHz,4H); 3.25 W O 95/06877 ~ 1 7 0 ~ ~ 3 PCTrUS94/09705 (m,lH); 1.23 (d,J=7.OHz,3H); 1.16 (t,J=7.OHz,6H);
MS (EI) calcd. for C~OzTe 439 (using I~Te); found 439 (M+,2~); 232 (M+-C~H5Te,100~); 217 (25%); 202 (35%);
W -Vis (toluene) 310 nm (3860); 330 nm (2400); 370 nm (510).

FxamplQ 3 Preparation of a Photoactive Indicator Precursor Br T~Br t--BuL: ~ THF

1~ C N ~ ] 7J 3~T e--T e~ B r 2 [~
~ N \ 7 ~ ~

A solution of p-bromo-N,N-dimethyl aniline (10.0 g, 50.0 mmol) in anhydrous THF (200 mL) was cooled to -78C under argon. Into this cooled solution was carefully added t-butyl lithium (56 mL of 1.8 M, 100 mmol) in pentane, and the resulting yellow sll~p~n~ion stirred for 1 hour at -78C.
Finely ground tellurium ~o./del (6.50 g, 50 mmol) was added under a stream of argon. The reaction mixture was then allowed to attain room temperature, by that time (-2 hours) most of the tellurium had dissolved.
The reaction mixture was ~l~n~he~ with water (20 mL) and poured into aqueous ~[Fe(CN) 6] solution (17 g in 200 mL, 0.052 mol). The mixture was stirred for 1 hour and then extracted with CH2Cl2 (3x200 mL). The cnmh;
organic portions were washed with brine (100 mL) and dried over anhydrous Na2SO4 (lOO g). The dried material was passed through a plug of silica (300 g) and the filtrate cnnc~n~ated to yield 12.2 g of an orange-red paste.
Cryst~ tion from ethanol gave 8.6 g of the ditelluride 7, a~ an orange red powder. Another batch (2.2 g) waR recovered from the mother liquor;
MS (FI) calcnlAte~ for C~ Te2, 500; found 500 (20%);
498 (40%); 496 (45~); 250 (100%); 240 (98~).
The ditelluride 7 (1.70 g, 3.4 mmol) was dissolved in a m;n~ lm amount of anhydrous THF and cooled to 0C. The solution was treated with bromine (175 ~L, 3.4 mmol) and the mixture stirred at 0C for 3 hours to yield a solution cont~;n;ng the desired product 8.

~ W 095/06877 2 ~ 7 0 ~ 7 ~ PCTrUS94109705 B. ~ N\

/ N ~

The product 8 was then cAnnlllAted under argon into a solution of the dihydro coumarin 2 (800 mg, 3.4 mmol) and lithium diisopropyl amide (3.5 mL
of 1.0 M, 3.5 mmol) in THF. m e resulting orange red mixture was allowed to attain room temperature and q~nrh~d with aqueous NH~Cl (lO mL of 1.0~).
The mixture was subsequently extracted with CH2Cl2 (3x50 mL) and the pooled organic portion dried with brine (50 mL) and anhydrous Na2SO4.
C~nc~n~ation followed by flash chromato~ ~hy (under subdued light) on silica with CH2Cl2 gave 510 mg of the coumarin 3-(4-dimethylamino)phenyl telluride 9, together with 110 mg of the starting dihydro coumarin 2. The yield of 2 was 37~ based upon recovered starting material;
H-NMR (CDCl3,250MHz) ~ 7.65 (d,J=8.0Hz,2H); 6.92 (d,J=8.5Hz,lH); 6.52 (d,J=8.0Hz,2H); 6.41 (dd,J=8.5Hz,1.5Hz,lH); 6.24 (d,J=1.5Hz,lH); 4.03 (d,2Hz,lH); 3.38 (q,J=7.OHz,4H); 3.25 (m,H);
2.95 (8, 6H); 1.19 (d,J=7.OHz,3H); 1.14 (t,J=7.OHz,6H).;
MS (EI) calcd. for C~N2~2Te, 482 (using ~30Te); found 482 (M~, 20~); 252 (20~); 232 (M~-C8HIOTe, 100~);
~V-Vi8 (toluene) 300 nm (18000); 320 nm (13600);
330 nm (8400).
E~ample 4 Preparation of Photoactive Indicator Precursor Particles (Acceptor Beads) A 0.3 ~ solution of coumarin-3-(4-dimethylamino)phenyl telluride 9 was prepared in degassed ethoxy ethanol by gentle warming. Ethylene glycol (1 mL) was heAte~ to 105-110C in a 4 mL vial. A stock latex suspension (200 ~L of 10~ solids in H20) was added to the vial and the mixture stirred magnetically under argon. Coumarin-3-(4-dimethylamino)phenyl telluride 2 (200 ~L, 0.3 M in ethoxyethanol) was added slowly to the mixture and the resulting mixture stirred for 5 'nntes~ then allowed to attain room temperature under argon. After cooling, the suspension was treated with ethanol (3 mL) and transferred to a centrifuge tube. The mixture was then centrifuged at 15,000 rpm (Sorval, SA 600 rotor) for 1 hour. The supe~n~tAnt was carefully decanted and the pellet resuspended in aqueous ethanol (4.0 mL) by sonication. The suspension was centrifuged at 15,000 W 095/06877 2 1 7 0 ~ 7 ~ PCTrUS94/0970~

rpm for 1 hour. The supernatant was once again removed and the pellet was resuspended in water (4 mL). Following a final centrifugation and removal of supernatant, the pellet was resuspended in water to a final volume of 2 mL to a yield of 10 mg/mL photoactive indicator precursor particle~
suspension.

E~ample 5 Preparation of Streptavidin-Photoactive Indicator Precursor Dyed Particles The photoactive indicator precursor particles (1 mL o~ 10 mg/mL) suspension prepared in Example 4 above was added to an EDAC solution (0.5 mg/mL, 1 mL of 0.02 M phosphAte buffer, pH 6.0) cooled to 0C. The suspension was stirred under argon for 30 minutes. After this time, the suspension was added dropwise into a streptavidin solution (5 mg/mL, 1 mL) in borate buffer (0.2 M, pH 9.0) kept at ~0C. The suspension was stirred for 1 hour and allowed to warm up to room temperature. Water (1 mL) was added and the mixture centrifuged at 15,000 rpm for 1 hour. The supernAtAnt was discarded and the pellet suspended in water (4 mL) by sonication. The sample was recentrifuged in water (4 mL) by sonication, and after a final centrifugation at 15,000 rpm for 30 minutes, the resultant pellet was suspended in water (5 mL). This gave a 2 mg/mL
suspension of streptavidin-photoactive indicator precursor particles. The presence of streptavidin was confirmed by 3H biotin h;n~;ng and quantitated to 2500 + 250 streptavidin/particle.
E~ample 6 Preparation of Maleimidated De~L-or. Photosensitizer Particles A. st~in;ng of particles.
A dye mixture of chlorophyll-a (2.0 mM) and tetrabutyl squarate (4.0 mM) in benzyl alcohol was prepared. Ethylene glycol (80 mL) was placed in a 125 mL Erlenmeyer flask and warmed to 125C on a laboratory hot plate.
The dye mixture in benzyl alcohol (8 mL) was then added followed immediately by stock latex suspension (10 mL of 10~ solids). ~t; ng was discnnt;nne~ and the flask and it~ cnnten~s allowed to attain room temperature. After cooling, the mixture was diluted with an equal volume of ethanol and i ~;~te~y centrifuged at 15,000 rpm for two hours. The bluish-green supernatant was discarded and the pellet suspended in 50 mL of ethanol by sonication. The suspension was centrifuged at 15,000 rpm for one hour and the faintly blue supernatant ~C~nte~. The pellet was resuspended in 50~ aqueous ethanol (50 mL) by sonication to disperse the particles. Centrifugation was repeated at 15,000 rpm for an hour. The sup~rn~t~nt wag decanted and the pellet resuspended in water by sonication.
Following a ~inal centrifugation, the pellets were resu6pended in water to 4S a final volume of 20 mL.

W O 95/06877 PCT~US94/09705 2~ 7Qg~3 B. Preparation of Maleimidated Dextran Photosensitizer Particles.
~ nO~xtran (500 mg) was partially maleimidated by reacting it with sulfo-SMCC (157 mg, 10 mL H2O). The sulfo-SMCC was added to a solution of the 2 ;nn~tran (in 40 mL, 0.05 M Na2HPO4, pH 7.5) and the resulting mixture was ;nCl~h2qte~ for 1.5 hr. The reaction mixture was then dialyzed against MES/NaCl (2x2L, 10 mM MES, 10 mM NaCl, pH 6.0, 4C). The maleimidated dextran was centrifuged at 15,000 rpm for 15 minutes and the sup~rn2~t2~nt collected. The sUpern2~t2~nt de~L ~, solution (54 mL) was then treated with imidazole (7 mL of 1.0 M ~olution) in MES buffer (pH 6.0) and into this stirred solution was added the stained photosensitizer particles (10 mL of lOmg/mL). After stirring for 10 m; nutes the suspension was treated with EDAC (7 mmol in 10 mM pH 6.0 MES) and the suspension stirred for 30 r;nntes After this time, SurfactAmps~ (Pierce) Tween-20 (10~, 0.780 mL) was added to the reaction mixture for a final concentration of 0.1~. The particles were then centrifuged at 15,000 rpm for 45 minutes and the supernatant discarded. The pellet was resuspended in MES/NaCl (pH 6.0, 10 mM, 100 mL) by sonication. Centrifugation at 15,000 rpm for 45 ;n~tes, followed by pellet resuspension after discarding the supernAt2~nt, was performed twice. The maleimidated de~L~- photosensitizer particles were stored in water as a 10 mg/mL suspension.

Ex2~mple 7 Preparation of GATTAG-Photosensitizer Particles.
Amino-GATTAG (180 ~L, 50 nmol) (prepared as described below in Example 8) in water was treated with 0.25M borax (50 ~L) to give a pH o~
9.2. SPDP (50 mg/mL in dry DMT) was added in four aliquots at 0, 10, 20 and 30 minutes (3~.8 ~mol total). The reaction mixture was allowed to stand for 2 hours. Ice cold ethanol (2.1 mL) was added and the product left in the freezer overnight. The cloudy product mixture was split into two Eppendorf tubes and centrifuged at ~; lm speed for 10 ;n~tes. The supern2~t2~nt was carefully removed and the pellet dissolved in 400 ~L H2O.
Into this solution was added 2.5 M acetate buffer (20 ~L, 2.5M, pH 5.3).
TCEP in distilled water (10 ~L, 20mM) was added and the reduction allowed to proceed for 30 minutes at room temperature. Absolute ethanol (1.2 mL) was added 2nd the reaction mixture put in the freezer for 2 hours.
The reaction mixture was centrifuged at full speed in the cold room and the precipitated GATTAG-SH oligonucleotide was removed as a pellet. m e pellet - was di~solved in 200 ~L of 50 mM Na2HPO4 buffer (pH 6.85) cnnt~;n;ng 20 mM
EDTA. The solution was degassed and kept under argon. This solution was then added to the maleimidated dextran photosensitizer particles (14.2 mg/1.5 mL) (prepared above in Example 6) and the reaction mixture allowed to stand overnight. The mixture was centrifuged at 15,000 rpm for 1 hour and the supernatant discarded. The pellet was resuspended in water (2 mL) and centrifuged at 15,000 rpm for 1 hour. The sUpernAtAnt was discarded and the pellet resuspended in water (2 mL). After a final W 095/06877 2 1 7 0 8 7 3 PCTrUS94/09705 centrifugation the GATTAG-photosensitizer particles were stored in 2 mL of water solution as a ~uspension.

Example 8 Assay for Detecting DNA
A. The target 65mer oligonucleotide with the sequence shown below:
5'-GTG-A~G-CGG-GCG-AGC-ATG-GCG-CAC-CGG-CAG-AGC-ATT-TTC-GAG-CGT-GA~-GGC-A~C-AAC-CTG-TAT-TG-3' (SBQ ID NO:5);
and CTA~TC-30mer and amino-CATTAG were prepared on a Milligen Biosearch DNA
synthesizer (Model #8750) using standard solid phase phosphoramidite methodology (Bee Oligonucleotide Synthese6 - A Practical Approach (1984), Gait M.J., Ed., IRL Press Oxford.) The protocol briefly consisted of (a) removal with dichloroactic acid of the 5'-dimethoxytrityl group on the nucleoside attached to the solid support; (b) coupling of the i nC~m; ng nucleoside, which contains a 5'-hyd~u~yl protecting group (preferably dimethoxytrityl) and a 3~ yd~u~yl protecting group (preferably N,N-diisopropylphssrhoramidite), using tetrazole as the catalyst; (c) a capping step with acetic anhydride; and (d) iodine oxidation to convert the ph~8phite triegter into a phosphAte triester. At the conclusion of the synthesis ammonium hyd~u~ide was used to (a) cleave the synthesized polynucleotide from the support; (b) remove the phosphoryl protecting groups (~-cyanoethyl); and (c) to remove the base protecting groups. The oligonucleotide was finally purified by HPLC.
B. Biotin-3Omer was prepared similarly as above except that the base of the last in~ ' ng nucleotide was a 5-methyl-cytosine with a protected amine-modifier (American Bionetics, #ABN2599) as shown below:

H~N~''~,'N ~ ~ ~ CF~

~

After deprotection, the free amine was reacted with biotin-LC-~HS
(Pierce, #21335G) at a 1:60 molar ratio of the two reagents in 0.1M NaHCO3, pH 9Ø Following incubation overnight at room temp, the resulting oligonucleotide was analyzed and purified on a 12~ ~n~tl~ing polyacrylamide gel.
C. The assay was performed by mixing various volumes (0-80 ~L of 21 nM) of target 65mer oligonucleotide with CTAATC-30mer (200 ~L of 15 nM) and biotin-30mer (200 ~ of 15 nM) in TRIS/EDTA/NaCl solution (pH 8.0, 100 mM, 0.1 mM, 0.30 M, respectively) cont~;n~d in a 1.5 mL Eppendorf tube. The ~ W 095/06877 2 1 7 0 8 7 3 PCTrUS94/09705 volumes were made up to 0.5 mL and the solution annealed at 55C for 30 minutes 80 that the 30mer probes could hybridize with their complements on the target 65mer oligonucleotide upon cooling. The reaction mixture was cooled to room temperature and then treated with the streptavidin-photoactive indicator precursor particles (100 ~L of 100 ~g/mL) followed by GATTAG-photosensitizer particle5 (400 ~L of 100 ~g/mL). m e mixture was gently vortexed and allowed to incubate for 2 hours at room temperature.
The suspension was then transferred to a 12x75 mm test tube and irradiated for 5 n~tes with a Dolan-Jenner lamp (tungsten) equipped with a 610 nm cutoff filter. The sample was treated with an equal volume (1 mL) of buffer and transferred to a fluorometer. The fluorescence units corresp~n~;ng to an excitation with a 360 NB filter and 420 NB filter emission were recorded. The resulting standard curves for two individual a~says are shown in Figure 1.
~lthough the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and underst~n~ ng, it will be obvious that certain changes or modifications may be practiced within the scope of the ~pp~n~ claims.

\~

W 095/06877 2 1 7 0 8 7 3 PCTrUS94/09705 SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT
(A) ADDRESSEE: SYNTEX (U.S.A.) INC.
(B STREET: 3401 HILLVIEW AVENUE
(C CITY: PALO ALTO
(Dl STATE CALIFORNIA
(E COUN~1~r: USA
(FJ ZIP: 94304 (ii) TITLE OF 1NV~N-11ON
FLUORESCBNT OXYGEN CHPNNRT.TNG TM~1N~SSAYS
(iii) Nr~URR~ OF S~U~N~S 5 (iv) CC ~ul~ READABLE FORM
(A) MEDIUM TYPE FLOPPY DISK
(B) C~-~U-1'~K: IBM PC COMPATIBLE
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(D) SOFTWARE: PA'1'~N1'1N RELEASE ~1.0, VERSION ~1. 25 (Vi) ~U~K~N1 APPLICATION DATA
(A) APPLICATION NrnMRR~ PCT/US/

(2) INFORMATION FOR SEQ ID NO:1:
(i) ~QU~N~ CH~RACTERISTICS:
~A~ LENGTH: 42 base pair~
~,B TYPE: nucleic acid C~ STR~NV~VN~SS: Bingle D TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iV) ANTI-SENSE: NO
(Vi) ~TGTNAL SOURCE:
(A) ORGANISN: synthetic (Xi) S~UU~N~ DESCRIPTION: SEQ ID NO:1:

(2) 1N~L-TION FOR SEQ ID NO: 2:
(i) S~UU~N~ CHP~TR~TSTICS:
~A) LENGTH: 30 base pairs ~B) TYPE: nucleic acid C) STR~N~ S: single ,D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) nY~O1~11CAL: NO
(iV) ANTI-SENSE: NO

W O 95/06877 2 1 7 0 8 7 3 PCTrUS94/0970S
.

(vi) ORIGINAL SOURCE:
(A) ORGANISM: synthetic (xi) S~YU~NU_ DESCRIPTION: SEQ ID NO:2:

(2) INFORMATION FOR SEQ ID NO:3:
(i) ~_yu_Nu_ CHARACTERISTICS:
'A' LENGTH: 66 base pairs B TYPE: nucleic acid C STRANnKnNRSS: single ,D, TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) ~Y~u~ CAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: synthetic (xi) S_YU_N~_ DESCRIPTION: SEQ ID NO:3:
CTGCCGGTGC GCCATGCTCG CCCG~l-l~AC CTAATCCTAA TCCTAATCCT A~TCCTAATC 60 CTA~TC 66 (2) lN~O~L!TION FOR SEQ ID NO:4:
(i) ~_yu_Nu_ CHARACTERISTICS:
'A LENGTH: 42 base pairs B TYPE: nucleic acid C STR~nRnN-~S: single ,D, TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) ~Y~ln_llCAL: NO
(iv) ANTI-SENSE: NO
(vi) O~TGTN~L SOURCE:
(A) ORGANISM: synthetic (xi) ~_UU_N~_ DESCRIPTION: SEQ ID NO:4:

(2) lN~O~L TION FOR SEQ ID NO:5:
uuKNrK CHARACTERISTICS:
~A' LENGTH: 65 base pairs B TYPE: nucleic acid C STRA~nRnNR~S: single ~D, TOPOLOGY: linear (ii) M~T-RCUT-R TYPE: DNA (genomic) (iii) ~Y~vln_llCAL: NO
(iv) ANTI-SENSE: NO

WO 95/06877 ~ ~ 7 o ~; 7 3 62- PCT/US94/09705 (vi ) ORIGI~L SOURCE:
(A) ORGANISN: synthetic (xi) ~U~N~ DESCRIPTION-: SEQ ID N-0:5:

Claims (12)

WHAT IS CLAIMED IS:

1. A method for determining an analyte which is a specific binding pair member (sbp), which method comprises:
(a) providing in combination:
(1) a medium suspected of containing an analyte;
(2) a photosensitizer capable in its excited state of generating singlet oxygen, wherein said photosensitizer is bound to an sbp member or is bound to or incorporated in a particle having said sbp member incorporated therein or bound thereto; and (3) a photoactive indicator precursor capable of forming a photoactive indicator upon reaction with singlet oxygen, wherein said photoactive indicator precursor is bound to an sbp member or is bound to or incorporated in a particle having said sbp member incorporated therein or bound thereto;
(b) exciting said photosensitizer by irradiation with light; and (c) measuring the fluorescence of said photoactive indicator;
wherein at least one of said sbp members is capable of binding directly or indirectly to said analyte or to an sbp member complementary to said analyte, and wherein said fluorescence is related to the amount of said analyte in said medium.

2. The method of Claim 1 wherein said photosensitizer is selected from the group consisting of ketones, polyaromatic compounds, cyanines, merocyanines, phthalocyanines, squarate dyes, porphyrins, xanthenes, thiazines and oxazines.

3. The method of Claim 1 wherein said photoactive indicator has an extinction coefficient of at least 10,000 M-1cm-1 at its absorption maximum and a fluorescence emission quantum yield of at least 0.1.

4. The method of Claim 1 wherein neither the photosensitizer nor the photoactive indicator precursor are bound to or incorporated in a particle and which further comprises prior to step (b) causing at least one of said photosensitizer and said photoactive indicator precursor to become bound to a surface by means of a specific binding pair binding.

5. The method of Claim 4 wherein a suspendible particle comprises said surface, said particle being selected from the group consisting of latex particles, lipid vesicles, oil droplets, silica particles, metal sols, and dye crystallites.

6. The method of Claim 1 wherein said analyte is selected from the group consisting of a drug, a protein, a polynucleotide, a receptor and a microorganism.

7. A method for determining an analyte, which method comprises:
(A) if the analyte is a polynucleotide (a) combining in an aqueous medium;
(1) said analyte;
(2) one or more polynucleotide probes, wherein each probe contains a nucleotide sequence complementary to a nucleotide sequence of said analyte and wherein at least one probe is bound to a specific binding pair (sbp) member, or is bound to or incorporated in a particle having said sbp member incorporated therein or bound thereto, said sbp member being different from said complementary nucleotide sequence;
(3) a photosensitizer capable in its excited state of generating singlet oxygen, wherein said photosensitizer is bound to, or is bound to or incorporated in a particle having incorporated therein or bound thereto, a nucleotide sequence complementary to a nucleotide sequence of said probe; and (4) a photoactive indicator precursor capable of forming a photoactive indicator upon reaction with singlet oxygen, wherein said photoactive indicator precursor is bound to, or is bound to or incorporated in a particle having incorporated therein or bound thereto, an sbp member complementary to said sbp member associated with said probe;
(b) irradiating said medium with light to excite said photosensitizer to generate singlet oxygen; and (c) measuring the fluorescence of said photoactive indicator;
wherein said fluorescence is related to the amount of said analyte in said medium; or (B) if the analyte is other than a polynucleotide (a) providing in combination:
(1) a medium suspected of containing an analyte;
(2) a photosensitizer capable in its excited state of generating singlet oxygen, wherein said photosensitizer is bound to, or is bound to or incorporated in a particle having incorporated therein or bound thereto, a first specific binding pair (sbp) member; and (3) a photoactive indicator precursor capable of forming a photoactive indicator upon reaction with singlet oxygen, wherein said photoactive indicator precursor is bound to, or is bound to or incorporated in a particle having incorporated therein or bound thereto, a second sbp member;

(b) irradiating said combination with light to excite said photosensitizer; and (c) measuring the fluorescence of said photoactive indicator;
wherein each sbp member is capable of binding directly or indirectly to said analyte or to an sbp member complementary to said analyte, and wherein said fluorescence is related to the amount of said analyte in said medium, and wherein said photosensitizer is optionally part of a suspendible particle to which said first sbp member is bound, and wherein said photoactive indicator precursor is optionally part of a suspendible particle to which said second sbp member is bound.

8. A composition for use in a method for determining an analyte which is a specific binding pair member (sbp) comprising suspendible particles of average diameter of 20 to 4000 nanometers having associated therewith a photoactive indicator precursor which reacts with singlet oxygen to form a phototactive indicator, wherein said photoactive indicator precursor contains a selenium or tellurium atom and said phototactive indicator does not contain a selenium or tellurium atom

9. A kit for conducting an assay for an analyte, which kit comprises, in packaged combination:
(a) a photoactive indicator precursor containing a selenium or tellurium atom, wherein said photoactive indicator precursor is bound to, or is bound to or incorporated in a particle having incorporated therein or bound thereto, a first specific binding pair (sbp) member; and (b) a photosensitizer capable in its excited state of activating oxygen to its singlet state, wherein said photosensitizer is bound to, or is bound to or incorporated in a particle having incorporated therein or bound thereto, a second sbp member, wherein said sbp members are capable of binding to said analyte or to an sbp member capable of binding said analyte, and wherein said photoactive indicator precursor is optionally part of a suspendible particle to which said first sbp member is bound, and wherein said photosensitizer is optionally part of a suspendible particle to which said second sbp member is bound.

10. A binding assay for an analyte that is a specific binding pair (sbp) member, which assay comprises:
(a) combining a medium suspected of containing said analyte with an sbp member capable of binding directly or indirectly to said analyte or to an sbp member complementary to said analyte; and (b) detecting the binding of said sbp member to said analyte or said complementary sbp member, wherein said detection comprises exposing a photoactive indicator precursor in said medium to singlet oxygen to produce a photoactive indicator and measuring the fluorescence of said photoactive indicator.

11. A compound for use in a method for determining an analyte which is a specific binding pair member (sbp) containing the following structure:

(I) wherein H is cis to the XR group;
X is a selenium or tellurium;
R is an organic or organometallic group bound to X through an unsaturated carbon atom, a silicon atom, or a tin atom; and A, when taken with the carbon-carbon group, forms an alicyclic ring (optionally fused to one or more aromatic rings) or a heterocyclic ring;
wherein, upon reaction of the compound with singlet oxygen, the H and the XR group are replaced by a carbon-carbon double bond to yield a fluorescent molecule having an extinction coefficient of at least 10,000 M-1cm-1 at its absorption maximum and a fluorescence emission quantum yield of at least 0.1.

12. The compound of Claim 11 of the following formula:

wherein R is an organic or organometallic group bound to X, through an unsaturated carbon atom, a silicon atom, or a tin atom;
and R1 is hydrogen or alkyl; and wherein up to four of the remaining hydrogen atoms may be replaced by alkyl or alkylene substituents which may be taken together to form one or more alicyclic or aromatic rings.