CA2178308C - Fluorescent labeling complexes with large stokes' shifts formed by coupling together cyanine and other fluorochromes capable of resonance energy transfer - Google Patents

Abstract

The present invention provides low molecular weight fluorescent labelling complexes with large wavelength shifts between absorption of one dye in the complex and emission from another dye in the complex. These complexes can be used, for example, for multiparameter fluorescence cell analysis using a single excitation wavelength. The low molecular weight of the complex permits materials labelled with the complex to penetrate cell structures for use as probes. The labelling complexes are synthesised by covalently attaching through linkers to form donor-acceptor complexes. Resonance energy transfer from an excited donor to fluorescent acceptor provides wavelength shifts up to 300nm. The fluorescent labelling complexes preferably contain reactive groups for the labelling of functional groups on target compounds, such as derivatised oxy and deoxy polynucleic acids, antibodies, enzymes, lipids, carbohydrates, proteins and other materials. The complexes may contain functional groups permitting covalent reaction with materials containing reactive groups.

Description

2 oa3oa FLUORESCENT LABELLING COMPLEXES WITH LARGE STOKES' SHIFTS
FORMED BY COUPLING TQ~,ETHER CYANINE AND OTHER FLUOROCHROMES
CAPABLE OF R.ESONANC~ ENERGY TRANSFER
The present invention relates to fluorescent labelling complexes, and more particularly to low molecular weight fluorescent complexes with large Stokes' shifts'. and to their use in the preparation of fluorescent derivatives of target materials.
Fluorescence labelling is an important technology for detecting biological molecules. For example, antibodies can be labelled with fluorescent dyes. The binding of antibodies to their specific target molecules can then be monitored on the basis of a fluorescence signal, which may be detected with a spectrometer, immunofluorescence instrument, flow cytometer, or fluorescence microscope. In a similar way DNA sequences can be detected with fluorescence detection instruments after the DNA has been hybridized with a complementary DNA sequence that has been labelled with a fluorescent dye.
Energy transfer complexes containing covalently linked donor and acceptor molecules are known. For example, a model system was developed by Stryer and Haugland for the study of the dependence of singlet-singlet energy transfer on distance (Stryer, L.
and Haugland, R..P., Proc.Nat.Acad.Sci., Vo1.58, pp.720-26, (1967)). The synthesis and properties of new photochemical model compounds containing a cyanine dye and a porphyrin has been reported (Lindsey et al, Tetrahedron, Vol. 45, No.lS, pp.4$45-66, (1989))., Complexes containing fluorescent donor and acceptor chromophores have been described as substrates for the kinetic study and assay of hydrolytic enzymes (Car-mel et al, FEBS Letters, Vo1.30, No.l, pll, (1.973)).
European Patent Application No.609894 discloses a labelling complex comprising a tri-nucleus dye represented by the general formula (1).
w w \ ,~ \ ~~ \
' ~ ' ~ ' ~La ~ +-Lb-, ' '\ . , '\
w--' \w_..' w_-' (1) where Xa, Xb and Xc are independently substituted or unsubstituted heterocyclic rings containing one to three heteroatoms and La and Lb are conjugated methine chains. One of La and Lb may be omitted so as to link the heterocycles directly. The compounds of structure ( 1 ) can include a reactive group for forming a covalent linkage between the trinucleus dye and a biological substance. Compounds of such a formula are reported to have a large Stokes' shift (SO-100nm). However, it is not thought that resonance energy transfer is involved in the process of fluorescence with those dyes.
,......,.. ,..","~,x,r.~~.,.,.,"..,....~~...".,.,., ........M..m. .......
......rc:~rxu~crt.

Multiparameter analysis using fluorescent labels with distinctly different emission wavelengths further increases the importance of this technology by providing a powerful tool for correlating multiple antigenic or genetic parameters in individual cells. In epifluorescence microscopy, a continuous light source with different sets of excitation and emission filters are used to excite and detect each fluorescent species. This approach works especially well if the absorption and emission wavelengths of each of the fluorophores are relatively close together (eg. Stokes' shifts of 15-30nm). Most of the highly fluorescent, low molecular weight fluorophors like the cyanines and xanthenes have narrow absorption and emission peaks and small Stokes' shifts.
i1p to 5 separate fluorescent labels have been analysed on the same specimen by microscopy using epifluorescence filter sets as described by DeBiasio et al, Journal of Cell Biology, Vol.105, pp.1613-1622, ( 1987).
While it is easy to find a single fluorophore that can be efficiently excited at a particular laser wavelength, it is difficult to find additional fluorescent labels with large enough Stokes' shifts to provide emission well separated from that of the first fluorophore. The naturally occurnng phycobiliproteins are a class of multichromophore fluorescent photosystem proteins that have large wavelength shifts; see Oi, V.T., Glazer, A.N. and Stryer, L., Journal of Cell Biology, Vo1.93, pp.981-986, (1982). These can be covalently coupled to antibodies and have been widely used in flow cytometry for 2-colour lymphocyte subset analysis. R-phycoerythrin (R-PE), a photosystem protein containing 34 bilin fluorophores which can be excited at 4$$nm with the widely available argon ion laser, has been especially useful. It fluoresces maximally at 575nm. R-PE and fluorescein can both be excited at 488nm, but R-PE can be readily discriminated with optical band pass intenerence filter sets from the fluorescein signal which appears at 525nm. Recently, 3-colour immunofluorescence by flow cytometry has become possible through the development of tandem conjugate labelling reagents that contain a reactive fluorescent dye which is excited at 488nm and fluoresces at 613nm, and is sold commercially under the name Duochrome, see: US Patent No.4876190. With another tandem fluorophore energy transfer from excited R-PE to the linked cyanine dye l~mown as Cy-5 leads to fluorescence at 670nm (Waggoner et al. Ann. N. Y.Acad. Sci., Vol.6'17, pp.185-193, (1993)).
'fhe phycobiliprotein-based labels are very fluorescent and provide excellent signals in 2- and 3-parameter experiments for detection of cell surface antigens. However these reagents have not been widely utilised for measurement of cytoplasmic antigens or for detection of chromosomal markers by fluorescence in situ hybridization because their large size (MW
210,000 Daltons) limits penetration into dense cell structures.
Notwithstanding the above, there is still a lack of low molecular weight fluorescent compounds which can be used as labels for the covalent labelling of target rrrolecules and which will provide multicolour fluorescence detection using single wavelength excitation.
There is also a requirement for several such fluorescent labels, each of which can be excited optimally at a particular laser wavelength but fluoresce at significantly different emission wavelengths. We have now found a class of low molecular weight fluorescent labels which will provide multicolour fluorescence detection using single wavelength excitation.
Accordingly, the present invention relates to a low molecular weight fluorescent labelling complex comprising:

- a first or donor fluorochrome having first absorption and emission spectra;
- a second or acceptor fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome;
- at least one linker for covalently attaching said first and second fluorochromes for transfer of resonance energy transfer between said first and second fluorochromes;
- a target bonding group capable of forming a covalent bond with a target compound;
wherein the combined molecular weight of said first and second fluorochromes and said linker is less than about 20,000 Daltons.
Preferably at least one of said first or second fluorochromes is a cyanine dye.
In accordance with one aspect of the present invention there is provided the complex according to claim 7 or 8 wherein said first fluorochrome is selected from the group consisting of monomethine rigidized cyanine dyes, a trimethine cyanine dye, fluorescein, pyrene trisulphonate, bispyrromethine boron difluoride dyes and said second and third fluorochromes are polymethine cyanine dyes.
In accordance with another aspect of the present invention there is provided a method of labelling a carrier material comprising incubating an aqueous sample containing a carrier material with a low molecular weight, water soluble fluorescent labelling complex comprised of: i) a first fluorochrome having first absorption and emission spectra covalently linked to a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and the absorption spectrum of said second fluorochrome overlapping the emission spectrum of said first fluorochrome for transfer of energy absorbed by said first fluorochrome upon excitation with light to said second fluorochrome, wherein at least one of said first or second fluochromes is a cyanine dye; ii) a target bonding group capable of forming a covalent bond with a complementary group of said carrier material, and iii) water solubilising constituents for conferring a polar characteristic to said complex, said water solubilising constituents being unreactive with said bonding group, for a period of time sufficient for covalently binding said bonding group of said complex to said complementary group of said carrier material.

In accordance with yet another aspect of the present invention there is provided a set of fluorescent labeling complexes each of said complexes comprising: i) a first fluorochrome having first absorption and emission spectra; ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome, the first and second fluorochromes being selected from the cyanine dyes, fluoresceins, rhodamines, pyrene trisulphonates and derivatives of the bispyrromethine boron difluoride dyes; iii) at least one linker group having between 2 and 20 bond lengths for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes; iv) at least one target bonding group capable of forming a covalent bond with a target compound wherein the target bonding group is a reactive group for reacting with a functional group on the target material; wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons, wherein at least two complexes of said set absorb at the same wavelength and emit (fluoresces) at significantly different emission wavelengths, and wherein at least one of said first or second fluorochromes in each of said complexes is a cyanine dye.
In accordance with a further aspect of the present invention there is provided a set of reagents each reagent comprising: A) a fluorescent water soluble labeling complex comprised of: i) one or more low molecular weight first fluorochromes, each having first absorption and emission spectra, covalently attached through a linker group to one or more low molecular weight second fluorochromes, each having second absorption and emission spectra, and wherein the wavelength of the emission maximum of at least one said second fluorochrome is longer than the wavelength of the emission maximum of at least one said first fluorochrome and a portion of the absorption spectrum of at least one said second fluorochrome overlaps a portion of the emission spectrum of at least one said first fluorochrome for transfer of energy absorbed by said first fluorochrome upon excitation with light to said second fluorochrome; ii) at least one target bonding group capable of forming a covalent bond with a carrier material; and, iii) at least one water solubilizing constituent attached to said complex, said water solubilizing constituent being unreactive with said at least one target bonding group; wherein at least one of said first or second fluorochromes is a cyanine dye and the linker group is chosen from the group consisting of alkyl chains containing from 1 to 15 carbon atoms, which may optionally include oxygen atoms as ether linkages, or nitrogen atoms as amine linkages, or CO--NH groups as amide linkages; B) a carrier material having a group that reacts with said target bonding group of said complex and is covalently bound thereto.
In accordance with yet a further aspect of the present invention there is provided a method of labeling a carrier material with one of a set of fluorescent labeling complexes, comprising incubating an aqueous sample containing a carrier material with a low molecular weight, water soluble fluorescent labeling complex comprised 3a of: i) a first fluorochrome having first absorption and emission spectra covalently linked through a linker group to a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and the absorption spectrum of said second fluorochrome overlapping the emission spectrum of said first fluorochrome for transfer of energy absorbed by said first fluorochrome upon excitation with light to said second fluorochrome, wherein at least one of said first or second fluorochromes in said complex is a cyanine dye; ii) a target bonding group attached to said complex capable of forming a covalent bond with a complementary group of said carrier material, and iii) water solubilizing constituents attached to said complex for conferring a polar characteristic to said complex, said water solubilizing constituents being unreactive with said bonding group; for a period of time sufficient for covalently binding said bonding group of said complex to said complementary group of said carrier material.
In accordance with one embodiment of the present invention there is provided use of a set of fluorescent labeling complexes for analysis or detection comprising incubating a fluorescent labeling complex of said set with at least one target material, each of said fluorescent labeling complexes comprising: i) a first fluorochrome having first absorption and emission spectra; ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome, the first and second fluorochromes being selected from the cyanine dyes, fluoresceins, rhodamines, pyrene trisulphonates and derivatives of the bispyrromethine boron difluoride dyes; iii) at least one linker group having between 2 and 20 bond lengths, which may optionally include oxygen atoms as ether linkages, or nitrogen atoms as amine linkages, or CONH groups as amide linkages, for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes; iv) at least one target bonding group capable of forming a covalent bond with a target compound wherein the target bonding group is a reactive group for reacting with a functional group on the target material; wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons, wherein at least one of said first and second fluorochromes in each of said complexes is a cyanine dye, and wherein at least two complexes of said set absorb at the same wavelength and emit (fluoresces) at significantly different emission wavelengths; and measuring and comparing the differences in fluorescent emission between said fluorescent labeling complexes.
In accordance with another embodiment of the present invention there is provided use of a set of fluorescent labeling complexes for analysis and detection comprising incubating a fluorescent labeling complex of said set with at least one target material, each of said fluorescent labeling complexes comprising: i) a first fluorochrome having first absorption and emission spectra; ii) a second fluorochrome having 3b second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome; iii) at least one linker group chosen from the group consisting of alkyl chains containing from 1 to 15 carbon atoms, which may optionally include oxygen atoms as ether linkages, or nitrogen atoms as amine linkages, or CONH
groups as amide linkages, for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes; iv) at least one target bonding group capable of forming a covalent bond with a target compound; wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons, and wherein at least one of said first or second fluorochromes in each of said complexes is a cyanine dye; and measuring and comparing the differences in fluorescent emission between said fluorescent labeling complexes.
In accordance with yet another embodiment of the present invention there is provided a set of fluorescent labeling complexes each of said complexes comprising:
i) a first fluorochrome having first absorption and emission spectra; ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome, the first and second fluorochromes being selected from the cyanine dyes, fluoresceins, rhodamines, pyrene trisulphonates and derivatives of the bispyrromethine boron difluoride dyes; iii) at least one linker group having between 2 and 20 bond lengths for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes; iv) at least one target bonding group capable of forming a covalent bond with a target compound wherein the target bonding group is a reactive group for reacting with a functional group on the target material; wherein said fluorochromes and said linker in each of said complexes, the relative orientation of the transition moments of the fluorochromes during the excited state lifetime of the first fluorochrome, and the proximity of the fluorochromes, are selected such that there is sufficient energy transfer; wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons, wherein at least two complexes of said set absorb at the same wavelength and emit (fluoresces) at significantly different emission wavelengths, and wherein at least one of said first or second fluorochromes in each of said complexes is a cyanine dye.
In accordance with a further embodiment of the present invention there is provided a method of analysis or detection of multiple target compounds comprising:
incubating a first reagent of a set of reagents with a first one of said multiple target materials, each of said reagents of said set of reagents comprising: A) a fluorescent water soluble labeling complex comprised of: i) one or more low molecular weight first fluorochromes, each having first absorption and emission spectra, covalently 3c attached through a linker group of 2 to 20 bond lengths to one or more low molecular weight second fluorochromes, each having second absorption and emission spectra, and wherein the wavelength of the emission maximum of at least one said second fluorochrome is longer than the wavelength of the emission maximum of at least one said first fluorochrome and a portion of the absorption spectrum of at least one said second fluorochrome overlaps a portion of the emission spectrum of at least one said first fluorochrome for transfer of energy absorbed by said first fluorochrome upon excitation with light to said second fluorochrome; ii) at least one reactive group capable of forming a covalent bond with a carrier material;
and, iii) at least one water solubilizing constituent attached to said complex, said water solubilizing constituent being unreactive with said at least one reactive group;
wherein at least one of said first or second fluorochromes is a cyanine dye;
B) a carrier material having a functional group that reacts with said reactive group of said complex and is covalently bound thereto, wherein said functional group is selected from the group consisting of amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate and said carrier material is selected from the group consisting of antibody, lipid, protein, carbohydrate, nucleotide that contains one of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups and oxy or deoxy polynucleic acids that contains one of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups; wherein said fluorescent labeling complexes of each of said set of reagents absorbs at the same wavelength and emits at significantly different emission wavelengths; wherein the incubating step continues for a period of time sufficient to permit said first reagent to bind to said first target compound to form a reagent labeled-first target compound; incubating a second reagent of said set of reagents with a second one of said multiple target materials, wherein the incubating step continues for a period of time sufficient to permit said second reagent to bind to said second target compound to form a reagent labeled-second target compound; and, measuring and comparing the differences in fluorescent emission between said labeling complexes of said reagent labeled-first target compound and said reagent labeled-second target compound.
In accordance with yet a further embodiment of the present invention there is provided a fluorescent detection and analytical method for detecting a first target material in a sample, comprising the steps of: a) contacting a first fluorescent labeling complex with a first sample; and b) detecting labeling of a target material in the first sample with the fluorescent labeling complex, the first fluorescent labeling complex having at least: i) a first fluorochrome having first absorption and emission spectra, covalently attached through a linker group of between 2 and 20 bond lengths to a low molecular weight second fluorochrome having second absorption and emission spectra, and wherein the wavelength of the emission maximum of the second fluorochrome is longer than the wavelength of the emission maximum of the first fluorochrome and a portion of the absorption spectrum of the second fluorochrome overlaps a portion of the emission spectrum of the first fluorochrome for transfer of energy absorbed by the first fluorochrome upon excitation with light to the second fluorochrome, wherein at least one of the first fluorochrome and the second fluorochrome is a cyanine dye and wherein said linker group is selected to 3d transfer resonance energy between said first fluorochrome and said second fluorochrome; ii) a water solubilizing group; and; iii) a carrier material covalently bound to portion (i) of said first fluorescent labeling complex and selected from the group consisting of an antibody, a lipid, a protein, a carbohydrate, a nucleotide and a nucleic acid.
The linker may be rigid or flexible to orientate the transition moments of the donor and acceptor chromophores. For optimal energy transfer to occur, the transition moments of the first and the second fluorochromes are orientated relative to each other in a non perpendicular direction, eg. positioned generally parallel or in tandem relative to each other. The transition moments of the flexibly linked fluorochromes will chap as the linker flexes, but provided that the donor and acceptor transition moments are non perpendicular during the excited state lifetime of the donor, energy transfer will occur. The complexes prepared and described herein show energy transfer ranging from 50% to 99%
efficiency. Energy transfer efficiency depends on several factors such as spectral overlap, spatial separation between donor and acceptor, relative orientation of donor and acceptor molecules, quantum yield of the donor and excited state lifetime of the donor. In a preferred embodiment, the fluorochromes may be separated by a distance that provides efficient energy transfer, preferably better than 75% .
Closer proximity of the donor and acceptor fluorophors would enhance energy transfer, since efficiency of energy transfer varies as the inverse 6te power of separation of the centres of the chromophores according to Forster's equation.
ET a KZ ~D J/R6 iD
where ET is the energy transfer rate constant, K is the relative orientation of donor and acceptor transition moments, ~D is the quantum yield of the donor molecule, R is the distance between the centres of the donor and acceptor fluorochromes, J is the overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor fluorochromes, and iD is the excited state lifetime of the donor molecule. See, Forster, T. "Intermolecular Energy Transfer and Fluorescence", Ann. Physik., Vol.2, p.55, (1948). The distance R
between the centres of the donor and acceptor fluorochromes may be preferably from 10 to 80 Angstroms. The linker should permit resonance energy transfer between the fluorochromes.
3e The fluorochromes should not interact chemically or form secondary bonds with each other.
The linker may be preferably from 2 to 20 bond lengths. For example, if the linker contains an alkyl charm, -(CF~s , the carboy number "n" may bo from 1 to about 15. The linker may include part of the constituents extending from the fluorochronu. In other words, the linlaer is attached to the dye chromophore but is not a part of it. Referring to the linkers shown in Table 2, some extend from the ring nitrogen is one cysaine to a functional group on the benzene ring of another cyanine. Some linkers extend between functional groups on the benzene rings of lixaoed dyes. However, in three examples, none of the linkers includes a network of double bonds that permit eoqjugation of the donor and acceptor.
With a relatively short linker cad optimal orientation, there may be efficient resonance energy transfer even when the spectral overlap becomes small. Therefore, it is possible to obtain large wavelength shifty even when only two chromophorea are used in the complex.
Suitable linloers are selected from the group consiatiag of alkyl chains containing front 1 to 20 cattioa grooms which may optionally include from 1 to 8 oxygen atoms as polyecher linkages, or from 1 to 8 aitmaen atoms as polyamine lin>cagea, or from 1 to 4 CO-NH
groups as polyamide linkages, up to 2 bicyclo[2,2,2]octyl groups and up to 10 nucleotide units.
The complexes of the present invention include a target bonding group capable of forming a covalent bond with a target compound to enable the complex to label the target, such as a carrier materistl or a biological compound. The target bonding group may be a reactive group for reacting with a functional group o~a the target materlat. Alternatively the complex may contain a functional group and the target may contain the reactive constituent.
Suitably, the rea~etive group is selected from the group consisting of succinimidyl ester, isothiocyanates; dichlorot<iaziae, isocyaaates, haloacetacnide, maleimide, sulphonyl halides, , acid halides, alkylimido esters, arylimido esters, substituted hydrazines, substituted hydroxylamines, earbodiimides, acylhalide, anhydride, acrylate, acrylamide and phosphoramidites.
Suitably, the functional group is selected from the group consisting of amino, sulphydryl, carboxyl, hydroxyl, carbonyl, thiophosphate.
Suitably, halo- and halide are selected from chloro, bromo and iodo, or chloride, bromide and iodide.
Suitable target materials may include antibodies, antigens, proteins, carbohydrates, lipids, nucleotides derivatized to contain one of amino, hydroxyl, sulphydryl, carboxyl, or carbonyl groups, and oxy or deoxy polynuclbic acids derivatized to contain one of amino, hydroxyl, thiophosphoryl, sulphydryl, carboxyl, or carbonyl groups, cells, polymer particles, or glass beads. Ia the alternative embodiment, the target may be derivatized to contain the reactive groups identified above to form covalent bonds with the functional groups on the complex.
In a second embodiment, the fluorescent complexes of the invention may contain a po~ymerizab1e group suitable far the formation of a polymer containing the complex. Suitable polymerlxable groups are selected from acrylate, merhacrylate and acrytamide.
polymerization 2 ~ ~a3o$
may be carried out with a suitably derivatized complex of this present invention used in <;onj unction with a second polymerizable monomer starting material, such as styrene or vinyltoluene, to form a copolymer containing the fluorescent complex.
Alternatively, the fluorescent complexes of the invention need not have a reactive group when used to non-covalently bind to another material. For example, the complex may be incorporated during polymerisation or particle formation or may be absorbed into or onto polymer particles.
The complex may also include water solubilising constituents attached thereto for conferring a hydrophilic characteristic to the complex. They are preferably attached to the aromatic ring system of the cyanine fluorochrome. If the cyanine dye does not contain the water solubilising constituent, then the other dye or the linker moiety can contain the water solubilising group.
The water solubilising constituents must be unreactive with the target bonding group of the complex. Suitable solubilising constituents may be selected from the group consisting of amide, sulphonate, sulphate, phosphate, quaternary ammonium, hydroxyl, guanidinium and phosphonate. Sulphonate or sulphonic acid groups attached directly to the aromatic ring of the c:yanine fluorochrome are particularly preferred. Water solubility may be necessary when labelling proteins and oxy and deoxy nucleic acids derivatized with amino groups or sulphydryl groups in aqueous solutions. Alternatively, a less hydrophilic polar form of the energy transfer compound may bind non-covalently to DNA by intercalation between the base pairs or by interaction in the minor groove of DNA. Such compounds may be useful for DNA
quantitation or localisation.
In addition to the embodiment of the invention which includes a single donor and a single acceptor fluorochrome, the fluorescent labelling complex may include further fluorochromes.
The further fluorochromes must have absorption or emission spectra which permit energy transfer to occur. For example, a third fluorochrome may be. attached to the second fluorochrome. In this example, the wavelength of the emission spectrum of the third fluorochrome is longer than the wavelength emission of the sea~nd fluorochrome, and a portion of the emission spectrum of the second fluorochrome overlaps a portion of the absorption spectrum of the third fluorochrome for transferring energy absorbed from the first fluorochrome to the second fluorochrome to the thud fluorachrome:.
In another embodiment of the present invention, the complex may include a plurality of the first fluorochromes, each covalently linked by a linker moiety to the second fluorochrome and each capable, upon excitation with light, of transferring energy to the second fluorochrome.
vi a further embodiment of the present invention, the complex may include a plurality of the second fluorochromes, each covalently linked by a linker moiety to a first fluorochrome and each capable of accepting energy from the first fluorochrome when the first fluorochrome is excited by light. The plurality of first and second fluorochromes may be the same molecule or may be different. For example, there may be several donor fluorcxhromes which are each excitable at different wavelengths to accommodate different excitation light sources.
In a still further embodiment of the present invention, the complex may include one or a plurality of the second fluorochromes, each covalently linked by a '.linker moiety to one or a 2~ 7a3oa plurality of the first fluorochrome and each covalently linked by a linker moiety to a third iluorochrome. Energy transfer proceeds in parallel in these embodiments.
'fhe first fluorochrome preferably has an extinction coefficient greater than 20,000 Litres/mole.cm and more preferably greater than 50,000 Litres/mole.em. The second fluorochrome has a fluorescence quantum yield greater than or equal to about 0.05. Quantum yield is generally related to a molecule's rigidity or planarity and indicates the molecule's propensity to fluoresce, ie. give off energy as light, rather than as heat when energy is provided to the molecule.
'Che complexes of the present invention preferably include at least one cyanine fluorochrome <md preferably at least one polymethine cyanine dye. The cyanines are particularly useful due to the wide range of structural variations and spectral properties available that may be obtained by varying the number of carbon atoms in the methine bridge, andL the heteroatoms or other constituents of the cyanine dyes. It is possible to synthesise dyes having particular excitation wavelengths to correspond to a particular excitation source, such as a laser, eg. a HeNe laser or a diode laser. Therefore, energy transfer labels can be made that absorb and emit efficiently at most wavelengths in the visible region of the spectrum. Commonly used sources of excitation excite at laser line 488nm. Whilst that excitation wavelength will be used for the purposes of the description of the invention, it is to be understood by those skilled in the art that other energy transfer labels can be made for specific excitation sources without departing from the scope of the invention.
Examples of dyes that can be used as donor and acceptor fluorochromes in the fluorescent labelling complexes of the present invention are shown in formulas'. 2 and 3, 03 S~ ~ ~CH2COOH OH
Os S 1SO3H
Cascade Blue FITC
(2) (3) and in formula (4), X
R~
R
(~HZ)n p P
(4) wherein X is selected from C(CH3)Z, sulphur and oxygen, R' and Rz are independently selected from the group consisting of CHzNH2, S03-, CHZCOOH and NCS., P is selected from S03-, NHz and COOH, and n is an integer from 1-5.
additional cyanines for use in complexes of the invention are the rigidized monomethine cyanines disclosed in the copending application of Waggoner ea al, entitled "Rigidized l4lonomethine Cyanines", filed on even date herewith. The monomethine rigidized dyes have the following general structure (5).
R~
_..

Z
.__ .__, (5) optionally substituted by one to six groups RZ to R' ;
where T is a linking group such that:
~T~~
is a six or seven membered ring;
X and Y are selected from bis-substituted carbon, oxygen, sulphur, selenium, -CH=CH-, and -N-W wherein N is nitrogen and W is selected from hydrogen and a group -(CHZ)~R~ where n is an integer from 1 to 26 and R8 is selected from hydrogen, amino, aldehyde, acetal, ketal, halo, cyano, aryl, heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate, substituted amino, quaternary amino, vitro, primary amide, substituted amide, and groups reactive with amino, 2~ ~a3oa hydroxyl, aldehyde, phosphoryl, or sulphydryl groups;
groups Z' and ZZ represent the atoms necessary to complete one., two fused or three fused aromatic rings each ring having five or six atoms, selected from carbon atoms and, optionally, no more than two oxygen, nitrogen and sulphur atoms; and RZ and R3 are attached to the carbon atoms of T when T contains carbon atoms.
The rigidized monomethine cyanine dyes have sharp distinct absorptive and emissive signals, which are photostable. Certain of the rigidized monomethine cyanine dyes maximally absorb and emit light at wavelengths between 300 and SOOnm.
Other low molecular weight fluorochromes in addition to the cyanine fluorochromes may be selected from the fluoresceins, pyrene trisulphonates (which are sold under the trade mark '"Cascade Blue"), rhodamines, and derivatives of the bis-pyrromethine boron difluoride dyes, such as 3,3',S,S'-tetramethyl-2,2'-pyrromethene-1,1'-boron difluoride, sold under the trademark BODIPY by Molecular Probes Inc. BODIPY analogues are disclosed in US
Patent Nos.4774339, 5187223, 5248782 and 5274113 (Haugland and lEtang), as well as in the "Handbook of Fluorescent Probes and Research Chemicals", published by Molecular Probes Inc.
For obtaining exceptionally large excitation-emission wavelength shifts, it is possible to use sequential energy transfer steps in the complex. For example, three chromophores have been linked to provide maximal emission at the wavelength of a cyanine dye, the heptamethine cyanine, CY7, (compound 4, X=C(CH3)Z, R', RZ=-SO3 , P=COOH, n=S, m=3), above 7$Onm with excitation at 4$8nm. The initial donor was fluorescein isothiocyanate and the intermediate fluorophore in the complex was the trimethine cyanine dye designated CY3 (compound 4, X=C(CH3)2, R'=Rz=CHZNH2, P=S03-, n=4, m==1). The fluorescein was excited at 488nm and transferred nearly 100% of its excited state energy to the trimethine cyanine, which in turn transferred about 90% of its excited state energy to the CY7 fluorescing at 782nm. The same efficiency was observed when a pentamethine cyanine CYS was used in place of CY7, with fluorescence at 667nm. The development of such multichromophore complexes is particularly useful for multicolour detection systems.
Although several of the complexes show efficient energy transfer, the overall quantum yield of these labelling complexes can be further improved. For example, the use of acceptor dyes with quantum yield higher than that of CYS would improve the overall brightness of the complex.
The fluorescent labelling complexes of the invention have low molecular weights and can be readily conjugated to antibodies, other proteins and DNA probes. lC.ow molecular weight as used herein shall mean that the combined molecular weight of the fluorochromes and linker of the complex is preferably between about S00 and 10000 Daltons, and for the two fluorochrome complex, preferably in the range of 1000 to 2500 L)altons.
Therefore these labelled species will have much greater penetration into intracellular environments than is possible with the large phycobiliprotein labels currently in use. The low molecular weight fluozesoesit complexes of the present invention should be valuable not only for flow cytometry, but also for laser confocal microscopy and for other detection systems requiring multicolour detection with single wavelength excitation.
The invention includes a reagent and a method for mating the reagent including incubating the fluorescent water soluble labelling complex described above with a, cattier material.
The present invention also provides processes for the preparation of the ~luoreacent labelling complexes which comprise covalently linking tluorxhromes such as cysnine fluorochromes to cyanines or other fluarnchtnmes, by methods well known to those skilled in the art to form energy transfer donor-acceptor complexes.
For example, complexes of the present invention wherein the linkage contains an amide or an ester may be prepared by the reaction of a compound of formula (6) with a compound of formula (7);
R-(11~-COA B-(l~-R' (6) (~) wherein R and R' are different fluorochromes; COA is an activated or activatable carboxyl group; B is NHz or OH; and M and N are independently aliphatic moieties containing C,.«
alkyl and optionally including one or more linldag phenyl, naphthyl, amide, aster, or ether functionalities. See for example, Mufjumdar, R.B. et al, Bioconjugate Chemistry, Vol.4, pp.105-111, (1993); US Patent No.5268486 to Waggoner et al.
Suitable groups A include halo, for example chloro or bmmo, pare-nitrophenoxyl, N hydrmcysuccinimido, or OCOR" wherein R" is C,~
alkyl.
Complexes of tire prgsertt invention wherein the linkage contzins an amino, ether or a thioether group, may be prepared by the reaction of a compound of formula, (8) with a compound of formula (9);
R-(N17-B' G(N) R~
(8) (9) wherein R, R', M and N are as defined above; B' is OH, NHz, or SH; and C is a displacable group for exempla iodo, or pare toluenesulphonate. The reaction is suitably carried out in the presence of a base.
Alternatively, complexes of the present invention may be prepared by first coupling together two dye precursors using a non-conjugated linlaer to give an intermediate represented by structure (10).

Xa-(L)-Xb (10) wherein Xa and Xb are independently substituted or unsubstituted hetarocyclic precursors and (L) is a non-conjugated linl~ group comprising Ct.,= a11cy1, optionally including one or more linking phenyl, tsaphthyl, bicyclo[2,2,2]oMyl, ether, amine, eater, or amide groups, or combinations thereof. Suitable heterocyclic precursors, Xa and Xb are shown in Table 1, Compounds I and II. By way of example, the synthesis of intercnediabe ( 10) wherein the linker consists of an alkyl chain linked to the nitrogen atoms of two indolenine units, may be accomplished by reaction with an a,~-dibaloalkane, such as 1,6-dibromohexane, either in a one or a two stage reaction process. Suitably the reaction is carried out at an elevated temperature such as about 100-110'C, in an inert solvent such as xylene. See for example, Hamer, F.M., "The Cyanine Dyes and Related Compounds", p.676, Wiley lnterscience (1964).
The intermediate (10) can then be used as a precursor in the formation, by methods known in the art, of complexes containing two different fluorophors c.~nacbed by the linker. See for example, Hamer, F.M., "The Cyanine Dyes and Related Compounds", p.118-119, Whey Interacience (1964).
The following examples serve to illustrate the preparstioa of complexes of the present invention and their spectral properties.
Facample 1.
a a ~ ~ ~a as Cyanuric chloride (trichlorotriazine) (Smg), sodium bieatbonate (2mg), and purified dimethylformamide (DMF) (0.25m1) were mixed at 0'C. To this solution was eddy Smg of amino-cyanine dye (Mujumdar of al, Cytometry, Vol.10, pp.11-19, (1989)), represented above by the box containing CYS and the trrixtute was stirrrd at 0°C for 10 minutes. Stirring was continued overnight at mom temperature. Thin layer chromatography (TLC) revealed one major spot end two minor spots; the latter spots were determined to be impurities.
The reaction mixture was worked up by precipitation with ether. A dark blue powder was obtain. DMF (0.3tn!) wrs added to dissolve the powder. Tv this solution was added sodium bicarbonate (Zr~ and 4.7mg of the amino-CY7 dye represented by the box oont$ining CY7.
The nuwcture was stirred at room temperature for Z4 hours, The product was precipitated and washed several tunes with ether, providi~,g a dark powder. The complex showed an absorption spectrum with peaks for the individual fluorochromes at 650nm (CYS) and 761nm (CY7), indicating that no new chromophore had been generated.
Example 2.
i) Sd~
a) Purification of the tluorochromea was performed on a Spectra-Phydcs model analytical HPI,C unit equipped with a C8-RP column. Purification could also be achieved by conventional or flash column chivmato8raphy on comanerrially available C18-RP
powder.
Water/methanol mixture: were used for eluti~oa in all experiments. Dyes were recovered from the fractions by rotary evaporation at 60-70'C without appreciable tos=. For further putzf~tion, tile fluornehrome, with undecGrmined eo~uarer ion composition wet passed through a Dowex-SOW (trade-mark) column (hydrogen form).
b) Ultra-violet/visibie spoctzs wer~a measured with a Flevvlett-Pa~clmrd IiP8452 diode array spectrophotometer. Proton Nl~ spectra were obtained with an IBM 300 FT-NlvBt meter using DzO, CD~OD or DMSO-d6 as solvents. NMR signals are described in a by the use of s for ringlet, d for doublet, t for triplet, q for quartet and m for multiplet.
Fluorescence measurements were performed using a SPBX Fluorolog Z System.
Quantum Yields were deeermin0d by latoMm techniques as described by Mqjumdar R.B.,et al, "Cyanine Dye Labelling Reagents Containing Iaothiocyanate Groups", Cytometry, Vo1.10, pp.ll-19 (1989).
c) Ce~~~l~~ti~ ~n~ Rlnv Mononuclear leulaocytw wem obtained by >;i'ubopaque, density 1.077, separation from healthy volunbxrs. The lymphocyte population was selected by flow cytometry based on forward and side scatter characteristiee. Sub-populations were identified using spoeific monoclonal 2~la3oa antibodies (CD4, staining T-helper cells and CD3, pan T-cell population).
Optimal <;oncentration of Complex 1-tagged antibody was determined by analysing the results of a dilution series. Direct immunofluorescence was accomplished by incubating the recommended amount of labelled antibody with 1-2 x 106 cells for 45 minutes at 4°C.
Samples were then washed twice in Hank's balanced salt solution (HBSS) containing 2% fetal bovine serum and 0.1 % sodium azide. After the final wash, the cells were resuspended in 1 ml of HBSS
containing 1 % paraformaldehyde and analysed within one vveek. Flow cytometry measurements were made with a Becton Dickinson FAGS 440 dual laser flow cytometer equipped with a Consort 40 data analysis system. The argon ion laser provided 400mW of excitation at 488nm. Fluorescence signals from Complex 1 arrd R-phycoerythrin were collected using 670/13.5nm and 575/26nm band pass filters respectively.
<i) Calculation of Donor Quenching Efficienc,~(DQE) Resonance energy transfer efficiencies were estimated from t:he quenching of donor fluorescence intensities. Absorption and fluorescence spectra of the donor (alone) and the fluorescent labelling complex were obtained in order to determine the relative concentrations of each in fluorescence experiments. Donor excitation was used to obtain emission spectra of both compounds. DQE was then calculated using:
DQE% _ (1 - F~A/FA~) x 100 where F is the fluorescence intensity of the donor alone, F~ is the fluorescence intensity of the complex at the donor wavelength, A is the absorbance at the wavelength of excitation of the donor alone and A~ is the absorbance at the wavelength of excitation of the fluorescent labelling complex.
e) Synthesis of Fluorochromes Amino cyanines (CY3NH2, CY3(NH~2 and CY3NHZS03) and carboxyalkyl cyanines (CYSCOOH, CY30(S03)2, CYS(S03)2 and CY7(S03)~ required as precursors for energy transfer fluorochromes were synthesised by the methods previously described in Ernst, L. A.
et al, "Cyanine Dye Labelling Reagents for Sulphydryl Groups", Cytcrmetry, Vol.10, pp.3-10, (1989), Hammer, F.M., "The Cyanine Dyes and Related Compounds", (Whey, pub.
New York 1964), Mujumdar, R.B.et al, "Cyanine Dye Reagents Containing Isothiocyanate Groups", Cytometry, Vo1.10, pp.ll-19, (1989); Mujumdar, R..B.et al, "Cyanine Dye Labelling Reagents: Sulphoindocyanine succinimidyl ester", Bioconjugate Chemistry, Vol.4, pp.105-111, ( 1993); Southwick, P. L. et al, "Cyanine Dye Labelling Reagents:
C:arboxymethylindocyanine succinimidyl esters", Cytometry, Vol.ll, pp.418-430, (1990).
The synthesis and properties of one amino-cyanine fluorochrome, CY3NH2S03 and its conjugation with the succinimidyl ester of CYS(S03)z to form Complex 1 is described below.
T'he spectral properties for all the fluorochromes are shown in Tables 3 and 4. The unsymmetrical trimethinecarbocyanine, CY3NHZS03, was synthesised in four steps. Refer to Table 1 for the structures (I) - (VI).

'Table I

Compound R' lf~z I H 1~I
II CHZPhth 1:~
III CHzPhth (CHz)SCOOH
IV S03 (CHZ)SCOOH
R
V S03- CH;,Phth VI 503 CHzNH., (CY3NHzS03) CHzPhth =

1.5. I Synthesis of 5-Phthalimidomethvl-1- E-card~vnent,rl)-2.3.3-trimgth,~rlindole ~III~
5-Phthalimidomethyl-2,3,3-trimethylindolenine (Ln was synthesised according to the procedure of Gale and Wilshire, "The Amidomethylation and Bromination of Fischer's Base.
The Preparation of Some New Polymethine Dyes", Aust.J.Chem., Vo1.30, pp.689-694, (1977).
Powdered N-hydroxymethylphthalimide (70g, 0.4mo1) was added in small portions over a period of 45 minutes to a stirred solution of 2,3,3-trimethyl-(3H)-indolenine (I) (70g, 0.44mo1) in concentrated sulphuric acid (360m1) at room temperature. The solution was stirred for 70 hrs at room temperature before being poured onto ice-water. Basification of the solution with conc. ammonium hydroxide gave a yellow powder which was filtered and dried ( 111 g, yield 80% , mp.180-182°C). 'H NMR (DMSO-db), 8, 7. $-7.95 (m, 4H, phthalimido), 7.4 (s, 1 H, 4-H), 7.38 (d, 1H, J=9.OHz, 6-H), 7.2 (d, 1H, J=9.OHz, 7-H), 4G.7 (s, 2H, -CHZ), 2.2 (s, 3H, CH3), 1.2 (s, 6H, -(CH3)2).
This dry powder (lOg, 0.03mo1) and 6-bromohexanoic acid (9.1g, 0.05mo1) were mixed in 1,2-dichlorobenzene (25m1) and heated at 125°C for 12 hours undc;r nitrogen. The mixture was cooled. 1,2-Dichlorobenzene was decanted and the solid mass was triturated with isopropanol until free powder was obtained (llg, yield 80% , mp.124-126°C). 'H NMR
(DMSO~, b, 7.8-7.95 (m, 4H, phthalimido), 7.4 (s, 1H, 4-H), 7.38 (d, 1H, J=9.OHz, 6-H), 7.2 (d, 1H, J=9.OHz, 7-H), 4.7 (s, 2H, -CHI, 4.5 (t, 2H, J=7.5Hz, a-CHI, 2.3 (t, 2H, J=7Hz, E-CHI, 1.99 (m, 2H, (i-CHI, 2.3-1.7 (m, 4H, Y-CH2 and. 8-CHZ merged with s of 6H-(CH3)~.
1.5.2 - s- i n IV
Compound (IV) was synthesised according to the procedure described previously by Mujumdar, R.B. et al, Bioconjugate Chemistry, (1993), supra. The potassium salt of 2,3,3-trimethylindoleninium-5-sulphonate ( 11 g, 0.04mo1) and 6-bromohexanoic acid (9. 8g, 0. 05 mol) were mixed in 1,2-dichlorobenzene, (100m1) and heated at 110°C for 12 hours under nitrogen.
The mixture was cooled. 1,2-Dichlorobenzene was decanted and the solid mass was triturated with isopropanol until free powder was obtained (llg, yield $0% ). a,max (water) 275nm: 'H-NMR (DZO), b, 8.13 (s, 1H, 4-H), 8.03 (dd, 1H, J=9.0, l.lHz, b-H:), 7.2 (d, 1H, J=9.OHz, T-H), 4.51 (t, 2H, J=7.5Hz, a-CHZ), 2.25 (t, 2H, J=7.5Hz, y-CH.), 1.99 (m, 2H, ~3-CHI, 1.35-1.66 (m, 4H, 8-CH2, Y-CHI, 1.61 (s, 6H, -(CH~~. Rf = 0.55 (C-18, water-methanol, 2.5 % ).
1.5.3 Synthesis of Intermediate (V) A solution of 1-(e-carboxypentyl)-2,3,3-trimethylindoleninium-5-sulphonate (IV) (lOg, 0.03mo1) and N,N-dimethylformamide (7.2g, 0.04mo1) in acetic acid (20m1) were heated to reflux for 1 hour. Acetic acid was removed on a rotary evaporator and the product was washed with ethyl acetate (3x50m1) whereupon a dark brown solid was obtained.
~. max (water) 415nm Rf = 0.32 (C-18, 25% methanol in water). The crude product thus obtained was used for the next reaction without further purification. The solid (3.$g) was dissolved in a mixture of acetic anhydride (lOml) and pyridine (5m1). 5-~Phthalimidomethyl-I-(E-carboxypentyl)-2,3,3-trimethylindole (III) (2.5g, 6mmo1) was added and the reaction mixture was heated to 110°C for 1 hour. The solution was cooled and .diluted with diethyl ether 1;500m1). Product separated in the form of a red powder from which supernatant fluid was removed by decanting. It was dissolved in a minimum volume of methanol and re-precipitated with 2-propanol. The product was collected on a filter paper and dried to yield 5.3g of compound (V). It was purified by flash column chromatography on reverse phase C-1$ using water methanol mixture as eluent, (1.6g, yield 30%). Amax (water) 554nm, E
1.3x105 T /mol.cm. 'H NMR (CD30D), 8, 8.5 (t, 1H, J = 14 Hz, p-proton of the bridge), 7.8-8.0 (m, 6H, 4 protons of the phthalimido group and 4-H and 6-H of the;
sulphoindole ring), 7.55 (s, 2H, 4'-H), 7.6 (d, 1H, J=l2Hz, 6'-H), 7.3 (two d, 2H, 7-H and 7'-H), 6.1-6.3 (t, 2H, cz, a'-protons of the bridge), 4.1 (m, 4H, a, a'-CHZ ), 2.9 (t, 2H, J = 7Hz, -CHZCOOH), l.4-2.0 (m, 21H, three -CHZ, one -CH3, and two -(CH3)2, methyl protons of the phthalimidomethyl group are merged in a water signal at 4.8.
1.5.4 HXdrolXsis d;~(Vl to dive I,VI) Compound (V) (l.g, l.lmmol) was dissolved in concentrated hydrochloric acid (5m1) and heated under reflux for 12 hours. After cooling, the crystalline phthalic acid was filtered off.
'fthe filtrate was concentrated with a rotary evaporator and then slowly neutralised with concentrated ammonium hydroxide while the temperature was kept below 30°C. Pure fluorochrome CY3NHzS03 (VI) was obtained by reverse phase column chromatography using a water-methanol mixture as eluent. 7~max (methanol) 552nm. 'H NMR (DMSO-d~, 8, 8.45 (t, J = 7.2Hz, 1H, 9-H), 7.3-7.9 (m, 6H, aromatic protons), 6.55 (dd, 2H, 8 and 8'-H), 4.5 {m, 4H, N-CHI, 4.1 (s, 2H, CHzNH~, 2.15 (t, 2H, CHZCOOH), a, a'-protons of the t>ridge), 4.1 (m, 4H, a, a'-CHZ ), 2.9 (t, 2H, J = 7Hz, -CHzCOOH), 1.25-1.8 (broad m, 24H, two -(CH~2 and 6-C-(CH3)~. Rf = 0.415 (RP C1$ 60% methanol in water).
1.5.5 Synthesis of Comvlex I
Dry powder of CY5(SO~2 succinimidyl ester (425mg, 0.26mmol) prepared by the method of Mujumdar et al, Bioconjugate Chemistry, Vol.4, pp.105-111, (1993), was added in small portions to a well stirred solution of CY3NHZS03 (200mg, 0.26mmo1) in lOml of carbonate -bicarbonate buffer (O.1M, pH 9.4). Stirring was continued for an additional 30 minutes after which the reaction was purified by flash column chromatography on C-1$ reverse powder using water-methanol (6.3:3.7) as eluent. 5ml fractions were collected and monitored by 7'LC. Fractions containing CY5(SO~2 acid and CY3NHxS03 were discarded. Violet coloured fractions were checked by ultraviolet light in methanol and the fractions containing Complex 1 fluorochrome (Table 2) were pooled. Evaporation of the solvent yielded Complex 1 as a violet powder, (yield 37%). Rf = 0.45 (RP 37% methanol-water). 1H NMR spectrum recorded in D20 showed broad signals and were difficult to assign. The fluorochrome was purified on a strongly acidic ion-exchange column (Dowex 50, H~~ form). High resolution F~AB mass spectrometry showed (M+H)+ ion at 1391.83 (C~3H91N5016S3 +H requires 1391.73).
1.5.6 Succinimidvl Ester of Energy Transfer ,~~ranine Dye C'.omplex 1 (60mg, 0.04mmol) was dissolved in a mixture of dry DMF ( 1 ml) and dry pyridine (O.OSmI). Disuocinimidyl carbonate (DSC) (46mg, 4.18mmol, 1.5 equiv/carboxyl group) was added and the mixture was stirred at 55-60°C for 90 minutes under nitrogen. After diluting the mixture with dry diethyl ether (20m1), the supernatant was decanted. The product was washed repeatedly with ether, filtered and dried under vacuum. The formation of the active succinimidyl ester was confirmed by its reaction with benzylamine in DMF or its reaction with taurine in a pH 9.4 bicmbonate buffer. Reversed phase C-18 TLC spotted with the conjugate, the succinimidyl ester and the hydrolysed carboxylate product fot comparison was developed with water-methanol (l : l) mixture. Rf = 0.78 (Acid), 0.3 (Benrylamine adduct).
1.5.7 Reaction of ccinirnidYl_ Ester ith Antibod~r and Stre tavidin A stock solution of Complex 1 tluorochrome succinimidyl active ester was made in dry DMF
(lmg/100p1). In one sample, one milligram sheep Y-globulin was dissolved in 0.2Sm1 carbonate/bicarbonate buffer (approximately 6.45nmoU0.25m1). In another example, streptavidin (Img) was dissolved in 0.25m1 of the carbonate/bicarbonate buffer. Appropriate volumes of ~e tluorochrome stock were added to 0.25m1 portions of each protein solution to produce the desired starting fluorochrvme to antibody ratios, and each reaction mixture was stirred at room temperature for 30 minutes. The protein conjugate was separated from unmacted fluorochrome in each sample by gel filtration chromatography over SephadeX'4'G-50 (0.7x20cm column), using PBS, pH 7.4, containing 0.196 azide. Dye conjugated proteins eluted as coloured bands well separated from the unreact~ fluaroehmme. The normalised excitation spectrum of the Complex I-stcnptavidin conjugate in PBS is shown in Figure S. The absorbance spectrum of Complex 1-Sheep IgI in PBS is shown is Pigure 6. Figure 7 shows the flow cytometry analysis of Complex 1-streptavidin used to detect CD3 antibody.
Furthei energy transfer donor r complexes according to the present invention were prepared from cyanine fluorochmmes in order oo investigate the energy transfer efficiency of such compounds. The structures of these analogues are shown in Table 2.
The spectral properties of the precursor cyanines are given in Table 3 and those of the complexes are shown in Table 4.

~~ so;
A
C:,Fi"
O~

_ _ p COOH ICOOH Complex 6 "A" designates the fluorochrome that acts as the energy acceptor and "D"
designates the fluorochrome that acts as the energy donor.
The energy transfer complexes shown in Table 2 are as follows: Complex l, CY3NHzS0, (Donor) + CYS(SO~z (Acceptor); Complex 2, CY3=O(SO~~ (Donor;) + CY3NHz (Acceptor);
Complex 3, CY3NH2 (Donor) + CYSCOOH (Acceptor); Complex 4, CY3NHz (Donor) +
CYS(S03)Z (Acceptor); Complex 5, CY3(NH~Z (Donor) + CY7(SO 3)2 (Acceptor);
Complex 6, ? CY3NHzSOj (Donor) + CYS(SO3)2 (Acceptor).

Table 3 Spectral Properties of Cy~nine D~ l~)sed as Precursors for the Fluorescent Energv Transfer Complexes of the Invention Dye Solvent Absorption Emission Quantum Yield Maximum (nm)Maximum (nm) (~) Amine containing Cyanine Dyes CY3NHz Methanol 552 569 0.05 PBS 548 563 0.05 CY3(NHZ)z Methanol 552 569 0.05 PBS 548 653 0.05 CY3NHZS03 Methanol 556 573 0.08 PBS I 548 I 65:3 0.09 Carboxyl containing Cyanine Dyes CYSCOOH Methanol 658 685 0.22 PBS 648 667 0,13 CY5(SO3)2 Methanol 658 677 0.4 PBS 650 667 0.27 CY3-O(S03)2 Methanol 492 506 0.2 PBS 486 500 0.09 CY7(S03)z Methanol 758 789 NDs 8 ND means not determined. PBS means phosphate-buffered saline.
The efficiency of energy transfer was estimated by calculating the amount of quenching of donor fluorescence that occurs (DQE) when the acceptor is attached. It is possible that some quenching could occur by pathways other than resonance energy transfer when the acceptor is bound. However, the cyanine donor preferred for the fluorescent labelling complexes of t1e present invention are relatively insensitive to their molecular environment, Furthermore, addition of large substituents to trimethine cyanines usually increases, rather than decreases, their fluorescence. Therefore, DQE may be equal to the efficiency of energy transfer. The estimated energy transfer efficiencies based on DQE measurements ranged 50% to 99% and flue wavelength shifts between the donor absorption maxima and the terminal acceptor emission maxima (DI) varied between 83nm and 294nm.

'Cwo of the complexes, 1 and 6, are capable of absorbing light at the argon laser wavelength, 488nm. Complex 1 contains a single donor and single acceptor, and Complex 6 contains 2 donors per acceptor. Complex 1 has 3 carboxyl groups and Complex 6 has 4 carboxyl groups.
'Chese are converted to succinimidyl active esters upon activation. Figure 2 shows the absorption spectra of Complex 1 and Complex 6 in methanol.
(Jomplex 1 was selected for further studies. As shown in Figures 3(a) and 3(b), the Wsorbance (solid line) of Complex 1 varies slightly in phosphate-buffered saline (Figure 3(b)) and methanol (Figure 3(a)) but fluorescence remains unchanged. The emission of the donor component at 572nm is very weak compared with the emission of the acceptor at 675nm, as would be expected when energy transfer is efficient.
Figure 5 demonstrates that sheep antibodies can be readily labelled with the activated Complex l . Conjugates made of Complex 1 conjugated to sheep IgG at various dye:protein ratios were tested. The lowest dye: protein ratio is represented by the line having its first peak (at about a!70nm) at 0.8 and the highest dye:protein ratio is represented by the; line having its first peak (at about 270nm) at a little less than 0.4. No dimer formation involving either the donor or the acceptor fluorochromes was observed with increasing dye: protein ratios.
Each Complex 1 contains up to 3 reactive groups. More reactive groups may be: used provided no cross-linking occurs. It is important to use labelling conditions that avoid protein cross-linking which quench the fluorescence. Cross-linking by doubly activated cyanines has been observed previously by Southwick, P. L. et al, "Cyanine Dye Labelling Reagents:
C'.arboxymethylindocyanine succinimidyl esters", Cytometry, Vol. l l, pp 418-430, (1990) and can be minimised by limiting the concentration of protein to be labelled to approximately 1 mg/ml.
Upon binding to antibodies, the quantum yield of the complex was enhanced three fold as shown in Table 4. It is believed that this occurs because the radiationless deactivation pathway of both the CY3 and CY5 components of Complex 1 are reduced because of their restricted mobility when bound to the surface of the protein. Other means of restricting conformational mobility are known to increase the fluorescence efficiency of cyanine fluorochromes, as described in Mujumdar, R.B. et al, "Cyanine Dye Labelling Reagents:
Sulphoindocyanine Succinimidyl Ester", Bioconjugate Chemistry, Vol.4, pp.105-111, (1993). In fact, when Complex 1 was dissolved in glycerine, the quantum yield increased by several fold, as shown in Table 4.
Activated Complex 1 can be used as a fluorescent label for 2 colour flow cytometry experiments with 488nm excitation. The scatter plot is shown in Figure 6.
Human T-lymphocytes were used to compare the Complex 1 label with another two-colour reagent, R-phycoerythrin, which also excites at 488nm and emits at 575nnn. Complex 1 labelled streptavidin (fluorochrome/protein ~4) was used to detect biotinylated CD3 antibody, which rr~arks all T-cells. In the same lymphocyte sample, phycoerythrin(PE)-labelled anti-CD4 was used to mark the Helper Cell subset of the T-cells. Thus, in the toW I
lymphocyte population there is a population of cells that contain neither CD3 nor CD4 (ie. CD3 and CD4 negative, 'Table 4 Spectral Properties of Energy Transfer Com l~ exes Dye Abs max Excitation Em Quantum Energy Wavelgth (nm) Wavelgth max Yield TransferredShift a (nm) (nm) {~) (%) {nm) Complex 556 (9.5),488 675 0.32 91 119 le 652 ( 14.3514 676 0.37 92 120 600 673 0.49 - -Complex 536 (16), 48$ 675 0.03 89 139 658 (16) 514 673 0.04 89 137 600 668 0.21 - -Complex 558, 658 488 674 0.11 95 116 1' (PBS) 514 673 0.13 95 116 600 676 0.14 - -Complex 562, 65$ 488 674 0.19 ND ND
ld 514 674 0.32 ND ND

600 674 0.39 ND ND

Complex 490 (13), 466 571 0.15 89 81 554 (9.5) , Complex 545 (9.5),514 679 0.08 83 133 658 (14.3) Complex 550 (9.4),514 674 0.2 96 124 4a 656 (14.2) Complex 445 (9.5),520 782 ND 99 226 5a 754 (14.4) Complex 556 (9.5),488 674 0.23 49 118 6a 652 ( 14.4)514 674 0. 24 50 118 600 674 0.34 - -Complex 548 20.0),488 566 0.05 43 118 652 ( 15.0)514 564 0.05 38 116 600 668 0.23 - -a - in methanol, b = in PBS, ° -- Complex 1 on streptavidin, d/p = 4 in glycerine, ' = difference between Em,~,~(A) - Ab",~(D) ND means not determined.

2~ ~a3o8 shown in the lower left population of the 2-dimensional scatter plot in Figure 6), a subset of Complex 1-labelled CD3-positive cells that do not have a phycoerythrin signal {ie. CD3 positive and CD4 negative, shown in the upper left population of Figure 6), and a third subset consisting of Complex 1-labelled cells that are phycoerythrin stained (ie. CD3 and CD4 positive, shown in the upper right population of Figure 6). It is clear that Complex 1 gave base-line separation of the positive and negative cell populations, and that there was minimal :pill over of Complex 1 fluorescence into the phycoerythrin channel. The Complex 1 ~luorochrome gave a three times brighter signal when the fluorochrome was excited at 514nm.
Ex 1 Several other complexes with the general structure shown in formula ( 10) below were synthesised. Table 5 shows their spectral properties in solution in methanol.

HR
( 10) 'I'hese series of spectra demonstrate efficient energy transfer with :long Stokes' shifts. Each emission spectrum shows substantially all of the emission coming from the final acceptor fluorochrome in each series with only minimal emission from either the donor fluorescein, or t:he intermediate cyanine.
lVlultiparameter analysis can be dune of multiple samples to detect the presence of target biological compounds. Each sample is labelled by well known labelling methods with a different complex. For example, one sample suspected of containing a target biological compound is incubated with a single fluorochrome, such as fluorescein, Cascade Blue, a BODIPY dye, or one of the monomethine rigidized dyes, or CY30(S03),, or CY3{SO,),, all emitting in the 500 - 575nm wavelength range (green to orange). A second sample suspected ~~H2~4 ~~~~4 U

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217a3~8 of containing the target biological compound (the same compound or a different compound as that in sample 1), is incubated with a complex of the invention, for example fluorescein-CY3NH2, which will absorb light at 48$nm and emits fluorescence at 574nm (orange).
Additional samples suspected of containing another target compound are incubated with other labelling complexes of the invention, such as fluorescein-CY3-CYS aaad fluorescein-CY3-CY7, hoth of which light at 4$8nm, but emit fluorescence at 672nm and 782nm respectively (red to near infra-red). After a suitable period to permit the fluorescent labels to bind with the target <;ompounds, unbound label is removed by washing and the labelled samples are mixed.
Detection is possible with a single wavelength excitation source, ie. a~.t laser line 488nm. Each differently labelled sample will fluoresce a different colour at the emission wavelength of its particular label. Those skilled in the; art will recognise that the fluorescent labelling complexes of the present invention can be used for a variety of immunofluoresc;ent techniques, including <iirect and indirect immunoassays, and other known fluorescent detection methods. The conditions of each incubation, eg. pH, temperature and time are known in the art, but generally room temperature is preferred. If reacting with an amine, pH 9.4 is preferred. The pH is adjusted depending on the optimum reaction conditions for the particular reactive groups according to known techniques.
The fluorescent labelling complexes may be used to form reagents by covalently binding the complexes to a carrier material, such as polymer particles, cells, glass beads, antibodies, proteins, enzymes, carbohydrates, lipids and nucleotides or nucleic acids (DNA
and RNA) and analogues which have been derivatised to include at least one first reactive group capable of forming a covalent bond with the functional group on the labelling complex (or a functional group capable of forming a covalent bond with a reactive group on the complex, as described above) and at least one second reactive group (or functional group, as the case may be), having specificity for, and being capable of forming a covalent bond with, a target biological compound, such as antibodies, cells, drugs, antigens, bactE;ria, viruses and other microorganisms. When the carrier has functional groups, it may be antibody or DNA suited for attachment to antigen or a complementary DNA sequence, respectively. When the carrier material has reactive groups on it, the carrier may be a polymer particle or an antigen suitable for attachment to DNA or an antibody for example. Techniques for covalently binding fluorochromes to carrier molecules such as those mentioned are well known in the art and readily available in the literature. The carrier material can further include nucleotides derivatised to contain one of amino, sulphydryl, carboxyl, carbonyl or hydroxyl groups, and oxy or deoxy polynucleic acids derivatised to contain one of amino, thiophosphoryl, sulphydryl, carboxyl, carbonyl or hydroxyl groups. The functional groups on the carrier material which are complementary to. ie. form covalent bonds with, t:he reactive groups of the labelling complexes of the invention include amino, sulphydryl, carboxyl, carbonyl and hydroxyl groups.
A comparison of the energy transfer complexes of the present invention to the conventional R-phycoerythrin dyes is shown in Table 6 below.

'Cable 6 Complex 2 vs R-Ph. cv oerXthrin R-PhvcQe~r~thrin y_, lp ex 2_ Excitation Wavelength4$8 488 (nm) Emission Wavelength 580 578 (nm) 488-laserline Flow- P>=; fluorescence Signals were stable was greatly Cytometer reduced at pH 8.5 throughout pH range.
and extinguished at pH

9.5.

M W 240000 166'7 Staining Do not penetrate readilyLabelled antibody into intracellular penfarates into intracellular tissues to reach target antigen.tissues to reach target antigen.

Binding Rate Rate of binding to Rapid binding.
antigen is low.

1'he energy transfer complexes of the present invention provide a valuable set of fluorescent labels which are particularly useful for multiparameter analysis and importantly, are sufficiently low in molecular weight to permit materials labelled with the fluorescent complexes to penetrate all structures. As such, the complexes are well suited for use as DNA
probes. The complexes of the invention and the reagents that can be made from them offer a wide variety of fluorescent labels with large Stokes' shifts. Those in the art will recognise that the complexes of the invention can be used in a variety of fluorescence applications over a wide range of the visible spectrum.

Fi ur s Figure 1 is a schematic illustration of the overlapping absorption and emission spectra of four cyanine fluorochromes that can be used in the energy transfer labelling complexes of the present invention.
Figure 2 illustrates the absorption spectra of two fluorescent labelling complexes, Complex 1 (solid line) in methanol, comprised of one cyanine donor and one cyanine acceptor, and (;omplex 6 (dotted line) in methanol, comprised of two cyanine donors and one cyanine acceptor.
Figures 3(a) and (b) illustrate the absorbance (solid line) and emission (dotted line) spectra of t~omplex 1 of the invention made of trimethine and pentamethine cyanine dyes in (a) methanol and (b) PBS.
Figure 4 illustrates the normalised excitation spectra of the Complex 1 in PBS
(solid line), methanol (- w-), glycerol (- - --), and Complex 1-streptavidin conjugate in PBS (-------).
Figure 5 illustrates the absorbance spectra in PBS of sheep IgG-Complex 1 conjugates at various dye molecule:protein ratios (1 - 4:1) demonstrating that no dimer formation involving either donor or acceptor is evident with increasing dye:protein ratios.
Figure 6 illustrates the two colour flow cytometry analysis of human lymphocytes labelled with anti-CD4-PE and anti-CD3-streptavidin-Complex 1 to mark the helper cell subset of T-cells and total T-cell subset, respectively, showing a subset of Complex 1 labelled cells without the I'E signal and a second subset of C'.omplex 1 labelled cells that is PE
stained.

Claims (93)

1. A low molecular weight fluorescent labelling complex, the complex comprising:
i) a first fluorochrome having first absorption and emission spectra;
ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome;
iii) at least one linker group for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes;
iv) at least one target bonding group capable of forming a covalent bond with a target compound.
wherein said target bonding group is a reactive group for reacting with a functional group on the target material and wherein at least one of said first or second fluorochromes is a cyanine dye and the combined molecular weight of said first and second fluorochromes and said linker group is less than 20,000 Daltons.

2. The complex according to claim 1 wherein said first and second fluorochromes are both cyanine dyes.

3. The complex according to claims 1 or 2 further comprising water solubilizing constituents attached thereto, said water solubilizing constituents being unreactive with said target bonding group.

4. The complex according to claim 3 wherein said water solubilizing constituents are selected from the group consisting of amide, sulphonate, sulphate, phosphate, quaternary ammonium, hydroxyl, guanidinium and phosphonate.

5. The complex according to claim 1 or 2 wherein said target bonding group is a reactive group selected from the group consisting of succinimidyl ester, isothiocyanate, isocyanate, haloacetamide, dichlorotriazine, maleimide, sulphonyl halide, alkylimidoester, arylimidoester, substituted hydrazine, substituted hydroxylamine, carbodiimide, acyl halide, anhydride, phosphoramidite, acrylate and acrylamide.

6. The complex according to claims 1 to 3 wherein the combine molecular weight of the said first and second fluorochromes and said linker is within the range of 500 to 10,000 Daltons.

7. The complex according to claim 1 further comprising a third fluorochrome having third absorption and emission spectra covalently attached to said second fluorochrome; the wavelength of the emission maximum of said third fluorochrome being longer than the wavelength of the emission maximum of said second fluorochrome and a portion of the emission spectrum of said second fluorochrome overlapping a portion of the absorption spectrum of said third fluorochrome such that excitation of said first fluorochrome produces fluorescence from said third fluorochrome.

8. The complex according to claim 7 further comprising water solubilizing constituents attached thereto, said water solubilizing constituents being unreactive with said target bonding group.

9. The complex according to claim 7 or 8 wherein said first fluorochrome is selected from the group consisting of monomethine rigidized cyanine dyes, a trimethine cyanine dye, fluorescein, pyrene trisulphonate, bispyrromethine boron difluoride dyes and said second and third fluorochromes are polymethine cyanine dyes.

10. The complex according to claim 1 further comprising either:
i) a plurality of said first fluorochromes each covalently attached through a linker to said second fluorochrome and each of said first fluorochromes being capable, upon excitation with light, of transferring energy to said second fluorochrome; or, ii) a plurality of said second fluorochromes each covalently attached through a linker to said first fluorochrome and each of said second fluorochromes being capable of accepting energy from said first fluorochrome when said first fluorochrome is excited by light; and at least one target bonding group capable of forming a covalent bond with a target compound.

11. The complex according to claim 10 further comprising water solubilizing constituents attached thereto, said water solubilizing constituents being unreactive with said target bonding group.

12. The complex according to claims 10 or 11 wherein said water solubilizing constituents are selected from the group consisting of amide, sulphonate, sulphate, phosphate, quaternary ammonium, hydroxyl, guanidinium and phosphonate.

13. The complex according to claims 10 or 11 wherein said target bonding group is a reactive group selected from the group consisting of succinimidyl ester, isothiocyanate, isocyanate, haloacetamide, dichlorotriazine, maleimide, sulphonyl halide, alkylimidoester, arylimidoester, substituted hydrazine, substituted hydroxylamine, carbodiimide, acyl halide, anhydride, phosphoramidite, acrylate and acrylamide.

14. A reagent comprising:

A) A fluorescent water soluble labelling complex comprised of:
i) one or more low molecular weight first fluorochromes, each having first absorption and emission spectra, covalently attached through a linkergroup to one or more low molecular weight second fluorochromes, each having second absorption and emission spectra, and wherein the wavelength of the emission maximum of at least one said second fluorochrome is longer than the wavelength of the emission maximum of at least one said first fluorochrome and a portion of the absorption spectrum of at least one said second fluorochrome overlaps a portion of the emission spectrum of at least one said first fluorochrome for transfer of energy absorbed by said first fluorochrome upon excitation with light to said second fluorochrome; ii) at least one bonding group capable of forming a covalent bond with a carrier material; and, iii) at least one water solubilizing constituent attached to said complex, said water solubilizing constituent being unreactive with said at least one bonding group, wherein at least one of said first or second fluochromes is a cyanine dye; and B) a carrier material having a group that reacts with said bonding group of said complex and is covalently bound thereto.

15. The reagent according to claim 14, wherein said first and second fluorochromes are both cyanine dyes.

16. The reagent according to claim 14 or 15, wherein said carrier material has a functional group selected from the group consisting of amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate and said carrier material is selected from the group consisting of antibody, lipid, protein, carbohydrate, nucleotide derivatized to contain one of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups and oxy or deoxy polynucleic acids derivatized to contain one of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups.

17. A method of labelling a carrier material comprising incubating an aqueous sample containing a carrier material with a low molecular weight, water soluble fluorescent labelling complex comprised of:
i) a first fluorochrome having first absorption and emission spectra covalently linked to a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and the absorption spectrum of said second fluorochrome overlapping the emission spectrum of said first fluorochrome for transfer of energy absorbed by said first fluorochrome upon excitation with light to said second fluorochrome, wherein at least one of said first or second fluochromes is a cyanine dye;

ii) a target bonding group capable of forming a covalent bond with a complementary group of said carrier material, and iii) water solubilising constituents for conferring a polar characteristic to said complex, said water solubilising constituents being unreactive with said bonding group, for a period of time sufficient for covalently binding said bonding group of said complex to said complementary group of said carrier material.

18. Use of the reagent according to any one of claims 14 to 16 for analysis or detection employing fluorescent emission.

19. Use of the complex according to any one of claims 1 to 13 in a reagent for analysis or detection employing fluorescent emission.

20. A set of fluorescent labeling complexes each of said complexes comprising:
i) a first fluorochrome having first absorption and emission spectra;
ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome, the first and second fluorochromes being selected from the cyanine dyes, fluoresceins, rhodamines, pyrene trisulphonates and derivatives of the bispyrromethine boron difluoride dyes;
iii) at least one linker group having between 2 and 20 bond lengths for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes;
iv) at least one target bonding group capable of forming a covalent bond with a target compound wherein the target bonding group is a reactive group for reacting with a functional group on the target material;
wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons, wherein at least two complexes of said set absorb at the same wavelength and emit (fluoresces) at significantly different emission wavelengths, and wherein at least one of said first or second fluorochromes in each of said complexes is a cyanine dye.

21. A set according to claim 20 wherein each of said complexes further comprises water solubilizing constituents attached thereto, said water solubilizing constituents being unreactive with said target bonding group.

22. A set according to claim 21 wherein said water solubilizing constituents are selected from the group consisting of amide, sulphonate, sulphate, phosphate, quaternary ammonium, hydroxyl, guanidinium and phosphonate.

23. A set according to claim 20, 21 or 22, wherein said target bonding group in each of said complexes is a reactive group selected from the group consisting of succinimidyl ester, isothiocyanate, isocyanate, haloacetamide, dichlorotriazine, maleimide, sulphonyl halide, alkylimidoester, arylimidoester, substituted hydrazine, substituted hydroxylamine, carbodiimide, acyl halide, anhydride, phosphoramidite, acrylate and acrylamide.

24. A set according to claim 20, 21, 22 or 23, wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is within the range of 500 to 10,000 Daltons.

25. A set according to claim 20, 21, 22, 23 or 24, wherein one or more of said fluorescent labeling complexes further comprises a third fluorochrome having third absorption and emission spectra covalently attached to said second fluorochrome; the wavelength of the emission maximum of said third fluorochrome being longer than the wavelength of the emission maximum of said second fluorochrome and a portion of the emission spectrum of said second fluorochrome overlapping a portion of the absorption spectrum of said third fluorochrome such that excitation of said first fluorochrome produces fluorescence from said third fluorochrome.

26. A set according to claim 25 wherein one or more of said fluorescent labeling complexes further comprise water solubilizing constituents attached thereto, said water solubilizing constituents being unreactive with said target bonding groups on each of said complexes.

27. A set according to claim 25 wherein said first fluorochromes are selected from the group consisting of monomethine rigidized cyanine dyes, a trimethine cyanine dye, fluorescein, pyrene trisulphonate, bispyrromethine boron difluoride dyes and said second and third fluorochromes are polymethine cyanine dyes.

28. A set of fluorescent labeling complexes each of said complexes comprising:
i) a first fluorochrome having first absorption and emission spectra;
ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome;
iii) at least one linker group chosen from the group consisting of alkyl chains containing from 1 to 15 carbon atoms, which may optionally include oxygen atoms as ether linkages, or nitrogen atoms as amine linkages, or CONH groups as amide linkages, for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochomes;
iv) at least one target bonding group capable of forming a covalent bond with a target compound;
wherein at least one of said first or second fluorochromes in each of said complexes is a cyanine dye and wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons.

29. A set of reagents each reagent comprising:
A) a fluorescent water soluble labeling complex comprised of:
i) one or more low molecular weight first fluorochromes, each having first absorption and emission spectra, covalently attached through a linker group to one or more low molecular weight second fluorochromes, each having second absorption and emission spectra, and wherein the wavelength of the emission maximum of at least one said second fluorochrome is longer than the wavelength of the emission maximum of at least one said first fluorochrome and a portion of the absorption spectrum of at least one said second fluorochrome overlaps a portion of the emission spectrum of at least one said first fluorochrome for transfer of energy absorbed by said first fluorochrome upon excitation with light to said second fluorochrome;
ii) at least one target bonding group capable of forming a covalent bond with a carrier material; and, iii) at least one water solubilizing constituent attached to said complex, said water solubilizing constituent being unreactive with said at least one target bonding group;
wherein at least one of said first or second fluorochromes is a cyanine dye and the linker group is chosen from the group consisting of alkyl chains containing from 1 to 15 carbon atoms, which may optionally include oxygen atoms as ether linkages, or nitrogen atoms as amine linkages, or CO--NH groups as amide linkages;
B) a Garner material having a group that reacts with said target bonding group of said complex and is covalently bound thereto.

30. A set according to claim 29 wherein said first fluorochrome is selected from the group consisting of monomethine rigidized cyanine dyes, a trimethine cyanine dye, fluorescein, pyrene trisulphonate, bispyrromethine boron difluoride dyes and said second fluorochrome is a polymethine cyanine dye.

31. A set according to claim 29 wherein said carrier material has a functional group selected from the group consisting of amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate and said carrier material is selected from the group consisting of antibody, lipid, protein, carbohydrate, nucleotide derivatized to contain one of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups and oxy or deoxy polynucleic acids derivatized to contain one of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups.

32. A method of labeling a carrier material with one of a set of fluorescent labeling complexes, comprising incubating an aqueous sample containing a carrier material with a low molecular weight, water soluble fluorescent labeling complex comprised of:
i) a first fluorochrome having first absorption and emission spectra covalently linked through a linker group to a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and the absorption spectrum of said second fluorochrome overlapping the emission spectrum of said first fluorochrome for transfer of energy absorbed by said first fluorochrome upon excitation with light to said second fluorochrome, wherein at least one of said first or second fluorochromes in said complex is a cyanine dye;
ii) a target bonding group attached to said complex capable of forming a covalent bond with a complementary group of said carrier material, and iii) water solubilizing constituents attached to said complex for conferring a polar characteristic to said complex, said water solubilizing constituents being unreactive with said bonding group;
for a period of time sufficient for covalently binding said bonding group of said complex to said complementary group of said carrier material.

33. Use of a set of fluorescent labeling complexes for analysis or detection comprising incubating a fluorescent labeling complex of said set with at least one target material, each of said fluorescent labeling complexes comprising:
i) a first fluorochrome having first absorption and emission spectra;
ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome, the first and second fluorochromes being selected from the cyanine dyes, fluoresceins, rhodamines, pyrene trisulphonates and derivatives of the bispyrromethine boron difluoride dyes;
iii) at least one linker group having between 2 and 20 bond lengths, which may optionally include oxygen atoms as ether linkages, or nitrogen atoms as amine linkages, or CONH groups as amide linkages, for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes;
iv) at least one target bonding group capable of forming a covalent bond with a target compound wherein the target bonding group is a reactive group for reacting with a functional group on the target material;
wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons, wherein at least one of said first and second fluorochromes in each of said complexes is a cyanine dye, and wherein at least two complexes of said set absorb at the same wavelength and emit (fluoresces) at significantly different emission wavelengths; and measuring and comparing the differences in fluorescent emission between said fluorescent labeling complexes.

34. Use of a set of fluorescent labeling complexes for analysis and detection comprising incubating a fluorescent labeling complex of said set with at least one target material, each of said fluorescent labeling complexes comprising:
i) a first fluorochrome having first absorption and emission spectra;
ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome;
iii) at least one linker group chosen from the group consisting of alkyl chains containing from 1 to 15 carbon atoms, which may optionally include oxygen atoms as ether linkages, or nitrogen atoms as amine linkages; or CONH groups as amide linkages, for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes;
iv) at least one target bonding group capable of forming a covalent bond with a target compound;
wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons, and wherein at least one of said first or second fluorochromes in each of said complexes is a cyanine dye; and measuring and comparing the differences in fluorescent emission between said fluorescent labeling complexes.

35. A carrier material or biological compound labeled with one of a set of fluorescent labeling complexes according to claim 20 in which the reactive group of said complex is covalently bound to a functional group on the target material.

36. A carrier material or biological compound covalently labeled with one of a set of fluorescent labeling complexes according to claim 28.

37. A method of labeling a carrier material or target compound by the use of one of a set of fluorescent labeling complexes according to claim 20, said method comprising incubating said complex with the carrier material or target compound under conditions suitable for the formation of a covalent linkage between said complex and said carrier material or target compound.

38. A method according to claim 37 wherein said Garner material or target compound is selected from the group consisting of antibodies, antigens, proteins, carbohydrates, lipids, nucleotides derivatized to contain one of amino, hydroxyl, sulphydryl, carboxyl or carbonyl groups, and oxy or deoxy polynucleic acids derivatized to contain one of amino, hydroxyl, phosphate, thiophosphoryl, sulphydryl, carboxyl or carbonyl groups, cells, polymer particles, or glass beads.

39. A method of labeling a carrier material or target compound by the use of one of a set of fluorescent labeling complexes according to claim 28, said method comprising incubating said complex with the carrier material or target compound under conditions suitable for the formation of a covalent linkage between said complex and said carrier material or target compound.

40. A method according to claim 39 wherein the carrier material or target compound is selected from the group consisting of antibodies, antigens, proteins, carbohydrates, lipids, nucleotides derivatized to contain one of amino, hydroxyl, sulphydryl, carboxyl or carbonyl groups, and oxy or deoxy polynucleic acids derivatized to contain one of amino, hydroxyl, phosphate, thiophosphoryl, sulphydryl, carboxyl or carbonyl groups, cells, polymer particles, or glass beads.

41. A set of reagents comprising fluorescent labeling complexes according to claim 20 each complex absorbing at the same wavelength and emitting at significantly different emission wavelengths, each complex being linked through a covalent bond to a carrier material or target compound selected from the group consisting of antibodies, antigens, proteins, carbohydrates, lipids, nucleotides derivatized to contain one of amino, hydroxyl, sulphydryl, carboxyl or carbonyl groups, and oxy or deoxy polynucleic acids derivatized to contain one of amino, hydroxyl, phosphate, thiophosphoryl, sulphydryl, carboxyl or carbonyl groups, cells, polymer particles, or glass beads.

42. A set of reagents comprising fluorescent labeling complexes according to claim 28 each complex absorbing at the same wavelength and emitting at significantly different emission wavelengths, each complex being linked through a covalent bond to a Garner material or target compound selected from the group consisting of antibodies, antigens, proteins, carbohydrates, lipids, nucleotides derivatized to contain one of amino, hydroxyl, sulphydryl, carboxyl or carbonyl groups, and oxy or deoxy polynucleic acids derivatized to contain one of amino, hydroxyl, phosphate, thiophosphoryl, sulphydryl, carboxyl or carbonyl groups, cells, polymer particles, or glass beads.

43. The method of claim 32, wherein there are different kinds of carrier materials and different kinds of labeling complexes within said set wherein the emission maximum of the second fluorochromes of each different kind of fluorescent labeling complex of said set is different from the at least one other kind of labeling complexes, and said labeling comprises covalently binding one kind of carrier material with one of said set of fluorescent labeling complexes and covalently binding a different kind of said carrier material with a different kind of said set of fluorescent labeling complexes.

44. The method of claim 33, wherein there are first and second carrier materials and wherein said incubating comprises covalently binding said first carrier material with of said set of fluorescent labeling complexes and covalently binding said second carrier material with the another of said set of fluorescent labeling complexes, wherein said set of fluorescent labeling complexes includes complexes that differ from each other in their respective emission wavelengths.

45. The method of claim 35, wherein there are first and second carrier materials and wherein said incubating comprises covalently binding said first carrier material with one of said set of fluorescent labeling complexes and covalently binding said second carrier material with the another of said set of fluorescent labeling complexes, wherein said set of fluorescent labeling complexes includes complexes that differ from each other in their respective emission wavelengths.

46. A set of fluorescent labeling complexes each of said complexes comprising:
i) a first fluorochrome having first absorption and emission spectra;
ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome, the first and second fluorochromes being selected from the cyanine dyes, fluoresceins, rhodamines, pyrene trisulphonates and derivatives of the bispyrromethie boron difluoride dyes;
iii) at least one linker group having between 2 and 20 bond lengths for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes;
iv) at least one target bonding group capable of forming a covalent bond with a target compound wherein the target bonding group is a reactive group for reacting with a functional group on the target material;

wherein said fluorochromes and said linker in each of said complexes, the relative orientation of the transition moments of the fluorochromes during the excited state lifetime of the first fluorochrome, and the proximity of the fluorochromes, are selected such that there is sufficient energy transfer;
wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons, wherein at least two complexes of said set absorb at the same wavelength and emit (fluoresces) at significantly different emission wavelengths, and wherein at least one of said first or second fluorochromes in each of said complexes is a cyanine dye.

47. A set of fluorescent labeling complexes each of said complexes comprising:
i) a first fluorochrome having first absorption and emission spectra;
ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome, the first and second fluorochromes being selected from the cyanine dyes, fluoresceins, rhodamines, pyrene trisulphonates and derivatives of the bispyrromethine boron difluoride dyes;
iii) at least one linker group having between 2 and 20 bond lengths for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes, wherein said linker group optimally orients the transition moments of said first and second fluorochromes;
iv) at least one target bonding group capable of forming a covalent bond with a target compound wherein the target bonding group is a reactive group for reacting with a functional group on the target material;
wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons, wherein at least two complexes of said set absorb at the same wavelength and emit (fluoresces) at significantly different emission wavelengths, and wherein at least one of said first or second fluorochromes in each of said complexes is a cyanine dye.

48. A set of fluorescent labeling complexes each of said complexes comprising:
i) a first fluorochrome having first absorption and emission spectra;
ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome, the first and second fluorochromes being selected from the cyanine dyes, fluoresceins, rhodamines, pyrene trisulphonates and derivatives of the bispyrromethine boron difluoride dyes;
iii) at least one linker group having between 2 and 20 bond lengths for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes, wherein said linker group is rigid;
iv) at least one target bonding group capable of forming a covalent bond with a target compound wherein the target bonding group is a reactive group for reacting with a functional group on the target material;
wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons, wherein at least two complexes of said set absorb at the same wavelength and emit (fluoresces) at significantly different emission wavelengths, and wherein at least one of said first or second fluorochromes in each of said complexes is a cyanine dye.

49. A set of fluorescent labelling complexes each of said complexes comprising:
i) a first fluorochrome having first absorption and emission spectra;
ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome;
iii) at least one linker group of between 2 and 20 bond lengths for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes;
iv) at least one bonding group capable of forming a covalent bond with a target compound;
wherein at least one of said first or second fluorochromes in each of said complexes is a cyanine dye and wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons.

50. A set of fluorescent labelling complexes according to claim 49 wherein each of said complexes further comprises water solubilizing constituents attached thereto, said water solubilizing constituents being unreactive with said bonging group of said complex.

51. A set of fluorescent labelling complexes according to claim 50 wherein said water solubilizing constituents are selected from the group consisting of amide, sulphonate, sulphate, phosphate, quaternary ammonium, hydroxyl, guanidinium and phosphonate.

52. A set of fluorescent labelling complexes according to claim 49 or 50, wherein said bonding group in each of said complexes is a reactive group selected from the group consisting of succinimidyl ester, isothiocyanate, isocyanate, haloacetamide, dichlorotriazine, maleimide, sulphonyl halide, alkylimidoester, arylimidoester, substituted hydrazine, substituted hydroxylamine, carbodiimide, acyl halide, anhydride, phosphoramidite, acrylate and acrylamide.

53. A set of fluorescent labelling complexes according to claim 49, 50 or 51, wherein the combined molecular weight of the said first and second fluorochromes and said linker group in each of said complexes is within the range of 500 to 10,000 Daltons.

54. A set of fluorescent labelling complexes according to claim 49 wherein one or more of said fluorescent labelling complexes of said set of fluorescent labeling complexes further comprises a third fluorochrome having third absorption and emission spectra covalently attached to said second fluorochrome; the wavelength of the emission maximum of said third fluorochrome being longer than the wavelength of the emission maximum of said second fluorochrome and a portion of the emission spectrum of said second fluorochrome overlapping a portion of the absorption spectrum of said third fluorochrome such that excitation of said first fluorochrome produces fluorescence from said third fluorochrome.

55. A set of fluorescent labelling complexes according to claim 54 wherein one or more of said fluorescent labelling complexes further comprise water solubilizing constituents attached thereto, said water solubilizing constituents being unreactive with said bonding groups on each of said complexes.

56. A set of fluorescent labelling complexes according to claim 54 wherein said first fluorochromes are selected from the group consisting of monomethine rigidized cyanine dyes, a trimethine cyanine dye, fluorescein, pyrene trisulphonate, bispyrromethine boron difluoride dyes and said second and third fluorochromes are polymethine cyanine dyes.

57. A set of fluorescent labelling complexes each of said complexes including:
i) a first fluorochrome having first absorption and emission spectra;
ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome, the first and second fluorochromes being selected from the cyanine dyes, fluoresceins, rhodamines, pyrene trisulphonates and derivatives of the bispyrromethine boron difluoride dyes;
iii) at least one rigid linker group having between 2 and 20 bond lengths for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes;
iv) at least one bonding group capable of forming a covalent bond with a target compound wherein the bonding group is a reactive group for reacting with a functional group on the target compound;
wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons, and wherein at least two complexes of said set absorbs at the same wavelength and emits at significantly different emission wavelengths.

58. A set of fluorescent labelling complexes according to claim 57 wherein each of said complexes further includes water solubilizing constituents attached thereto, said water solubilizing constituents being unreactive with said bonding group.

59. A set of fluorescent labelling complexes according to claim 58 wherein said water solubilizing constituents are selected from the group consisting of amide, sulphonate, sulphate, phosphate, quaternary ammonium, hydroxyl, guanidinium and phosphonate.

60. A set of fluorescent labelling complexes according to claim 57, 58 or 59, wherein said bonding group in each of said complexes is a reactive group selected from the group consisting of succinimidyl ester, isothiocyanate, isocyanate, haloacetamide, dichlorotriazine, maleimide, sulphonyl halide, alkylimidoester, arylimidoester, substituted hydrazine, substituted hydroxylamine, carbodiimide, acyl halide, anhydride, phosphoramidite, acrylate and acrylamide.

61. A set of fluorescent labelling complexes according to claim 57 wherein the combined molecular weight of the said first and second fluorochromes and said linker group in each of said complexes is within the range of 500 to 10,000 Daltons.

62. A set of fluorescent labelling complexes according to claim 57 wherein one or more of said fluorescent labelling complexes further includes a third fluorochrome having third absorption and emission spectra covalently attached to said second fluorochrome; the wavelength of the emission maximum of said third fluorochrome being longer than the wavelength of the emission maximum of said second fluorochrome and a portion of the emission spectrum of said second fluorochrome overlapping a portion of the absorption spectrum of said third fluorochrome such that excitation of said first fluorochrome produces fluorescence from said third fluorochrome.

63. A set of fluorescent labelling complexes according to claim 62 wherein one or more of said fluorescent labelling complexes further include water solubilizing constituents attached thereto, said water solubilizing constituents being unreactive with said bonding groups on each of said complexes.

64. A set of fluorescent labelling complexes according to claim 62 wherein said first fluorochromes are selected from the group consisting of monomethine rigidized cyanine dyes, a trimethine cyanine dye, fluorescein, pyrene trisulphonate, bispyrromethine boron difluoride dyes and said second and third fluorochromes are polymethine cyanine dyes.

65. A set of reagents, each reagent of the set comprising:
A) a fluorescent water soluble labelling complex having at least:
i) one or more low molecular weight first fluorochromes, each having first absorption and emission spectra, covalently attached through a linker group of to 20 bond lengths to one or more low molecular weight second fluorochromes, each having second absorption and emission spectra, and wherein the Wavelength of the emission maximum of at least one said second fluorochrome is longer than the wavelength of the emission maximum of at least one said first fluorochrome and a portion of the absorption spectrum of at least one said second fluorochrome overlaps a portion of the emission spectrum of at least one said first fluorochrome for transfer of energy absorbed by said first fluorochrome upon excitation with light to said second fluorochrome;
ii) at least one bonding group capable of forming a covalent bond with a carrier material; and, iii) at least one water solubilizing constituent attached to said complex, said water solubilizing constituent being unreactive with said at least one bonding group;
and, B) a carrier material having one of a functional group or a reactive group that reacts with said bonding group of said complex and is covalently bound thereto;
wherein the bonding group of each said complex is one of a reactive group for reacting with a functional group on said carrier material or a functional group for reacting with a reactive group on said carrier material, and at least one of said first or second fluorochromes is a cyanine dye.

66. A set of reagents according to claim 65 wherein said first fluorochrome is selected from the group consisting of monomethine rigidized cyanine dyes, a trimethine cyanine dye, fluorescein, pyrene trisulphonate, bispyrromethine boron difluoride dyes and said second fluorochrome is a polymethine cyanine dye.

67. A set of reagents according to claim 65 wherein said carrier material has a functional group selected from the group consisting of amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate and said carrier material is selected from the group consisting of antibody, lipid, protein, carbohydrate, nucleotide derivatized to contain one of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups and oxy or deoxy polynucleic acids derivatized to contain one of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups.

68. A method of analysis or detection of multiple target compounds comprising:
incubating a first reagent of a set of reagents with a first one of said multiple target materials, each of said reagents of said set of reagents comprising:
A) a fluorescent water soluble labeling complex comprised o~
i) one or more low molecular weight first fluorochromes, each having first absorption and emission spectra, covalently attached through a linker group of to 20 bond lengths to one or more low molecular weight second fluorochromes, each having second absorption and emission spectra, and wherein the wavelength of the emission maximum of at least one said second fluorochrome is longer than the wavelength of the emission maximum of at least one said first fluorochrome and a portion of the absorption spectrum of at least one said second fluorochrome overlaps a portion of the emission spectrum of at least one said first fluorochrome for transfer of energy absorbed by said first fluorochrome upon excitation with light to said second fluorochrome;
ii) at least one reactive group capable of forming a covalent bond with a carrier material; and, iii) at least one water solubilizing constituent attached to said complex, said water solubilizing constituent being unreactive with said at least one reactive group;
wherein at least one of said first or second fluorochromes is a cyanine dye;
B) a carrier material having a functional group that reacts with said reactive group of said complex and is covalently bound thereto, wherein said functional group is selected from the group consisting of amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate and said carrier material is selected from the group consisting of antibody, lipid, protein, carbohydrate, nucleotide that contains one of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups and oxy or deoxy polynucleic acids that contains one of an amino, sulphydryl, carbonyl, hydroxyl and carboxyl, phosphate and thiophosphate groups;
wherein said fluorescent labeling complexes of each of said set of reagents absorbs at the same wavelength and emits at significantly different emission wavelengths;

wherein the incubating step continues for a period of time sufficient to permit said first reagent to bind to said first target compound to form a reagent labeled-first target compound;
incubating a second reagent of said set of reagents with a second one of said multiple target materials, wherein the incubating step continues for a period of time sufficient to permit said second reagent to bind to said second target compound to form a reagent labeled-second target compound; and, measuring and comparing the differences in fluorescent emission between said labeling complexes of said reagent labeled-first target compound and said reagent labeled-second target compound.

69. A carrier material labelled with one of a set of fluorescent labelling complexes according to claim 49 in which the reactive group of said complex is covalently bound to a functional group on the carrier material.

70. A carrier material covalently labelled with one of a set of fluorescent labelling complexes according to claim 57.

71. A method comprising incubating one complex of a set of fluorescent labelling complexes according to claim 49 with one of a carrier material or a target compound under conditions suitable for the formation of a covalent linkage between said one complex and one of said carrier material or target compound.

72. A method comprising incubating one complex of a set of fluorescent labelling complexes according to claim 57 with one of a carrier material or a target compound under conditions suitable for the formation of a covalent linkage between said one complex and one of said carrier material or target compound.

73. A method according to claim 71 wherein said carrier material or target compound is selected from the group consisting of antibodies, antigens, proteins, carbohydrates, lipids, nucleotides that contain one of amino, hydroxyl, sulphydryl, carboxyl or carbonyl groups, and oxy or deoxy polynucleic acids that contain one of amino, hydroxyl, phosphate, thiophosphoryl, sulphydryl, carboxyl or carbonyl groups, cells, polymer particles, or glass beads.

74. A method according to claim 72 wherein said carrier material or target compound is selected from the group consisting of antibodies, antigens, proteins, carbohydrates, lipids, nucleotides that contain one of amino, hydroxyl, sulphydryl, carboxyl or carbonyl groups, and oxy or deoxy polynucleic acids that contain one of amino, hydroxyl, phosphate, thiophosphoryl, sulphydryl, carboxyl or carbonyl groups, cells, polymer particles, or glass beads.

75. A set of reagents comprising the fluorescent labelling complexes according to claim 49, each complex absorbing at the same wavelength and emitting at significantly different emission wavelengths, each complex being linked through a covalent bond to a carrier material selected from the group consisting of antibodies, antigens, proteins, carbohydrates, lipids, nucleotides that contain one of amino, hydroxyl, sulphydryl, carboxyl or carbonyl groups, and oxy or deoxy polynucleic acids that contain one of amino, hydroxyl, phosphate, thiophosphoryl, sulphydryl, carboxyl or carbonyl groups, cells, polymer particles, or glass beads.

76. A set of reagents comprising fluorescent labelling complexes according to claim 49, each complex absorbing at the same wavelength and emitting at significantly different emission wavelengths, each complex being linked through a covalent bond to a carrier material selected from the group consisting of antibodies, antigens, proteins, carbohydrates, lipids, nucleotides that contain one of amino, hydroxyl, sulphydryl, carboxyl or carbonyl groups, and oxy or deoxy polynucleic acids that contain one of amino, hydroxyl, phosphate, thiophosphoryl, sulphydryl, carboxyl or carbonyl groups, cells, polymer particles, or glass beads.

77. A method of analysis or detection of multiple target compounds comprising:
incubating a first one of a fluorescent labeling complex of a set of a fluorescent labeling complexes with a first one of multiple target compounds, each complex of said set of fluorescent labeling complexes having at least:
i) a first fluorochrome having first absorption and emission spectra;
ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome, the first and second fluorochromes being selected from the cyanine dyes, fluoresceins, rhodamines, pyrene trisulphonates and derivatives of the bispyrromethine boron difluoride dyes;
iii) at least one linker group having between 2 and 20 bond lengths for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes;
iv) at least one reactive group capable of forming a covalent bond with a functional group on said target compound;
wherein at least one of said first or second fluorochromes in each of said complexes is a cyanine dye;
wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons, and wherein at least two complexes of said set absorbs at the same wavelength and emits at significantly different emission wavelengths;

wherein the incubating step continues for a period of time sufficient to permit said reactive group of said first complex to covalently bind to the functional group of said first target compound to form a labeled-first target compound;
incubating a second one of said fluorescent labeling complexes of said set of a fluorescent labeling complexes with a second one of said multiple target compounds, wherein the incubating step continues for a period of time sufficient to permit said reactive group of said second complex to covalently bind to the functional group of said second target compound to form a labeled-second target compound;
removing any unbound fluorescent labeling complexes; and, measuring and comparing the differences in fluorescent emission between fluorescent labeling complexes of said labeled-first target compound and said labeled-second target compound.

78. A method of analysis or detection of multiple target compounds comprising:
incubating a first reagent of a set of reagents with a first one of multiple target compounds, each reagent of said set of reagents comprising:
A) a fluorescent water soluble labeling complex comprised of:
i) one or more low molecular weight first fluorochromes, each having first absorption and emission spectra, covalently attached through a linker group of to 20 bond lengths to one or more low molecular weight second fluorochromes, each having second absorption and emission spectra, and wherein the wavelength of the emission maximum of at least one said second fluorochrome is longer than the wavelength of the emission maximum of at least one said first fluorochrome and a portion of the absorption spectrum of at least one said second fluorochrome overlaps a portion of the emission spectrum of at least one said first fluorochrome for transfer of energy absorbed by said first fluorochrome upon excitation with light to said second fluorochrome;
ii) at least one bonding group capable of forming a covalent bond with a carrier material; and, iii) at least one water solubilizing constituent attached to said complex, said water solubilizing constituent being unreactive with said at least one bonding group;
wherein the bonding group is one of a reactive group for reacting with a functional group on said carrier material or a functional group for reacting with a reactive group on said carrier, and, at least one of said first or second fluorochromes is a cyanine dye;
B) a carrier material having one of a functional group that reacts with a reactive group on said complex or a reactive group that reacts with a functional group on said complex and is covalently bound thereto;

wherein said fluorescent labeling complexes of each of Said set of reagents absorbs at the same wavelength and emits at significantly different emission wavelengths;

wherein the incubating step continues for a period of time sufficient to permit said first reagent to bind to said first target compound to form a reagent labeled-first target compound;

incubating a second reagent of said set of reagents with a second one of said multiple target compounds, wherein the incubating step continues for a period of time sufficient to permit said second reagent to bind to said second target compound to form a reagent labeled-second target compound;

removing any unbound reagent; and;

measuring and comparing the differences in fluorescent emission between said fluorescent water soluble labeling complexes of said reagent labeled-first target compound and said reagent labeled-second target compound.

79. A fluorescent labeling complex, comprising:

i) a first fluorochrome having first absorption and emission spectra;

ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome;

iii) a rigid linker group of 2 to 20 bond lengths covalently attaching said first fluorochrome and said second fluorochromes selected to transfer resonance energy between said first fluorochrome and said second fluorochrome; and iv) a bonding group capable of forming a covalent bond with a carrier material, wherein at least one of said first fluorochrome and said second fluorochrome is a cyanine dye and wherein the combined molecular weight of said first fluorochrome and said second fluorochrome and said linker group in each of said complexes is less than 20,000 Daltons.

80. A fluorescent labeling complex according to claim 79, comprising one of a plurality of said first fluorochrome and a plurality of said second fluorochrome, each of said fluorochromes being attached to said other fluorochromes by a linker group of 2 to 20 bond lengths selected to transfer resonance energy between said first fluorochrome and said second fluorochrome.

81. A fluorescent labeling complex according to claim 79, further comprising a water solubilizing group that is unreactive with said bonding group of said complex.

82. A fluorescent labeling complex according to claim 79, wherein said water solubilizing group is selected from the group consisting of amide, sulphonate, sulphate, phosphate, quaternary ammonium, hydroxyl, guanidinium and phosphonate.

83. A fluorescent labeling complex according to claim 79, wherein said bonding group is a reactive group selected from the group consisting of succinimidyl ester, isothiocyanate, isocyanate, haloacetamide, dichlorotriazine, maleimide, sulphonyl halide, alkylimidoester, arylimidoester, substituted hydrazine, substituted hydroxylamine, carbodiimide, acyl halide, anhydride, phosphoramidite, acrylate and acrylamide.

84. A set having a plurality of fluorescent labelling complexes, wherein each of said complexes has:

i) a first fluorochrome having first absorption and emission spectra;

ii) a second fluorochrome having second absorption and emission spectra, the wavelength of the emission maximum of said second fluorochrome being longer than the wavelength of the emission maximum of said first fluorochrome, and a portion of the absorption spectrum of said second fluorochrome overlapping a portion of the emission spectrum of said first fluorochrome, the first and second fluorochromes being selected from the cyanine dyes, fluoresceins, rhodamines, pyrene trisulphonates and derivatives of the bispyrromethine boron difluoride dyes;

iii) at least one linker group having between 2 and 20 bond lengths for covalently attaching said first and second fluorochromes for transfer of resonance energy between said first and second fluorochromes;

iv) at least one reactive group capable of forming a covalent bond with a functional group of a target compound, said reactive group being selected from the group consisting of succinimidyl ester, isothiocyanate, isocyanate, haloacetamide, dichlorotriazine, maleimide, sulphonyl halide, alkylimidoester, arylimidoester, substituted hydrazine, substituted hydroxylamine, carbodiimide, acyl halide, anhydride, phosphoramidite, acrylate and acrylamide;

v) an optionally present water solubilizing constituent being unreactive with said reactive group, and being selected from the group consisting of amide, sulphonate, sulphate, phosphate, quaternary ammonium, hydroxyl, guanidinium and phosphonate;

wherein the combined molecular weight of said first and second fluorochromes and said linker group in each of said complexes is less than 20,000 Daltons, and wherein at least two complexes of said set absorbs at the same wavelength and emits at significantly different emission wavelengths.

85. The set of fluorescent labelling complexes according to claim 84 wherein one or more of said fluorescent labelling complexes further comprises a third fluorochrome having third absorption and emission spectra covalently attached to one of said first or said second fluorochrome; the wavelength of the emission maximum of said third fluorochrome being longer than the wavelength of the emission maximum of said fluorochrome to which it is attached, and a portion of the emission spectrum of said attached fluorochrome overlapping a portion of the absorption spectrum of said third fluorochrome such that excitation of said attached fluorochrome produces fluorescence from said third fluorochrome.

86. The set of fluorescent labelling complexes according to claim 85 wherein said first fluorochromes are selected from the group consisting of monomethine rigidized cyanine dyes, a trimethine cyanine dye, fluorescein, pyrene trisulphonate, bispyrromethine boron difluoride dyes and said second and third fluorochromes are polymethine cyanine dyes.

87. The set of fluorescent labelling complexes according to claim 84, wherein the functional group of said carrier material is selected from the group consisting of amino, sulfhydryl, carbonyl, hydroxyl, phosphate and thiophosphate groups.

88. The set of fluorescent labelling complexes according to claim 84, wherein said first fluorochrome is selected from the group consisting of a monomethine rigidized cyanine dye, a trimethine cyanine dye, a fluorescein, a pyrene trisulphonate, a bispyrromethine boron difluoride dye and said second fluorochrome is a polymethine cyanine dye.

89. The set of fluorescent labelling complexes according to claim 84, wherein said carrier material is selected from the group consisting of an antibody, a lipid, a protein, a carbohydrate, a nucleotide and a nucleic acid.

90. A fluorescent detection and analytical method for detecting a first target material in a sample, comprising the steps of:

a) contacting a first fluorescent labeling complex with a first sample; and b) detecting labeling of a target material in the first sample with the fluorescent labeling complex, the first fluorescent labeling complex having at least:

i) a first fluorochrome having first absorption and emission spectra, covalently attached through a linker group of between 2 and 20 bond lengths to a low molecular weight second fluorochrome having second absorption and emission spectra, and wherein the wavelength of the emission maximum of the second fluorochrome is longer than the wavelength of the emission maximum of the first fluorochrome and a portion of the absorption spectrum of the second fluorochrome overlaps a portion of the emission spectrum of the first fluorochrome for transfer of energy absorbed by the first fluorochrome upon excitation with light to the second fluorochrome, wherein at least one of the first fluorochrome and the second fluorochrome is a cyanine dye and wherein said linker group is selected to transfer resonance energy between said first fluorochrome and said second fluorochrome;

ii) a water solubilizing group; and;

iii) a carrier material covalently bound to portion (i) of said first fluorescent labeling complex and selected from the group consisting of an antibody, a lipid, a protein, a carbohydrate, a nucleotide and a nucleic acid.

91. The fluorescent detection and analytical method according to claim 90, further comprising the steps of:

a) contacting a second fluorescent labeling complex with a second sample to detect a second target material; and b) detecting labeling of the second target material with the second fluorescent labeling complex, the second fluorescent labeling complex having at least:

i) a first fluorochrome having first absorption and emission spectra, covalently attached through a linker group to a low molecular weight second fluorochrome having second absorption and emission spectra, wherein the wavelength of the emission maximum of the second fluorochrome is longer than the wavelength of the emission maximum of the first fluorochrome and a portion of the absorption spectrum of the second fluorochrome overlaps a portion of the emission spectrum of the first fluorochrome for transfer of energy absorbed by the first fluorochrome upon excitation with light to the second fluorochrome, wherein at least one of the first fluorochrome and the second fluorochrome is a cyanine dye and said linker group is selected to transfer resonance energy between the first fluorochrome and the second fluorochrome and wherein the second fluorochrome of the second fluorescent labeling complex has an emission spectrum different from the emission spectrum of the second fluorochrome of the first labeling complex;

ii) a water solubilizing group; and;

iii) a carrier material covalently bound to portion (i) of said second fluorescent labeling complex and selected from the group consisting of an antibody, a lipid, a protein, a carbohydrate, a nucleotide and a nucleic acid.

92. A method of labeling a compound or a carrier material, comprising the step of conjugating a fluorescent labeling complex according to claim 79 with the compound or carrier material under conditions suitable for the formation of a covalent linkage between the complex and the compound or carrier material.

93. A method according to claim 92, wherein the compound or carrier material is selected from the group consisting of an antibody, an antigen, a protein, a carbohydrate, a lipid, a nucleotide, a nucleic acid, a cell, a polymer particle and a glass bead.