Fluorochromes labelling complexes with large stokes shift formed by coupling together cyanine and other fluorochromes capable of resonance energy transfer
First Claim
1. 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.
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Abstract
The present invention provides low molecular weight fluorescent labeling 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 labeled with the complex to penetrate cell structures for use as probes. The labeling complexes are synthesized by covalently attaching through linkers at least one cyanine fluorochrome to another low molecular weight fluorochrome to form energy donor-acceptor complexes. Resonance energy transfer from an excited donor to fluorescent acceptor provides wavelength shifts up to 300 nm. The fluorescent labeling complexes preferably contain reactive groups for the labeling of functional groups on target compounds, such as derivatized oxy and deoxy polynucleic acids, antibodies, enzymes, proteins and other materials. The complexes may also contain functional groups permitting covalent reaction with materials containing reactive groups.
81 Citations
45 Claims
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1. A set of fluorescent labelling complexes each of said complexes comprising:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 21, 23, 25, 27)
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9. A set of fluorescent labelling complexes each of said complexes including:
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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. - View Dependent Claims (10, 11, 12, 13, 14, 15, 16, 22, 24, 26, 28)
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17. A set of reagents, each reagent of the set comprising:
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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 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 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. - View Dependent Claims (18, 19)
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20. A method of analysis or detection of multiple target compounds comprising:
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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 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.
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29. A method of analysis or detection of multiple target compounds comprising:
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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.
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30. A method of analysis or detection of multiple target compounds comprising:
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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 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 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
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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.
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31. A fluorescent labeling complex, comprising:
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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. - View Dependent Claims (32, 33, 34, 35, 44, 45)
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36. A set having a plurality of fluorescent labelling complexes, wherein each of said complexes has:
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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. - View Dependent Claims (37, 38, 39, 40, 41)
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42. A fluorescent detection and analytical method for detecting a first target material in a sample, comprising the steps of:
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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
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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. - View Dependent Claims (43)
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
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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.
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Specification