Reagents including a carrier and fluorescent labeling complexes with large stokes shift formed by coupling together cyanine and other fluorochromes capable of resonance energy transfer
First Claim
1. A reagent comprising:
- a fluorescent water soluble labeling complex consisting of;
(i) one or more low molecular weight first fluorochromes, each having first absorption and emission spectra, covalently attached through a linker to one or more low molecular weight second fluorochromes, each having second absorption and emission spectra, said linker being between 2 to 20 bond lengths and wherein at least one of said first and second fluorochromes is a cyanine dye 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,having covalently bonded thereto a carrier material having a group that reacts with said bonding group of said complex.
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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.
126 Citations
11 Claims
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1. A reagent comprising:
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a fluorescent water soluble labeling complex consisting of; (i) one or more low molecular weight first fluorochromes, each having first absorption and emission spectra, covalently attached through a linker to one or more low molecular weight second fluorochromes, each having second absorption and emission spectra, said linker being between 2 to 20 bond lengths and wherein at least one of said first and second fluorochromes is a cyanine dye 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, having covalently bonded thereto a carrier material having a group that reacts with said bonding group of said complex. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
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Specification