Bright fluorochromes based on multimerization of fluorescent dyes
View Patent ↗The invention is directed to a fluorescent dye according to the general formula I: with C is a core moiety comprising 20 to 200 atoms; S same or different ether residues comprising 1 to 10 carbon atoms; n is an integer ranging from 2 to 500; m is an integer ranging from 0 to 500; x is an integer ranging from 2 to 50; y is an integer ranging from 1 to 50; R same or different residue comprising a reactive group capable of forming a covalent bond with a biomolecule; F same or different fluorophores covalently bound to (S) n . The fluorescent dyes can be conjugated to a biomolecule and used for flow cytometry and/or by fluorescence microscopy.
1. A fluorescent dye according to the formula III:
wherein
p is an integer ranging from 10 to 200;
Z is at least one of F and R, with
F comprises same or different fluorophores covalently bound to the residues CH 2 CH 2 O—
R comprises a reactive group selected from the group consisting of N-hydroxysuccinimid ester, tetrafluorophenyl ester, pentafluorophenyl ester, sulfodichlorophenyl ester, imido ester, isothiocyanate, isocyanate, sulfonyl halides, acyl halides, acyl azide, monochlorotriazine, dichlorotriazine, aldehyde, glyoxal, maleimide, iodoacetamide, hydrazine, azidonitrophenyl, phosphoramidite, alkyne, alkyl azide, dienes and allyl groups;
with the provision that general formula (III) comprises at least two F and at least one group R.
2. The fluorescent dye according to claim 1 , wherein the fluorophore F is selected from the group consisting of xanthene dyes, rhodamine dyes, coumarine dyes, cyanine dyes, pyrene dyes, oxazine dyes, pyridyl oxazole dyes and pyrromethene dyes.
3. The fluorescent dye according to claim 1 , wherein the fluorophore F is substituted with one more water solubility imparting substituents selected from the group consisting of sulfonates, phosphonates, phosphates, sulfonamides, polyethers and carbonates.
4. A fluorescent biomolecule conjugate comprising at least one fluorescent dye according claim 1 , wherein a biomolecule is conjugated via group R to the at least one fluorescent dye, and the biomolecule is selected from group consisting of immunoglobulin, antibody, fragmented antibody, Fab, Fab′, F(ab′) 2 , sdAb, scFv, di-scFv each of naturally or recombinant origin.
5. A fluorescent biomolecule conjugate comprising at least one fluorescent dye according claim 1 , wherein a biomolecule is conjugated via group R to the at least one fluorescent dye, and the biomolecule is selected from the group consisting of peptide/MHC-complexes, receptors for cell adhesion or costimulatory molecules, receptor ligands, antigens, hapten binders, avidin, streptavidin, neutravidin, aptamers, primers and ligase substrates.
6. A fluorescent biomolecule conjugate comprising at least one fluorescent dye according claim 1 , wherein a biomolecule is conjugated with 2 to 20 fluorescent dyes according to claim 1 , each fluorescent dye comprising between 1 and 10 fluorophores F, inclusive.
7. A method of analyzing cells or tissue, comprising:
labeling with the fluorescent biomolecule conjugate according to claim 4 ; and
performing at least one of flow cytometry and fluorescence microscopy.