MODIFIED CARBOCYANINE DYES AND THEIR CONJUGATES

1 - 17 . (canceled)

18 . A compound of Formula:

wherein,

δ is 0 or 1;

n is 1, 2 or 3;

W represents the atoms necessary to form one fused aromatic ring having 6 atoms, which atoms are selected from —CH and —CR 1′ ;

each R 1′ is sulfo;

R 2 is C 1 -C 6 alkyl, which is optionally substituted by sulfo;

R 3 is C 1 -C 6 alkyl;

R 4 is C 1 -C 6 alkyl;

R 6 is H or sulfo;

R 7 is H or sulfo;

R 8 is H or sulfo;

R 9 is H or sulfo;

R 12 is -L-R x or -L-S c , or a C 1 -C 6 alkyl, which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, or amino;

R 13 is -L-R x or -L-S c ;

R 14 is C 1 -C 6 alkyl;

R x is the reactive group;

S c is a conjugated substance; and

L is a covalent linkage.

19 . The compound of claim 18 , wherein W is substituted with one sulfo group.

20 . The compound of claim 19 , wherein R 7 is sulfo.

21 . The compound of claim 18 , wherein δ is 0.

22 . The compound of claim 18 , wherein n is 2.

23 . The compound of claim 18 , wherein R 6 , R 8 an R 9 are H.

24 . The compound of claim 23 , wherein R x is an activated ester of a carboxylic acid.

25 . The compound of claim 18 , wherein R 3 and R 4 are methyl.

26 . The compound of claim 18 , wherein R 2 is sulfobutyl.

27 . The compound of claim 18 , wherein R 14 is methyl.

28 . The compound of claim 23 , wherein R 13 is -alkyl-R x .

29 . The compound of claim 28 , wherein R x is a carboxylic acid or a succinimidyl ester of a carboxylic acid.

30 . The compound of claim 18 , wherein R 13 is -L-S c .

31 . The compound of claim 18 , wherein S c is an antigen, steroid, vitamin, drug, hapten, metabolite, toxin, environmental pollutant, amino acid, peptide, protein, nucleic acid, nucleic acid polymer, carbohydrate, lipid, ion-complexing moiety, stable free radical or glass, plastic or other non-biological polymer.

32 . The compound of claim 18 , wherein R x is an amine, amide, thiol, alcohol, phenol, aldehyde, ketone, phosphate, imidazole, hydrazine, hydroxylamine, disubstituted amine, halide, epoxide, carboxylate ester, sulfonate ester, purine, pyrimidine, carboxylic acid, olefinic bond, a succinimidyl ester of a carboxylic acid, an acyl azide, an acyl nitrile, an alkyl halide, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a diazoalkane, a haloacetamide, a halotriazine, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, or a sulfonyl halide.

33 . The compound of claim 18 , wherein L is comprised of single, double, triple or aromatic carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds, phosphorus-oxygen bonds, phosphorus-nitrogen bonds, and nitrogen-platinum bonds.

34 . A compound of Formula:

wherein,

R 2 is C 1 -C 6 alkyl, which is substituted by sulfo;

R 3 is C 1 -C 6 alkyl;

R 4 is C 1 -C 6 alkyl;

R 12 is C 1 -C 6 alkyl;

R 13 is -L-R x or -L-S c ;

R 14 is C 1 -C 6 alkyl;

δ is 0 or 1; and

n is 1, 2, or 3.

35 . The compound of claim 34 , wherein δ is 0 and R 3 , R 4 and R 14 are methyl.

36 . The compound of claim 34 , wherein δ is 0 and R 13 is -alkyl-R x .

37 . The compound of claim 36 , wherein R x is a carboxylic acid or a succinimidyl ester of a carboxylic acid.

38 . The compound of claim 34 , wherein δ is 0 and R 13 is -L-S c .

39 . The compound of claim 34 , wherein S c is an antigen, steroid, vitamin, drug, hapten, metabolite, toxin, environmental pollutant, amino acid, peptide, protein, nucleic acid, nucleic acid polymer, carbohydrate, lipid, ion-complexing moiety, stable free radical or glass, plastic or other non-biological polymer.

40 . The compound of claim 34 , wherein R x is an amine, amide, thiol, alcohol, phenol, aldehyde, ketone, phosphate, imidazole, hydrazine, hydroxylamine, disubstituted amine, halide, epoxide, carboxylate ester, sulfonate ester, purine, pyrimidine, carboxylic acid, olefinic bond, a succinimidyl ester of a carboxylic acid, an acyl azide, an acyl nitrile, an alkyl halide, an anhydride, an aniline, an aryl halide, an azide, an aziridine, a boronate, a diazoalkane, a haloacetamide, a halotriazine, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, or a sulfonyl halide.

41 . A carbocyanine dye according to the formula:

A-BRIDGE-B

wherein A and B are indolium rings, wherein the substituent on the 3-carbon of one of the indolium rings contains a chemically reactive group or a conjugated substance; and

BRIDGE is a polymethine linker.

42 . The carbocyanine dye of claim 41 , wherein both indolium rings are substituted with a sulfo group.

43 . The carbocyanine dye of claim 41 , wherein the chemically reactived group comprises a carboxylic acid, an activated ester of a carboxylic acid, an amine, an azide, a hydrazide, a haloacetamide, an alkyl halide, an isothiocyanate, or a maleimide group.

44 . The carbocyanine dye of claim 41 , wherein the conjugated substance is an antibody, a peptide, a lectin, a polysaccharide, a nucleotide, a nucleoside, an oligonucleotide, a nucleic acid polymer, an ion-complexing moiety, a lipid, or a non-biological organic polymer or polymeric microparticle.

45 . The carbocyanine dye of claim 41 , wherein A is:

wherein,

Y represents the atoms necessary to form one fused aromatic ring having 6 atoms selected from —CH or —CR 1 and each R 1 is sulfo;

X is —CR 3 R 4 , where R 3 and R 4 are independently C 1 -C 6 alkyl, -L-R x or -L-S c ;

R 2 is C 1 -C 6 alkyl; and

α is 0 or 1.

46 . The carbocyanine dye of claim 41 , wherein B is:

wherein,

W represents the atoms necessary to form one fused aromatic ring having 6 atoms selected from —CH, or —CR 1 , and each R 1′ is sulfo;

Z is —CR 13 R 14 , where R 13 and R 14 are independently -L-R x , -L-S c or C 1 -C 6 alkyl;

R 12 is C 1 -C 6 alkyl; and

δ is 0 or 1.

47 . The carbocyanine dye of claim 45 , wherein R 3 and R 4 are methyl.

48 . The carbocyanine dye of claim 46 , wherein R 13 is -L-R x or -L-S c .

49 . The carbocyanine dye of claim 48 , wherein R 14 is methyl.

50 . The carbocyanine dye of claim 41 , wherein BRIDGE is:

wherein,

each of R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , and R 27 , independently H, F, Cl, alkyl having 1-6 carbons, alkoxy having 1-6 carbons, aryloxy, a N-heteroaromatic moiety, or an iminium ion; and

a and b are independently 0 or 1.

51 . The carbocyanine dye of claim 50 , wherein a is 1 and b is 0.

52 . The carbocyanine dye of claim 50 , wherein each of R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , and R 27 are H,

53 . A method of staining a biological sample, comprising:

combining a dye solution comprising a compound having the formula:

wherein,

δ is 0 or 1;

n is 1, 2 or 3;

W represents the atoms necessary to form one fused aromatic ring having 6 atoms, which atoms are selected from —CH and —CR 1′ ;

each R 1′ is sulfo;

R 2 is C 1 -C 6 alkyl, which is substituted by sulfo;

R 3 is C 1 -C 6 alkyl;

R 4 is C 1 -C 6 alkyl;

R 6 is H or sulfo;

R 7 is H or sulfo;

R 8 is H or sulfo;

R 9 is H or sulfo;

R 12 is -L-R x or -L-S c , or a C 1 -C 6 alkyl, which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, or amino;

R 13 is -L-R x or -L-S c ;

R 14 is C 1 -C 6 alkyl;

R x is the reactive group;

S c is a conjugated substance; and

L is a covalent linkage;

with a biological sample in a concentration sufficient to yield a detectable optical response under desired conditions.

54 . The method according to claim 53 , wherein the sample comprises cells, proteins, or nucleic acid polymers.

55 . The method according to claim 53 , wherein the sample is contained in or on a solid or semi-solid matrix that is a membrane, an elctrophoretic gel, silicon chip, a glass slide, a microwell plate or a microfluidic chip.

56 . A method of staining a biological sample, comprising:

combining a dye solution comprising a compound having the formula:

A-BRIDGE-B

wherein A and B are indolium rings, wherein the substituent on the 3-carbon of one of the indolium rings contains a chemically reactive group or a conjugated substance; and

BRIDGE is a polymethine linker;

with a biological sample in a concentration sufficient to yield a detectable optical response under desired conditions.

57 . A method for forming a dye-conjugate of a protein, peptide or a nucleic acid polymer, said method comprising:

combining a dye solution comprising a compound of the formula

wherein,

δ is 0 or 1;

n is 1, 2 or 3;

W represents the atoms necessary to form one fused aromatic ring having 6 atoms, which atoms are selected from —CH and —CR 1′ ;

each R 1′ is sulfo;

R 2 is C 1 -C 6 alkyl, which is substituted by sulfo;

R 3 is C 1 -C 6 alkyl;

R 4 is C 1 -C 6 alkyl;

R 6 is H or sulfo;

R 7 is H or sulfo;

R 8 is H or sulfo;

R 9 is H or sulfo;

R 12 is -L-R x or -L-S c , or a C 1 -C 6 alkyl, which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, or amino;

R 13 is -L-R x or -L-S c ;

R 14 is C 1 -C 6 alkyl;

R x is the reactive group;

S c is a conjugated substance; and

L is a covalent linkage;

with a sample comprising a protein, peptide or a nucleic acid polymer whereby a dye conjugate is formed.

58 . A method for forming a dye-conjugate of a protein, peptide or a nucleic acid polymer, said method comprising:

combining a dye solution comprising a compound of the formula

A-BRIDGE-B

wherein A and B are indolium rings, wherein the substituent on the 3-carbon of one of the indolium rings contains a chemically reactive group or a conjugated substance; and

BRIDGE is a polymethine linker;

with a sample comprising a protein, peptide or a nucleic acid polymer whereby a dye conjugate is formed.

59 . A kit for fluorescent labeling of a biological sample, comprising:

a dye solution comprising a compound having the formula:

wherein,

δ is 0 or 1;

n is 1, 2 or 3;

W represents the atoms necessary to form one fused aromatic ring having 6 atoms, which atoms are selected from —CH and —CR 1′ ;

each R 1′ is sulfo;

R 2 is C 1 -C 6 alkyl, which is substituted by sulfo;

R 3 is C 1 -C 6 alkyl;

R 4 is C 1 -C 6 alkyl;

R 6 is H or sulfo;

R 7 is H or sulfo;

R 8 is H or sulfo;

R 9 is H or sulfo;

R 12 is -L-R x or -L-S c , or a C 1 -C 6 alkyl, which is optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxy, sulfo, or amino;

R 13 is -L-R x or -L-S c ;

R 14 is C 1 -C 6 alkyl;

R x is the reactive group;

S c is a conjugated substance; and

L is a covalent linkage; and

a buffer.

60 . A kit for fluorescent labeling of a biological sample, comprising:

a dye solution comprising a compound having the formula:

A-BRIDGE-B

wherein A and B are indolium rings, wherein the substituent on the 3-carbon of one of the indolium rings contains a chemically reactive group or a conjugated substance; and

BRIDGE is a polymethine linker; and

a buffer.

61 . A carbocyanine dye comprising at least one substituted indolium ring system wherein a substituent on the 3-carbon of the indolium ring contains a chemically reactive group or a conjugated substance.

62 . The carbocyanine dye of claim 62 , comprising two substituted indolium ring systems wherein a substituent on the 3-carbon of one of the indolium ring contains a chemically reactive group or a conjugated substance.

63 . A compound comprising 3-(5-carboxypentyl)-2,3-dimethyl-5-sulfoindolium or a salt thereof.