Poly-sarcosine solubilized dyes and methods for using the same

Abstract

Dyes, including tandem dyes, are provided. Aspects of embodiments of the dyes include a fluorophore and a water-solubilizing poly-sarcosine group. Also provided are methods of using the dyes, as well as kits that include such dyes.

Description

[0001] POLY-SARCOSINE SOLUBILIZED DYES AND METHODS FOR USING THE SAME

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 63 / 524,017 filed June 29, 2023; the disclosure of which application is incorporated herein by reference in its entirety.

[0004] INTRODUCTIO

[0005] Fluorescent dyes are compounds which, when irradiated with light of a wavelength which they absorb, emit light of a (usually) different wavelength. Fluorescent dyes find use in a variety of applications in biochemistry, biology and medicine, e.g., in diagnostic kits, in microscopy or in drug screening. Fluorescent dyes are characterized by a number of parameters allowing a user to select a suitable dye depending on the desired purpose. Parameters of interest include the excitation wavelength maximum, the emission wavelength maximum, the Stokes shift, the extinction coefficient, the fluorescence quantum yield and the fluorescence lifetime. Dyes may be selected according to the application of interest in order to, e.g., allow penetration of exciting radiation into biological samples, to minimize background fluorescence and / or to achieve a high signal-to-noise ratio.

[0006] Molecular recognition involves the specific binding of two molecules. Molecules which have binding specificity for a target biomolecule find use in a variety of research and diagnostic applications, such as the labelling and separation of analytes, flow cytometry, in situ hybridization, enzyme-linked immunosorbent assays (ELISAs), western blot analysis, magnetic cell separations and chromatography. Target biomolecules may be detected by labelling with a fluorescent dye.

[0007] Polyethylene glycol (PEG) groups have been attached to fluorescent dyes (as well as small molecules, nucleotides, peptides, proteins, liposomes, and nanoparticles) to improve solubility, stability, and pharmacokinetic properties. However, some naive individuals can have pre-existing antibodies that can bind to PEG and induce an immune response (Chen et al., above). Many common methods of synthesizing PEG and other polymers generate polydisperse compositions with broad distributions of molecular weights. Such polydispersity can disadvantageously inhibit control over the properties of the polymers.

[0008] SUMMARY

[0009] Dyes, including tandem dyes, are provided. Aspects of embodiments of the dyes include a fluorophore and a water-solubilizing poly-sarcosine group. Also provided are methods of using the dyes, as well as kits that include such dyes.

[0010] BRIEF DESCRIPTION OF THE FIGURES

[0011] FIG. 1 shows chemical structures of polysarcosine, N-modified polysarcosine, and a polysarcosine copolymer.

[0012] FIG. 2 shows the chemical structures of polysarcosine and methyl-polyethylene glycol.

[0013] FIG. 3 shows the chemical structures of exemplary cyanine, rhodamine, coumarin, and BODIPY dyes.

[0014] FIG. 4 shows examples of a dye and a tandem dye that include a polymeric donor chromophore, poly-sarcosine groups, and an antibody. The bottom structure includes an acceptor dye, thereby making the structure a tandem dye.

[0015] FIG. 5 shows examples of structures having a dye connected to a biomolecule through a linker that includes a poly-sarcosine group.

[0016] FIG. 6 shows a solid-phase synthesis of poly-sarcosine groups.

[0017] FIG. 7 shows synthetic options wherein a hydroxyl group attached to a polysarcosine group is converted to different substituted amine groups.

[0018] FIG. 8 shows different options for the placement of poly-sarcosine groups within a compound.

[0019] FIG. 9 shows poly-sarcosine groups attached to a polyfluorene or BODIPY chromophore.

[0020] FIG. 10 shows SSC data for donor-acceptor complexes that used PEGylated dyes and Sarcosine-functionalized dyes covalently attached to hCD4 and used as a cell stain. The sarcosine-substituted dyes provide roughly 10% greater signal.

[0021] FIG. 11 shows pendant poly-sarcosine groups attached to a BODIPY dye or a cyanine dye. DEFINITIONS

[0022] "Alkyl" refers to a monoradical, branched or linear, non-cyclic, saturated hydrocarbon group. Exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, n- butyl, isobutyl, t-butyl, octyl, decyl, cyclopentyl, and cyclohexyl. In some cases, the alkyl group has 1 to 24 carbon atoms, e.g., 1 to 12, 1 to 6, or 1 to 3.

[0023] "Alkenyl" refers to a monoradical, branched or linear, non-cyclic hydrocarbonyl group that comprises a carbon-carbon double bond. Exemplary alkenyl groups include ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, and tetracosenyl.

[0024] “Alkynyl" refers to a monoradical, branched or linear, non-cyclic hydrocarbonyl group that comprises a carbon-carbon triple bond. Exemplary alkynyl groups include ethynyl and n-propynyl.

[0025] "Cycloalkyl” refers to a monoradical, cyclic, saturated hydrocarbon group. Similarly, “cycloalkenyl” refers to a monoradical and cyclic group having carbon-carbon double bond whereas "cycloalkynyl” refers to a monoradical and cyclic group having carbon-carbon triple bond.

[0026] "Heterocyclyl” refers to a monoradical, cyclic group that contains a heteroatom (e.g., O, S, N) as a ring atom and that is not aromatic (i.e., distinguishing heterocyclyl groups from heteroaryl groups). Exemplary heterocyclyl groups include piperidinyl, tetrahydrofuranyl, dihydrofuranyl, and thiocanyl.

[0027] "Aryl" refers to an aromatic group containing at least one aromatic ring, wherein each of the atoms in the ring are carbon atoms, i.e., none of the ring atoms are heteroatoms (e.g., O, S, N). In some cases, the aryl group has a second aromatic ring, e.g., that is fused to the first aromatic ring. Exemplary aryl groups are phenyl, naphthyl, biphenyl, diphenylether, diphenylamine, and benzophenone.

[0028] "Heteroaryl” refers to an aromatic group containing at least one aromatic ring, wherein at least one of the atoms in the aromatic ring is a heteroatom (e.g., O, S, N). Exemplary heteroaryl groups include those obtained from removing a hydrogen atom from pyridine, pyrimidine, furan, thiophene, or benzothiophene.

[0029] The term “substituted” refers the removal of one or more hydrogens from an atom (e.g., from a C or N atom) and their replacement with a different group. For instance, a hydrogen atom on a phenyl (-CeH5) group can be replaced with a methyl group to form a -C6H4CH3 group. Thus, the -C6H4CH3 group can be considered a substituted aryl group. As another example, two hydrogen atoms from the second carbon of a propyl (- CH2CH2CH3) group can be replaced with an oxygen atom to form a -CH2C(O)CH3group, which can be considered a substituted alkyl group. However, replacement of a hydrogen atom on a propyl (-CH2CH2CH3) group with a methyl group (e.g. giving -CH2CH(CH3)CH3) is not considered a “substitution” as used herein since the starting group and the ending group are both alkyl groups. However, if the propyl group was substituted with a methoxy group, thereby giving a -CH2CH(OCH3)CH3group, the overall group can no long be considered “alkyl”, and thus is “substituted alkyl”. Thus, in order to be considered a substituent, the replacement group is a different type than the original group. In addition, groups are presumed to be unsubstituted unless described as substituted. For instance, the term “alkyl” and “unsubstituted alkyl” are used interchangeably herein.

[0030] Exemplary substituents include alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, acyl, alkoxy, amino, azido, carbonyl, carboxy, cyano, ether, halo, hydroxy, nitro, sulfonate, and substituted versions thereof.

[0031] In some cases, the substitutions can themselves be further substituted with one or more groups. For example, the group -C6H4CH2CH3 can be considered as substituted aryl, i.e., an aryl group substituted with the ethyl, which is an alkyl group. Furthermore, the ethyl group can itself be substituted with a pyridyl group to form -C6H4CH2CH2C5H5N, wherein -C6H4CH2CH2C5H5N can also be considered as a substituted aryl group as the term is used herein. In some cases, the substituents are not substituted with any other groups.

[0032] Diradical groups are also described herein, i.e., in contrast to the monoradical groups such as alkyl and aryl described above. The term "alkylene” refers to the diradical version of an alkyl group, i.e., an alkylene group is a diradical, branched or linear, cyclic or non-cyclic, saturated hydrocarbon group. Exemplary alkylene groups include diylmethane (-CH2-, which is also known as a methylene group), 1 ,2-diylethane (-CH2CH2- ), and 1 ,1-diylethane (i.e., a CHCH3fragment where the first atom has two single bonds to other two different groups). The term “arylene” refers to the diradical version of an aryl group, e.g., 1 ,4-diylbenzene refers to a C6H4 fragment wherein two hydrogens that are located para to one another are removed and replaced with single bonds to other groups. The terms “alkenylene”, “alkynylene”, “heteroarylene”, and “heterocyclene” are also used herein. "Acyl” refers to a group of formula -C(O)R wherein R is alkyl, alkenyl, alkynyl, or substituted versions thereof. For example, the acetyl group has formula -C(O)CH3. “Carbonyl” refers to a diradical group of formula -C(O)-.

[0033] “Alkoxy" refers to a group of formula -O(alkyl). Similar groups can be derived from alkenyl, alkynyl, aryl, heteroaryl, and other groups.

[0034] “Amino" refers to the group -NRXRYwherein Rxand RYare each independently H or a non-hydrogen substituent. Exemplary non-hydrogen substituents include alkyl groups (e.g., methyl, ethyl, and isopropyl).

[0035] “Carbonyl” refers to a diradical group of formula -C(O)-.

[0036] “Carboxy” is used interchangeably with carboxyl and carboxylate to refer to the - CO2H group and salts thereof.

[0037] “Ether” refers to a diradical group of formula -O-. For instance, if the ether group is connected to an alkyl group, then the overall group is an alkoxy group (e.g. -OCH3or methoxy). If the ether is connected to a carbonyl group, then the overall group is an ester group of formula -OC(O)-.

[0038] “Halo” and “halogen” refer to the chloro, bromo, fluoro, and iodo groups.

[0039] “Nitro” refers to the group of formula -NO2.

[0040] Unless otherwise specified, reference to an atom is meant to include all isotopes of that atom. For example, reference to H includes1H,2H (i.e., D or deuterium) and3H (i.e., tritium), and reference to C includes both12C and all other isotopes of carbon (e.g.,13C). Unless specified otherwise, groups include all possible stereoisomers.

[0041] The terms “reactive moiety”, “chemoselective functional group”, “chemoselective tag”, and “conjugation tag” are used interchangeably and refer to a functional group that can selectively react with another compatible functional group to form a covalent bond, in some cases, after optional activation of one of the functional groups. Chemoselective functional groups of interest include, but are not limited to, thiols and maleimide or iodoacetamide, amines and carboxylic acids or active esters thereof, as well as groups that can react with one another via Click chemistry, e.g., azide and alkyne groups (e.g., cyclooctyne groups), tetrazine, transcyclooctene, dienes and dienophiles, and azide, sulfur(VI) fluoride exchange chemistry (SuFEX), sulfonyl fluoride, as well as hydroxyl, hydrazido, hydrazino, aldehyde, ketone, azido, alkyne, phosphine, epoxide, succinimide and the like.

[0042] As used herein, the term “sample” relates to a material or mixture of materials, in some cases in liquid form, containing one or more analytes of interest. In some embodiments, the term as used in its broadest sense, refers to any plant, animal or bacterial material containing cells or producing cellular metabolites, such as, for example, tissue or fluid isolated from an individual (including without limitation plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) or from in vitro cell culture constituents, as well as samples from the environment. The term “sample” may also refer to a “biological sample”. As used herein, the term “a biological sample” refers to a whole organism or a subset of its tissues, cells or component parts (e.g., body fluids, including, but not limited to, blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). A “biological sample” can also refer to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors and organs. In certain embodiments, the sample has been removed from an animal or plant. Biological samples may include cells. The term “cells” is used in its conventional sense to refer to the basic structural unit of living organisms, both eukaryotic and prokaryotic, having at least a nucleus and a cell membrane. In certain embodiments, cells include prokaryotic cells, such as from bacteria. In other embodiments, cells include eukaryotic cells, such as cells obtained from biological samples from animals, plants or fungi.

[0043] The terms “support bound” and “linked to a support” are used interchangeably and refer to a moiety (e.g., a specific binding member) that is linked covalently or non- covalently to a support of interest. Covalent linking may involve the chemical reaction of two compatible functional groups (e.g., two chemoselective functional groups, an electrophile and a nucleophile, etc.) to form a covalent bond between the two moieties of interest (e.g., a support and a specific binding member). In some cases, non-covalent linking may involve specific binding between two moieties of interest (e.g., two affinity moieties such as a hapten and an antibody or a biotin moiety and a streptavidin, etc.). In certain cases, non-covalent linking may involve absorption to a substrate.

[0044] The terms “peptide” and “polypeptide” are used interchangeably herein to refer to a polymeric form of amino acids of any length, including peptides that range from 2 to 500 amino acids in length, such as from 2 to 350 amino acids, from 2 to 200 amino acids, from 2 to 100 amino acids, from 2 to 50 amino acids, and from 2 to 25 amino acids. In some cases, the peptide has 3 or more amino acids, such as 5 or more, 10 or more, or 20 or more. The terms peptide and polypeptide are also used interchangeably with the term “protein”. The amino acids in the peptide can be coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and peptides having a modified backbone in which the conventional backbone has been replaced with non- naturally occurring or synthetic backbones.

[0045] The terms “polyethylene oxide”, “PEO”, "polyethylene glycol” and “PEG” are used interchangeably and refer to a polymeric group including a chain described by the formula -(CH2— -O-)n- or a derivative thereof. In some embodiments, "n" is 5000 or less, such as 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, such as 3 to 15, or 10 to 15. It is understood that the PEG polymeric group may be of any convenient length and may include a variety of terminal groups and / or further substituent groups, including but not limited to, alkyl, aryl, hydroxyl, amino, acyl, acyloxy, and amido terminal and / or substituent groups. PEG groups are also described by S. Zalipsky in “Functionalized polyethylene glycol) for preparation of biologically relevant conjugates”, Bioconjugate Chemistry 1995, 6 (2), 150-165; by Zhu et al in “Water-Soluble Conjugated Polymers for Imaging, Diagnosis, and Therapy”, Chem. Rev., 2012, 112 (8), pp 4687-4735, “Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications”, J. M. Harris, Ed., Plenum Press, New York, N.Y. (1992); and “Poly(ethylene glycol) Chemistry and Biological Applications”, J. M. Harris and S. Zalipsky, Eds., ACS (1997); and International Patent Applications: WO 90 / 13540, WO 92 / 00748, WO 92 / 16555, WO 94 / 04193, WO 94 / 14758, WO 94 / 17039, WO 94 / 18247, WO 94 / 28937, WO 95 / 11924, WO 96 / 00080, WO 96 / 23794, WO 98 / 07713, WO 98 / 41562, WO 98 / 48837, WO 99 / 30727, WO 99 / 32134, WO 99 / 33483, WO 99 / 53951 , WO 01 / 26692, WO 95 / 13312, WO 96 / 21469, WO 97 / 03106, WO 99 / 45964, and U.S. Pat. Nos. 4,179,337; 5,075,046; 5,089,261 ; 5,100,992; 5,134,192; 5,166,309; 5,171 ,264; 5,213,891 ; 5,219,564; 5,275,838; 5,281 ,698; 5,298,643; 5,312,808; 5,321 ,095; 5,324,844; 5,349,001 ; 5,352,756; 5,405,877; 5,455,027; 5,446,090; 5,470,829; 5,478,805; 5,567,422; 5,605,976; 5,612,460; 5,614,549; 5,618,528; 5,672,662; 5,637,749; 5,643,575; 5,650,388; 5,681 ,567; 5,686,110; 5,730,990; 5,739,208; 5,756,593; 5,808,096; 5,824,778; 5,824,784; 5,840,900; 5,874,500; 5,880,131 ; 5,900,461 ; 5,902,588; 5,919,442; 5,919,455; 5,932,462; 5,965,119; 5,965,566; 5,985,263; 5,990,237; 6,011 ,042; 6,013,283; 6,077,939; 6,1 13,906; 6,127,355; 6,177,087; 6,180,095; 6,194,580; 6,214,966).

[0046] As used herein the term “isolated,” refers to a moiety of interest that is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free, and even at least 99% free from other components with which the moiety is associated with prior to purification.

[0047] As used herein, the terms “evaluating”, “determining,” “measuring,” and “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.

[0048] The term “separating”, as used herein, refers to physical separation of two elements (e.g., by size or affinity, etc.) as well as degradation of one element, leaving the other intact.

[0049] The term “linker” or “linkage” refers to a linking moiety that connects two groups and has a backbone of 100 atoms or less in length. A linker or linkage may be a covalent bond that connects two groups or a chain of between 1 and 100 atoms in length, for example a chain of 1 , 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more carbon atoms in length, where the linker may be linear, branched, cyclic or a single atom. In some cases, the linker is a branching linker that refers to a linking moiety that connects three or more groups. In certain cases, one, two, three, four or five or more carbon atoms of a linker backbone may be optionally substituted with a sulfur, nitrogen or oxygen heteroatom. In some cases, the linker backbone includes a linking functional group, such as an ether, thioether, amino, amide, sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester or imine. The bonds between backbone atoms may be saturated or unsaturated, and in some cases not more than one, two, or three unsaturated bonds are present in a linker backbone. The linker may include one or more substituent groups, for example with an alkyl, aryl or alkenyl group. A linker may include, without limitations, polyethylene glycol; ethers, thioethers, tertiary amines, alkyls, which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1 -methylethyl (iso-propyl), n-butyl, n-pentyl, 1 ,1 -dimethylethyl (t-butyl), and the like. The linker backbone may include a cyclic group, for example, an aryl, a heterocycle or a cycloalkyl group, where 2 or more atoms, e.g., 2, 3, or 4 atoms, of the cyclic group are included in the backbone. A linker may be cleavable or non-cleavable.

[0050] As used herein, the terms “water solubilizing group”, “water soluble group” and WSG are used interchangeably and refer to a group or substituent that is well solvated in aqueous environments e.g., under physiological conditions, and which imparts improved water solubility upon the molecule to which it is attached relative to the same molecule lacking the WSG(s). A WSG can increase the solubility of a tandem dye or component thereof, e.g., donor or acceptor fluorophore, in a predominantly aqueous solution, as compared to a control tandem dye or component thereof which lacks the WSG. In some cases, the WSG can increase the solubility of a compound (e.g., a dye, a tandem dye, or a labeled specific binding member) as compared to a control compound wherein the WSG is replaced with a hydrogen atom. In some cases, the WSG increases the solubility in an aqueous medium, e.g., distilled water, by 1% or more, such as by 10% or more, 25% or more, 50% or more, 100% or more, or 500% or more. The water solubilizing groups may be any convenient hydrophilic group that is well solvated in aqueous environments.

[0051] The water soluble group (WSG)(s) can be capable of imparting solubility in water in excess of 10 mg / mL to the subject dye or polymeric tandem dye, such as in excess of 20 mg / mL, in excess of 30 mg / mL, in excess of 40 mg / mL, in excess of 50 mg / mL, in excess of 60 mg / mL, in excess of 70 mg / mL, in excess of 80 mg / mL, in excess of 90 mg / mL or in excess of 100 mg / mL. In certain cases, the water soluble group (WSG)(s) is capable of imparting solubility in water (e.g., an aqueous buffer) of 20 mg / mL or more to the subject dye or polymeric tandem dye, such as 30 mg / mL or more, 40 mg / mL or more, 50 mg / mL or more, 60 mg / mL or more, 70 mg / mL or more, 80 mg / mL or more, 90 mg / mL or more, 100 mg / mL or more, or even more.

[0052] The term "specific binding" refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bond interactions, including interactions such as salt bridges and water bridges. A specific binding member describes a member of a pair of molecules which have binding specificity for one another. The members of a specific binding pair may be naturally derived or wholly or partially synthetically produced. One member of the pair of molecules has an area on its surface, or a cavity, which specifically binds to and is therefore complementary to a particular spatial and polar organization of the other member of the pair of molecules. Thus, the members of the pair have the property of binding specifically to each other. Examples of pairs of specific binding members are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. Specific binding members of a binding pair exhibit high affinity and binding specificity for binding with each other. Typically, affinity between the specific binding members of a pair is characterized by a Kd (dissociation constant) of 106M or less, such as 107M or less, including 108M or less, e.g., 10-9M or less, 10-10M or less, 10'11M or less, 10‘12M or less, 10-13M or less, 10-14M or less, including 10-15M or less. "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower KD. In an embodiment, affinity is determined by surface plasmon resonance (SPR), e.g., as used by Biacore systems. The affinity of one molecule for another molecule is determined by measuring the binding kinetics of the interaction, e.g., at 25°C. "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower KD. In an embodiment, affinity is determined by surface plasmon resonance (SPR), e.g., as used by Biacore systems. The affinity of one molecule for another molecule is determined by measuring the binding kinetics of the interaction, e.g., at 25°C.

[0053] Specific binding members may vary, as desired. In some instances, the specific binding members are biomolecules, such as carbohydrates, lipids, nucleic acids (e.g., oligonucleotides), and proteins or binding fragments thereof.

[0054] The specific binding member can be proteinaceous. As used herein, the term “proteinaceous” refers to a moiety that is composed of amino acid residues. A proteinaceous moiety can be a polypeptide. In certain cases, the proteinaceous specific binding member is an antibody. In certain embodiments, the proteinaceous specific binding member is an antibody fragment, e.g., a binding fragment of an antibody that specifically binds to a target analyte. As used herein, the terms “antibody” and “antibody molecule” are used interchangeably and refer to a protein consisting of one or more polypeptides substantially encoded by all or part of the recognized immunoglobulin genes. The recognized immunoglobulin genes, for example in humans, include the kappa (k), lambda (I), and heavy chain genetic loci, which together comprise the myriad variable region genes, and the constant region genes mu (u), delta (d), gamma (g), sigma (e), and alpha (a) which encode the IgM, IgD, IgG, IgE, and IgA isotypes respectively. An immunoglobulin light or heavy chain variable region consists of a “framework” region (FR) interrupted by three hypervariable regions, also called “complementarity determining regions” or “CDRs”. The extent of the framework region and CDRs have been precisely defined (see, “Sequences of Proteins of Immunological Interest,” E. Kabat et al., U.S. Department of Health and Human Services, (1991 )). The numbering of all antibody amino acid sequences discussed herein conforms to the Kabat system. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope of an antigen. The term antibody is meant to include full length antibodies and may refer to a natural antibody from any organism, an engineered antibody, or an antibody generated recombinantly for experimental, therapeutic, or other purposes as further defined below. Antibody fragments of interest include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, or other antigen-binding subsequences of antibodies, either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies. Antibodies may be monoclonal or polyclonal and may have other specific activities on cells (e.g., antagonists, agonists, neutralizing, inhibitory, or stimulatory antibodies). It is understood that the antibodies may have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other antibody functions.

[0055] In certain embodiments, the specific binding member is a Fab fragment, a F(ab')2 fragment, a scFv, a diabody or a triabody. In certain embodiments, the specific binding member is an antibody. In some cases, the specific binding member is a murine antibody or binding fragment thereof. In certain instances, the specific binding member is a recombinant antibody or binding fragment thereof."

[0056] "Active pharmaceutical ingredient” (API), “active agent”, “pharmacologically active agent”, and “drug” are used interchangeably herein to refer to a chemical material or compound which, when administered to an organism (e.g., a human or non-human animal) induces a desired pharmacologic and / or physiologic effect by local and / or systemic action.

[0057] A "pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," and "pharmaceutically acceptable adjuvant" means an excipient, diluent, carrier, and adjuvant that are useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use as well as human pharmaceutical use. "A pharmaceutically acceptable excipient, diluent, carrier and adjuvant" as used in the specification and claims includes both one and more than one such excipient, diluent, carrier, and adjuvant.

[0058] A “plurality” contains at least 2 members. In certain cases, a plurality may have 5 or more, such as 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 300 or more, 1000 or more, 3000 or more, 10,000 or more, 100,000 or more members.

[0059] Numeric ranges are inclusive of the numbers defining the range. DETAILED DESCRIPTION

[0060] Dyes, including tandem dyes, are provided. Aspects of embodiments of the dyes include a fluorophore and a water-solubilizing poly-sarcosine group. Also provided are methods of using the dyes, as well as kits that include such dyes.

[0061] Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0062] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0063] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0064] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0065] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0066] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0067] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0068] While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. §1 12, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §112 are to be accorded full statutory equivalents under 35 U.S.C. §112.

[0069] DYES

[0070] The present disclosure provides dyes that include a fluorophore and at least one water-solubilizing poly-sarcosine group. The "at least one water-solubilizing polysarcosine group" can increase the solubility of the dye in water. As used herein, the terms “water-solubilizing group”, “water-soluble group” and WSG are used interchangeably and refer to a group or substituent that is well-solvated in aqueous environments e.g., under physiological conditions, and which imparts improved water solubility upon the molecule to which it is attached. A WSG can increase the solubility of a tandem dye or component thereof, e.g., donor or acceptor fluorophore, in a predominantly aqueous solution, as compared to a control tandem dye or component thereof which lacks the WSG.

[0071] The magnitude of increase in solubility imparted to the dye by the one or more poly-sarcosine groups (e.g., collectively by a plurality of poly-sarcosine groups bonded to the dye) may vary, and in some instances is 5-fold or more, such as 10-fold or more, including 50-fold or more, relative to control dye that lacks water solubilizing polysarcosine groups. The water soluble group (WSG) can be capable of imparting solubility in water in excess of 10 mg / mL to the subject dye or polymeric tandem dye, such as in excess of 20 mg / mL, in excess of 30 mg / mL, in excess of 40 mg / mL, in excess of 50 mg / mL, in excess of 60 mg / mL, in excess of 70 mg / mL, in excess of 80 mg / mL, in excess of 90 mg / mL or in excess of 100 mg / mL. In certain cases, the branched non-ionic water soluble group (WSG) is capable of imparting solubility in water (e.g., an aqueous buffer) of 20 mg / mL or more to the subject dye or polymeric tandem dye, such as 30 mg / mL or more, 40 mg / mL or more, 50 mg / mL or more, 60 mg / mL or more, 70 mg / mL or more, 80 mg / mL or more, 90 mg / mL or more, 100 mg / mL or more, or even more. It is understood that water-soluble dipyrromethene-based dye may, under certain conditions, form discrete water solvated nanoparticles in aqueous systems. In certain cases, the water solvated nanoparticles are resistant to aggregation and find use in a variety of biological assays.

[0072] The number of water-solubilizing poly-sarcosine groups that is bonded to a given dye may vary, where the number may be chosen based on the desired water solubility of the dye. While the number of water-solubilizing poly-sarcosine groups that is bonded to a given dye may vary, in some instances the number ranges from 1 to 20, such as 2 to 15 and including 2 to 10.

[0073] Water-solubilizing poly-sarcosine groups of embodiments of the invention are polymers that include a plurality of (i.e., 2 or more) sarcosine residues. The term "sarcosine residue" as used here refers to a monomeric residue that is formed from sarcosine or derivative thereof, e.g., an N-modified sarcosine. While the length of a given water-solubilizing poly-sarcosine group may vary, in some instances the length ranges from 2 to 40 monomeric residues, such as 3 to 20 monomeric residues, including 3 to 10 monomeric residues. A given water-solubilizing poly-sarcosine group may be made up of identical sarcosine residues, such that it is a homopolymer, or different sarcosine residues, such that it is heteropolymer. In some instances, the water-solubilizing poly-sarcosine group may be made of sarcosine residues and non-sarcosine residues, e.g., residues derived from non-sarcosine amino acids or other types of monomers, as desired, such as where the water-solubilizing poly-sarcosine group is a copolymer of sarcosine and non- sarcosine residues. As such, in some instances a given water-solubilizing poly-sarcosine group may be made up entirely of sarcosine residues, such that it may be referred to as a poly-sarcosine polymer. In other instances, a given water-solubilizing poly-sarcosine group may be made up of N-modified sarcosine residues, such that it may be referred to as an N-modified poly-sarcosine polymer. In yet other instances, a given water-solubilizing poly-sarcosine group may be made up of different types of sarcosine residues (e.g., sarcosine and N-modified sarcosine residues) or sarcosine and non-sarcosine residues, such that it may be referred to as a poly-sarcosine-type co-polymer. In some embodiments, the one or more water-solubilizing poly-sarcosine groups bonded to a dye are poly-sarcosine polymers.

[0074] Sarcosine residues found in water-solubilizing poly-sarcosine groups of embodiments of the invention include one or more sarcosine residues described by the following formula: wherein Ri is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl.

[0075] For example, in some cases Ri is methyl, i.e., wherein the residue is sarcosine. In some cases, Ri is a non-methyl moiety, e.g., where the sarcosine residue is a N-modified sarcosine. In some instances, Ri is a substituted alkyl, e.g., a water-solubilizing group, e.g., an alkyl group substituted with a polyethylene glycol group or an alkyl substituted with an amino group, e.g., as described in greater detail below.

[0076] As such, in some instances the water-solubilizing poly-sarcosine group is a polymer described by the formula: wherein R is H or a non-hydrogen substituent and n is an integer of 2 or more. Thus, the polymer of formula (II) can be considered a polymer of residues of formula (I) wherein Ri is methyl. In some instances, n is an integer ranging from 2 to 40, such as 3 to 20, including 3 to 10.

[0077] Where Ri is other than methyl, Ri may vary as desired. In some instances, Ri may be selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, amino, azido, carboxy, cyano, ether, halo, hydroxy, nitro, thiol, thioether, thioketo, borate, -SO2, -SO3-, a reactive moiety, and a reactive moiety protecting group.

[0078] In some instances, Ri may be a non-poly-sarcosine water-solubilizing group. When present, these water solubilizing groups may be any convenient hydrophilic group that is well-solvated in aqueous environments. A variety of water-soluble polymer groups can be adapted for use in the WSG of the subject dyes. Any convenient water solubilizing groups (WSG’s) may be included in the dyes described herein to provide for increased water-solubility. While the increase in solubility may vary, in some instances the increase (as compared to the compound without the WSG(s)) is 2-fold or more, e.g., 5-fold, 10-fold, 25-fold, 50-fold, 100-fold or more. In some cases, the hydrophilic water solubilizing group is charged, e.g., positively or negatively charged. In certain cases, the hydrophilic water solubilizing group is a neutral hydrophilic group. In some embodiments, the WSG is branched (e.g., as described herein). In certain instances, the WSG is linear. In some embodiments, the WSG is a hydrophilic polymer, e.g., a polyethylene glycol, a modified PEG, a peptide sequence, a peptoid, a carbohydrate, an oxazoline, a polyol, a dendron, a dendritic polyglycerol, a cellulose, a chitosan, or a derivative thereof. Water solubilizing groups of interest include, but are not limited to, carboxylate, phosphonate, phosphate, sulfonate, sulfate, sulfinate, sulfonium, ester, polyethylene glycols (PEG) and modified PEGs, hydroxyl, amine, amino acid, ammonium, guanidinium, pyridinium, polyamine and sulfonium, polyalcohols, straight chain or cyclic saccharides, primary, secondary, tertiary, or quaternary amines and polyamines, phosphonate groups, phosphinate groups, ascorbate groups, glycols, including, polyethers, -COOM', -SO3M', -PO3M', -NR3+, Y', (CH2CH2O)PR and mixtures thereof, where Y' can be any halogen, sulfate, sulfonate, or oxygen containing anion, p can be 1 to 500, each R can be independently H or an alkyl (such as methyl) and M' can be a cationic counterion or hydrogen, - (CH2CH2O)yyCH2CH2XRyy, --(CH2CH2O)yyCH2CH2X--, -X(CH2CH2O)yyCH2CH2-, glycol, and polyethylene glycol, wherein yy is selected from 1 to 1000, X is selected from O, S, and NRZZ, and Rzzand RYYare independently selected from H and C1-3 alkyl. In some cases, a WSG is (CH2)x(OCH2CH2)yOCH3 where each x is independently an integer from 0-20, each y is independently an integer from 0 to 50. In some cases, the water solubilizing group includes a non-ionic polymer (e.g., a PEG polymer) substituted at the terminal with an ionic group (e.g., a sulfonate).

[0079] In some embodiments of the formulae, the pendant group of interest includes a substituent selected from (CH2)x(OCH2CH2)yOCH3 where each x is independently an integer from 0-20, each y is independently an integer from 0 to 50; and a benzyl optionally substituted with one or more halogen, hydroxyl, Ci-Ci2alkoxy, or (OCH2CH2)zOCH3where each z is independently an integer from 0 to 50. In some instances, the substituent is (CH2)3(OCH2CH2)HOCH3. In some embodiments, one or more of the substituents is a benzyl substituted with at least one WSG groups (e.g., one or two WSG groups) selected from (CH2)x(OCH2CH2)yOCH3 where each x is independently an integer from 0-20 and each y is independently an integer from 0 to 50.

[0080] Multiple WSGs may be included at a single location in the subject dyes via a branching linker. In certain embodiments, the branching linker is an aralkyl substituent, further di-substituted with water solubilizing groups. As such, in some cases, the branching linker group is a substituent of the dye that connects the dye to two or more water solubilizing groups. In certain embodiments, the branching linker is an amino acid, e.g., a lysine amino acid that is connected to three groups via the amino and carboxylic acid groups. In some cases, the incorporation of multiple WSGs via branching linkers imparts a desirable solubility on the dye. The subject water soluble polymers can be adapted to include any convenient linking groups. It is understood that in some cases, the water soluble polymer can include some dispersity with respect to polymer length, depending on the method of preparation and / or purification of the polymeric starting materials. In some instances, the water soluble polymers are monodisperse.

[0081] The water soluble polymer can include one or more spacers or linkers. Examples of spacers or linkers include linear or branched moieties comprising one or more repeat units employed in a water-soluble polymer, diamino and or diacid units, natural or unnatural amino acids or derivatives thereof, as well as aliphatic moieties, including alkyl, aryl, heteroalkyl, heteroaryl, alkoxy, and the like, which can contain, for example, up to 18 carbon atoms or even an additional polymer chain. The water soluble polymer moiety, or one or more of the spacers or linkers of the polymer moiety when present, may include polymer chains or units that are biostable or biodegradable. For example, polymers with repeat linkages have varying degrees of stability under physiological conditions depending on bond lability. Polymers with such bonds can be categorized by their relative rates of hydrolysis under physiological conditions based on known hydrolysis rates of low molecular weight analogs, e.g., from less stable to more stable, e.g., polyurethanes (-NH-C(O)-O-) > polyorthoesters (-0- C((OR)(R’))-O-) > polyamides (-C(O)-NH-). Similarly, the linkage systems attaching a water-soluble polymer to a target molecule may be biostable or biodegradable, e.g., from less stable to more stable: carbonate (-O-C(O)-O-) > ester (-C(O)-O-) > urethane (-NH- C(O)-O-) > orthoester (-O-C((OR)(R’))-O-) > amide (-C(O)-NH-). In general, it may be desirable to avoid use of a sulfated polysaccharide, depending on the lability of the sulfate group. In addition, it may be less desirable to use polycarbonates and polyesters. These bonds are provided by way of example, and are not intended to limit the types of bonds employable in the polymer chains or linkage systems of the water-soluble polymers useful in the WSGs disclosed herein.

[0082] An example of a water-solubulizing poly-sarcosine group that is made of up of N- modified sarcosine residues is described the following formula: wherein R is hydrogen or a non-hydrogen substitutent and n is an integer of 2 or more. In some instances, n is an integer ranging from 2 to 40, such as 3 to 20, including 3 to 10.

[0083] As mentioned above, in some instances the water-solubilizing group may be copolymer of sarcosine and non-sarcosine residues. In such cases, the polymer may include two or more different repeating residues (i.e., units), e.g., when the polymer is a multiblock polymer, a random arrangement of units or a defined sequence, each block may define a distinct repeating unit. It is understood that a variety of arrangements of repeating units or blocks are possible and that in the water-solubilizing poly-sarcosine polymers described herein any convenient linear arrangements of various lengths can be included within the structure of the overall polymer. It is understood that the polymer may also be represented by a formula in terms of mol% values of each unit in the polymer and that such formula may represent a variety of arrangements of repeat unit, such as random or multiblock polymer or a defined sequence of residues. In some cases, a repeating unit of the polymer includes a single monomer group. In certain instances, a repeating unit of the polymer includes two or more monomer groups, i.e., co-monomer groups, such as two, three, four or more co-monomer groups. The term “co-monomer” or “co-monomer group” refers to a structural unit of a polymer that may itself be part of a repeating unit of the polymer. Co-monomers that may be present in embodiments of the invention may vary, as desired, and include, amino acid monomers, peptoid monomers, polyakylene oxide monomers, etc.

[0084] An example of a water-solubulizing poly-sarcosine group that is coplymer is described the following formula: wherein R is hydrogen or a non-hydrogen substituent, wherein n may vary, and in some instances is 1 to 20, such as 1 to 15, m may vary, and in some instances is 1 to 20, such as 1 to 15, and p may vary, and in some instances is 1 to 20, such as 1 to 15.

[0085] As summarized above, aspects of the invention include dyes to which one or more water-solubilizing poly-sarcosine groups are bonded. In some cases, the dye comprises an organic dye. Organic dyes may vary, wherein organic dyes that may be modified with the water-solubilizing peptides with sulfonate groups of the invention include, but are not limited to: cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethines, pyrenes, dipyrromethene borondifluorides, napthalimides, thiazine dyes, and acridine dyes. Organic dyes of interest include, but are not limited to, fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP (peridinin chlorophyll-a Protein), PerCP-Cy5.5, JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, 7-amino-4- methylcoumarin-3-acetic acid, BODIPY FL, BODIPY FL-Br2, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591 , BODIPY 630 / 650, BODIPY 650 / 665, BODIPY R6G, BODIPY TMR, BODIPY TR, Dyonomics dyes (e.g. DY 431 , DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701 , DY 704, DY 730, DY 731 , DY 732, DY 734, DY 752, DY 778, DY 782, DY 800, DY 831 ), dipyrromethene borondifluoride (BODIPY), Biotium CF 555, diethylamino coumarin, and derivatives thereof. In some cases, the water-solubilizing peptide is bound to the organic dye.

[0086] Examples of specific organic dyes solubilized with water-solubilizing polysarcosine groups include, but are not limited to, the following: a rhodamine, a perylene, a diimide, a coumarin, a xanthene, a cyanine, a polymethine, a pyrene, a thiazine, an acridine, a dipyrromethene borondifluoride, a napthalimide, a phycobiliprotein, a peridinum chlorophyll protein, conjugates thereof, and combinations thereof. In certain embodiments, the acceptor fluorophore (A) is a cyanine dye, a xanthene dye, a coumarin dye, a thiazine dye or an acridine dye. In some instances, the acceptor fluorophore (A) is selected from DY 431 , DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701 , DY 704, DY 730, DY 731 , DY 732, DY 734, DY 752, DY 778, DY 782, DY 800, DY 831 , Biotium CF 555, Cy 3.5 and diethylamino coumarin. Fluorescent dyes of interest include, but are not limited to, fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP (peridinin chlorophyll-a Protein), PerCP-Cy5.5, JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy- X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY FL, BODIPY FL-Br.sub.2, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591 , BODIPY 630 / 650, BODIPY 650 / 665, BODIPY R6G, BODIPY TMR, BODIPY TR, conjugates thereof, and combinations thereof. Lanthanide chelates of interest include, but are not limited to, europium chelates, terbium chelates and samarium chelates. In some embodiments, the polymeric tandem dye includes a multichromophore linked to an acceptor fluorophore selected from Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Alexa488, Alexa 647 and Alexa700. In certain embodiments, the polymeric tandem dye includes a multichromophore linked to an acceptor fluorophore selected from Dyomics dyes (such as DY 431 , DY 485XL, DY 500XL, DY 530, DY 610, DY 633, DY 640, DY 651 , DY 654, DY 682, DY 700, DY 701 , DY 704, DY 730, DY 731 , DY 732, DY 734, DY 752, DY 754, DY 778, DY 782, DY 800 or DY 831), Biotium CF 555, Cy 3.5, and diethylamino coumarin. In certain cases, the acceptor fluorophore (A) is selected from fluorescein, 6-FAM, rhodamine, Texas Red, California Red, iFluor594, tetramethylrhodamine, a carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy7®, Cy-Chrome, DyLight 350, DyLight 405, DyLight 488, DyLight 549, DyLight 594, DyLight 633, DyLight 649, DyLight 680, DyLight 750, DyLight 800, phycoerythrin, PerCP (peridinin chlorophyll-a Protein), PerCP-Cy5.5, JOE (6-carboxy- 4',5'-dichloro-2',7'-dimelhoxyfluorescein), NED, ROX (5-(and -6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor® 350, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY® FL, BODIPY® FL-Br2, BODIPY® 530 / 550, BODIPY® 558 / 568, BODIPY® 564 / 570, BODIPY® 576 / 589, BODIPY® 581 / 591 , BODIPY® 630 / 650, BODIPY® 650 / 665, BODIPY® R6G, BODIPY® TMR, BODIPY® TR, conjugates thereof and combinations thereof.

[0087] In some cases, the organic dye solubilized with water-solubilizing poly-sarcosine groups can be selected from the group consisting of a rhodamine, a perylene, a diimide, a coumarin, a xanthene, a cyanine, a polymethine, a pyrene, a thiazine, an acridine, a dipyrromethene borondifluoride, a napthalimide, a phycobiliprotein, a peridinum chlorophyll protein. In some instances, the dye is not a dye employed in medical imaging applications.

[0088] For example, in some embodiments the dye is not a cyanine dye, such as indocyanine green.

[0089] In some embodiments, the dye further comprises a non-conjugated polymeric backbone comprising non-conjugated repeat units. By “non-conjugated” is meant that at least a portion of the repeat unit includes a saturated backbone group (e.g., a group having two or more consecutive single covalent bonds) which precludes pi conjugation or an extended delocalized electronic structure along the polymeric backbone from one repeat unit to the next. It is understood that even though one repeat unit may not be conjugated to an adjacent repeat unit, such a repeat unit may include one or more isolated unsaturated groups including an unsaturated bond (e.g., of an alkenylene group or an alkynylene group) and / or an aryl or heteroaryl group, which groups can be a part of the backbone. In some cases, each repeat unit of the polymeric backbone includes one sidechain including a linked pendant group or a chemo-selective tag for linking to a pendant group.

[0090] In certain embodiments, the polymeric backbone is a linear polymer. In certain cases, the linear polymer is selected from a peptide, a peptoid, a hydrocarbon polymer, and a PEG polymer. In certain cases, the linear polymer is a peptide. In certain cases, the linear polymer is a peptoid. In certain cases, the polymer is a hydrocarbon polymer. In certain other cases, the non-conjugated polymer is a PEG polymer. Further details regarding non-conjugated polymeric backbones that may be employed in embodiments of the invention are found in PCT application serial no. PCT / US2019 / 024662 published as WO 2019 / 191482 and PCT application serial no. PCT / US2020 / 019510 published as WO 2020 / 222894; the disclosures of which applications are herein incorporated by reference.

[0091] In certain instances, the non-conjugated polymeric backbone is a peptide of from 2 to 100 amino acids, such as 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40 or 2 to 30 amino acids. In some cases, the linear peptide backbone includes 2 or more amino acids, such as 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, up to a maximum of 100 amino acids. In certain cases, the tandem dye includes a linear peptide backbone of from 5 to 30 amino acids, such as 5 to 25, 5 to 20, 5 to 15, or 5 to 10 amino acids.

[0092] The non-conjugated repeat units may have any convenient configuration, such as a linear, branched or dendrimer configuration. The polymeric backbone can be a linear polymer. The polymeric backbone can be branched. In some instances, the dye includes a plurality of pendant chromophore groups each independently linked to a non-conjugated repeat unit of the polymeric backbone. The configuration of pendant groups can be installed during or after synthesis of the non-conjugated polymeric backbone. The incorporation of pendant groups can be achieved with a random configuration, a block configuration, or in a sequence-specific manner via stepwise synthesis, depending on the particular method of synthesis utilized.

[0093] In some instances, the non-conjugated repeat units comprise a plurality of amino acid residues. In some instances, the dye comprises an organic dye bound to the nonconjugated polymeric backbone. The water-solubilizing peptide can be bound to the nonconjugated polymeric backbone and / or to the organic dye.

[0094] In some instances, the dye is a polymeric dye (e.g., a fluorescent polymeric dye). Fluorescent polymeric dyes that find use in the subject methods and systems are varied. In some instances of the method, the polymeric dye includes a conjugated polymer. Conjugated polymers (CPs) are characterized by a delocalized electronic structure which includes a backbone of alternating unsaturated bonds (e.g., double and / or triple bonds) and saturated (e.g., single bonds) bonds, where TT-electrons can move from one bond to the other. As such, the conjugated backbone may impart an extended linear structure on the polymeric dye, with limited bond angles between repeat units of the polymer. For example, proteins and nucleic acids, although also polymeric, in some cases do not form extended-rod structures but rather fold into higher-order three-dimensional shapes. In addition, CPs may form "rigid-rod” polymer backbones and experience a limited twist (e.g., torsion) angle between monomer repeat units along the polymer backbone chain. In some instances, the polymeric dye includes a CP that has a rigid rod structure. The structural characteristics of the polymeric dyes can have an effect on the fluorescence properties of the molecules.

[0095] Any convenient polymeric dye may be utilized in the subject devices and methods. In some instances, a polymeric dye is a multichromophore that has a structure capable of harvesting light to amplify the fluorescent output of a fluorophore. In some instances, the polymeric dye is capable of harvesting light and efficiently converting it to emitted light at a longer wavelength. In some cases, the polymeric dye has a light-harvesting multichromophore system that can efficiently transfer energy to nearby luminescent species (e.g., a “signaling chromophore”). Mechanisms for energy transfer include, for example, resonant energy transfer (e.g., Forster (or fluorescence) resonance energy transfer, FRET), quantum charge exchange (Dexter energy transfer), and the like. In some instances, these energy transfer mechanisms are relatively short range; that is, close proximity of the light harvesting multichromophore system to the signaling chromophore provides for efficient energy transfer. Under conditions for efficient energy transfer, amplification of the emission from the signaling chromophore occurs when the number of individual chromophores in the light harvesting multichromophore system is large; that is, the emission from the signaling chromophore is more intense when the incident light (the “excitation light”) is at a wavelength which is absorbed by the light harvesting multichromophore system than when the signaling chromophore is directly excited by the pump light.

[0096] The multichromophore may be a conjugated polymer. Conjugated polymers (CPs) are characterized by a delocalized electronic structure and can be used as highly responsive optical reporters for chemical and biological targets. Because the effective conjugation length is substantially shorter than the length of the polymer chain, the backbone contains a large number of conjugated segments in close proximity. Thus, conjugated polymers are efficient for light harvesting and enable optical amplification via Forster energy transfer.

[0097] Polymeric dyes of interest include, but are not limited to, those dyes described by Gaylord et al. in U.S. Publication Nos. 20040142344, 20080293164, 20080064042, 20100136702, 20110256549, 201 10257374, 20120028828, 20120252986,

[0098] 20130190193, the disclosures of which are herein incorporated by reference in their entirety; and Gaylord et al., J. Am. Chem. See., 2001 , 123 (26), pp 6417-6418; Feng et al., Chem. Sec. Rev., 2010,39, 241 1 -2419; and Traina et al., J. Am. Chem. Sec., 2011 , 133 (32), pp 12600-12607, the disclosures of which are herein incorporated by reference in their entirety.

[0099] In some embodiments, the polymeric dye includes a conjugated polymer including a plurality of first optically active units forming a conjugated system, having a first absorption wavelength (e.g., as described herein) at which the first optically active units absorbs light to form an excited state. The conjugated polymer (CP) may be polycationic, polyanionic and / or a charge-neutral conjugated polymer.

[0100] The polymeric dye may have any convenient length. In some cases, the particular number of monomeric repeat units or segments of the polymeric dye may fall within the range of 2 to 500,000, such as 2 to 100,000, 2 to 30,000, 2 to 10,000, 2 to 3,000 or 2 to 1 ,000 units or segments, or such as 100 to 100,000, 200 to 100,000, or 500 to 50,000 units or segments. The polymeric dyes may be of any convenient molecular weight (MW). In some cases, the MW of the polymeric dye may be expressed as an average molecular weight. In some instances, the polymeric dye has an average molecular weight of from 500 to 500,000, such as from 1 ,000 to 100,000, from 2,000 to 100,000, from 10,000 to 100,000 or even an average molecular weight of from 50,000 to 100,000. In certain embodiments, the polymeric dye has an average molecular weight of 70,000.

[0101] In certain instances, the polymeric dye includes the following structure: where CPi, CP2, CP3 and CP4 are independently a conjugated polymer segment or an oligomeric structure, wherein one or more of CP1, CP2, CP3 and CP4 are bandgaplowering n-conjugated repeat units, and each n and each m are independently 0 or an integer from 1 to 10,000 and p is an integer from 1 to 100,000.

[0102] In some instances, the polymeric dye includes the following structure: where each R1is independently a solubilizing group (e.g., a water-solubilizing polysarcosine group) or a linker-dye; L1and L2are optional linkers; each R2is independently H or an aryl substituent; each A1and A2is independently H, an aryl substituent or a fluorophore; G1and G2are each independently selected from the group consisting of a terminal group, a n-conjugated segment, a linker and a linked specific binding member; each n and each m are independently 0 or an integer from 1 to 10,000; and p is an integer from 1 to 100,000. Solubilizing groups of interest include water-solubilizing poly-sarcosine groups as described herein.

[0103] In some cases, the polymeric dye includes, as part of the polymeric backbone, a conjugated segment having one of the following structures: where each R3is independently an optionally substituted alkyl or aryl group; Ar is an optionally substituted aryl or heteroaryl group; and each n is an integer from 1 to 10,000. In certain embodiments, R3is an optionally substituted alkyl group. In certain embodiments, R3is an optionally substituted aryl group. In some cases, R3is substituted with a water-solubilizing poly-sarcosine group, a polyethyleneglycol, a dye, a chemoselective functional group or a specific binding moiety. In some cases, Ar is substituted with a polyethyleneglycol, a dye, a chemoselective functional group or a specific binding moiety.

[0104] In some instances, the polymeric dye includes the following structure: where each R1is independently a solubilizing group (e.g., a water-solubilizing polysarcosine group) or a linker-dye group; each R2is independently H or an aryl substituent; each L1and L3are independently optional linkers; each A1and A3are independently H, a fluorophore, a functional group or a specific binding moiety (e.g., an antibody); and n and m are each independently 0 or an integer from 1 to 10,000, wherein n+m>1 .

[0105] The polymeric dye may have one or more desirable spectroscopic properties, such as a particular absorption maximum wavelength, a particular emission maximum wavelength, extinction coefficient, quantum yield, and the like (see e.g., Chattopadhyay et al., “Brilliant violet fluorophores: A new class of ultrabright fluorescent compounds for immunofluorescence experiments.” Cytometry Part A, 81A(6), 456-466, 2012). In some embodiments, the polymeric dye has an absorption curve between 280 nm and 475 nm. In certain embodiments, the polymeric dye has an absorption maximum (excitation maximum) in the range 280 nm and 475 nm. In some embodiments, the polymeric dye absorbs incident light having a wavelength in the range between 280 nm and 475 nm. In some embodiments, the polymeric dye has an emission maximum wavelength ranging from 400 nm to 850 nm, such as 415 nm to 800 nm, where specific examples of emission maxima of interest include, but are not limited to: 421 nm, 510 nm, 570 nm, 602 nm, 650 nm, 71 1 nm and 786 nm. In some instances, the polymeric dye has an emission maximum wavelength in a range selected from the group consisting of 410 nm to 430nm, 500 nm to 520nm, 560 nm to 580nm, 590 nm to 610nm, 640 nm to 660nm, 700 nm to 720nm, and 775 nm to 795nm. In certain embodiments, the polymeric dye has an emission maximum wavelength of 421 nm. In some instances, the polymeric dye has an emission maximum wavelength of 510 nm. In some cases, the polymeric dye has an emission maximum wavelength of 570 nm. In certain embodiments, the polymeric dye has an emission maximum wavelength of 602 nm. In some instances, the polymeric dye has an emission maximum wavelength of 650 nm. In certain cases, the polymeric dye has an emission maximum wavelength of 71 1 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 786 nm. In certain instances, the polymeric dye has an emission maximum wavelength of 421 nm ± 5 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 510 nm ± 5 nm. In certain instances, the polymeric dye has an emission maximum wavelength of 570 nm ± 5 nm. In some instances, the polymeric dye has an emission maximum wavelength of 602 nm ± 5 nm. In some embodiments, the polymeric dye has an emission maximum wavelength of 650 nm ± 5 nm. In certain instances, the polymeric dye has an emission maximum wavelength of 71 1 nm ± 5 nm. In some cases, the polymeric dye has an emission maximum wavelength of 786 nm ± 5 nm. In certain embodiments, the polymeric dye has an emission maximum selected from the group consisting of 421 nm, 510 nm, 570 nm, 602 nm, 650 nm, 71 1 nm and 786 nm.

[0106] In some instances, the polymeric dye has an extinction coefficient of 1 x 106cm1M-1or more, such as 2 x 106cm-1M'1or more, 2.5 x 106cm1M‘1or more, 3 x 106cm-1M-1or more, 4 x 106cm-1M'1or more, 5 x 106cnr1M'1or more, 6 x 106cnr1M'1or more, 7 x 106cm-1M'1or more, or 8 x 106cm-1M'1or more. In certain embodiments, the polymeric dye has a quantum yield of 0.05 or more, such as 0.1 or more, 0.15 or more, 0.2 or more, 0.25 or more, 0.3 or more, 0.35 or more, 0.4 or more, 0.45 or more, 0.5 or more, or even more. In certain cases, the polymeric dye has a quantum yield of 0.1 or more. In certain cases, the polymeric dye has a quantum yield of 0.3 or more. In certain cases, the polymeric dye has a quantum yield of 0.5 or more. In some embodiments, the polymeric dye has an extinction coefficient of 1 x 106or more and a quantum yield of 0.3 or more. In some embodiments, the polymeric dye has an extinction coefficient of 2 x 106or more and a quantum yield of 0.5 or more. Specific polymeric dyes that may be employed include, but are not limited to, BD Horizon Brilliant™ Dyes, such as BD Horizon Brilliant™ Violet Dyes (e.g., BV421 , BV510, BV605, BV650, BV711 , BV786); BD Horizon Brilliant™ Ultraviolet Dyes (e.g., BUV395, BUV496, BUV737, BUV805); and BD Horizon Brilliant™ Blue Dyes (e.g., BB515) (BD Biosciences, San Jose, CA).

[0107] In some cases, the water solubilizing water-solubilizing poly-sarcosine group is a pendant group, i.e., it is located at an end of the dye. In such cases, the pendant water solubilizing water-solubilizing poly-sarcosine group will be a monoradical. In other cases, the water solubilizing poly-sarcosine group is a linker group, i.e., it is located between two or more groups. For example, the linker water solubilizing water-solubilizing polysarcosine group can be a diradical that links two different groups, e.g., a dye to a polymer backbone, e.g., a non-conjugated or conjugated polymer, such as described above.

[0108] TANDEM DYES

[0109] The present disclosure provides tandem dyes that include a donor fluorophore, and acceptor fluorophore and at least one water-solubilizing poly-sarcosine group. The at least one water-solubilizing poly-sarcosine group can increase the solubility of the tandem dye in water, e.g., as described above.

[0110] Tandem dyes are compounds having two different, covalently linked fluorophores, which fluorophores may be covalently linked to each other directly or through a linking group, e.g., a poly-sarcosine water-solubilizing group or another polymer, e.g., a nonconjugated polymeric backbone, etc., such as described above. In the tandem dye, one of the fluorophores serves as donor fluorophore and the other fluorophore acts as acceptor fluorophore. The donor and acceptor fluorophores together form a fluorescenceresonance energy transfer (FRET) pair. Such FRET pairs behave as a unique dye that has the excitation properties of the donor fluorophore and the emission properties of the acceptor fluorophore.

[0111] Excitation of the donor can lead to energy transfer to, and emission from, the covalently attached acceptor fluorophore. Mechanisms for energy transfer between the donor chromophores to a linked acceptor signaling fluorophore include, for example, resonant energy transfer (e.g., Forster (or fluorescence) resonance energy transfer, FRET), quantum charge exchange (Dexter energy transfer) and the like. These energy transfer mechanisms can be relatively short range; that is, close proximity of chromophores of the light harvesting multichromophore system to each other and / or to an acceptor fluorophore provides for efficient energy transfer. Under conditions for efficient energy transfer, amplification of the emission from the acceptor fluorophore can occur where the emission from the luminescent acceptor fluorophore is more intense when the incident light (the "pump light") is at a wavelength which is absorbed by, and transferred from, the chromophores of the light harvesting chromophore than when the luminescent acceptor fluorophore is directly excited by the pump light. By ‘‘efficient’’ energy transfer is meant 10% or more, such as 20% or more or 30% or more, 40% or more, 50 % or more, of the energy harvested by the donor chromophores is transferred to the acceptor. By “amplification” is meant that the signal from the acceptor fluorophore is 1.5x or greater when excited by energy transfer from the donor light harvesting chromophore system as compared to direct excitation of the acceptor fluorophore with incident light of an equivalent intensity. The signal may be measured using any convenient method. In some cases, the 1.5x or greater signal refers to an intensity of emitted light. In certain cases, the 1.5x or greater signal refers to an increased signal to noise ratio. In certain embodiments of the tandem dye, the acceptor fluorophore emission is 1 .5 fold greater or more when excited by the chromophore as compared to direct excitation of the acceptor fluorophore with incident light, such as 2-fold or greater, 3-fold or greater, 4-fold or greater, 5-fold or greater, 6-fold or greater, 8-fold or greater, 10-fold or greater, 20-fold or greater, 50-fold or greater, 100-fold or greater, or even greater as compared to direct excitation of the acceptor fluorophore with incident light.

[0112] In some instances, the tandem dye exhibits an effective Stokes shift ranging from 25 nm to 300 nm, such as from 50 nm to 250 nm or from 75 nm to 200 nm. In some cases the effective Stokes shift is 25 nm or more, such as 50 nm or more, 75 nm or more, 100 nm or more, such as 110 nm or more, 120 nm or more, 130 nm or more, 140 nm or more, 150 nm or more, 160 nm or more, 170 nm or more, 180 nm or more, 190 nm or more, 200 nm or more, 250 nm or more when the light harvesting chromophore is directly excited with incident light.

[0113] The emission of the tandem dye can have a quantum yield of 0.03 or more, such as a quantum yield of 0.04 or more, 0.05 or more, 0.06 or more, 0.07 or more, 0.08 or more, 0.09 or more, 0.1 or more, 0.15 or more, 0.2 or more, 0.3 or more or even more. In some instances, the polymeric tandem dye has an extinction coefficient of 5 x 105cm-1M'

[0114] 1or more, such as 6 x 105cm'1M-1or more, 7 x 105cm-1M'1or more, 8 x 105cm-1M'1or more, 9 x 105cm-1M'1or more, such as 1 x 106cm-1M’1or more, 1.5 x 106cm'1M-1or more,

[0115] 2 x 106cm'1M'1or more, 2.5 x 106cm1M'1or more, 3 x 106cm1M'1or more, 4 x 106cm'1M-1or more, 5 x 106crn ’M-1or more, 6 x 106crn ’M-1or more, 7 x 106cm ’M-1or more, or 8 x 106cm’1M’1or more. In some embodiments, the tandem dye has a molar extinction coefficient of 5 x 105M'1crrr1or more. In certain embodiments, the tandem dye has a molar extinction coefficient of 1 x 106M’1cm-1or more.

[0116] In some embodiments, one of the donor and acceptor fluorophore, and in some cases both of the donor fluorophore and the acceptor fluorophore, comprise an organic dye. For instance, the organic dye of the donor fluorophore can be selected from the group consisting of cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethines, pyrenes, dipyrromethene borondifluorides, napthalimides, thiazine dyes, and acridine dyes. In some cases, the organic dye of the acceptor fluorophore can be selected from the group consisting of cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethines, pyrenes, dipyrromethene borondifluorides, napthalimides, thiazine dyes, and acridine dyes.

[0117] Organic dyes of interest that may be employed as donors or acceptors include, but are not limited to, fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine 1 10, Cascade Blue, Cascade Yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP (peridinin chlorophyll-a Protein), PerCP-Cy5.5, JOE (6-carboxy- 4',5'-dichloro-2',7'-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY FL, BODIPY FL-Br.sub.2, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591 , BODIPY 630 / 650, BODIPY 650 / 665, BODIPY R6G, BODIPY TMR, BODIPY TR, Dyonomics dyes (e.g. DY 431 , DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701 , DY 704, DY 730, DY 731 , DY 732, DY 734, DY 752, DY 778, DY 782, DY 800, DY 831 ), dipyrromethene borondifluoride (BODIPY), Biotium CF 555, diethylamino coumarin, and derivatives thereof.

[0118] In some instances, a tandem dye can include a non-conjugated polymeric backbone, e.g., a polymer made up of non-conjugated repeat units, such as described above. In some embodiments the non-conjugated repeat units include a plurality of amino acid residues. In such instances, the water-solubilizing poly-sarcosine group can be bound to the donor fluorophore, the acceptor fluorophore, and / or the non-conjugated polymeric backbone, and may even be incorporated into the non-conjugated polymeric backbone.

[0119] In some instances, dyes can include a fluorophore that is a conjugated polymer. As such, in some cases the donor fluorophore of the tandem dye can comprise a conjugated polymer. As described above in relation to dyes, in some cases the conjugated polymer can include a series of optionally-substituted aryl and / or heteroaryl groups. Aryl groups of interest include fluorene and phenyl whereas heteroaryl groups of interest include thiophene, pyridine, and BODIPY groups. In such cases, the water-solubilizing peptide can be bound to the conjugated polymer and / or the acceptor fluorophore.

[0120] In some cases, the water-solubilizing poly-sarcosine group is a pendant group, i.e. , it is located at an end of the tandem dye. In such cases, the pendant water-solubilizing poly-sarcosine group will be a monoradical. In other cases, the water-solubilizing polysarcosine group is a linker group, i.e., it is located between two or more groups. For example, the linker water-solubilizing poly-sarcosine group can be a diradical that links two different groups. For instance, the water-solubilizing poly-sarcosine group can be bonded to the acceptor fluorophore and to a non-conjugated backbone, or the watersolubilizing poly-sarcosine group is bonded to a donor fluorophore and to a nonconjugated backbone.

[0121] LABELED SPECIFIC BINDING MEMBERS

[0122] Aspects of the present disclosure also include labeled specific binding members that include a dye or tandem dye, e.g., as described above, along with a specific binding member. Stated in another manner, the specific binding member is labeled with the dye or tandem dye, e.g., to allow for detection of a target analyte that binds to the specific binding member.

[0123] As discussed above, the water-solubilizing poly-sarcosine group can be a pendant group. As such, the water-solubilizing poly-sarcosine group can be a terminal, monoradical group bonded to a fluorophore or a specific binding member. In other cases, the water-solubilizing poly-sarcosine group can be a linker that connects two or more groups. For instance, the water-solubilizing poly-sarcosine group can connect an acceptor chromophore to the remainder of a tandem dye, e.g., wherein the water-solubilizing polysarcosine group is bonded to the acceptor fluorophore and to a non-conjugated backbone. In other instances, the water-solubilizing poly-sarcosine group peptide is bonded to a donor fluorophore and a non-conjugated backbone, or the water-solubilizing polysarcosine group is bonded to a specific binding member and a non-conjugated backbone.

[0124] For example, in some cases the labeled specific binding member has formula (V): wherein:

[0125] D is a dye;

[0126] A is absent or an acceptor dye;

[0127] Li is absent or a linker that optionally comprises a poly-sarcosine group;

[0128] L2is a linker that optionally comprises a poly-sarcosine group; x is 0 or an integer ranging from 1 to 10; each Y is independently a water solubilizing group comprising a poly-sarcosine group; and

[0129] SBM is a specific binding member.

[0130] In some embodiments, the labeled specific binding member is a dye, i.e., wherein A is absent and l_2is absent. As such, D is a dye, as discussed above. Exemplary dyes that can be used at the D group include those described above, e.g., cyanine dyes, rhodamine dyes, coumarin dyes, BODIPY dyes, cyanine dyes, and a polyfluorene dye.

[0131] In some cases, the labeled specific binding member is a tandem dye, i.e., wherein A is present. As such, D can be considered a donor dye and A is the acceptor dye that accepts energy transfer from D. With tandem dyes, L2can be absent or L2can be a linker, e.g., a linker that comprises a poly-sarcosine group, which can increase water solubility, a non-conjugated polymeric backbone, etc. In some instances, D is a conjugated polymer, e.g., as described above.

[0132] Li is absent or a linker that optionally comprises a poly-sarcosine group, e.g., that increases water solubility. Additionally, x can be an integer from 1 to 10 and therefore the labeled specific binding member can include one to ten Y groups. As discussed above, the Y groups are each independently a water solubilizing group comprising a polysarcosine group.

[0133] METHODS OF LABELING A TARGET MOLECULE Also provided are methods of labeling a target molecule. Such methods include contacting the target molecule with a dye or tandem dye as described herein to covalently bond the target molecule to a reactive moiety of the dye or tandem dye, thereby producing the labelled target molecule.

[0134] Methods of interest for labelling a target, include but are not limited to, those methods and reagents described by Hermanson, Bioconjugate Techniques, Third edition, Academic Press, 2013. The contacting step may be performed in an aqueous solution. In some instances, the reactive moiety includes an amino functional group and the target molecule includes an activated ester functional group, such as a NHS ester or sulfo-NHS ester, or vice versa. In certain instances, the reactive moiety includes a maleimide functional group and the target molecule includes a thiol functional group, or vice versa. In certain instances, the reactive moiety includes an alkyne (e.g., a cyclooctyne group) functional group and the target molecule includes an azide functional group, or vice versa, which can be conjugated via Click chemistry.

[0135] Any convenient target molecules may be selected for labelling utilizing the subject methods. Target molecules of interest include, but are not limited to, a nucleic acid, such as an RNA, DNA, PNA, CNA, HNA, LNA or ANA molecule, a protein, such as a fusion protein, a modified protein, such as a phosphorylated, glycosylated, ubiquitinated, SUMOylated, or acetylated protein, or an antibody, a peptide, an aggregated biomolecule, a cell, a small molecule, a vitamin and a drug molecule. As used herein, the term “a target protein” refers to all members of the target family, and fragments thereof. The target protein may be any protein of interest, such as a therapeutic or diagnostic target, including but not limited to: hormones, growth factors, receptors, enzymes, cytokines, osteoinductive factors, colony stimulating factors and immunoglobulins. The term “target protein” is intended to include recombinant and synthetic molecules, which can be prepared using any convenient recombinant expression methods or using any convenient synthetic methods, or purchased commercially. In some embodiments, the target molecule is a specific binding member (e.g., as described herein). In certain instances, the specific binding member is an antibody. In some instances, the specific binding member is an antibody fragment or binding derivative thereof. In some case, the antibody fragment or binding derivative thereof is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a scFv, a diabody and a triabody.

[0136] In some cases, the method includes a separating step where the labelled target molecule is separated from the reaction mixture, e.g., excess reagents or unlabeled target. A variety of methods may be utilized to separate a target from a sample, e.g., via immobilization on a support, precipitation, chromatography, and the like.

[0137] In some instances, the method further includes detecting and / or analyzing the labelled target molecule. In some instances, the method further includes fluorescently detecting the labelled target molecule. Any convenient methods may be utilized to detect and / or analyze the labelled target molecule in conjunction with the subject methods and compositions. Methods of analyzing a target of interest that find use in the subject methods, include but are not limited to, flow cytometry, fluorescence microscopy, in-situ hybridization, enzyme-linked immunosorbent assays (ELISAs), western blot analysis, magnetic cell separation assays and fluorochrome purification chromatography. Detection methods of interest include but are not limited to fluorescence spectroscopy, fluorescence microscopy, nucleic acid sequencing, fluorescence in-situ hybridization (FISH), protein mass spectroscopy, flow cytometry, and the like.

[0138] Detection may be achieved directly via the polymeric tandem dye, or indirectly by a secondary detection system. The latter may be based on any one or a combination of several different principles including, but not limited to, antibody labelled anti-species antibody and other forms of immunological or non-immunological bridging and signal amplification systems (e.g., biotin-streptavidin technology, protein-A and protein-G mediated technology, or nucleic acid probe / anti-nucleic acid probes, and the like). Suitable reporter molecules may be those known in the field of immunocytochemistry, molecular biology, light, fluorescence, and electron microscopy, cell immunophenotyping, cell sorting, flow cytometry, cell visualization, detection, enumeration, and / or signal output quantification. More than one antibody of specific and / or non-specific nature might be labelled and used simultaneously or sequentially to enhance target detection, identification, and / or analysis.

[0139] The methods described herein may include multiple steps. Each step may be performed after a predetermined amount of time has elapsed between steps, as desired. As such, the time between performing each step may be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more and including 5 hours or more. In certain embodiments, each subsequent step is performed immediately after completion of the previous step. In other embodiments, a step may be performed after an incubation or waiting time after completion of the previous step, e.g., a few minutes to an overnight waiting time. METHODS OF EVALUATING A SAMPLE FOR PRESENCE OF A TARGET ANALYTE

[0140] Also provided are methods of evaluating a sample for the presence of a target analyte by using a labeled specific binding member that includes a water-solubilizing peptide comprising a sulfonate group. In some embodiments the methods include:

[0141] (a) contacting the sample with a labeled specific binding member that specifically binds the target analyte to produce a labeled sample; and

[0142] (b) assaying the labeled composition for the presence of a labelled specific binding member-target analyte binding complex to evaluate whether the target analyte is present in the sample.

[0143] The labeled specific binding member employed in embodiments of methods of the invention includes a specific binding member conjugated to a dye or tandem dye, e.g., as described above. In the following section, the target analyte may be a target molecule of interest or reagent, e.g., primary antibody, bound to the target moleulce, depending on whether the labeled specific binding member is employed as a primary or secondary label. Any convenient method may be used to contact the sample with a labeled specific binding member that specifically binds to the target analyte to produce the assay composition. In some instances, the sample is contacted with the labeled specific binding member under conditions in which the labeled specific binding member specifically binds to the target analyte, if present. For specific binding of the labeled specific binding member with the target analyte, an appropriate medium may be used that maintains the biological activity of the components of the sample and the singal domain antibody. The medium may be a balanced salt solution, e.g., normal saline, PBS, Hank’s balanced salt solution, etc., conveniently supplemented with fetal calf serum, human platelet lysate or other factors, in conjunction with an acceptable buffer at low concentration, such as from 5-25 mM. Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc. Various media are commercially available and may be used according to the nature of the target analyte, including dMEM, HBSS, dPBS, RPMI, Iscove’s medium, etc., in some cases supplemented with fetal calf serum or human platelet lysate. The final components of the medium, which may be a solution, may be selected depending on the components of the sample which are included. The temperature at which specific binding of the labeled specific binding member to the target analyte takes place may vary, and in some instances may range from 5 °C to 50 °C, such as from 10 °C to 40 °C, 15 °C to 40 °C, 20 °C to 40 °C, e.g., 20 °C, 25 °C, 30 °C, 35 °C or 37 °C (e.g., as described above). In some instances, the temperature at which specific binding takes place is selected to be compatible with the biological activity of the specific binding member and / or the target analyte. In certain instances, the temperature is 25 °C, 30 °C, 35 °C, or 37 °C. In certain cases, the temperature at which specific binding takes place is room temperature (e.g., 25 °C), 30 °C, 35 °C, or 37 °C. Any convenient incubation time for specific binding may be selected to allow for the formation of a desirable amount of binding complex, and in some instances, may be 1 minute (min) or more, such as 2 min or more, 10 min or more, 30 min or more, 1 hour or more, 2 hours or more, or even 6 hours or more.

[0144] Any convenient specific binding members may be utilized in the labeled specific binding members employed in methods of the invention. Specific binding members of interest include, but are not limited to, those specific binding members that specifically bind cell surface proteins of a variety of cell types, including but not limited to, stem cells, e.g., pluripotent stem cells, hematopoietic stem cells, T cells, T regulator cells, dendritic cells, B Cells, e.g., memory B cells, antigen specific B cells, granulocytes, leukemia cells, lymphoma cells, virus cells (e.g., HIV cells) NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythroid cells. Target cells of interest include cells that have a convenient cell surface marker or antigen that may be captured by a convenient specific binding member conjugate. In some embodiments, the target cell is selected from HIV containing cell, a Treg cell, an antigen-specific T -cell populations, tumor cells or hematopoetic progenitor cells (CD34+) from whole blood, bone marrow or cord blood. Any convenient cell surface proteins or cell markers may be targeted for specific binding to the conjugates employed in the subject methods. In some embodiments, the target cell includes a cell surface marker selected from a cell receptor and a cell surface antigen. In some cases, the target cell may include a cell surface antigen such as CD11 b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61 , CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor alpha / beta, T cell receptor gamma / delta, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-Terminal), SSEA-3, TRA-1 -60 Antigen, Disialoganglioside GD2 and CD71 .

[0145] Any convenient targets may be selected for evaluation utilizing the subject methods. Targets of interest include, but are not limited to, a nucleic acid, such as an RNA, DNA, PNA, CNA, HNA, LNA or ANA molecule, a protein, such as a fusion protein, a modified protein, such as a phosphorylated, glycosylated, ubiquitinated, SUMOylated, or acetylated protein, or an antibody, a peptide, an aggregated biomolecule, a cell, a small molecule, a vitamin and a drug molecule. As used herein, the term “a target protein” refers to all members of the target family, and fragments thereof. The target protein may be any protein of interest, such as a therapeutic or diagnostic target, including but not limited to: hormones, growth factors, transcription factor, receptors, enzymes, cytokines, osteoinductive factors, colony stimulating factors and immunoglobulins. The term “target protein” is intended to include recombinant and synthetic molecules, which can be prepared using any convenient recombinant expression methods or using any convenient synthetic methods, or purchased commercially. In some embodiments, the polymeric dye conjugates include an antibody or antibody fragment. Any convenient target analyte that specifically binds an antibody or antibody fragment of interest may be targeted in the subject methods.

[0146] In some embodiments, the target analyte is associated with a cell. In certain instances, the target analyte is a cell surface marker of the cell. In certain cases, the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen. In some instances, the target analyte is an intracellular target, and the method further includes treating the cell so as to provide access of the labeled specific binding memberto the intracellular target, e.g., by permeabilizing or lysing the cell. As such, a labeled specific binding member employed in methods of the invention may target a cell surface or intracellular antigen. Alternatively, a labeled specific binding member employed in methods of the invention may target a primary antibody that in turn specifically binds to a target cell surface or intracellular antigen.

[0147] In some embodiments, the sample may include a heterogeneous cell population from which target cells are isolated. In some instances, the sample includes peripheral whole blood, peripheral whole blood in which erythrocytes have been lysed prior to cell isolation, cord blood, bone marrow, density gradient-purified peripheral blood mononuclear cells or homogenized tissue. In some cases, the sample includes hematopoetic progenitor cells (e.g., CD34+ cells) in whole blood, bone marrow or cord blood. In certain embodiments, the sample includes tumor cells in peripheral blood. In certain instances, the sample is a sample including (or suspected of including) viral cells (e.g., HIV).

[0148] The labeled specific binding members find use in the subject methods, e.g., for labeling a target cell, particle, target or analyte with a polymeric tandem fluorescent dye. For example, labeled specific binding members find use in labeling cells to be processed (e.g., detected, analyzed, and / or sorted) in a flow cytometer. The labeled specific binding members may include specific binding members, e.g., antibodies or binding fragments thereof, that specifically bind to, e.g., cell surface proteins of a variety of cell types (e.g., as described herein). The labeled specific binding members may be used to investigate a variety of biological (e.g., cellular) properties or processes such as cell cycle, cell proliferation, cell differentiation, DNA repair, T cell signaling, apoptosis, cell surface protein expression and / or presentation, and so forth. Labelled specific binding members may be used in any application that includes (or may include) antibody-mediated labeling of a cell, particle or analyte.

[0149] Aspects of the methods include assaying the assay composition, i.e., labeled specific binding member contacted sample, for the presence of a labeled specific binding member-target analyte binding complex to evaluate whether the target analyte is present in the sample. Once the sample has been contacted with the labeled specific binding member, any convenient method may be utilized in assaying the assay composition that is produced for the presence of a labeled specific binding member-target analyte binding complex. The labeled specific binding member-target analyte binding complex is the binding complex that is produced upon specific binding of the labeled specific binding member to the target analyte (or primary binding member, e.g., primeary antibody, to the target antigent depending on the embodiment), if present. Assaying the assay composition may include detecting a fluorescent signal from the binding complex, if present. In some cases, the assaying includes a separating step where the target analyte, if present, is separated from the sample. A variety of methods can be utilized to separate a target analyte from a sample, e.g., via immobilization on a support. Assay methods of interest include, but are not limited to, any convenient methods and assay formats where pairs of specific binding members such as avidin- biotin or hapten-anti-hapten antibodies find use, are of interest. Methods and assay formats of interest that may be adapted for use with the subject compositions include, but are not limited to, flow cytometry methods, in-situ hybridization methods, enzyme-linked immunosorbent assays (ELISAs), western blot analysis, magnetic cell separation assays and fluorochrome purification chromatography.

[0150] In certain embodiments, the method further includes contacting the sample with a second specific binding member that specifically binds the target analyte. In certain instances, the second specific binding member is support bound. Any convenient supports may be utilized to immobilize a component of the subject methods (e.g., a second specific binding member). In certain instances, the support is a particle, such as a magnetic particle. In some instances, the second specific binding member and the polymeric dye conjugate produce a sandwich complex that may be isolated and detected, if present, using any convenient methods. In some embodiments, the method further includes flow cytometrically analyzing the polymeric dye conjugate-target analyte binding complex, i.e. , a fluorescently labelled target analyte. Assaying for the presence of a labeled specific binding member -target analyte binding complex may provide assay results (e.g., qualitative or quantitative assay data) which can be used to evaluate whether the target analyte is present in the sample.

[0151] Any convenient supports may be utilized in the subject methods to immobilize any convenient component of the methods, e.g., labelled specific binding member, target, secondary specific binding member, etc. Supports of interest include, but are not limited to: solid substrates, where the substrate can have a variety of configurations, e.g., a sheet, bead, or other structure, such as a plate with wells; beads, polymers, particle, a fibrous mesh, hydrogels, porous matrix, a pin, a microarray surface, a chromatography support, and the like. In some instances, the support is selected from the group consisting of a particle, a planar solid substrate, a fibrous mesh, a hydrogel, a porous matrix, a pin, a microarray surface and a chromatography support. The support may be incorporated into a system that it provides for cell isolation assisted by any convenient methods, such as a manually-operated syringe, a centrifuge or an automated liquid handling system. In some cases, the support finds use in an automated liquid handling system for the high throughput isolation of cells, such as a flow cytometer.

[0152] In some embodiments of the method, the separating step includes applying an external magnetic field to immobilize a magnetic particle. Any convenient magnet may be used as a source of the external magnetic field (e.g., magnetic field gradient). In some cases, the external magnetic field is generated by a magnetic source, e.g. by a permanent magnet or electromagnet. In some cases, immobilizing the magnetic particles means the magnetic particles accumulate near the surface closest to the magnetic field gradient source, i.e. the magnet.

[0153] The separating may further include one or more optional washing steps to remove unbound material of the sample from the support. Any convenient washing methods may be used, e.g., washing the immobilized support with a biocompatible buffer which preserves the specific binding interaction of the polymeric dye and the specific binding member. Separation and optional washing of unbound material of the sample from the support provides for an enriched population of target cells where undesired cells and material may be removed. In certain embodiments, the method includes detecting the labeled target analyte. Detecting the labeled target analyte may include exciting the polymeric fluorescent tandem dye with one or more lasers and subsequently detecting fluorescence emission from the polymeric fluorescent tandem dye using one or more optical detectors. Detection of the labeled target can be performed using any convenient instruments and methods, including but not limited to, flow cytometry, FACS systems, fluorescence microscopy; fluorescence, luminescence, ultraviolet, and / or visible light detection using a plate reader; high performance liquid chromatography (HPLC); and mass spectrometry. When using fluorescently labeled components in the methods and compositions of the present disclosure, it is recognized that different types of fluorescence detection systems can be used to practice the subject methods. In some cases, high throughput screening can be performed, e.g., systems that use 96 well or greater microtiter plates. A variety of methods of performing assays on fluorescent materials can be utilized, such as those methods described in, e.g., Lakowicz, J. R., Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983); Herman, B., Resonance energy transfer microscopy, in: Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed. Taylor, D. L. & Wang, Y.-L., San Diego: Academic Press (1989), pp. 219-243; Turro, N.J., Modern Molecular Photochemistry, Menlo Park: Benjamin / Cummings Publishing Col, Inc. (1978), pp. 296-361.

[0154] Fluorescence in a sample can be measured using a fluorimeter. In some cases, excitation radiation, from an excitation source having a first wavelength, passes through excitation optics. The excitation optics cause the excitation radiation to excite the sample. In response, fluorescently labelled targets in the sample emit radiation which has a wavelength that is different from the excitation wavelength. Collection optics then collect the emission from the sample. The device can include a temperature controller to maintain the sample at a specific temperature while it is being scanned. In certain instances, a multi-axis translation stage moves a microtiter plate holding a plurality of samples in order to position different wells to be exposed. The multi-axis translation stage, temperature controller, auto-focusing feature, and electronics associated with imaging and data collection can be managed by an appropriately programmed digital computer. The computer also can transform the data collected during the assay into another format for presentation.

[0155] In some embodiments, the method of evaluating a sample for the presence of a target analyte further includes detecting fluorescence in a flow cytometer. In some embodiments, the method of evaluating a sample for the presence of a target analyte further includes imaging the labelling composition contacted sample using fluorescence microscopy. Fluorescence microscopy imaging can be used to identify a polymeric dye conjugate-target analyte binding complex in the contacted sample to evaluate whether the target analyte is present. Microscopy methods of interest that find use in the subject methods include laser scanning confocal microscopy.

[0156] The methods described herein may include multiple steps. Each step may be performed after a predetermined amount of time has elapsed between steps, as desired. As such, the time between performing each step may be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more and including 5 hours or more. In certain embodiments, each subsequent step is performed immediately after completion of the previous step. In other embodiments, a step may be performed after an incubation or waiting time after completion of the previous step, e.g., a few minutes to an overnight waiting time.

[0157] SYSTEMS

[0158] Aspects of the invention further include systems for use in practicing the subject methods and compositions. A sample analysis system can include sample field of view or a flow channel loaded with a sample and labeled specific binding member of the invention, e.g., as described above. In some embodiments, the system is a flow cytometric system including: a flow cytometer including a flow path; a composition in the flow path, wherein the composition includes: a sample and a labled specific binding member (e.g., as described herein). In some embodiments, the system for analyzing a sample is a fluorescence microscopy system, including: a fluorescence microscope comprising a sample field of view; and a composition disposed in the sample field of view, wherein the composition comprises a sample; and a labelled specific binding member (e.g., as described herein).

[0159] In certain embodiments of the systems, the composition further includes a second specific binding member that is support bound and specifically binds the target analyte. In some cases, the support includes a magnetic particle. As such, in certain instances, the system may also include a controllable external paramagnetic field configured for application to an assay region of the flow channel.

[0160] The sample may include a cell. In some instances, the sample is a cell-containing biological sample. In some instances, the sample includes a labelled specific binding member specifically bound to a target cell. In certain instances, the target analyte that is specifically bound by the specific binding member is a cell surface marker of the cell. In certain cases, the cell surface marker is selected from a cell receptor and a cell surface antigen.

[0161] In certain aspects, the system may also include a light source configured to direct light to an assay region of the flow channel or sample field of view. The system may include a detector configured to receive a signal from an assay region of the flow channel or a sample field of view, wherein the signal is provided by the fluorescent composition. Optionally further, the sample analysis system may include one or more additional detectors and / or light sources for the detection of one or more additional signals.

[0162] In certain aspects, the system may further include computer-based systems configured to detect the presence of the fluorescent signal. A “computer-based system" refers to the hardware means, software means, and data storage means used to analyze the information of the present invention. The minimum hardware of the computer-based systems of the present invention includes a central processing unit (CPU), input means, output means, and data storage means. A skilled artisan can readily appreciate that any one of the currently available computer-based system are suitable for use in the subject systems. The data storage means may include any manufacture including a recording of the present information as described above, or a memory access means that can access such a manufacture.

[0163] To "record" data, programming or other information on a computer readable medium refers to a process for storing information, using any such methods as known in the art. Any convenient data storage structure may be chosen, based on the means used to access the stored information. A variety of data processor programs and formats can be used for storage, e.g., word processing text file, database format, etc.

[0164] A “processor” references any hardware and / or software combination that will perform the functions required of it. For example, any processor herein may be a programmable digital microprocessor such as available in the form of an electronic controller, mainframe, server or personal computer (desktop or portable). Where the processor is programmable, suitable programming can be communicated from a remote location to the processor, or previously saved in a computer program product (such as a portable or fixed computer readable storage medium, whether magnetic, optical or solid state device based). For example, a magnetic medium or optical disk may carry the programming, and can be read by a suitable reader communicating with each processor at its corresponding station.

[0165] In addition to the sensor device and signal processing module, e.g., as described above, systems of the invention may include a number of additional components, such as data output devices, e.g., monitors and / or speakers, data input devices, e.g., interface ports, keyboards, etc., fluid handling components, power sources, etc.

[0166] In certain aspects, the system includes a flow cytometer. Suitable flow cytometry systems may include, but are not limited to, those described in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91 , Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan;49(pt 1 ):17-28; Linden, et. al., Semin Throm Hemost. 2004 Oct;30(5):502- 1 1 ; Alison, et al. J Pathol, 2010 Dec; 222(4) :335-344; and Herbig, et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255; the disclosures of which are incorporated herein by reference. In certain instances, flow cytometry systems of interest include BD Biosciences FACSCanto™ flow cytometer, BD Biosciences FACSCanto™ II flow cytometer, BD Accuri™ flow cytometer, BD Accuri™ C6 Plus flow cytometer, BD Biosciences FACSCelesta™ flow cytometer, BD Biosciences FACSLyric™ flow cytometer, BD Biosciences FACSVerse™ flow cytometer, BD Biosciences FACSymphony™ flow cytometer, BD Biosciences LSRFortessa™ flow cytometer, BD Biosciences LSRFortessa™ X-20 flow cytometer, BD Biosciences FACSPresto™ flow cytometer, BD Biosciences FACSVia™ flow cytometer and BD Biosciences FACSCalibur™ cell sorter, a BD Biosciences FACSCount™ cell sorter, BD Biosciences FACSLyric™ cell sorter, BD Biosciences Via™ cell sorter, BD Biosciences Influx™ cell sorter, BD Biosciences Jazz™ cell sorter, BD Biosciences Aria™ cell sorter, BD Biosciences FACSAria™ II cell sorter, BD Biosciences FACSAria™ III cell sorter, BD Biosciences FACSAria™ Fusion cell sorter and BD Biosciences FACSMelody™ cell sorter, BD Biosciences FACSymphony™ S6 cell sorter or the like.

[0167] In some embodiments, the subject systems are flow cytometric systems, such those described in U.S. Patent Nos. 10,663,476; 10,620,111 ; 10,613,017; 10,605,713; 10,585,031 ; 10,578,542; 10,578,469; 10,481 ,074; 10,302,545; 10,145,793; 10,113,967; 10,006,852; 9,952,076; 9,933,341 ; 9,726,527; 9,453,789; 9,200,334; 9,097,640; 9,095,494; 9,092,034; 8,975,595; 8,753,573; 8,233,146; 8,140,300; 7,544,326; 7,201 ,875; 7,129,505; 6,821 ,740; 5,620,842; 5,602,039; 4,987,086; 4,498,766; the disclosures of which are herein incorporated by reference in their entirety.

[0168] In certain instances, flow cytometry systems of the invention are configured for imaging particles in a flow stream by fluorescence imaging using radiofrequency tagged emission (FIRE), such as those described in Diebold, et al. Nature Photonics Vol. 7(10); 806-810 (2013) as well as described in U.S. Patent Nos. 9,423,353; 9,784,661 ; 9,983,132; 10,006,852; 10,078,045; 10,036,699; 10,222,316; 10,288,546; 10,324,019; 10,408,758; 10,451 ,538; 10,620,1 11 ; and U.S. Patent Publication Nos. 2017 / 0133857; 2017 / 0328826; 2017 / 0350803; 2018 / 0275042; 2019 / 0376895 and 2019 / 0376894 the disclosures of which are herein incorporated by reference.

[0169] Other systems may find use in practicing the subject methods. In certain aspects, the system may be a fluorimeter or microscope loaded with a sample having a fluorescent composition of any of the embodiments discussed herein. The fluorimeter or microscope may include a light source configured to direct light to the assay region of the flow channel or sample field of view. The fluorimeter or microscope may also include a detector configured to receive a signal from an assay region of the flow channel or field of view, wherein the signal is provided by the fluorescent composition.

[0170] KITS

[0171] Aspects of the invention further include kits for use in practicing the subject methods. The dyes, tandem dyes, labeled specific binding members, or a combination thereof can be included as reagents in kits either as starting materials or provided for use in, for example, the methodologies described above. Such dyes, tandem dyes, and labeled specific binging members can be provided with a container. Any convenient containers can be utilized, such as tubes, bottles, or wells in a multi-well strip or plate, a box, a bag, an insulated container, and the like. The subject kits can further include one or more components selected from a primer specific binding member for a given target analyte, a support bound specific binding member, a cell, a support, a biocompatible aqueous elution buffer, a control (positive and / or negative), etc., and instructions for use, as desired. A given kit may include reagents suitable for detection of a single target analyte, or multiple reagents suitable for detection of two or more different target analytes, e.g., where a given kit is configured for multiplex detection applications.

[0172] In certain embodiments, the kit finds use in evaluating a sample for the presence of a target analyte, such as an intracellular target. As such, in some instances, the kit includes one or more components suitable for permeabilizing or lysing cells. The one or more additional components of the kit may be provided in separate containers (e.g., separate tubes, bottles, or wells in a multi-well strip or plate).

[0173] In certain aspects, the kit further includes reagents for performing a flow cytometric assay. Reagents of interest include, but are not limited to, buffers for reconstitution and dilution, buffers for contacting a cell sample the chromophore, wash buffers, control cells, control beads, fluorescent beads for flow cytometer calibration and combinations thereof. The kit may also include one or more cell fixing reagents such as paraformaldehyde, glutaraldehyde, methanol, acetone, formalin, or any combinations or buffers thereof. Further, the kit may include a cell permeabilizing reagent, such as methanol, acetone or a detergent (e.g., triton, NP-40, saponin, tween 20, digitonin, leucoperm, or any combinations or buffers thereof. Other protein transport inhibitors, cell fixing reagents and cell permeabilizing reagents familiar to the skilled artisan are within the scope of the subject kits.

[0174] The compositions of the kit may be provided in a liquid composition, such as any suitable buffer. Alternatively, the compositions of the kit may be provided in a dry composition (e.g., may be lyophilized), and the kit may optionally include one or more buffers for reconstituting the dry composition. In certain aspects, the kit may include aliquots of the compositions provided in separate containers (e.g., separate tubes, bottles, or wells in a multi-well strip or plate).

[0175] In addition, one or more components may be combined into a single container, e.g., a glass or plastic vial, tube or bottle. In certain instances, the kit may further include a container (e.g., such as a box, a bag, an insulated container, a bottle, tube, etc.) in which all of the components (and their separate containers) are present. The kit may further include packaging that is separate from or attached to the kit container and upon which is printed information about the kit, the components of the and / or instructions for use of the kit.

[0176] In addition to the above components, the subject kits may further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, DVD, portable flash drive, etc., on which the information has been recorded. Yet another means that may be present is a website address which may be used via the Internet to access the information at a removed site. Any convenient means may be present in the kits.

[0177] UTILITY

[0178] The dyes, tandem dyes and labeled specific binding members comprising the same, compositions, methods and systems as described herein may find use in a variety of applications, including diagnostic and research applications, in which the labelling, detection and / or analysis of a target of interest is desirable. Such applications include methodologies such as cytometry, microscopy, immunoassays (e.g. competitive or noncompetitive), assessment of a free analyte, assessment of receptor bound ligand, and so forth. The compositions, system and methods described herein may be useful in analysis of any of a number of samples, including but not limited to, biological fluids, cell culture samples, and tissue samples. In certain aspects, the compositions, system and methods described herein may find use in methods where analytes are detected in a sample, if present, using fluorescent labels, such as in fluorescent activated cell sorting or analysis, immunoassays, immunostaining, and the like. In certain instances, the compositions and methods find use in applications where the evaluation of a sample for the presence of a target analyte is of interest. In certain aspects, the green excitable tandem dyes can be excited by a green laser, e.g., with a wavelength of 532 nm.

[0179] In some cases, the methods and compositions find use in any assay format where the detection and / or analysis of a target from a sample is of interest, including but not limited to, flow cytometry, fluorescence microscopy, in-situ hybridization, enzyme-linked immunosorbent assays (ELISAs), western blot analysis, magnetic cell separation assays and fluorochrome purification chromatography. In certain instances, the methods and compositions find use in any application where the fluorescent labelling of a target molecule is of interest. The subject compositions may be adapted for use in any convenient applications where pairs of specific binding members find use, such as biotinstreptavidin and hapten-anti-hapten antibody.

[0180] The following examples are offered by way of illustration and not by way of limitation. EXPERIMENTAL

[0181] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed.

[0182] Example 1 : Synthesis of Compounds

[0183] FIG. 6 shows the synthesis of a poly-sarcosine moiety by use of a SPPS resin support. First, a sarcosine monomer is reacted through a transesterification reaction (e.g., acid or base catalyzed) to form a covalent bond between the two components. An FMOC protecting group is present on the other end of the sarcosine residue. Next, the resin with a single sarcosine residue can be reacted with piperdine to deprotect the amino group, and thereafter the compound can be reacted with a second sarcosine residue in the presence of HATU to attach a second sarcosine group through a peptide synthesis reaction to the SPPS resin. This attachment of additional sarcosine residues can be repeated to give a poly-sarcosine group. Thereafter, the bond between the poly-sarcosine and the resin can be cleaved, thereby giving monofunctional or bifunctional poly-sarcosine residues.

[0184] Additional methods of both solid-phase and solution-phase peptide synthesis can be used to synthesize the poly-sarcosine groups. For example, such methods can be found in: Steward et al., in “Solid Phase Peptide Synthesis”, W.H. Freeman Co., San Francisco, 1969; Bodanszky et al., in “Peptide Synthesis”, John Wiley & Sons, Second Edition, 1976 and Meienhofer, in “Hormonal Proteins and Peptides”, Vol. 2, p.46, Academic Press (New York), 1983; and Kent, Ann. Rev. Biochem., 57, 957, 1988, for solid phase peptide synthesis, and Schroder et al., in “The Peptides”, Vol. 1 , Academic Press (New York), 1965 for solution synthesis. Any convenient protecting group strategy may be used such as, but not limited to, Fmoc solid-phase peptide synthesis and Boc solid-phase peptide synthesis strategies. In Boc solid-phase peptide synthesis a Boc-amino protecting group is used at the amino terminal and benzyl or benzyl-based or other convenient protecting groups may be used for the protection of sidechain functional groups. In Fmoc solid-phase peptide synthesis a Fmoc-amino protecting group is used at the amino terminal and tert-butyl or benzyl-based or other convenient protecting groups may be used for protection of sidechain functional groups.

[0185] Various methods including those known in the art can be used to covalently attach these poly-sarcosine groups to dyes and tandem dyes. For example, the monofunctional and bifunctional poly-sarcosine groups of FIG. 6 have terminal carboxy groups. These carboxy groups can be reacted with a nucleophilic group on a dye to cause a nucleophilic displacement reaction, thereby covalently bonding the polysarcosine to the dye. For example, the carboxy groups can be reacted in transesterification reactions or with EDC ((1 -Ethyl-3-(3- dimethylaminopropyl)carbodiimide)) or DCC (N,N’-dicyclohexane carbodiimide) coupling reactions, which are known in the art. Additionally, FIG. 7 shows conversion of a hydroxy-terminated poly-sarcosine group to an amino group, an OTos group, and an alkyne group. Such groups can be reacted with corresponding groups on the target molecule to form a covalent bond therebetween, e.g., the alkyne can be reacted with an azide group through a Click reaction. Additionally, the amino group can be a nucleophile and the OTos group can be a leaving group in nucleophilic displacement reactions.

[0186] Additionally, syntheses of poly-sarcosine along with co-polymers thereof are described in Huesmann et al. (Polymer 67 (2015) 240-248, doi:

[0187] 10.1016 / j.polymer.2015.04.070) and Chan et al. (Biopolymers, 2018;109:e23070, doi:10.1002 / bip.23070). Such synthetic methods can also be applied to the presently described sarcosine compounds.

[0188] Convenient protecting groups that may be used in such synthetic methods are described in the above references and by McOmic in “Protective Groups in Organic Chemistry”, Plenum Press, New York, 1973; and Greene and Wuts, “Protective Groups in Organic Synthesis”, John Wiley & Sons, 4th Edition, 2006.

[0189] Example 2: Embodiments

[0190] FIG. 1 shows poly-sarcosine, N-modified poly-sarcosine, and poly-sarcosine copolymer groups that can be attached to dyes and tandem dyes. FIG. 3 shows examples of dyes that are functionalized with poly-sarcosine groups, such as a cyanine dye, rhodamine dye, coumarin dye, and BODIPY dye. FIG. 4 shows polyfluorene dyes that are functionalized with antibody specific binding members and poly-sarcosine water solubilizing groups. FIG. 5 shows poly-sarcosine groups being used as linkers between a dye and a biomolecule, e.g., that functions as a specific binding member. FIG. 7 shows the conversion of terminal hydroxy group on a poly-sarcosine to other functional groups, thereby allowing for covalent attachment to a dye or tandem dye. FIGS. 8, 9, and 11 show poly-sarcosines as terminal groups and linkers in a labeled specific binding member.

[0191] Example 3: Experimental Properties of Dyes

[0192] A PEGylated dye was synthesized that contained polyethylene glycol (PEG) water solubilizing groups attached to the dye. Similarly, in a separate synthesis, the dye was functionalized sarcosine groups. The dyes were also attached to the hCD4 antibody as a specific binding member, thereby generating a labeled specific binding member. The compound was used as a cell stain. Additionally, side scatter (SSC) experiments were conducted to measure the optical properties of the two dyes, as shown in FIG. 10. It was found that the sarcosine-substituted dyes provided approximately 10% greater signal.

[0193] Notwithstanding the appended claims, the disclosure is also defined by the following clauses:

[0194] 1. A tandem dye comprising: a donor fluorophore; an acceptor fluorophore; a water-solubilizing poly-sarcosine group.

[0195] 2. The tandem dye according to Clause 1 , wherein the water-solubilizing polysarcosine group comprises a polymer having from 2 to 40 residues.

[0196] 3. The tandem dye according to Clause 2, wherein the polymer is selected from the group consisting of a poly-sarcosine polymer, an N-modified poly-sarcosine polymer and a poly-sarcosine-type co-polymer.

[0197] 4. The tandem dye according to Clause 3, wherein the polymer is a poly-sarcosine polymer.

[0198] 5. The tandem dye according to any of the preceding clauses, wherein at least one of the donor and acceptor fluorophore comprises an organic dye.

[0199] 6. The tandem dye according to any of the preceding clauses, wherein both the donor fluorophore and acceptor fluorophore comprise an organic dye.

[0200] 7. The tandem dye according to Clause 5, wherein the organic dye is selected from the group consisting of cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethines, pyrenes, dipyrromethene borondifluorides, napthalimides, thiazine dyes, and acridine dyes. 8. The tandem dye according to any of Clauses 1 to 7, wherein at least one of the donor and acceptor fluorophore comprises a pendant water-solubilizing poly-sarcosine group.

[0201] 9. The tandem dye according to Clause 8, wherein the donor fluorophore comprises a pendant water-solubilizing poly-sarcosine group.

[0202] 10. The tandem dye according to Clause 8, wherein the acceptor fluorophore comprises a pendant water-solubilizing poly-sarcosine group.

[0203] 11 . The tandem dye according to Clause 8, wherein both the donor fluorophore and acceptor fluorophore comprise a pendant water-solubilizing poly-sarcosine group.

[0204] 12. The tandem dye according to any of the preceding clauses, further comprising a non-conjugated polymeric backbone comprising non-conjugated repeat units, wherein the donor and acceptor fluorophore are linked to the non-conjugated polymeric backbone.

[0205] 13. The tandem dye according to Clause 12, wherein the non-conjugated repeat units comprise a plurality of amino acid residues.

[0206] 14. The tandem dye according to Clauses 12 or 13, wherein the non-conjugated polymeric backbone comprises a water-solubilizing poly-sarcosine group bound thereto.

[0207] 15. The tandem dye according to any of Clauses 12 to 14, wherein at least one of the donor and acceptor fluorophores is linked to the non-conjugated polymeric backbone by a water-solubilizing poly-sarcosine group.

[0208] 16. The tandem dye according to Clause 15, wherein the donor fluorophore is linked to the non-conjugated polymeric backbone by a water-solubilizing poly-sarcosine group.

[0209] 17. The tandem dye according to Clause 15, wherein the acceptor fluorophore is linked to the non-conjugated polymeric backbone by a water-solubilizing poly-sarcosine group.

[0210] 18. The tandem dye according to Clause 15, wherein both the donor fluorophore and the acceptor fluorophore are linked to the non-conjugated polymeric backbone by a water-solubilizing poly-sarcosine group.

[0211] 19. The tandem dye according to any of Clauses 12 to 18, wherein the nonconjugated polymeric backbone comprises a water-solubilizing poly-sarcosine group.

[0212] 20. The tandem dye according to any of Clauses 1 to 3, wherein the donor chromophore comprises a conjugated polymer.

[0213] 21 . The tandem dye according to Clause 20, wherein the conjugated polymer comprises a pendant water-solubilizing poly-sarcosine group. 22. The tandem dye according to Clauses 20 or 21 , wherein the conjugated polymer is linked to the acceptor chromophore by a water-solubilizing poly-sarcosine group.

[0214] 23. The tandem dye according to any of Clauses 20 to 22, wherein the acceptor fluorophore comprises a pendant water-solubilizing poly-sarcosine group.

[0215] 24. A labeled specific binding member comprising: a tandem dye according to any of the preceding clauses; and a specific binding member.

[0216] 25. The labeled specific binding member according to Clause 24, wherein the specific binding member is a biomolecule.

[0217] 26. The labeled specific binding member according to Clause 24, wherein the biomolecule is selected from the group consisting of carbohydrates, lipids, nucleic acids, and proteins or fragments thereof.

[0218] 27. The labeled specific binding memember according to any of Clauses 24 to 26, wherein the specific binding member is an antibody, an antibody fragment or binding derivative thereof.

[0219] 28. The labeled specific binding member according to any of Clauses 24 to 27, wherein the specific binding member is linked to the tandem dye by a water-solubilizing poly-sarcosine group.

[0220] 29. The labeled specific binding member according to Clause 28, wherein the tandem dye comprises a non-conjugated polymeric backbone and the specific binding member is linked to the non-conjugated backbone by the water-solubilizing polysarcosine group.

[0221] 30. A method comprising contacting a sample with a tandem dye according to any of Clauses 24 to 29.

[0222] 31 . The method according to Clause 30, wherein method comprises assaying the sample for a target analyte to which the specific binding member binds.

[0223] 32. The method according Clause 31 , wherein the analyte is associated with a cell.

[0224] 33. The method according to Clause 32, wherein the analyte is a cell surface marker of the cell.

[0225] 34. The method according to Clause 33, wherein the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen.

[0226] 35. The method according to Clause 32, wherein the analyte is an intracellular target.

[0227] 36. The method according to Clause 35, wherein the method comprises permeabilizing or lysing the cell. 37. The method according to any of Clauses 30 to 36, wherein the method comprises flow cytometrically analyzing the sample.

[0228] 38. A method of labeling a specific binding member, the method comprising: bonding a tandem dye according to any of Clauses 1 -23 with the specific binding member to label the specific binding member.

[0229] 39. The method according to Clause 38, wherein the specific binding member is a biomolecule.

[0230] 40. The method according to Clause 39, wherein the biomolecule is selected from the group consisting of carbohydrates, lipids, nucleic acids, and proteins or fragments thereof.

[0231] 41 . The method according to any of Clauses 38 to 40, wherein the specific binding member is an antibody, an antibody fragment or binding derivative thereof.

[0232] 42. A kit comprising: a tandem dye according to an of Clauses 1 to 23 or a labeled specific binding member according to any of Clauses 24 to 29.

[0233] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0234] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0235] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0236] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1 -3 articles refers to groups having 1 , 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1 , 2, 3, 4, or 5 articles, and so forth.

[0237] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0238] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0239] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §1 12(f) or 35 U.S.C. §1 12(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §1 12(6) is not invoked.

Claims

What is claimed is:1 . A tandem dye comprising: a donor fluorophore; an acceptor fluorophore; a water-solubilizing poly-sarcosine group.

2. The tandem dye according to Claim 1 , wherein the water-solubilizing polysarcosine group comprises a polymer having from 2 to 40 residues.

3. The tandem dye according to Claim 2, wherein the polymer is selected from the group consisting of a poly-sarcosine polymer, an N-modified polysarcosine polymer and a poly-sarcosine-type co-polymer.

4. The tandem dye according to Claim 3, wherein the polymer is a polysarcosine polymer.

5. The tandem dye according to any of the preceding claims, wherein at least one of the donor and acceptor fluorophore comprises an organic dye.

6. The tandem dye according to any of the preceding claims, wherein both the donor fluorophore and acceptor fluorophore comprise an organic dye.

7. The tandem dye according to any of the preceding claims, wherein at least one of the donor and acceptor fluorophore comprises a pendant watersolubilizing poly-sarcosine group.

8. The tandem dye according to any of the preceding claims, further comprising a non-conjugated polymeric backbone comprising non-conjugated repeat units, wherein the donor and acceptor fluorophore are linked to the nonconjugated polymeric backbone.

9. The tandem dye according to Claim 8, wherein the non-conjugated repeat units comprise a plurality of amino acid residues.

10. The tandem dye according to Claims 8 or 9, wherein the non-conjugated polymeric backbone comprises a water-solubilizing poly-sarcosine group bound thereto.1 1 . The tandem dye according to any of Claims 8 to 10, wherein at least one of the donor and acceptor fluorophores is linked to the non-conjugated polymeric backbone by a water-solubilizing poly-sarcosine group.

12. The tandem dye according to any of Claims 8 to 11 , wherein the nonconjugated polymeric backbone comprises a water-solubilizing poly-sarcosine group.

13. A labeled specific binding member comprising: a tandem dye according to any of the preceding claims; and a specific binding member.

14. A method comprising contacting a sample with a labeled specific binding member according to Claim 13.

15. A kit comprising: a tandem dye according to an of Claims 1 to 12 or a labeled specific binding member according to Claim 13.

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