Polysarcosine solubilized dyes and methods of use thereof

Abstract

Dyes are provided, including tandem dyes. Aspects of embodiments of these dyes include fluorophores and water-solubilizing polysarcosine groups. Also provided are methods of using the dyes, as well as kits comprising such dyes.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 524,017, filed June 29, 2023, pursuant to 35 USC §119(e), the entire contents of which are incorporated herein by reference.

[0003] introduction

[0004] Fluorescent dyes are compounds that emit light of (typically) different wavelengths when illuminated by light of wavelengths they absorb. Fluorescent dyes are used in a wide range of applications in biochemistry, biology, and medicine, such as in diagnostic kits, microscopy, or drug screening. Fluorescent dyes are characterized by many parameters, allowing users to select the appropriate dye for the desired purpose. Target parameters include maximum excitation wavelength, maximum emission wavelength, Stokes shift, extinction coefficient, fluorescence quantum yield, and fluorescence lifetime. Dyes can be selected based on the target application to, for example, allow excitation radiation to penetrate biological samples, minimize background fluorescence, and / or achieve a high signal-to-noise ratio.

[0005] Molecular recognition involves the specific binding of two molecules. Molecules that bind specifically to a target biomolecule can be used in a variety of research and diagnostic applications, such as labeling and separating analytes, flow cytometry, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blotting, magnetic cell separation, and chromatography. Target biomolecules can be detected by labeling them with fluorescent dyes.

[0006] Polyethylene glycol (PEG) groups have been linked to fluorescent dyes (as well as small molecules, nucleotides, peptides, proteins, liposomes, and nanoparticles) to improve solubility, stability, and pharmacokinetic properties. However, some initial individuals may already have pre-existing antibodies that can bind to PEG and induce an immune response (Chen et al., ibid.). Many common methods for synthesizing PEG and other polymers produce polydisperse compositions with broad molecular weight distributions. This polydispersity can adversely inhibit control over polymer properties.

[0007] summary

[0008] Dyes, including tandem dyes, are provided. Aspects of embodiments of these dyes include fluorophores and water-soluble polysarcosine groups. Methods for using these dyes, as well as kits including such dyes, are also provided.

[0009] Brief description of the attached figures

[0010] Figure 1 The chemical structures of polysarcosine, N-modified polysarcosine, and polysarcosine copolymers are shown.

[0011] Figure 2 The chemical structures of polysarcosine and methyl-polyethylene glycol are shown.

[0012] Figure 3 The chemical structures of exemplary anthocyanin, rhodamine, coumarin, and BODIPY dyes are shown.

[0013] Figure 4 Examples of dyes and tandem dyes comprising a polymer donor chromophore, a polysarcosine group, and an antibody are shown. The bottom structure contains an acceptor dye, thus making the structure a tandem dye.

[0014] Figure 5 An example of a structure is shown that links a dye to a biomolecule via a linker containing a polysarcosine group.

[0015] Figure 6 The solid-phase synthesis of polysarcosine groups is shown.

[0016] Figure 7 Synthetic options for converting hydroxyl groups attached to polysarcosine groups into different substituted amine groups are shown.

[0017] Figure 8 Different options for the position of the polysarcosine group within the compound are shown.

[0018] Figure 9 Polysarcosine groups attached to polyfluorene or BODIPY chromophores are shown.

[0019] Figure 10 SSC data for a donor-receptor complex using PEGylated and sarcosine-functionalized dyes covalently linked to hCD4 and used as cellular dyes are shown. The sarcosine-substituted dyes provided approximately 10% stronger signal.

[0020] Figure 11 Polysarcosine side groups attached to BODIPY dyes or cyanine dyes are shown.

[0021] definition

[0022] "Alkyl" refers to a monoradical, branched or linear, acyclic, saturated hydrocarbon group. Exemplary alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, octyl, decyl, cyclopentyl, and cyclohexyl. In some cases, alkyl groups have 1 to 24 carbon atoms, such as 1 to 12 carbon atoms, 1 to 6 carbon atoms, or 1 to 3 carbon atoms.

[0023] "Alkenyl" refers to a single-radical, branched or linear, acyclic hydrocarbon group containing a carbon-carbon double bond. Exemplary alkenyl groups include vinyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosene, and 24-carbonenyl.

[0024] "Alynyl" refers to a single-radical, branched or straight-chain, acyclic hydrocarbon group containing a carbon-carbon triple bond. Exemplary alkynyl groups include ethynyl and n-propynyl.

[0025] "Cycloalkyl" refers to a single-radical, cyclic, saturated hydrocarbon group. Similarly, "cycloalkenyl" refers to a single-radical cyclic group with a carbon-carbon double bond, while "cycloalkynyl" refers to a single-radical cyclic group with a carbon-carbon triple bond.

[0026] A "heterocyclic group" is a single-radical cyclic group that contains a heteroatom (e.g., O, S, N) as a ring atom and is not aromatic (i.e., distinguishing heterocyclic groups from heteroaryl groups). Exemplary heterocyclic groups include piperidinyl, tetrahydrofuranyl, dihydrofuranyl, and thiocanyl.

[0027] "Aryl" refers to an aromatic group containing at least one aromatic ring, wherein each atom in the ring is a carbon atom, i.e., there are no heteroatoms (e.g., O, S, N) in the ring. In some cases, the aryl group has a second aromatic ring, for example, fused with the first aromatic ring. Exemplary aryl groups are phenyl, naphthyl, biphenyl, diphenyl ether, diphenylamine, and benzophenone.

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

[0029] The term "substituted" refers to the removal of one or more hydrogen atoms from an atom (e.g., from a C or N atom) and their replacement with different groups. For example, a hydrogen atom on a phenyl ((-C6H5) group can be replaced by a methyl group to form a -C6H4CH3 group. Therefore, the -C6H4CH3 group can be considered a substituted aryl group. As another example, the two hydrogen atoms on the second carbon of a propyl (-CH2CH2CH3) group can be replaced by an oxygen atom to form a -CH2C(O)CH3 group, which can be considered a substituted alkyl group. However, replacing a hydrogen atom on a propyl ((-CH2CH2CH3) group with a methyl group (e.g., obtaining -CH2CH(CH3)CH3) is not considered a "substitution" as used herein because both the starting and ending groups are alkyl. However, if the propyl group is replaced with a methoxy group to obtain a -CH2CH(OCH3)CH3 group, the entire group can no longer be considered an "alkyl" group, and is therefore a "substituted alkyl" group. Therefore, for a group to be considered a substituent, the substituent group is a group of a different type from the original group. Furthermore, unless described as substituted, a group is assumed to be unsubstituted. For example, the terms "alkyl" and "unsubstituted alkyl" are used interchangeably herein.

[0030] Exemplary substituents include alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclic, aryl, heteroaryl, acyl, alkoxy, amino, azide, carbonyl, carboxyl, cyano, ether, halogen, hydroxyl, nitro, sulfonate / ester, and their substituted forms.

[0031] In some cases, the substituent itself may be further substituted by one or more groups. For example, the group -C6H4CH2CH3 can be considered a substituted aryl group, i.e., an aryl group substituted with an ethyl group (an alkyl group). Furthermore, the ethyl group itself can be substituted with a pyridyl group to form -C6H4CH2CH2C5H5N, which, according to the terminology used herein, can also be considered a substituted aryl group. In some cases, the substituent is not substituted by any other group.

[0032] This document also describes diradical groups, which are the opposite of the monoradical groups described above, such as alkyl and aryl groups. The term "alkylene" refers to the diradical form of an alkyl group, meaning that the alkylene is a diradical, branched or linear, cyclic or acyclic, saturated hydrocarbon group. Exemplary alkylenes include diylmethane (-CH2-, also known as methylene), 1,2-diylethane ((-CH2CH2-), and 1,1-diylethane (i.e., the CHCH3 fragment, where the first atom has two single bonds attached to two other distinct groups). "Arylene" refers to the diradical form of an aryl group; for example, 1,4-diphenylbenzene refers to the C6H4 fragment, where the two hydrogens at the para position are removed and replaced by single bonds to other groups. The terms "alkenylene," "alkynylene," "heteroarylene," and "heterocyclic alkylene" are also used herein.

[0033] "Acyl" refers to a group of the formula -C(O)R, where R is an alkyl, alkenyl, alkynyl, or a substituted form thereof. For example, the acetyl group has the formula -C(O)CH3. "Carbonyl" refers to a diradical group of the formula -C(O)-.

[0034] "Alkoxy" refers to a group with the formula -O (alkyl). Similar groups can be derived from alkenyl, alkynyl, aryl, heteroaryl, and other groups.

[0035] "Amino" refers to -NR X R Y Group, wherein R X and R Y Each can be an H or non-hydrogen substituent independently. Exemplary non-hydrogen substituents include alkyl groups (e.g., methyl, ethyl, and isopropyl).

[0036] "Carbonyl" refers to a diradical group with the formula -C(O)-.

[0037] The term "carboxyl group" is used interchangeably with carboxylic acid and carboxylate, referring to the -CO2H group and its salts.

[0038] "Ether" refers to a diradical group of the formula -O-. For example, if the ether group is attached to an alkyl group, the entire group is an alkoxy group (e.g., -OCH3 or methoxy). If the ether is attached to a carbonyl group, the entire group is an ester group of the formula -OC(O)-.

[0039] "Halogen" and "halogenated" refer to chlorine, bromine, fluorine, and iodine groups.

[0040] "Nitro" refers to a group with the formula -NO2.

[0041] Unless otherwise stated, mentioning an atom is intended to include all isotopes of that atom. For example, mentioning H includes... 1 H, 2H (i.e., D or deuterium) and 3 H (i.e., tritium), C includes 12 C and all other carbon isotopes (e.g.) 13 C). Unless otherwise stated, the group includes all possible stereoisomers.

[0042] The terms “reaction portion,” “chemoselective functional group,” “chemoselective tag,” and “combination tag” are used interchangeably to refer to a functional group capable of selectively reacting with another compatible functional group to form a covalent bond. In some cases, this refers to a functional group that reacts to form a covalent bond after the optional activation of one of the functional groups. Target chemoselective functional groups include, but are not limited to: thiols with maleimide or iodoacetamide, amines with carboxylic acids or their active esters, and groups capable of reacting with each other via click chemistry, such as azide and alkyne groups (e.g., cyclooctyne), tetrazides, trans-cyclooctene, dienes and dienophiles, as well as azides, sulfonyl(VI) fluoride exchange chemistry (SuFEX), sulfonyl fluorides, and hydroxyl, hydrazide, hydrazine, aldehyde, ketone, azide, alkyne, phosphine, epoxide, succinimide, etc.

[0043] As used herein, the term "sample" refers to a material or mixture of materials containing one or more target analytes, in some cases in liquid form. In some embodiments, the term, in its broadest sense, refers to any plant, animal, or bacterial material containing cells or producing cellular metabolites, such as tissues or fluids isolated from an individual (including, but not limited to, plasma, serum, cerebrospinal fluid, lymph, tears, saliva, and tissue sections) or components from in vitro cell cultures, as well as samples from the environment. The term "sample" can also refer to "biological sample." As used herein, the term "biological sample" refers to a subset of a whole organism or its tissues, cells, or components (e.g., bodily fluids, including but not limited to blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen). "Biological sample" can also refer to a homogenate, lysate, or extract prepared from a whole organism or its tissues, cells, or components or fractions thereof, including but not limited to plasma, serum, cerebrospinal fluid, lymph, external skin, respiratory tract, intestinal and genitourinary tract, tears, saliva, breast milk, blood cells, tumors, and organs. In some embodiments, the sample has been removed from the animal or plant. Biological samples may contain cells. The term "cell" is used in its conventional sense to refer to the basic structural unit of a living organism, including eukaryotes and prokaryotes, that has at least a nucleus and a cell membrane. In some embodiments, cells include prokaryotic cells, such as cells derived from bacteria. In other embodiments, cells include eukaryotic cells, such as cells obtained from biological samples of animals, plants, or fungi.

[0044] The terms "support-bound" and "attached to a support" are used interchangeably to refer to portions (e.g., specific binding members) covalently or nonvalently attached to a target support. Covalent attachment may involve a chemical reaction of two compatible functional groups (e.g., two chemically selective functional groups, an electrophile and a nucleophile, etc.) to form a covalent bond between two target portions (e.g., the support and the specific binding member). In some cases, nonvalent attachment may involve specific binding between two target portions (e.g., two affinity portions, such as a hapten and antibody, or a biotinylate and streptavidin, etc.). In some cases, nonvalent attachment may involve uptake of the matrix.

[0045] The terms “peptide” and “polypeptide” are used interchangeably herein to refer to a polymer of amino acids of any length, including peptides ranging from 2 to 500 amino acids in length, such as 2 to 350, 2 to 200, 2 to 100, 2 to 50, and 2 to 25 amino acids. In some cases, peptides have 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 a peptide can be coding amino acids and non-coding amino acids, chemically or biochemically modified or derived amino acids, and peptides having a modified backbone in which a non-naturally occurring or synthetic backbone replaces the conventional backbone.

[0046] The terms "polyethylene oxide," "PEO," "polyethylene glycol," and "PEG" are used interchangeably to refer to compounds containing the following: (CH2O) n- The polymer group of the chain shown 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 should be understood that the PEG polymer group can be of any convenient length and can include a variety of end groups and / or other substituents, including but not limited to alkyl, aryl, hydroxyl, amino, acyl, acyloxy, and amino end groups and / or substituents. The PEG group is also described in the following literature: S. Zalipsky in “Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates”, Bioconjugate Chemistry 1995, 6 (2), 150-165; 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”, JM Harris, editor, Plenum Press, New York, NY (1992); and “Poly(ethylene glycol) Chemistry and Biological Applications”, JM Harris and S.Edited by Zalipsky, ACS (1997); 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, hereafter: 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; 5,321,0 95; 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; 6,113,906; 6,127,355; 6,177,087; 6,180,095; 6,194,580; 6,214,966).

[0047] As used herein, “isolated” means that the target fraction is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free, or even at least 99% free from other components bound to it prior to purification.

[0048] As used herein, the terms “assessment,” “determination,” “measurement,” “evaluation,” and “determination” are used interchangeably and include both quantitative and qualitative determinations.

[0049] As used in this article, the term “separation” refers to the physical separation of two elements (e.g., by size or affinity) and the degradation of one element while the other remains intact.

[0050] The term "linker" or "linkage" refers to a linker that connects two groups and has a skeleton length of 100 atoms or less. A linker or linkage can be a covalent bond connecting two groups or a chain of 1 to 100 atoms in length, such as a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, or more than 20 carbon atoms. Linkers can be straight-chain, branched, cyclic, or monoatomic. In some cases, linkers are branched linkers, referring to a linker connecting three or more groups. In some cases, one, two, three, four, or five or more carbon atoms in the linker skeleton may optionally be replaced by sulfur, nitrogen, or oxygen heteroatoms. In some cases, the linker skeleton contains linking functional groups, such as ethers, thioethers, amino groups, amides, sulfonamides, carbamates, thiocarbamates, ureas, thioureas, esters, thioesters, or imines. The bonds between skeleton atoms can be saturated or unsaturated, and in some cases, the linker skeleton contains no more than one, two, or three unsaturated bonds. The linker can contain one or more substituents, such as alkyl, aryl, or alkenyl groups. Linkers can include, but are not limited to: polyethylene glycol; ethers, thioethers, tertiary amines, and alkyl groups, which can be linear or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), etc. The linker skeleton can contain cyclic groups, such as aryl, heterocyclic, or cycloalkyl groups, wherein the skeleton contains two or more atoms of the cyclic group, such as two, three, or four atoms. The linker can be cleavable or non-cleavable.

[0051] As used herein, the terms “water-solubilizing group,” “water-soluble group,” and “WSG” are used interchangeably to refer to a group or substituent that is readily solubilized in an aqueous environment, such as under physiological conditions, and that gives the molecule to which it is attached improved water solubility relative to the same molecule lacking one or more of the WSG. WSG can increase the solubility of a tandem dye or its components, such as a donor fluorophore or acceptor fluorophore, in primarily aqueous solutions compared to a control tandem dye or its components lacking the WSG. In some cases, WSG can increase the solubility of a compound (e.g., a dye, tandem dye, or a labeled specific binding member) compared to a control compound in which the WSG is replaced by a hydrogen atom. In some cases, the solubility of WSG in an aqueous medium, such as distilled water, increases by 1% or more, for example, by 10% or more, 25% or more, 50% or more, 100% or more, or 500% or more. The water-solubilizing group can be any hydrophilic group that readily solubilizes in an aqueous environment.

[0052] Water-soluble groups (WSGs) enable the target dye or polymer tandem dye to have a solubility in water exceeding 10 mg / mL, such as exceeding 20 mg / mL, exceeding 30 mg / mL, exceeding 40 mg / mL, exceeding 50 mg / mL, exceeding 60 mg / mL, exceeding 70 mg / mL, exceeding 80 mg / mL, exceeding 90 mg / mL, or exceeding 100 mg / mL. In some cases, the water-soluble group (WSG) can enable the target dye or polymer tandem dye to achieve a solubility of 20 mg / mL or more in water (e.g., aqueous buffer), 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.

[0053] The term "specific binding" refers to the direct binding between two molecules due to interactions such as covalent, electrostatic, hydrophobic, ionic, and / or hydrogen bonding, including interactions such as salt bridges and water bridges. Specific binding members describe the members of a pair of molecules that have specific binding affinity to each other. Members of a specific binding pair can be naturally derived or wholly or partially synthetically produced. One member of a pair of molecules has a region or cavity on its surface that specifically binds to and is therefore complementary to the specific spatial and polar organization of the other member in the pair. Thus, the members of the pair possess the property of specifically binding to each other. Examples of specific binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, and enzyme-substrate. Specific binding members of a binding pair exhibit high affinity and binding specificity to bind to each other. Typically, the affinity between a pair of specific binding members is within 10... -6 M or below 10 -6 M of K d Characterized by (dissociation constant), for example, 10 -7 M or below 10 -7 M, including 10 -8 M or below 10 -8 M, for example, 10 -9 M or below 10 -9 M, 10 -10 M or below 10 -10 M, 10 -11 M or below 10 -11 M, 10 -12 M or below 10 -12 M, 10 -13 M or below 10 -13 M, 10 -14 M or below 10 -14 M, including 10 -15 M or below 10 -15 M. “Affinity” refers to binding strength, and increased binding affinity is associated with a lower KD. In one embodiment, affinity is determined by surface plasmon resonance (SPR), for example, using a Biacore system. The affinity of one molecule to another is determined by measuring the binding kinetics of the interaction, for example, at 25°C. “Affinity” refers to binding strength, and increased binding affinity is associated with a lower KD. In one embodiment, affinity is determined by surface plasmon resonance (SPR), for example, using a Biacore system. The affinity of one molecule to another is determined by measuring the binding kinetics of the interaction, for example, at 25°C.

[0054] The specific binding member can vary as needed. In some cases, the specific binding member is a biomolecule, such as carbohydrates, lipids, nucleic acids (e.g., oligonucleotides), and proteins or their binding fragments.

[0055] The specific binding member can be proteinaceous. As used herein, the term "proteinaceous" refers to a portion consisting of amino acid residues. A protein portion can be a polypeptide. In some cases, the specific binding member of a protein is an antibody. In some embodiments, the specific binding member of a protein is an antibody fragment, such as 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 to refer to a protein consisting essentially of one or more polypeptides encoded by all or part of a recognized immunoglobulin gene. Recognized immunoglobulin genes, for example in humans, include κ(k), λ(l), and heavy chain loci, which together constitute numerous variable region genes; and constant region genes μ(u), δ(d), γ(g), σ(e), and α(a), which encode IgM, IgD, IgG, IgE, and IgA isotypes, respectively. The variable region of an immunoglobulin light or heavy chain consists of a "framework" region (FR) interrupted by three hypervariable regions (also known as "complementarity-determining regions" or "CDRs"). The scope of the frame region and CDR has been precisely defined (see, “Sequences of Proteins of Immunological Interest,” E. Kabat et al., USDepartment of Health and Human Services, (1991)). The amino acid sequences of all antibodies discussed herein are numbered according to the Kabat system. The sequences of the frame regions of different light or heavy chains are relatively conserved within the same species. The frame region of an antibody, that is, the combination of frame regions that make up the light and heavy chains, is used to locate and align the CDR. The CDR is primarily responsible for binding epitopes of the antigen. The term antibody is intended to include full-length antibodies and can refer to naturally occurring antibodies from any organism, engineered antibodies, or antibodies recombinantly produced for experimental, therapeutic, or other purposes as further defined below.

[0056] Antibody fragments of interest include, but are not limited to, Fab, Fab′, F(ab′)2, Fv, scFv, or other antigen-binding subsequences of the antibody, which can be generated de novo by modifying the complete antibody or using recombinant DNA technology. Antibodies can be monoclonal or polyclonal and can possess other cell-specific activities (e.g., antagonists, agonists, neutralizing antibodies, inhibitory antibodies, or stimulating antibodies). It should be understood that antibodies may have additional conserved amino acid substitutions that substantially do not affect antigen binding or other antibody functions.

[0057] In some embodiments, the specific binding member is a Fab fragment, an F(ab′)2 fragment, scFv, a bispecific antibody (diabody), or a trispecific antibody (triabody). In some embodiments, the specific binding member is an antibody. In some cases, the specific binding member is a murine antibody or a binding fragment thereof. In some cases, the specific binding member is a recombinant antibody or a binding fragment thereof.

[0058] The terms “active pharmaceutical ingredient” (API), “active agent”, “pharmacologically active agent” and “drug” are used interchangeably herein and refer to a chemical material or compound that, when administered to an organism (e.g., a human or non-human animal), induces a desired pharmacological and / or physiological effect through local and / or systemic action.

[0059] "Pharmaceutically acceptable excipients," "pharmaceutical acceptable diluents," "pharmaceutical acceptable carriers," and "pharmaceutical acceptable adjuvants" refer to excipients, diluents, carriers, and adjuvants useful in the preparation of pharmaceutical compositions, which are generally safe, non-toxic, and have no adverse effects in biological or other respects, and include excipients, diluents, carriers, and adjuvants suitable for veterinary use as well as for human pharmaceutical use. As used in the specification and claims, "pharmaceutical acceptable excipients, diluents, carriers, and adjuvants" includes one or more such excipients, diluents, carriers, and adjuvants.

[0060] "Multiple" includes at least 2 members. In some cases, "multiple" can have 5 or more members, 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, 10000 or more, 10000 or more, 100000 or more, 100000 or more.

[0061] The numerical range includes the numbers that define the range. Summary of the Invention

[0062] Dyes, including tandem dyes, are provided. Embodiments of these dyes include fluorophores and water-soluble polysarcosine groups. Methods for using these dyes, as well as kits containing such dyes, are also provided.

[0063] Before describing the invention in more detail, it should be understood that the invention is not limited to the specific embodiments described, which may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the invention will be limited only by the appended claims.

[0064] When a numerical range is provided, it should be understood that every intermediate value between the upper and lower limits of the range, as well as any other value or intermediate value within the range, is included in this invention, unless the context clearly indicates otherwise, each intermediate value should be as low as one-tenth of the lower limit unit. The upper and lower limits of these smaller ranges may be independently included within the smaller ranges and also within this invention, but are subject to any specific exclusions within the range. Where the range includes one or two limits, the range excluding any one or both of those included limits is also included in this invention.

[0065] Certain ranges presented in this paper are preceded by the term "approximately". The purpose of using the term "approximately" is to provide literal support for the exact number that follows and for numbers that are close to or approximate to the number following the term. In determining whether a number is close to or approximates a specifically referenced number, the close or approximate unreferenced number may be a number substantially equivalent to the specifically referenced number in its existing context.

[0066] Unless otherwise defined, 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 application pertains. While any methods and materials similar or equivalent to those described herein may be used in the practice or testing of this invention, representative illustrative methods and materials are described below.

[0067] All publications and patents referenced in this specification are incorporated herein 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 methods and / or materials relating to said publication. Any reference to a publication is due to its disclosure prior to the filing date and should not be construed as an admission that the invention is not entitled to precede that publication by prior inventiveness. Furthermore, the publication dates provided may differ from the actual publication dates and may require independent verification.

[0068] It should be noted that, unless the context clearly specifies otherwise, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended as a basis for precedent in relating the use of exclusive terms such as “alone,” “only,” or the use of “negative” to restrict the reference to elements of a claim.

[0069] Upon reading this disclosure, the following will be apparent to those skilled in the art: each individual embodiment described and illustrated herein has its own components and features that can be readily separated from or combined with features of any other several embodiments without departing from the scope or spirit of the invention. Any methods referenced may be performed in the order of the events referenced or in any other logically possible order.

[0070] Although, for the sake of grammatical fluency, functional interpretations have been or will be used to describe apparatuses and methods, it should be clearly understood that, unless expressly stated in 35 U.S.C. 112, a claim should not be construed in any way as necessarily limited to a structure that is “method” or “step,” but should be given the full meaning and scope of equivalents as defined by the judicial principle of equivalence, and where the claim is expressly stated in 35 U.S.C. 112, it should be given a full legal equivalent under 35 U.S.C. 112.

[0071] dye

[0072] This disclosure provides dyes comprising a fluorophore and at least one water-soluble polysarcosine group. "At least one water-soluble polysarcosine group" increases the dye's solubility in water. As used herein, the terms "water-soluble group," "water-soluble group," and WSG are used interchangeably and refer to a group or substituent that is well solubilized in an aqueous environment, such as under physiological conditions, and which imparts improved water solubility to the molecule to which it is attached. Compared to a control tandem dye or a component thereof lacking this WSG, the WSG can increase the solubility of the tandem dye or a component thereof, such as a donor or acceptor fluorophore, in primarily aqueous solutions.

[0073] The increase in solubility of a dye conferred by one or more polysarcosine groups (e.g., by multiple polysarcosine groups bonded to the dye) relative to a control dye lacking a water-soluble polysarcosine group may vary and may be 5 times or more in some cases, such as 10 times or more, including 50 times or more. Water-soluble groups (WSG) enable the target dye or polymeric tandem dye to achieve solubility in water exceeding 10 mg / mL, such as exceeding 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, or 100 mg / mL. In certain cases, branched nonionic water-soluble groups (WSGs) can enable target dyes or polymeric tandem dyes to achieve solubility in water (e.g., aqueous buffers) of 20 mg / mL or higher, such as 30 mg / mL or higher, 40 mg / mL or higher, 50 mg / mL or higher, 60 mg / mL or higher, 70 mg / mL or higher, 80 mg / mL or higher, 90 mg / mL or higher, 100 mg / mL or higher, or even higher. It should be understood that water-soluble dipyrrole methylene-based dyes may form discrete, water-solubilized nanoparticles in aqueous systems under certain conditions. In some cases, water-solubilized nanoparticles are resistant to aggregation and can be used in various bioassays.

[0074] The number of water-soluble polysarcosine groups bonded to a given dye can vary, and this number can be selected according to the desired water solubility of the dye. Although the number of water-soluble polysarcosine groups bonded to a given dye can vary, in some cases the number ranges from 1 to 20, for example from 2 to 15, including 2 to 10.

[0075] The water-soluble polysarcosine group of embodiments of the present invention is a polymer comprising a plurality of (i.e., two or more) sarcosine residues. As used herein, the term "sarcosine residue" refers to a monomeric residue formed from sarcosine or its derivatives, such as N-modified sarcosine. While the length of a given water-soluble polysarcosine group can vary, in some cases it is from 2 to 40 monomeric residues, for example from 3 to 20 monomeric residues, including 3 to 10 monomeric residues. A given water-soluble polysarcosine group can consist of the same sarcosine residues, thus it is a homopolymer, or it can consist of different sarcosine residues, thus it is a heteropolymer. In some cases, the water-soluble polysarcosine group can consist of sarcosine residues and non-sarcosine residues, for example residues derived from non-sarcosine amino acids or other types of monomers, as needed, for example, where the water-soluble polysarcosine group is a copolymer of sarcosine and non-sarcosine residues. Therefore, in some cases, a given water-soluble polysarcosine group can consist entirely of sarcosine residues and can therefore be referred to as a polysarcosine polymer. In other cases, a given water-soluble polysarcosine group may consist of N-modified sarcosine residues, and thus may be referred to as an N-modified polysarcosine polymer. In still other cases, a given water-soluble polysarcosine group may consist of different types of sarcosine residues (e.g., sarcosine and N-modified sarcosine residues) or sarcosine and non-sarcosine residues, and thus may be referred to as a polysarcosine-type copolymer. In some embodiments, one or more water-soluble polysarcosine groups bonded to the dye are polysarcosine polymers.

[0076] The sarcosine residues found in the water-soluble polysarcosine groups of embodiments of the present invention include one or more sarcosine residues described by the following formula:

[0077]

[0078] Wherein R1 is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl.

[0079] For example, in some cases R1 is methyl, i.e., where the residue is sarcosine. In some cases, such as when the sarcosine residue is N-modified sarcosine, R1 is a non-methyl moiety. In some instances, R1 is a substituted alkyl group, such as a water-soluble group, such as an alkyl group substituted with a polyethylene glycol group or an alkyl group substituted with an amino group, as described in more detail below.

[0080] Therefore, in some cases, water-soluble polysarcosine groups are polymers described by the following formula:

[0081]

[0082] Where R is an H or a non-hydrogen substituent, and n is an integer of 2 or greater than 2. Therefore, a polymer of formula (II) can be considered as a polymer composed of residues of formula (I), where R1 is a methyl group. In some instances, n is an integer from 2 to 40, for example from 3 to 20, including 3 to 10.

[0083] When R1 is not methyl, R1 can be varied as needed. In some instances, R1 can be selected from the group consisting of: alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, heterocyclic, substituted heterocyclic, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, amino, azide, carboxyl, cyano, ether, halogen, hydroxyl, nitro, thiol, thioether, thion, borate ester, -SO2, -SO3-, the reaction moiety, and the protecting group of the reaction moiety.

[0084] In some instances, R1 may be a non-polysarcosine water-soluble group. When present, these water-soluble groups can be any convenient hydrophilic group that readily solubilizes in an aqueous environment. A variety of water-soluble polymeric groups are suitable for the WSG of the target dye. Any convenient water-soluble group (WSG) may be included in the dyes described herein to provide increased water solubility. While the increase in solubility may vary, in some cases, the increase (compared to compounds without one or more WSGs) is 2-fold or more, for example, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, or more than 100-fold. In some cases, the hydrophilic water-soluble group is charged, for example, positively or negatively charged. In some cases, the hydrophilic water-soluble group is a neutral hydrophilic group. In some embodiments, the WSG is branched (e.g., as described herein). In some instances, the WSG is linear. In some embodiments, WSG is a hydrophilic polymer, such as polyethylene glycol, modified PEG, peptide sequences, peptide-like substances, carbohydrates, oxazoline, polyols, dendrons, dendritic polyglycerols, cellulose, chitosan, or derivatives thereof. The target water-solubilizing groups include, but are not limited to: carboxylates, phosphonates, phosphates, sulfonates, sulfates, sulfinates, sulfonium, esters, polyethylene glycol (PEG) and modified PEG, hydroxyl groups, amines, amino acids, ammonium groups, guanidine groups, pyridyl groups, polyamines and sulfonyl groups, polyols, linear or cyclic sugars, primary amines, secondary amines, tertiary or quaternary amines and polyamines, phosphonic acid groups, phosphonic acid groups, ascorbic acid groups, glycols including polyethers, -COOM′, -SO3M′, -PO3M′, -NR3 + Y′, (CH2CH2O) pR and mixtures thereof, wherein Y′ can be any halogen, sulfate, sulfonate or oxyanion, p can be 1 to 500, each R can be independently H or alkyl (e.g. methyl), and M′ can be a cationic counterion or hydrogen--(CH2CH2O). yy CH2CH2XR yy --(CH2CH2O) yy CH2CH2X--、--X(CH2CH2O) yy CH2CH2--, ethylene glycol and polyethylene glycol, wherein yy is selected from 1 to 1000, and X is selected from O, S and NR. ZZ And R ZZ and R YY Independently selected from H and C 1-3 Alkyl group. In some cases, WSG is (CH2). x (OCH2CH2) y OCH3, where each x is an integer from 0 to 20 independently, and each y is an integer from 0 to 50 independently. In some cases, the water-solubilizing group is contained in a nonionic polymer (e.g., PEG polymer) whose ends are replaced by ionic groups (e.g., sulfonate groups).

[0085] In some implementations, the target side group comprises a substituent selected from (CH2). x (OCH2CH2) y OCH3, wherein each x is an independent integer from 0 to 20, and each y is an independent integer from 0 to 50; and is optionally divided by one or more halogens, hydroxyl groups, C1 to C2 groups. 12 Alkyl group or (OCH2CH2) z OCH3-substituted benzyl groups, where each z is an independent integer from 0 to 50. In some instances, the substituent is (CH2)3(OCH2CH2). 11 OCH3. In some embodiments, one or more substituents are benzyl groups substituted with at least one WSG group (e.g., one or two WSG groups), said WSG group being selected from (CH2). x (OCH2CH2) y OCH3, where each x is an integer from 0 to 20 independently, and each y is an integer from 0 to 50 independently.

[0086] Multiple WSGs can be contained at a single position in the target dye via branched linkers. In some embodiments, the branched linker is an aralkyl substituent that is further disubstituted with a water-soluble group. Thus, in some cases, the branched linker group is a substituent of the dye that links the dye to two or more water-soluble groups. In some embodiments, the branched linker is an amino acid, such as lysine, linked to three groups via amino and carboxylic acid groups. In some cases, the introduction of multiple WSGs via branched linkers imparts the desired solubility to the dye. The target water-soluble polymer can be suitable for containing any convenient linking group. It should be understood that in some cases, the water-soluble polymer may have some dispersibility in terms of polymer length, depending on the preparation and / or purification methods of the polymer feedstock. In some instances, the water-soluble polymer is monodisperse.

[0087] Water-soluble polymers may contain one or more spacers or linkers. Examples of spacers or linkers include straight-chain or branched portions containing one or more repeating units used in the water-soluble polymer, diamino and / or diacid units, natural or non-natural amino acids or derivatives thereof, and aliphatic portions including alkyl, aryl, heteroalkyl, heteroaryl, alkoxy, etc., which may contain, for example, up to 18 carbon atoms or even other polymer chains.

[0088] The water-soluble polymer moiety, or when present with one or more spacer groups or linkers of the polymer moiety, may contain biostable or biodegradable polymer chains or units. For example, polymers with repeating linkages exhibit varying degrees of stability under physiological conditions, depending on the bond instability. Polymers with such linkages can be classified according to their relative hydrolysis rates under physiological conditions based on the known hydrolysis rates of their low molecular weight analogs, for example, from less unstable to more stable: polyurethane (-NH-C(O)-O-) > polyorthoester (-OC((OR)(R'))-O-) > polyamide (-C(O)-NH-). Similarly, the linking system that connects the water-soluble polymer to the target molecule can be biostable or biodegradable, for example, from less unstable to more stable: carbonate (-OC(O)-O-) > ester (-C(O)-O-) > urethane (-NH-C(O)-O-) > orthoester (-OC((OR)(R'))-O-) > amide (-C(O)-NH-). Typically, due to the instability of sulfate groups, it may be desirable to avoid using sulfated polysaccharides. Furthermore, the use of polycarbonates and polyesters may be less desirable. These bonds are provided by way of example and are not intended to limit the types of bonds in polymer chains or linking systems that can be used in the water-soluble polymers useful in the WSGs disclosed herein.

[0089] An example of a water-soluble polysarcosine group composed of N-modified sarcosine residues is described by the following formula:

[0090]

[0091] Where R is a hydrogen or non-hydrogen substituent, and n is an integer of 2 or greater than 2. In some instances, n is an integer from 2 to 40, such as 3 to 20, including 3 to 10.

[0092] As described above, in some cases, the water-soluble group can be a copolymer of sarcosine and non-sarcosine residues. In this case, the polymer can contain two or more distinct repeating residues (i.e., units), for example, when the polymer is a multiblock polymer, a random arrangement of units, or a specific sequence, each block can define a distinct repeating unit. It should be understood that the arrangement of repeating units or blocks is diverse, and in the water-soluble polysarcosine polymers described herein, any convenient linear arrangement of various lengths can be included in the overall polymer structure. It should be understood that the polymer can also be represented by a formula for the mole % of each unit in the polymer, and such a formula can represent various arrangements of repeating units, such as random or multiblock polymers or specific residue sequences. In some cases, the repeating unit of the polymer contains a single monomer group. In some instances, the repeating unit of the polymer contains two or more monomer groups, i.e., comonomer groups, such as two, three, four, or more than four comonomer groups. The term "comonomer" or "comonomer group" refers to a structural unit of the polymer that can itself be part of the repeating unit of the polymer. The comonomers that may be present in the embodiments of the present invention can be varied as needed, including amino acid monomers, peptide monomers, polyethylene oxide monomers, etc.

[0093] Examples of water-soluble polysarcosine groups in copolymers are described by the following formula:

[0094]

[0095] Where R is a hydrogen or non-hydrogen substituent, and n can vary, in some instances being 1 to 20, for example 1 to 15; m can vary, in some instances being 1 to 20, for example 1 to 15; p can vary, in some instances being 1 to 20, for example 1 to 15.

[0096] As summarized above, aspects of the present invention include dyes bonded with one or more water-soluble polysarcosine groups. In some cases, the dyes comprise organic dyes. The organic dyes can vary, and the organic dyes that can be modified with the water-soluble peptides having sulfonic acid groups of the present invention include, but are not limited to: cyanide dyes, rhodamine dyes, xanthan dyes, coumarin dyes, polymethine, pyrene, dipyrrole methylene boron difluoride, naphthimide, thiazide dyes, and acridine dyes.Target organic dyes include, but are not limited to: fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodamine alcohol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP (polydoxyphylla-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, Dyomics dyes (e.g., DY 431, DY 485XL, DY...) 500XL, DY 610, DY640, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY778, DY 782, DY 800, DY 831), dipyrrole methylene boron difluoride (BODIPY), Biotium CF 555, diethylaminocoumarin and its derivatives. In some cases, water-soluble peptides are bonded to organic dyes.

[0097] Examples of specific organic dyes that are water-soluble and solubilized by polysarcosine groups include, but are not limited to, the following: rhodamine, perylene, diimide, coumarin, xanthannaphthalene, anthocyanin, polymethystylene, pyrene, thiazide, acridine, dipyrrole methylene boron difluoride, naphthimide, phycobiliprotein, polydinophyll chlorophyll, their conjugates, and combinations thereof. In some embodiments, the acceptor fluorophore (A) is anthocyanin dye, xanthannaphthalene dye, coumarin dye, thiazide dye, or acridine dye. In some instances, the receptor 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 diethylaminocoumarin. Target fluorescent dyes include, but are not limited to: fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodamine alcohol, carboxyrhodamine 110, cascade blue, cascade yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP (polydinophyll 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, BODIPY558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY650 / 665, BODIPY R6G, BODIPY TMR, BODIPY TR, their conjugates and combinations thereof.Target lanthanide chelates include, but are not limited to, europium chelates, terbium chelates, and samarium chelates. In some embodiments, the polymer tandem dye comprises multiple chromophores linked to acceptor fluorophores selected from Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Alexa488, Alexa 647, and Alexa700. In some embodiments, the polymer tandem dye comprises a polychromatic group linked to an acceptor fluorophore selected from Dyomics dyes (e.g., DY 431, DY485XL, DY 500XL, DY 530, DY 610, DY 633, DY 640, DY 651, DY 654, DY 682, DY 700, DY701, DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 754, DY 778, DY 782, DY 800, or DY831), Biotium CF 555, Cy 3.5, and diethylaminocoumarin.In some cases, the receptor fluorophore (A) is selected from fluorescein, 6-FAM, rhodamine, Texas Red, California Red, iFluor594, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodamine alcohol, 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 (Polydoxyphyll-chlorophyll-a protein), PerCP-Cy5.5, JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, Fluorescein, Aquamarine, 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, their conjugates and combinations thereof.

[0098] In some cases, water-soluble polysarcosine-based organic dyes may be selected from the following substances: rhodamine, perylene, diimide, coumarin, xaton, anthocyanin, polymethyst, pyrene, thiazide, acridine, dipyrrole methylene borodifluoride, naphthaleneimide, phycobiliprotein, polydinoflavin chlorophyll protein.

[0099] In some instances, the dye is not used for medical imaging applications. For example, in some implementations, the dye is not anthocyanin dye, but rather indocyanine green.

[0100] In some embodiments, the dye further comprises a non-conjugated polymer backbone containing non-conjugated repeating units. "Non-conjugated" means that at least a portion of the repeating units contains saturated backbone groups (e.g., groups having two or more consecutive single covalent bonds), which prevents π-conjugation or extended delocalized electronic structures along the polymer backbone from one repeating unit to the next. It should be understood that even if a repeating unit may not be conjugated with adjacent repeating units, such a repeating unit may also contain one or more isolated unsaturated groups containing unsaturated bonds (e.g., unsaturated bonds of alkenyl or alkyneyl groups) and / or aryl or heteroaryl groups, which may be part of the backbone. In some cases, each repeating unit of the polymer backbone contains a side chain containing a connecting side group or a chemically selective tag for attachment to the side group.

[0101] In some embodiments, the polymer backbone is a linear polymer. In some cases, the linear polymer is selected from peptides, peptide-like polymers, hydrocarbon polymers, and PEG polymers. In some cases, the linear polymer is a peptide. In some cases, the linear polymer is a peptide-like polymer. In some cases, the polymer is a hydrocarbon polymer. In some other cases, the non-conjugated polymer is a PEG polymer. Further details regarding the non-conjugated polymer backbone that can be used in embodiments of the present invention can be found in PCT application serial number PCT / US2019 / 024662 (published as WO 2019 / 191482) and PCT application serial number PCT / US2020 / 019510 (published as WO 2020 / 222894); the contents of these applications are incorporated herein by reference.

[0102] In some instances, the non-conjugated polymer backbone is a peptide consisting of 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 contains 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, or up to 100 amino acids. In some cases, the tandem dye comprises a linear peptide backbone consisting of 5 to 30 amino acids, such as 5 to 25, 5 to 20, 5 to 15, or 5 to 10 amino acids.

[0103] The non-conjugated repeating unit can have any convenient configuration, such as linear, branched, or dendritic. The polymer backbone can be linear. The polymer backbone can be branched. In some instances, the dye comprises multiple side chromophores, each independently linked to a non-conjugated repeating unit of the polymer backbone. The configuration of the side groups can be introduced during or after the synthesis of the non-conjugated polymer backbone. The introduction of side groups can be achieved through random configurations, block configurations, or through stepwise synthesis in a sequence-specific manner, depending on the specific synthetic method used.

[0104] In some instances, the non-conjugated repeating unit contains multiple amino acid residues. In some instances, the dye comprises an organic dye bonded to the non-conjugated polymer backbone. Water-soluble peptides may be bonded to the non-conjugated polymer backbone and / or to an organic dye.

[0105] In some instances, the dye is a polymer dye (e.g., a fluorescent polymer dye). A wide variety of fluorescent polymer dyes are available for use in targeted methods and systems. In some instances of methods, the polymer dye comprises a conjugated polymer. Conjugated polymers (CPs) are characterized by a delocalized electronic structure consisting of a backbone of alternating unsaturated bonds (e.g., double and / or triple bonds) and saturated bonds (e.g., single bonds), where π electrons can move from one bond to another. Therefore, the conjugated backbone allows the polymer dye to have an extended linear structure with finite bond angles between the polymer repeating units. For example, proteins and nucleic acids, while also polymers, do not form extended rod-like structures in some cases, but rather fold into more advanced three-dimensional shapes. Furthermore, CPs may form “rigid rod-like” polymer backbones and undergo finite twist (e.g., torsion) angles along the polymer backbone between monomer repeating units. In some instances, the polymer dye comprises CPs with a rigid rod-like structure. The structural characteristics of the polymer dye can influence the fluorescence properties of the molecule.

[0106] Any readily available polymer dye can be used in the target device and method. In some instances, the polymer dye is a multi-chromophore with a structure capable of harvesting light to amplify the fluorescence output of a fluorophore. In some instances, the polymer dye is capable of collecting light and efficiently converting it into emitted light at longer wavelengths. In some cases, the polymer dye has a light-harvesting multi-chromophore system that can efficiently transfer energy to a nearby luminescent substance (e.g., a “signal chromophore”). Mechanisms of energy transfer include, for example, resonant energy transfer (e.g., Förster (or fluorescence) resonant energy transfer, FRET), quantum charge exchange (Dexter energy transfer), etc. In some instances, these energy transfer mechanisms are relatively short-range; that is, the close proximity of the light-harvesting multi-chromophore system to the signal chromophore provides efficient energy transfer. Under conditions of efficient energy transfer, the emission of the signal chromophore is amplified when the number of individual chromophores in the light-harvesting system is large; that is, the emission of the signal chromophore is stronger when the incident light (“excitation light”) is at a wavelength absorbed by the light-harvesting multi-chromophore system than when the signal chromophore is directly excited by the pump light.

[0107] Multichromophores can be conjugated polymers. Conjugated polymers (CPs) are characterized by their delocalized electronic structure and can be used as highly responsive optical reporter molecules for chemical and biological targets. Because the effective conjugation length is much smaller than the length of the polymer chain, the backbone contains a large number of closely adjacent conjugated segments. Therefore, conjugated polymers are highly efficient for light harvesting and can achieve optical amplification through Förster energy transfer.

[0108] The target polymer dyes include, but are not limited to, those described by Gaylord et al. in U.S. Publications 20040142344, 20080293164, 20080064042, 20100136702, 20110256549, 20110257374, 20120028828, 20120252986, and 20130190193, the entire contents of which are incorporated herein by reference; and Gaylord et al., J. Am. Chem. Soc., 2001, 123 (26), pp 6417–6418; Feng et al., Chem. Soc. Rev., 2010, 39, 2411–2419; and Traina et al., J. Am. Chem. Soc., 2011, 133(32). Pages 12600 to 12607, the entire contents of which are incorporated herein by reference.

[0109] In some embodiments, the polymeric dye comprises a conjugated polymer containing a plurality of first optically active units forming a conjugated system. The first optically active units have a first absorption wavelength (e.g., as described herein) at which they absorb light to form an excited state. The conjugated polymer (CP) can be a polycationic conjugated polymer, a polyanionic conjugated polymer, and / or a charge-neutral conjugated polymer.

[0110] Polymer dyes can have any suitable length. In some cases, the number of repeating units or segments of a particular monomer in a polymer dye may range from 2 to 500,000, for example, 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 for example, 100 to 100,000, 200 to 100,000, or 500 to 50,000 units or segments.

[0111] Polymer dyes can have any suitable molecular weight (MW). In some cases, the MW of a polymer dye can be expressed as its average molecular weight. In some instances, the average molecular weight of the polymer dye is from 500 to 500,000, for example from 1,000 to 100,000, from 2,000 to 100,000, from 10,000 to 100,000, or even from 50,000 to 100,000. In some embodiments, the average molecular weight of the polymer dye is 70,000.

[0112] In some instances, polymer dyes comprise the following structures:

[0113]

[0114] CP1, CP2, CP3 and CP4 are each independently a conjugated polymer segment or oligomeric structure, wherein one or more of CP1, CP2, CP3 and CP4 are n-conjugated repeating units that reduce the band gap, and each n and each m are independently 0 or an integer from 1 to 10000, and p is an integer from 1 to 100000.

[0115] In some instances, polymer dyes comprise the following structures:

[0116]

[0117] Each R 1 Independently a solubilizing group (e.g., a water-soluble polysarcosine group) or a linker-dye; L 1 and L 2 It is an optional connector; each R 2 Independently H or aryl substituents; each A 1and A 2 Independently, it is H, aryl substituent, or fluorophore; G 1 and G 2 Each n and m is independently selected from end groups, π-conjugated segments, linkers, and linker-specific binding members; each n and each m is independently an integer from 0 or 1 to 10000; p is an integer from 1 to 100000. Target solubilizing groups include water-soluble polysarcosine groups as described herein.

[0118] In some cases, polymer dyes include conjugated segments that are part of the polymer backbone and have one of the following structures:

[0119]

[0120] Each R 3 Independently, it is 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 10000. In some embodiments, R 3 It is an optionally substituted alkyl group. In some embodiments, R 3 The aryl group is optionally substituted. In some cases, R 3 Ar is replaced by water-soluble polysarcosine groups, polyethylene glycol, dyes, chemically selective functional groups, or specifically bound moieties. In some cases, Ar is replaced by polyethylene glycol, dyes, chemically selective functional groups, or specifically bound moieties.

[0121] In some instances, polymer dyes comprise the following structures:

[0122]

[0123] Each R 1 Independently a solubilizing group (e.g., a water-soluble polysarcosine group) or a linker-dye group; each R 2 Independently H or aryl substituents; each L 1 and L 3 Independently optional connectors; each A 1 and A 3 Independently, it can be H, a fluorophore, a functional group, or a specific binding moiety (e.g., an antibody); n and m are each independently an integer from 0 or 1 to 10000, where n+m>1.

[0124] Polymer dyes may possess one or more desired spectral properties, such as a specific maximum absorption wavelength, a specific maximum emission wavelength, extinction coefficient, quantum yield, etc. (see, for example, Chattopadhyay et al., “Brilliantviolet fluorophores: A new class of ultrabright fluorescent compounds for immunofluorescence experiments.” Cytometry Part A, 81A(6), 456-466, 2012). In some embodiments, the polymer dye has an absorption profile ranging from 280 nm to 475 nm. In some embodiments, the polymer dye has a maximum absorption (maximum excitation) in the range of 280 nm to 475 nm. In some embodiments, the polymer dye has an incident light with an absorption wavelength in the range of 280 nm to 475 nm. In some embodiments, the maximum emission wavelength range of the polymer dye is 400 nm to 850 nm, for example, 415 nm to 800 nm, wherein specific examples of the target maximum emission wavelength include, but are not limited to, 421 nm, 510 nm, 570 nm, 602 nm, 650 nm, 711 nm, and 786 nm. In some examples, the maximum emission wavelength of the polymer dye is selected from the following ranges: 410 nm to 430 nm, 500 nm to 520 nm, 560 nm to 580 nm, 590 nm to 610 nm, 640 nm to 660 nm, 700 nm to 720 nm, and 775 nm to 795 nm. In some embodiments, the maximum emission wavelength of the polymer dye is 421 nm. In some examples, the maximum emission wavelength of the polymer dye is 510 nm. In some cases, the maximum emission wavelength of the polymer dye is 570 nm. In some embodiments, the maximum emission wavelength of the polymer dye is 602 nm. In some examples, the maximum emission wavelength of the polymer dye is 650 nm. In some cases, the maximum emission wavelength of the polymer dye is 711 nm. In some embodiments, the maximum emission wavelength of the polymer dye is 786 nm. In some instances, the maximum emission wavelength of the polymer dye is 421 nm ± 5 nm. In some embodiments, the maximum emission wavelength of the polymer dye is 510 nm ± 5 nm. In some instances, the maximum emission wavelength of the polymer dye is 570 nm ± 5 nm. In some instances, the maximum emission wavelength of the polymer dye is 602 nm ± 5 nm. In some embodiments, the maximum emission wavelength of the polymer dye is 650 nm ± 5 nm. In some instances, the maximum emission wavelength of the polymer dye is 711 nm ± 5 nm. In some cases, the maximum emission wavelength of the polymer dye is 786 nm ± 5 nm.In some embodiments, the maximum emission value of the polymer dye is selected from 421 nm, 510 nm, 570 nm, 602 nm, 650 nm, 711 nm and 786 nm.

[0125] In some instances, the extinction coefficient of the polymer dye is 1 x 10⁻⁶. 6 cm -1 M -1 or higher than 1 x 10 6 cm -1 M -1 For example, 2 x 10 6 cm -1 M -1 or higher than 2 x 10 6 cm -1 M -1 2.5 x 10 6 cm -1 M -1 or higher than 2.5 x 10 6 cm -1 M -1 3 x 10 6 cm -1 M -1 or higher than 3 x 10 6 cm -1 M -1 4 x 10 6 cm -1 M -1 or higher than 4 x 10 6 cm -1 M -1 5 x 10 6 cm - 1 M -1 or higher than 5 x 10 6 cm -1 M -1 6 x 10 6 cm -1 M -1 or higher than 6 x 10 6 cm -1 M -1 7 x 10 6 cm -1 M -1 or higher than 7x 10 6 cm -1 M -1 or 8 x 10 6 cm -1 M -1 or higher than 8 x 106 cm -1 M -1 In some embodiments, the quantum yield of the polymer dye is 0.05 or higher, for example, 0.1 or higher, 0.15 or higher, 0.15 or higher, 0.2 or higher, 0.25 or higher, 0.25 or higher, 0.3 or higher, 0.35 or higher, 0.35 or higher, 0.4 or higher, 0.45 or higher, 0.45 or higher, 0.5 or higher, or even higher. In some cases, the quantum yield of the polymer dye is 0.1 or higher. In some cases, the quantum yield of the polymer dye is 0.3 or higher. In some cases, the quantum yield of the polymer dye is 0.5 or higher. In some embodiments, the extinction coefficient of the polymer dye is 1 x 10⁻⁶. 6 or higher than 1 x 10 6 cm -1 M -1 Furthermore, the quantum yield is 0.3 or higher. In some embodiments, the extinction coefficient of the polymer dye is 2 x 10⁻⁶. 6 or higher than 2 x 10 6 cm -1 M -1 And the quantum yield is 0.5 or higher.

[0126] Specific polymer dyes that may be used 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).

[0127] In some cases, the water-soluble polysarcosine group is a side group, i.e., it is located at the end of the dye. In this case, the side-group water-soluble polysarcosine group will be a single radical. In other cases, the water-soluble polysarcosine group is a linker group, i.e., it is located between two or more groups. For example, the linker water-soluble polysarcosine group can be a diradical linking two different groups, such as when the dye is attached to the polymer backbone, for example, in non-conjugated or conjugated polymers as described above.

[0128] Tandem dyes

[0129] This disclosure provides a tandem dye comprising a donor fluorophore, an acceptor fluorophore, and at least one water-soluble polysarcosine group. The at least one water-soluble polysarcosine group can increase the solubility of the tandem dye in water, for example, as described above.

[0130] Tandem dyes are compounds having two distinct, covalently linked fluorophores. These fluorophores can be directly covalently linked to each other or linked via a linking group, such as a polysarcosine water-soluble group or another polymer, such as the non-conjugated polymer backbone as described above. In tandem dyes, one fluorophore acts as a donor fluorophore, and the other as an acceptor fluorophore. The donor and acceptor fluorophores together form a fluorescence resonance energy transfer (FRET) pair. Such FRET pairs exhibit unique characteristics of dyes, possessing the excitation properties of the donor fluorophore and the emission properties of the acceptor fluorophore.

[0131] Excitation of the donor chromophore leads to energy transfer to the covalently linked acceptor fluorophore, which then emits energy. Energy transfer mechanisms from the donor chromophore to the linked acceptor signaling fluorophore include, for example, resonant energy transfer (e.g., Förster (or fluorescence) resonant energy transfer, FRET), quantum charge exchange (Dexter energy transfer), etc. These energy transfer mechanisms can be relatively short-range; that is, close proximity between the chromophores in a light-harvesting multi-chromophore system and / or with the acceptor fluorophore provides efficient energy transfer. Under conditions of efficient energy transfer, when the incident light ("pump light") is at the wavelength absorbed and transferred by the light-harvesting chromophore, the emission of the acceptor fluorophore is more intense than when the luminescent acceptor fluorophore is directly excited by the pump light, thus amplifying the acceptor fluorophore emission. "Efficient" energy transfer refers to 10% or more of the energy collected by the donor chromophore, such as 20% or more, 30% or more, 40% or more, or 50% or more, being transferred to the acceptor. "Amplification" refers to the signal of the acceptor fluorophore being 1.5 times higher or greater when excited by energy transfer from a donor light-harvesting chromophore system compared to direct excitation with incident light of the same intensity. The signal can be measured using any suitable method. In some cases, 1.5 times or greater refers to the intensity of the emitted light. In other cases, 1.5 times or greater refers to the increased signal-to-noise ratio. In certain embodiments of tandem dyes, the emission of the acceptor fluorophore upon excitation by the chromophore is 1.5 times or greater than that of the acceptor fluorophore when excited by incident light, for example, 2 times or greater than 12 times, 3 times or greater than 3 times, 4 times or greater than 4 times, 5 times or greater than 5 times, 6 times or greater than 6 times, 8 times or greater than 8 times, 10 times or greater than 10 times, 20 times or greater than 20 times, 50 times or greater than 50 times, 100 times or greater than 100 times, or even more.

[0132] In some instances, tandem dyes exhibit effective Stokes shifts of 25 nm to 300 nm, such as 50 nm to 250 nm or 75 nm to 200 nm. In other cases, when the photoharvesting chromophore is directly excited by incident light, the effective Stokes shift is 25 nm or greater, such as 50 nm or greater; 75 nm or greater; 100 nm or greater, such as 110 nm or greater; 120 nm or greater; 130 nm or greater; 140 nm or greater; 150 nm or greater; 160 nm or greater; 170 nm or greater; 180 nm or greater; 190 nm or greater; 200 nm or greater; and 250 nm or greater.

[0133] The emission quantum yield of tandem dyes can be 0.03 or higher, for example, 0.04 or higher, 0.05 or higher, 0.06 or higher, 0.07 or higher, 0.08 or higher, 0.09 or higher, 0.1 or higher, 0.15 or higher, 0.2 or higher, 0.3 or higher, or even higher. In some instances, the extinction coefficient of polymeric tandem dyes is 5 x 10⁻⁶. 5 cm -1 M -1 or higher than 5 x 10 5 cm -1 M -1 For example, 6 x 10 5 cm -1 M -1 or higher than 6 x 10 5 cm -1 M -1 7 x 10 5 cm -1 M -1 or higher than 7 x 10 5 cm -1 M -1 8 x 10 5 cm -1 M -1 or higher than 8 x 10 5 cm -1 M -1 9 x 10 5 cm -1 M -1 or higher than 9 x 10 5 cm-1 M -1 For example, 1 x 10 6 cm -1 M -1 or higher than 1 x 10 6 cm -1 M -1 1.5 x 10 6 cm -1 M -1 or higher than 1.5 x 10 6 cm -1 M -1 2 x 10 6 cm -1 M -1 or higher than 2 x 10 6 cm -1 M -1 2.5 x 10 6 cm -1 M -1 or higher than 2.5 x 10 6 cm -1 M -1 3 x 10 6 cm -1 M -1 or higher than 3 x 10 6 cm -1 M -1 4 x 10 6 cm -1 M -1 or higher than 4 x 10 6 cm -1 M -1 5 x 10 6 cm -1 M -1 or higher than 5 x 10 6 cm -1 M -1 6 x 10 6 cm -1 M -1 or higher than 6 x 10 6 cm -1 M -1 7 x 10 6 cm -1 M -1 or higher than 7 x 10 6 cm -1 M -1 or 8 x 10 6 cm -1 M -1or higher than 8 x 10 6 cm -1 M -1 In some implementations, the molar extinction coefficient of the tandem dye is 5 x 10⁻⁶. 5 cm -1 M -1 or higher than 5 x 10 5 cm -1 M -1 In some embodiments, the molar extinction coefficient of the tandem dye is 1 x 10⁻⁶. 6 cm -1 M -1 or higher than 1 x 10 6 cm -1 M -1 .

[0134] In some embodiments, one of the donor and acceptor fluorophores, and in some cases, both the donor and acceptor fluorophores contain organic dyes. For example, the organic dye for the donor fluorophore may be selected from anthocyanin dyes, rhodamine dyes, xanthan dyes, coumarin dyes, polymethystylene, pyrene, dipyrrole methylene borosilicate difluoride, naphthimide dyes, thiazide dyes, and acridine dyes. In some cases, the organic dye for the acceptor fluorophore may be selected from anthocyanin dyes, rhodamine dyes, xanthan dyes, coumarin dyes, polymethystylene, pyrene, dipyrrole methylene borosilicate difluoride, naphthimide dyes, thiazide dyes, and acridine dyes.

[0135] Target organic dyes that can be used as donors or acceptors include, but are not limited to: fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodamine 110, cascade blue, cascade yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP (polydoxyphylla-chlorophyll-a protein), PerCP-Cy5.5, JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, fluorescein yellow, aquamarine, 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, BODIPYTMR, BODIPY TR, Dyomics 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), dipyrrole methylene boron difluoride (BODIPY), Biotium CF 555, diethylaminocoumarin and its derivatives.

[0136] In some instances, the tandem dye may comprise a non-conjugated polymer backbone, such as a polymer composed of non-conjugated repeating units as described above. In some embodiments, the non-conjugated repeating units comprise multiple amino acid residues. In this case, water-soluble polysarcosine groups may be bonded to, or even incorporated into, the donor fluorophore, acceptor fluorophore and / or the non-conjugated polymer backbone.

[0137] In some instances, the dye may contain a fluorophore as a conjugated polymer. Therefore, in some cases, the donor fluorophore of a tandem dye may contain a conjugated polymer. As described above regarding dyes, in some cases, the conjugated polymer may contain a series of optionally substituted aryl and / or heteroaryl groups. Target aryl groups include fluorene and phenyl, while target heteroaryl groups include thiophene, pyridine, and BODIPY groups. In this case, the water-soluble peptide may be bonded to the conjugated polymer and / or the acceptor fluorophore.

[0138] In some cases, the water-soluble polysarcosine group is a side group, i.e., it is located at the end of the tandem dye. In this case, the side-group water-soluble polysarcosine group will be a single radical. In other cases, the water-soluble polysarcosine group is a linker group, i.e., it is located between two or more groups. For example, the linker water-soluble polysarcosine group can be a diradical linking two different groups. For example, the water-soluble polysarcosine group can be bonded to the acceptor fluorophore and a non-conjugated backbone, or the water-soluble polysarcosine group can be bonded to the donor fluorophore and a non-conjugated backbone.

[0139] Labeled specific binding members

[0140] This disclosure also includes labeled specific binding members comprising, for example, dyes or tandem dyes as described above, and the specific binding member itself. In other words, the specific binding member is labeled with a dye or tandem dye, for example, to enable the detection of target analytes bound to said specific binding member.

[0141] As described above, the water-soluble polysarcosine group can be a side group. Therefore, the water-soluble polysarcosine group can be a single radical group bonded to the end of a fluorophore or a specific binding member. In other cases, the water-soluble polysarcosine group can be a linker connecting two or more groups. For example, the water-soluble polysarcosine group can link the acceptor chromophore to the remainder of a tandem dye, such as where the water-soluble polysarcosine group is bonded to the acceptor fluorophore and a non-conjugated backbone. In other examples, the water-soluble polysarcosine peptide is bonded to the donor fluorophore and a non-conjugated backbone, or the water-soluble polysarcosine group is bonded to a specific binding member and a non-conjugated backbone.

[0142] For example, in certain cases, the labeled specific binding member has formula (V):

[0143]

[0144] in:

[0145] D is a dye;

[0146] A is absent or is a receptor dye;

[0147] L1 either does not exist or optionally contains a linker with a polysarcosine group;

[0148] L2 is a linker that optionally contains a polysarcosine group;

[0149] x is 0 or an integer from 1 to 10;

[0150] Each Y is independently a water-soluble group containing a polysarcosine group; and

[0151] SBM is a specific binding member.

[0152] In some embodiments, the labeled specific binding member is a dye, i.e., where A is absent and L2 is absent. Therefore, D is a dye as described above. Exemplary dyes that can be used with the D group include those described above, such as cyanine dyes, rhodamine dyes, coumarin dyes, BODIPY dyes, and polyfluorene dyes.

[0153] In some cases, the labeled specific binding member is a tandem dye, i.e., where A is present. Therefore, D can be considered the donor dye, and A is the acceptor dye that receives energy transfer from D. For tandem dyes, L2 may be absent, or L2 may be a linker, such as a linker containing a polysarcosine group (which increases water solubility), a non-conjugated polymer backbone, etc. In some instances, D is a conjugated polymer, as described above.

[0154] L1 may be a linker that does not exist or optionally contains a polysarcosine group (e.g., to increase water solubility). Furthermore, x can be an integer from 1 to 10, so the labeled specific binding member can contain one to ten Y groups. As described above, each Y group is independently a water-soluble group containing a polysarcosine group.

[0155] Methods for labeling target molecules

[0156] The present invention also provides a method for labeling target molecules. Such a method involves contacting the target molecule with a dye or tandem dye described herein to covalently bond the target molecule to the reactive moiety of the dye or tandem dye, thereby producing a labeled target molecule.

[0157] Methods of interest for target labeling include, but are not limited to, those described in Hermanson, Bioconjugate Techniques, Third edition, Academic Press, 2013. The contact step can be carried out in aqueous solution. In some instances, the reaction moiety contains an amino functional group, and the target molecule contains an activated ester functional group, such as an NHS ester or a sulfonyl-NHS ester, or vice versa. In some instances, the reaction moiety contains a maleimide functional group, and the target molecule contains a thiol functional group, or vice versa. In some instances, the reaction moiety contains an alkyne (e.g., a cyclooctyne group) functional group, and the target molecule contains an azide functional group, or vice versa; these can be conjugated via click chemistry.

[0158] Any suitable target molecule can be selected for labeling using the targeted approach. Target molecules include, but are not limited to: nucleic acids, such as RNA, DNA, PNA, CNA, HNA, LNA, or ANA molecules; proteins, such as fusion proteins; modified proteins, such as phosphorylated proteins, glycosylated proteins, ubiquitinated proteins, SUMOylated proteins, or acetylated proteins; or antibodies, peptides, aggregated biomolecules, cells, small molecules, vitamins, and drug molecules. As used herein, the term "target protein" refers to all members of the target family and fragments thereof. The target protein can be any target protein, such as therapeutic or diagnostic targets, including but not limited to: hormones, growth factors, receptors, enzymes, cytokines, bone-inducing factors, colony-stimulating factors, and immunoglobulins. The term "target protein" is intended to include recombinant and synthetic molecules that can be prepared using any suitable recombinant expression method or using any suitable synthetic method, or are commercially available. In some embodiments, the target molecule is a specifically binding member (e.g., as described herein). In some instances, the specifically binding member is an antibody. In some instances, the specifically binding member is an antibody fragment or a binding derivative thereof. In some cases, the antibody fragment or its binding derivative is selected from Fab fragments, F(ab')2 fragments, scFv, bispecific antibodies, and trispecific antibodies.

[0159] In some cases, the method includes a separation step in which labeled target molecules are separated from the reaction mixture (e.g., excess reagent or unlabeled target). A variety of methods can be used to separate the target from the sample, such as immobilization on a support, precipitation, chromatography, etc.

[0160] In some instances, the method also includes the detection and / or analysis of labeled target molecules. In some instances, the method also includes the fluorescence detection of labeled target molecules. Any suitable method can be used in conjunction with the target method and composition to detect and / or analyze labeled target molecules. Analytical methods for the target targets that can be used in the target method include, but are not limited to, flow cytometry, fluorescence microscopy, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blotting, magnetic cell separation assay, and fluorescent dye purification chromatography. Target detection methods include, but are not limited to, fluorescence spectroscopy, fluorescence microscopy, nucleic acid sequencing, fluorescence in situ hybridization (FISH), protein mass spectrometry, flow cytometry, etc.

[0161] Detection can be achieved directly via polymer-tandem dyes or indirectly via a secondary detection system. The latter can be based on any of several different principles or combinations thereof, including but not limited to antibody-labeled anti-species antibodies and other forms of immunological or non-immunological bridging and signal amplification systems (e.g., biotin-streptavidin technology, protein A and protein G-mediated technologies, or nucleic acid probes / anti-nucleic acid probes, etc.). Suitable reporter molecules can be those known in the fields of immunocytochemistry, molecular biology, optical microscopy, fluorescence microscopy and electron microscopy, cellular immunophenotyping, cell sorting, flow cytometry, cell visualization, detection, counting, and / or signal output quantification. Antibodies with more than one specific and / or non-specific property can be simultaneously or sequentially labeled and used to enhance target detection, recognition, and / or analysis.

[0162] The method described herein may include multiple steps. Each step may be executed after a predetermined time interval between steps, as needed. Therefore, the time between 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, including 5 hours or more. In some embodiments, each subsequent step is executed immediately after the completion of the preceding step. In other embodiments, a step may be executed after an incubation or waiting period following the completion of the preceding step, for example, after a waiting period of several minutes to overnight.

[0163] Methods for assessing the presence of target analytes in samples

[0164] A method is also provided for assessing the presence of a target analyte in a sample by using a labeled, specifically binding member comprising a water-soluble peptide having a sulfonic acid group. In some embodiments, the method includes:

[0165] (a) Contacting the sample with a labeled, specifically binding member that specifically binds to the target analyte to produce a labeled sample; and

[0166] (b) Determine whether a labeled specific binding member-target analyte binding complex is present in the labeled composition to assess the presence of the target analyte in the sample.

[0167] The labeled specific binding member used in embodiments of the method of the present invention comprises a specific binding member that binds to a dye or tandem dye (e.g., as described above). In the following sections, the target analyte can be a target molecule or reagent, such as a primary antibody binding to a target molecule, depending on whether the labeled specific binding member is used as a primary antibody label or a secondary antibody label. Any suitable method can be used to contact the sample with the labeled specific binding member that specifically binds to the target analyte to generate the assay composition. In some instances, the sample is contacted with the labeled specific binding member, if present, under conditions where the labeled specific binding member specifically binds to the target analyte. To ensure specific binding of the labeled specific binding member to the target analyte, a suitable medium that maintains the bioactivity of the sample components and the signal domain antibody can be used. This medium can be a balanced salt solution, such as physiological saline, PBS, Hank's balanced salt solution, etc., typically supplemented with fetal bovine serum, human platelet lysate, or other factors, and a low concentration (e.g., 5 mM to 25 mM) of acceptable buffer. Suitable buffers include HEPES, phosphate buffer, lactate buffer, etc. Various culture media are commercially available and can be used depending on the properties of the target analyte, including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., sometimes supplemented with fetal bovine serum or human platelet lysate. The final composition of the culture medium (which may be a solution) can be selected based on the sample components contained therein. The temperature at which the labeled specific binding member specifically binds to the target analyte can vary and, in some cases, can be in the range of 5°C to 50°C, for example, from 10°C to 40°C, 15°C to 40°C, 20°C to 40°C, for example, 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 occurs is selected to be compatible with the bioactivity of the specific binding member and / or the target analyte. In some instances, the temperature is 25°C, 30°C, 35°C, or 37°C. In some cases, the temperature at which specific binding occurs is room temperature (e.g., 25°C), 30°C, 35°C, or 37°C. Any suitable specific binding incubation time can be selected to allow the formation of the desired amount of binding complex, and in some cases, it can be 1 minute or more, such as 2 minutes or more, 10 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, or even 6 hours or more.

[0168] Any suitable specific binding member can be used as a labeled specific binding member in the methods of this invention. Target specific binding members include, but are not limited to, those that specifically bind to cell surface proteins of various cell types, including, but not limited to: stem cells, such as pluripotent stem cells and hematopoietic stem cells; T cells, regulatory T cells, dendritic cells; B cells, such as memory B cells and antigen-specific B cells; granulocytes, leukemia cells, lymphoma cells, viral cells (e.g., HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythroid cells. Target cells include cells with suitable cell surface markers or antigens that can be captured by suitable specific binding member conjugates. In some embodiments, the target cells are selected from HIV-containing cells, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+) derived from whole blood, bone marrow, or umbilical cord blood. Any suitable cell surface protein or cell marker can be targeted to specifically bind to the conjugate used in the target method. In some embodiments, the target cells contain cell surface markers selected from cell receptors and cell surface antigens. In some cases, target cells may contain cell surface antigens such as CD11b, 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 α / β, T cell receptor γ / δ, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminus), SSEA-3, TRA-1-60 antigen, disialiacoganglioside GD2, and CD71.

[0169] Targeted methods can be used to select any suitable target for evaluation. Target targets include, but are not limited to: nucleic acids, such as RNA, DNA, PNA, CNA, HNA, LNA, or ANA molecules; proteins, such as fusion proteins; modified proteins, such as phosphorylated proteins, glycosylated proteins, ubiquitinated proteins, SUMOylated proteins, or acetylated proteins; or antibodies, peptides, aggregated biomolecules, cells, small molecules, vitamins, and drug molecules. As used herein, the term "target protein" refers to all members of a target family and fragments thereof. The target protein can be any protein of interest, such as therapeutic or diagnostic targets, including but not limited to: hormones, growth factors, transcription factors, receptors, enzymes, cytokines, bone-inducing factors, colony-stimulating factors, and immunoglobulins. The term "target protein" is intended to include recombinant and synthetic molecules that can be prepared using any suitable recombinant expression method or any suitable synthetic method, or are commercially available. In some embodiments, the polymer dye conjugate comprises an antibody or antibody fragment. In targeted methods, any suitable target analyte that specifically binds to the target antibody or antibody fragment can be targeted.

[0170] In some embodiments, the target analyte binds to cells. In some instances, the target analyte is a cell surface marker. In some cases, the cell surface marker is selected from cell receptors and cell surface antigens. In some instances, the target analyte is an intracellular target, and the method further includes treating the cells to bring the labeled, specifically binding member into contact with the intracellular target, for example, by permeabilization or cell lysis. Thus, the labeled, specifically binding member used in the method of the present invention can target cell surface or intracellular antigens. Alternatively, the labeled, specifically binding member used in the method of the present invention can target a primary antibody that specifically binds to the target cell surface or intracellular antigen.

[0171] In some embodiments, the sample may contain a heterogeneous cell population from which target cells have been isolated. In some instances, the sample includes peripheral whole blood, peripheral whole blood with lysed red blood cells prior to cell separation, cord blood, bone marrow, density gradient purified peripheral blood mononuclear cells, or homogenate tissue. In some cases, the sample contains hematopoietic progenitor cells (e.g., CD34+ cells) from whole blood, bone marrow, or cord blood. In some embodiments, the sample contains tumor cells from peripheral blood. In some instances, the sample is a sample containing (or suspected of containing) viral cells (e.g., HIV).

[0172] Labeled specific binding members can be used in targeted methods, such as for labeling target cells, particles, targets, or analytes with polymer-tandem fluorescent dyes. For example, labeled specific binding members can be used to label cells to be processed (e.g., detected, analyzed, and / or sorted) in flow cytometry. Labeled specific binding members may comprise specific binding members, such as antibodies or binding fragments thereof, that specifically bind to cell surface proteins of multiple cell types (e.g., as described herein). Labeled specific binding members can be used to study 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, etc. Labeled specific binding members can be used in any application that includes (or may include) the labeling of antibody-mediated cells, particles, or analytes.

[0173] Aspects of the method include determining the presence of a labeled specific binding member-target analyte binding complex in the assay composition (i.e., a sample in contact with a labeled specific binding member) to assess the presence of a target analyte in the sample. Once the sample has been contacted with the labeled specific binding member, any suitable method can be used to determine the presence of a labeled specific binding member-target analyte binding complex in the resulting assay composition. A labeled specific binding member-target analyte binding complex is a binding complex (if present) formed when a labeled specific binding member specifically binds to a target analyte (or a primary antibody binding member, such as a primary antibody, with a target antigen, depending on the embodiment). Determining the assay composition may include detecting a fluorescent signal from the binding complex (if present). In some cases, the assay includes a separation step in which the target analyte (if present) is separated from the sample. Various methods can be used to separate the target analyte from the sample, such as by immobilization on a support. Target assay methods include, but are not limited to, any suitable method and assay form in which specific binding member pairs, such as avidin-biotin or hapten-anti-hapten antibody, can be used. The target methods and assays applicable to the target composition include, but are not limited to, flow cytometry, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assay, and fluorescent dye purification chromatography.

[0174] In some embodiments, the method further includes contacting the sample with a second specific binding member that specifically binds to the target analyte. In some instances, the second specific binding member is bound to a support. Any suitable support can be used to immobilize a component of the target method (e.g., the second specific binding member). In some instances, the support is a particle, such as a magnetic particle. In some instances, the second specific binding member and the polymer dye conjugate form a sandwich complex, which can be separated and detected (if present) using any suitable method. In some embodiments, the method further includes flow cytometry analysis of the polymer dye conjugate-target analyte binding complex, i.e., the fluorescently labeled target analyte. Determining the presence of a labeled specific binding member-target analyte binding complex can provide assay results (e.g., qualitative or quantitative assay data) that can be used to assess the presence of the target analyte in the sample.

[0175] Any suitable support can be used in the target method to immobilize any suitable component of the method, such as labeled specific binding members, targets, secondary specific binding members, etc. Target supports include, but are not limited to: solid matrices, which can have various configurations, such as sheet-like, bead-like, or other structures, such as porous plates; beads, polymers, particles, fibrous webs, hydrogels, porous matrices, needle-like structures, microarray surfaces, chromatographic supports, etc. In some instances, the support is selected from particles, planar solid matrices, fibrous webs, hydrogels, porous matrices, needle-like structures, microarray surfaces, and chromatographic supports. The support can be integrated into a system that assists cell separation by any suitable method, such as manually operated syringes, centrifuges, or automated liquid handling systems. In some cases, the support is used in automated liquid handling systems, such as flow cytometers, for high-throughput cell separation.

[0176] In some embodiments of the method, the separation step includes applying an external magnetic field to immobilize the magnetic particles. Any suitable magnet can be used as a source of the external magnetic field (e.g., a magnetic field gradient). In some cases, the external magnetic field is generated by a magnetic source, such as a permanent magnet or an electromagnet. In some cases, immobilizing the magnetic particles means that the magnetic particles accumulate near the surface closest to the source of the magnetic field gradient (i.e., the magnet).

[0177] Separation may also include one or more optional washing steps to remove unbound material from the sample onto the support. Any suitable washing method can be used, for example, washing the immobilized support with a biocompatible buffer that preserves the specific binding interactions between the polymer dye and the specific binding member. Separation from the support and optional washing of unbound material from the sample can provide an enriched population of target cells from which unwanted cells and material can be removed.

[0178] In some embodiments, the method includes detecting a labeled target analyte. Detection of the labeled target analyte may include exciting a polymeric fluorescent tandem dye with one or more lasers, followed by detecting fluorescence emission from the polymeric fluorescent tandem dye using one or more optical detectors. The labeled target can be detected using any suitable instrument and method, including but not limited to: flow cytometry, FACS systems, fluorescence microscopy; fluorescence, luminescence, UV and / or visible light detection using a microplate reader; high-performance liquid chromatography (HPLC); and mass spectrometry. When fluorescently labeled components are used in the methods and compositions of this disclosure, it is considered that different types of fluorescence detection systems can be used to practice the target method. In some cases, high-throughput screening can be performed, for example, using systems with 96 or more wells in a microplate. A variety of methods can be used to determine fluorescent materials, such as those described in Lakowicz, JR, 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, edited by Taylor, DL & Wang, Y.-L., San Diego: Academic Press (1989), pp. 219-243; Turro, NJ, Modern Molecular Photochemistry, Menlo Park: Benjamin / Cummings Publishing Col, Inc. (1978), pp. 296-361.

[0179] Fluorometers can be used to measure fluorescence in samples. In some cases, excitation radiation from an excitation source having a first wavelength passes through an excitation optics. The excitation optics excite the sample with the excitation radiation. In response, fluorescently labeled targets in the sample emit radiation with a wavelength different from the excitation wavelength. A collection optics then collects the emission from the sample. The device may include a temperature controller to maintain the sample at a specific temperature during scanning. In some instances, a multi-axis translation stage moves a microplate containing multiple samples to position different wells to be exposed. The multi-axis translation stage, temperature controller, autofocus function, and electronics associated with imaging and data acquisition can be managed by a suitably programmed digital computer. The computer can also convert the data collected during the measurement into another format for presentation.

[0180] In some embodiments, the method for assessing the presence of the target analyte in a sample further includes detecting fluorescence in a flow cytometer. In some embodiments, the method for assessing the presence of the target analyte in a sample further includes imaging the sample in contact with the labeled composition using a fluorescence microscope. Fluorescence microscopy imaging can be used to identify polymer dye conjugate-target analyte binding complexes in the contact sample to assess the presence of the target analyte. Target microscopy methods that can be used for target methods include laser scanning confocal microscopy.

[0181] The method described herein may include multiple steps. Each step may be performed after a predetermined time interval between steps, as needed. Therefore, the time between 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, including 5 hours or more. In some embodiments, each subsequent step is performed immediately after the completion of the preceding step. In other embodiments, a step may be performed after an incubation or waiting period following the completion of the preceding step, for example, after a waiting period of several minutes to overnight.

[0182] system

[0183] The invention also includes systems for carrying out the target methods and compositions. Sample analysis systems may include a sample field of view or flow channel loaded with the sample of the invention and a labeled specific binding member (e.g., as described above). In some embodiments, the system is a flow cytometry system comprising: a flow cytometer including a flow channel; and a composition in the flow channel, wherein the composition comprises: a sample and a labeled specific binding member (e.g., as described herein). In some embodiments, the system for analyzing samples is a fluorescence microscopy system comprising: a fluorescence microscope containing a sample field of view; and a composition disposed in the sample field of view, wherein the composition contains a sample; and a labeled specific binding member (e.g., as described herein).

[0184] In some embodiments of the system, the composition further includes a second specific binding member bound to the support, which specifically binds to the target analyte. In some cases, the support comprises magnetic particles. Therefore, in some instances, the system may also include a controllable external paramagnetic field configured to be applied to the measurement region of the flow channel.

[0185] Samples may contain cells. In some instances, the sample is a biological sample containing cells. In some instances, the sample contains a labeled, specific-binding member that specifically binds to target cells. In some instances, the target analyte that specifically binds to the specific-binding member is a cell surface marker. In some cases, the cell surface marker is selected from cell receptors and cell surface antigens.

[0186] In some aspects, the system may also include a light source configured to direct light to a measurement region of the flow channel or sample field of view. The system may include a detector configured to receive a signal from the measurement region of the flow channel or sample field of view, wherein the signal is provided by a fluorescent composition. Optionally, the sample analysis system may also include one or more additional detectors and / or light sources for detecting one or more additional signals.

[0187] In some aspects, the system may also include a computer-based system configured to detect the presence of fluorescence signals. "Computer-based system" refers to hardware devices, software devices, and data storage devices used to analyze the information of this invention. The minimum hardware of the computer-based system of this invention includes a central processing unit (CPU), input devices, output devices, and data storage devices. Those skilled in the art will readily understand that any currently available computer-based system is suitable for the target system. The data storage device may include any article of manufacture that includes the above-described record of the information of this invention, or a memory access device capable of accessing such article of manufacture.

[0188] Recording data, programs, or other information on a computer-readable medium refers to the process of storing information using any such methods known in the art. Any suitable data storage structure can be chosen, based on the means used to access the stored information. Various data processing programs and formats can be used for storage, such as word processing text files, database formats, etc.

[0189] The term "processor" refers to any combination of hardware and / or software that will perform its required functions. For example, any processor described herein may be a programmable digital microprocessor, such as a microprocessor provided in the form of an electronic controller, mainframe, server, or personal computer (desktop or portable). If the processor is programmable, suitable programming may be transferred to the processor from a remote location or pre-stored in a computer program product (e.g., a portable or fixed computer-readable storage medium, whether based on magnetic, optical, or solid-state devices). For example, a magnetic medium or optical disc may carry the programming and may be read by communicating with a suitable reader at each processor's respective site.

[0190] In addition to sensor devices and signal processing modules (e.g., as described above), the system of the present invention may also include many additional components, such as data output devices (e.g., displays and / or speakers), data input devices (e.g., interface ports, keyboards, etc.), fluid processing components, power supplies, etc.

[0191] In some respects, the system includes flow cytometers. Suitable flow cytometry systems may include, but are not limited to, Ormerod (eds.), 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 edition (eds.), 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):502-11; Alison et al. JPathol, 2010 Dec; 222(4):335-344; and Herbig et al. (2007) Crit Rev Ther DrugCarrier Syst. Those described in 24(3):203-255; the contents of these documents are incorporated herein by reference. In some instances, flow cytometry systems of interest include BD Biosciences FACSCanto TM Flow cytometer, BDBiosciences FACSCanto TM II flow cytometer, BD Accuri TM Flow cytometer, BD Accuri TM C6 Plus flow cytometer, BD Biosciences FACSCelesta TM Flow cytometer, BD Biosciences FACSLyric TM Flow cytometer, BD Biosciences FACSVerse TM Flow cytometer, BD Biosciences FACSymphony TM Flow cytometer, BD Biosciences LSRFortessa TM Flow cytometer, BD Biosciences LSRFortessa TM X-20 flow cytometer, BD Biosciences FACSPresto TMFlow cytometer, BD Biosciences FACSVia TM Flow cytometer and BD Biosciences FACSCalibur TM Cell sorter, BD Biosciences FACSCount TM Cell sorting instrument, BD Biosciences FACSLyric TM Cell sorter, BD Biosciences Via TM Cell sorter, BD Biosciences Influx TM Cell sorter, BD Biosciences Jazz TM Cell sorting instrument, BDBiosciences Aria TM Cell sorting instrument, BD Biosciences FACSAria TM II Cell Sorter, BDBiosciences FACSAria TM III Cell Sorter, BD Biosciences FACSAria TM Fusion Cell Sorter and BD Biosciences FACSMelody TM Cell sorting instrument, BD Biosciences FACSymphony TM S6 cell sorter, etc.

[0192] In some implementations, the target system is a flow cytometry system, such as 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, Those described in 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; 6,813,017; 6,809,804; 6,372,506; 5,700,692; 5,643,796; 5,627,040; 5,620,842; 5,602,039; 4,987,086; 4,498,766; the disclosures of these patents are incorporated herein by reference in their entirety.

[0193] In some instances, the flow cytometry system of the present invention is configured to image particles in a flowing stream using fluorescence imaging with radio frequency labeled emission (FIRE), for example, Diebold et al., Nature Photonics Vol. 7(10); 806-810 (2013) and 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; 10,620,111; and U.S. Patent Publication No. 2017 / 0133857; Those described in 2017 / 0328826; 2017 / 0350803; 2018 / 0275042; 2019 / 0376895 and 2019 / 0376894; the public content of these documents is incorporated herein by reference.

[0194] Other systems may also be used to implement the target method. In some aspects, the system may be a fluorometer or microscope loaded with a sample having any of the fluorescent compositions discussed herein. The fluorometer or microscope may include a light source configured to direct light to a measurement region of a flow channel or a sample field of view. The fluorometer or microscope may also include a detector configured to receive a signal from a measurement region of the flow channel or field of view, wherein the signal is provided by the fluorescent composition.

[0195] Reagent test kit

[0196] The invention also includes kits for practicing the target method. Dyes, tandem dyes, labeled specific binding members, or combinations thereof can be included as reagents in the kit, either as starting materials or for use in, for example, the methodologies described above. Such dyes, tandem dyes, and labeled specific binding members can be provided with a container. Any suitable container can be used, such as tubes, bottles, wells in multiwell strips or plates, boxes, bags, insulated containers, etc. The target kit may also include one or more components selected from: primer-specific binding members for a given target analyte, support-specific binding members, cells, support, biocompatible aqueous elution buffer, controls (positive and / or negative), etc., and instructions for use as needed. A given kit may include reagents suitable for detecting a single target analyte, or multiple reagents suitable for detecting two or more different target analytes, for example, when a given kit is configured for multiplex detection applications.

[0197] In some implementations, the kit is used to assess the presence of a target analyte (e.g., an intracellular target) in a sample. Therefore, in some instances, the kit includes one or more components suitable for permeabilizing or lysing cells. 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).

[0198] In some respects, the kit also includes reagents for performing flow cytometry assays. Target reagents include, but are not limited to: buffers for recovery and dilution, buffers for contacting cell samples with chromophores, wash buffers, control cells, control beads, fluorescent beads for flow cytometry calibration, and combinations thereof. The kit may also include one or more cell fixation reagents, such as paraformaldehyde, glutaraldehyde, methanol, acetone, formalin, or any combination thereof or buffers thereof. Furthermore, the kit may include cell permeation reagents, such as methanol, acetone, or surfactants (e.g., triton, NP-40, saponins, Tween 20, digitalis saponins, leucoperm, or any combination thereof or buffers thereof). Other protein transport inhibitors, cell fixation reagents, and cell permeation reagents familiar to those skilled in the art are also within the scope of the target kit.

[0199] The kit compositions may be provided as liquid compositions, such as in any suitable buffer. Alternatively, the kit compositions may be provided as dry compositions (e.g., lyophilized), and the kit may optionally include one or more buffers for reconstitution of the dry compositions. In some 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).

[0200] In addition, one or more components may be combined into a single container, such as a glass or plastic vial, tube, or bottle. In some instances, the kit may also include containers (e.g., boxes, bags, insulated containers, bottles, tubes, etc.) containing all components (and their individual containers). The kit may also include separate or attached packaging from the kit container, printed with information about the kit, the components of the kit, and / or instructions for use of the kit.

[0201] In addition to the components mentioned above, the target kit may also include instructions for practicing the target method. These instructions may exist in various forms within the target kit, one or more of which may be present. One form of these instructions may be printed information on a suitable medium or substrate, such as printed paper, kit packaging, packaging inserts, etc. Another form may be computer-readable media, such as disks, CDs, DVDs, portable flash drives, etc., on which the information has been recorded. Yet another possible form is a website address, allowing access to the information at a remote site via the internet. Any suitable means may be present in the kit.

[0202] use

[0203] The dyes, tandem dyes, and labeled specific binding members, compositions, methods, and systems described herein can be used in a variety of applications, including diagnostic and research applications, where the labeling, detection, and / or analysis of a target is required. Such applications include methods such as cytometry, microscopy, immunoassays (e.g., competitive or non-competitive), free analyte evaluation, receptor-binding ligand evaluation, etc. The compositions, systems, and methods described herein can be used to analyze any number of samples, including but not limited to biofluids, cell culture samples, and tissue samples. In some aspects, the compositions, systems, and methods described herein can be used in methods for detecting analytes (if present) in samples using fluorescent labeling, such as in fluorescence-activated cell sorting or analysis, immunoassays, immunostaining, etc. In some instances, the compositions and methods are used for applications that assess the presence of a target analyte in a sample. In some aspects, green excitable tandem dyes can be excited by green lasers, such as lasers with a wavelength of 532 nm.

[0204] In some cases, the methods and compositions can be used for any assay requiring the detection and / or analysis of a target in a sample, including but not limited to: flow cytometry, fluorescence microscopy, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blotting, magnetic cell separation assay, and fluorescent dye purification chromatography. In some instances, the methods and compositions can be used for any application requiring fluorescently labeled target molecules. Target compositions are suitable for any suitable application where paired, specific binding members (e.g., biotin-streptavidin and hapten-anti-hapten antibody) function.

[0205] The following examples are intended to illustrate, not limit, the invention.

[0206] Experimental Section

[0207] The following embodiments are intended to provide a complete disclosure and description for those skilled in the art, illustrating how to make and use the invention, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to represent that the following experiments are all or only the experiments performed.

[0208] Example 1: Synthesis of the compound

[0209] Figure 6The synthesis of polysarcosine moieties using SPPS resin supports is illustrated. First, sarcosine monomers undergo a transesterification reaction (e.g., acid- or base-catalyzed) to form a covalent bond between two components. An FMOC protecting group is present at the other end of the sarcosine residue. Next, the resin with a single sarcosine residue can be reacted with piperidine to deprotect the amino group, and then the compound can be reacted with a second sarcosine residue in the presence of HATU to attach the second sarcosine group to the SPPS resin via a peptide synthesis reaction. This attachment of additional sarcosine residues can be repeated to obtain polysarcosine groups. Subsequently, the bond between the polysarcosine and the resin can be cleaved to obtain monofunctional or bifunctional polysarcosine residues.

[0210] Other solid-phase and liquid-phase peptide synthesis methods can be used to synthesize polysarcosine groups. For example, such methods can be found in: Steward et al. in "Solid Phase Peptide Synthesis", WH 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 liquid-phase synthesis. Any suitable protecting group strategy can 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 terminus, and benzyl or benzyl-based or other suitable protecting groups can be used to protect the side chain functional groups. In Fmoc solid-phase peptide synthesis, an Fmoc-amino protecting group is used at the amino terminus, and tert-butyl or benzyl-based or other suitable protecting groups can be used to protect the side chain functional groups.

[0211] Various methods known in the art can be used to covalently link these polysarcosine groups to dyes and tandem dyes. For example, Figure 6The monofunctional and bifunctional polysarcosine groups have terminal carboxyl groups. These carboxyl groups can react with nucleophilic groups on the dye to initiate nucleophilic substitution reactions, thereby covalently bonding the polysarcosine to the dye. For example, the carboxyl groups can react in transesterification reactions or in coupling reactions with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) or DCC (N,N'-dicyclohexylcarbodiimide), reactions known in the art. Furthermore, Figure 7 This demonstrates the conversion of hydroxyl-terminated polysarcosine groups into amino, OTos, and alkynyl groups. These groups can react with corresponding groups on the target molecule to form covalent bonds between them; for example, alkynes can react with azide groups via click reactions. Furthermore, the amino group can be a nucleophile, and the OTos group can be a leaving group in a nucleophilic substitution reaction.

[0212] Furthermore, Huesmann et al. (Polymer 67 (2015) 240-248, doi: 10.1016 / j.polymer.2015.04.070) and Chan et al. (Biopolymers, 2018;109:e23070, doi:10.1002 / bip.23070) described the synthesis of polysarcosine and its copolymers. Such synthetic methods can also be applied to the sarcosine compounds described herein.

[0213] Convenient protecting groups that can be used in such synthetic methods are described in the above references, as well as in McOmic's "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.

[0214] Example 2: Implementation Plan

[0215] Figure 1 Polysarcosine, N-modified polysarcosine, and polysarcosine copolymer groups that can be attached to dyes and tandem dyes are shown. Figure 3 Examples of dyes functionalized with polysarcosine groups are shown, such as cyanine dyes, rhodamine dyes, coumarin dyes, and BODIPY dyes. Figure 4 Polyfluorene dyes functionalized with antibody-specific binding members and polysarcosine water-soluble groups are shown. Figure 5 The polysarcosine group is shown to be used as a linker between dyes and biomolecules (e.g., acting as a specific binding member). Figure 7 This demonstrates the conversion of terminal hydroxyl groups on polysarcosine to other functional groups, thereby allowing covalent attachment to dyes or tandem dyes. Figure 8 , Figure 9 and Figure 11 Polysarcosine, as a terminal group and linker, is shown in the labeled specific binding member.

[0216] Example 3: Experimental Properties of Dyes

[0217] PEGylated dyes were synthesized, each containing a polyethylene glycol (PEG) water-soluble group attached to the dye. Similarly, in a separate synthesis, the dye was functionalized with a sarcosine group. These dyes were also linked to an hCD4 antibody as a specific binding member, resulting in labeled specific binding members. These compounds were used as cell staining agents. Furthermore, side-scattering (SSC) experiments were performed to measure the optical properties of both dyes, such as… Figure 10 As shown, the sarcosine-substituted dyes were found to provide approximately 10% stronger signal.

[0218] Notwithstanding the appended claims, this disclosure is also defined by the following provisions:

[0219] 1. A tandem dye comprising:

[0220] Donor fluorophore;

[0221] Receptor fluorophores;

[0222] Water-soluble polystyrene groups.

[0223] 2. The tandem dye according to Clause 1, wherein the water-soluble polysarcosine group comprises a polymer having 2 to 40 residues.

[0224] 3. The tandem dye according to Clause 2, wherein the polymer is selected from polysarcosine polymers, N-modified polysarcosine polymers, and polysarcosine-type copolymers.

[0225] 4. The tandem dye according to Clause 3, wherein the polymer is a polysarcosine polymer.

[0226] 5. The tandem dye according to any one of the preceding clauses, wherein at least one of the donor fluorophore and the acceptor fluorophore comprises an organic dye.

[0227] 6. The tandem dye according to any one of the preceding clauses, wherein both the donor fluorophore and the acceptor fluorophore comprise organic dyes.

[0228] 7. The tandem dye according to Clause 5, wherein the organic dye is selected from cyanide dyes, rhodamine dyes, xanthan dyes, coumarin dyes, polymethyst, pyrene, dipyrrole methylene borodifluoride, naphthalimide, thiazide dyes and acridine dyes.

[0229] 8. The tandem dye according to any one of clauses 1 to 7, wherein at least one of the donor fluorophore and the acceptor fluorophore comprises a water-soluble polysarcosine side group.

[0230] 9. The tandem dye according to Clause 8, wherein the donor fluorophore comprises a water-soluble polysarcosine group.

[0231] 10. The tandem dye according to Clause 8, wherein the receptor fluorophore comprises a water-soluble polysarcosine side group.

[0232] 11. The tandem dye according to Clause 8, wherein both the donor fluorophore and the acceptor fluorophore contain a water-soluble polysarcosine side group.

[0233] 12. The tandem dye according to any one of the preceding clauses further comprises a non-conjugated polymer backbone, the non-conjugated polymer backbone comprising non-conjugated repeating units, wherein the donor fluorophore and the acceptor fluorophore are connected to the non-conjugated polymer backbone.

[0234] 13. The tandem dye according to Clause 12, wherein the non-conjugated repeating unit comprises a plurality of amino acid residues.

[0235] 14. The tandem dye according to Clause 12 or 13, wherein the non-conjugated polymer backbone comprises a water-soluble polysarcosine group bonded thereto.

[0236] 15. A tandem dye according to any one of clauses 12 to 14, wherein at least one of the donor fluorophore and the acceptor fluorophore is linked to the non-conjugated polymer backbone via a water-soluble polysarcosine group.

[0237] 16. The tandem dye according to Clause 15, wherein the donor fluorophore is linked to the non-conjugated polymer backbone via a water-soluble polysarcosine group.

[0238] 17. The tandem dye according to Clause 15, wherein the acceptor fluorophore is linked to the non-conjugated polymer backbone via a water-soluble polysarcosine group.

[0239] 18. The tandem dye according to Clause 15, wherein both the donor fluorophore and the acceptor fluorophore are linked to the non-conjugated polymer backbone via water-soluble polysarcosine groups.

[0240] 19. The tandem dye according to any one of Clauses 12 to 18, wherein the non-conjugated polymer backbone comprises a water-soluble polysarcosine group.

[0241] 20. A tandem dye according to any one of clauses 1 to 3, wherein the donor chromophore comprises a conjugated polymer.

[0242] 21. The tandem dye according to Clause 20, wherein the conjugated polymer comprises a water-soluble polysarcosine side group.

[0243] 22. The tandem dye according to clause 20 or 21, wherein the conjugated polymer is linked to the receptor chromophore via a water-soluble polysarcosine group.

[0244] 23. The tandem dye according to any one of clauses 20 to 22, wherein the receptor fluorophore comprises a water-soluble polysarcosine side group.

[0245] 24. A labeled, specifically binding member comprising:

[0246] Tandem dyes according to any one of the foregoing clauses; and

[0247] Specific binding member.

[0248] 25. The labeled specific binding member as described in Clause 24, wherein the specific binding member is a biomolecule.

[0249] 26. The labeled specific binding member as described in Clause 24, wherein the biomolecule is selected from carbohydrates, lipids, nucleic acids, and proteins or fragments thereof.

[0250] 27. A labeled specific binding member according to any one of clauses 24 to 26, wherein the specific binding member is an antibody, an antibody fragment, or a binding derivative thereof.

[0251] 28. A labeled specific binding member according to any one of clauses 24 to 27, wherein the specific binding member is linked to the tandem dye via a water-soluble polysarcosine group.

[0252] 29. The labeled specific binding member according to Clause 28, wherein the tandem dye comprises a non-conjugated polymer backbone, and the specific binding member is linked to the non-conjugated backbone via the water-soluble polysarcosine group.

[0253] 30. A method comprising contacting a sample with a tandem dye according to any one of clauses 24 to 29.

[0254] 31. The method according to Clause 30, wherein the method comprises determining a target analyte in the sample that binds to the specific binding member.

[0255] 32. The method according to Clause 31, wherein the analyte binds to the cell.

[0256] 33. The method according to Clause 32, wherein the analyte is a cell surface marker of the cell.

[0257] 34. The method according to Clause 33, wherein the cell surface marker is selected from cell receptors and cell surface antigens.

[0258] 35. The method according to Clause 32, wherein the analyte is an intracellular target.

[0259] 36. The method according to Clause 35, wherein the method includes permeabilizing or lysing the cells.

[0260] 37. The method according to any one of clauses 30 to 36, wherein the method includes analyzing the sample by flow cytometry.

[0261] 38. A method for labeling a specific binding member, the method comprising:

[0262] The tandem dye according to any one of clauses 1 to 23 is bonded to the specific binding member to label the specific binding member.

[0263] 39. The method according to Clause 38, wherein the specific binding member is a biomolecule.

[0264] 40. The method according to Clause 39, wherein the biomolecule is selected from carbohydrates, lipids, nucleic acids, and proteins or fragments thereof.

[0265] 41. The method according to any one of clauses 38 to 40, wherein the specific binding member is an antibody, an antibody fragment, or a binding derivative thereof.

[0266] 42. A kit comprising: a tandem dye according to any one of clauses 1 to 23 or a labeled specific binding member according to any one of clauses 24 to 29.

[0267] In at least some of the previously described embodiments, one or more elements used in the embodiments may be used interchangeably in another embodiment, unless such substitution is technically impractical. Those skilled in the art will understand that various other omissions, additions, and modifications can be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to fall within the scope of the subject matter defined in the appended claims.

[0268] Those skilled in the art will understand that, in general, the terminology used herein, particularly in the appended claims (e.g., the body of the appended claims), is typically intended as “open-ended” terms (e.g., the term “comprising” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “at least having,” the term “including” should be interpreted as “comprising but not limited to,” etc.). Those skilled in the art will further understand that if a specific number of claims is intended to be introduced, such intent will be explicitly stated in the claims, and if no such statement is present, such intent does not exist. For example, to aid understanding, the appended claims below may contain the use of the introductory phrases “at least one” and “one or more” to introduce the claims. However, the use of such phrases should not be construed as implying that introducing a claim statement with the indefinite article "a" or "an" limits any specific embodiment containing such an introduced claim statement to an embodiment containing only one such statement, even if the same claim includes the introductory phrase "a or more than one" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and / or "an" should be interpreted as meaning "at least one" or "a or more than one"); the same applies to the use of definite articles used to introduce a claim statement. Furthermore, even if a specific number of introduced claim statements is explicitly stated, those skilled in the art will recognize that such statements should be interpreted as meaning at least the number of stated statements (e.g., simply stating "two statements" without other modifiers means at least two statements, or two or more statements). Furthermore, when using conventions such as "at least one of A, B, and C," this structure is generally intended to have the meaning of the convention as understood by a person skilled in the art (e.g., "a system having at least one of A, B, and C" will include, but is not limited to, systems having a single A, a single B, a single C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). When using conventions such as "at least one of A, B, or C," this structure is generally intended to have the meaning of the convention as understood by a person skilled in the art (e.g., "a system having at least one of A, B, or C" will include, but is not limited to, systems having a single A, a single B, a single C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). A person skilled in the art will further understand that any extractive terms and / or phrases presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to include the possibility of including one term, any term, or both terms. For example, the phrase "A or B" should be understood to include the possibility of including "A" or "B" or "A and B."

[0269] Furthermore, when features or aspects of this disclosure are described in accordance with the Markush Group, those skilled in the art will recognize that this disclosure is therefore also described in accordance with any individual member or subgroup of the Markush Group.

[0270] As those skilled in the art will understand, for any and all purposes, such as providing a written description, all scopes disclosed herein also encompass any and all possible subscopes and combinations thereof. Any listed scope can be readily considered to be sufficiently descriptive and inspiring of decomposing the same scope into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each scope discussed herein can be readily decomposed into lower thirds, middle thirds, and upper thirds, etc. As those skilled in the art will also understand, all language such as “highest,” “at least,” “greater than,” “less than,” etc., includes the numbers stated and refers to a scope that can subsequently be decomposed into subscopes as described above. Finally, as those skilled in the art will understand, a scope includes each individual member. Thus, for example, a group having 1 to 3 items means a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items means a group having 1, 2, 3, 4, or 5 items, and so on.

[0271] Although the foregoing invention has been described in detail by way of illustration and example for the purpose of clarity, it will be readily apparent to those skilled in the art, based on the teachings of the invention, that certain changes and modifications may be made therein without departing from the spirit or scope of the appended claims.

[0272] Therefore, the foregoing merely illustrates the principles of the invention. It should be understood that those skilled in the art will be able to devise various arrangements that, while not explicitly described or shown herein, embody the principles of the invention and are contained within its spirit and scope. Furthermore, all examples and conditional language cited herein are primarily intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to advance the field, and should be interpreted as not being limited to these specifically cited examples and conditions. Moreover, all statements herein recounting the principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to cover their structural and functional equivalents. Additionally, it is intended that such equivalents include both currently known equivalents and those developed in the future, i.e., any element developed to perform the same function, regardless of its structure. Furthermore, nothing disclosed herein is intended for public use only, whether such disclosure is expressly stated in the claims.

[0273] Therefore, the scope of the invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied by the appended claims. In the claims, 35 USC §112(f) or 35 USC §112(6) is explicitly defined as referring to only when the exact phrase “means for…” or the exact phrase “steps for…” is recited at the beginning of the limitation in the claim; if such an exact phrase is not used in the limitation in the claim, then 35 USC §112(f) or 35 USC §112(6) is not referred to.

Claims

1. A tandem dye comprising: Donor fluorophore; Receptor fluorophores; Water-soluble polystyrene groups.

2. The tandem dye of claim 1, wherein the water-soluble polysarcosine group comprises a polymer having 2 to 40 residues.

3. The tandem dye according to claim 2, wherein the polymer is selected from polysarcosine polymers, N-modified polysarcosine polymers, and polysarcosine-type copolymers.

4. The tandem dye of claim 3, wherein the polymer is a polysarcosine polymer.

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

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

7. The tandem dye according to any one of the preceding claims, wherein at least one of the donor fluorophore and the acceptor fluorophore comprises a water-soluble polysarcosine side group.

8. The tandem dye according to any one of the preceding claims further comprises a non-conjugated polymer backbone, the non-conjugated polymer backbone comprising non-conjugated repeating units, wherein the donor fluorophore and the acceptor fluorophore are connected to the non-conjugated polymer backbone.

9. The tandem dye of claim 8, wherein the non-conjugated repeating unit comprises a plurality of amino acid residues.

10. The tandem dye according to claim 8 or 9, wherein the non-conjugated polymer backbone comprises a water-soluble polysarcosine group bonded thereto.

11. The tandem dye according to any one of claims 8 to 10, wherein at least one of the donor fluorophore and the acceptor fluorophore is linked to the non-conjugated polymer backbone via a water-soluble polysarcosine group.

12. The tandem dye according to any one of claims 8 to 11, wherein the non-conjugated polymer backbone comprises water-soluble polysarcosine groups.

13. A labeled, specifically binding member comprising: Tandem dyes according to any one of the preceding claims; and Specific binding member.

14. A method comprising contacting a sample with a labeled specific binding member as claimed in claim 13.

15. A reagent kit comprising: The tandem dye according to any one of claims 1 to 12 or the labeled specific binding member according to claim 13.

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