Green excitable tandem dyes and methods of making and using same

Abstract

Green excitable tandem dyes are provided. A tandem dye of an embodiment of the present invention comprises: a non-conjugated polymer backbone; one or more green excitable donor fluorophores attached to the non-conjugated polymer backbone; and one or more acceptor fluorophores attached to the non-conjugated polymer backbone; wherein the donor fluorophore and the acceptor fluorophore are in an energy transfer relationship. Also provided are methods of making and using the tandem dyes, as well as kits that include the dyes and can be used in the method embodiments.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 USC §119(e) to the filing date of U.S. Provisional Patent Application Serial No. 63 / 347,854, filed on June 1, 2022, the disclosure of which is incorporated herein by reference in its entirety. Background Art

[0003] Fluorescent dye is such a compound, when irradiated with the light of the wavelength absorbed by it, it emits (usually) light of different wavelengths. Fluorescent dye has multiple applications in biochemistry, biology and medicine, for example, for diagnostic kits, microscopy or drug screening. Fluorescent dye is characterized by many parameters, allowing the user to select suitable dye according to the desired purpose. The parameters of interest include excitation wavelength maximum, emission wavelength maximum, Stokes shift, extinction coefficient, fluorescence quantum yield and fluorescence lifetime. Dye can be selected according to the application of interest, so as to allow, for example, excitation radiation to penetrate into the biological sample, thereby minimizing background fluorescence and / or realizing high signal-to-noise ratio.

[0004] Molecular recognition involves the specific binding of two molecules. Molecules with binding specificity for a target biomolecule can be used in a variety of research and diagnostic applications, such as labeling and separation of analytes, flow cytometry, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation, and chromatography. Target biomolecules can be detected by labeling with fluorescent dyes.

[0005] Some applications (e.g., flow cytometry applications) use a combination or panel of different fluorescently labeled specific binding members (e.g., antibodies) to detect multiple different targets, such as internal or cell surface markers. In such applications, a variety of different fluorescent dyes are used for the same sample, wherein the multiple different fluorescent dyes are distinguishable from each other in terms of excitation and / or emission maxima. A class of fluorescent dyes useful in such applications is tandem fluorescent dyes. Tandem fluorescent dyes are compounds with two covalently linked different fluorophores that can be covalently linked to each other directly or through a linking group. One of the fluorophores is used as a donor fluorophore and the other is used as an acceptor fluorophore. The donor fluorophore and the acceptor fluorophore together form a fluorescence resonance energy transfer (FRET) pair. Such FRET pairs are characterized by a unique dye having the excitation characteristics of a donor fluorophore and the emission characteristics of an acceptor fluorophore. Summary of the invention

[0006] The inventors have recognized a need for tandem fluorescent dyes in which the donor fluorophore is excited in the green wavelength range, for example, so as to be able to be excited by a commonly used green laser. Embodiments of the present invention meet this need.

[0007] Green excitable tandem dyes are provided. The tandem dyes of embodiments of the present invention include: a non-conjugated polymer backbone; one or more green excitable donor fluorophores connected to the non-conjugated polymer backbone; and one or more acceptor fluorophores connected to the non-conjugated polymer backbone; wherein the donor fluorophore and the acceptor fluorophore are in an energy transfer relationship. Also provided are methods of preparing and using the tandem dyes, and kits that include the dyes and can be used in embodiments of the methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The present invention can be better understood by reading the following detailed description in conjunction with the accompanying drawings, which include the following figures:

[0009] Figure 1 Shown are UV-visible absorption spectra of exemplary polymer tandem dyes according to embodiments of the present invention.

[0010] Figure 2 Shown are normalized emission spectra of exemplary polymeric tandem dyes according to embodiments of the present invention.

[0011] Figure 3 Shown is an overlay of the absorption and emission spectra of an exemplary BODIPY-type donor polymer tandem dye according to an embodiment of the present invention.

[0012] Figure 4A and 4B Shown is an overlay of the absorption and emission spectra of an exemplary polymeric BODIPY-type donor tandem dye according to an embodiment of the present invention.

[0013] Figure 5A and 5B Shown is an overlay of the absorption and emission spectra of an exemplary polymeric rhodamine-type donor tandem dye according to an embodiment of the present invention.

[0014] Figures 6A to 6G Shown is an overlay of the absorption and emission spectra of an exemplary polymer tandem dye according to an embodiment of the present invention. DETAILED DESCRIPTION

[0015] definition

[0016] As used herein, the terms "chemoselective functional group" and "chemoselective tag" are used interchangeably and refer to a functional group that, in certain cases, can selectively react with another compatible functional group to form a covalent bond after optionally activating one of the functional groups. Chemoselective functional groups of interest include, but are not limited to, thiols and maleimides or iodoacetamides, amines and carboxylic acids or their active esters, and groups that can react with each other via click chemistry, such as azides and alkynyls (e.g., cyclooctynyl), tetrazines, trans-cyclooctenes, dienes and dienophiles, as well as azides, sulfur (VI) fluoride exchange chemistry (SuFEX), sulfonyl fluorides, and hydroxyls, hydrazidos, hydrazines, aldehydes, ketones, azidos, alkynes, phosphines, epoxides, and the like.

[0017] As used herein, the term "sample" refers to a material or mixture of materials containing one or more analytes of interest, in some cases in liquid form. In some embodiments, the term, when used in its broadest sense, refers to any plant, animal, or bacterial material containing cells or producing cell metabolites, such as tissues or fluids separated from individuals (including but not limited to plasma, serum, cerebrospinal fluid, lymph, tears, saliva, and tissue sections) or tissues or fluids separated from in vitro cell culture components, as well as samples from the environment. The term "sample" may also refer to a "biological sample". As used herein, the term "biological sample" refers to a whole organism or a subset of its tissues, cells, or components (e.g., body fluids, including but not limited to blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic blood, urine, vaginal fluid, and semen). "Biological sample" can also refer to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or components or its fractions or parts, including but not limited to plasma, serum, spinal fluid, lymph fluid, external parts of the skin, respiratory tract, intestinal and urogenital tracts, tears, saliva, milk, blood cells, tumors and organs. In certain embodiments, the sample has been taken out from an animal or plant. The biological sample may include cells. The term "cell" is used in its conventional sense to refer to the basic structural unit of a living organism (both eukaryotic and prokaryotic organisms), having at least a nucleus and a cell membrane. In certain embodiments, cells include prokaryotic cells, such as prokaryotic cells from bacteria. In other embodiments, cells include eukaryotic cells, such as cells obtained from biological samples from animals, plants or fungi.

[0018] The terms "support binding" and "connected to a support" are used interchangeably and refer to a structural moiety (e.g., a specific binding member) that is covalently or non-covalently connected to a support of interest. Covalent bonding can involve a chemical reaction of two compatible functional groups (e.g., two chemoselective functional groups, an electrophilic group, and a nucleophilic group, etc.) to form a covalent bond between two structural moieties of interest (e.g., a support and a specific binding member). In some cases, non-covalent bonding can involve specific binding between two structural moieties of interest (e.g., two affinity structural moieties, such as a hapten and an antibody or a biotin structural moiety and a streptavidin, etc.). In some cases, non-covalent bonding can involve the absorption of a substrate.

[0019] The term "polypeptide" refers to a polymeric form of amino acids of any length, including peptides with a length range of 2-50 amino acids and polypeptides with a length greater than 50 amino acids. The terms "polypeptide" and "protein" are used interchangeably herein. The term "polypeptide" includes chemically or biochemically modified or derived amino acids, polypeptides with modified peptide backbones (wherein the conventional backbone has been replaced by a non-naturally occurring or synthetic backbone), polymers of coded amino acids and non-coded amino acids. Polypeptides can have any convenient length, for example, 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, 50 or more amino acids, 100 or more amino acids, 300 or more amino acids, such as up to 500 or 1000 or more amino acids." Peptide" can be 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, such as up to 50 amino acids. In some embodiments, the length of the peptide is 5 to 30 amino acids.

[0020] As used herein, the term "isolated" refers to a moiety of interest that is at least 60%, at least 75%, at least 90%, at least 95%, at least 98%, or even at least 99% free from other components with which it was associated prior to purification.

[0021] A "plurality" contains at least 2 members. In some cases, a plurality can have 5 or more, e.g., 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 300 or more, 1000 or more, 3000 or more, 10,000 or more, 100,000 or more members.

[0022] Numeric ranges are inclusive of the numbers defining the range.

[0023] The term "specific binding" refers to a direct association between two molecules, for example due to covalent, electrostatic, hydrophobic and ionic and / or hydrogen bond interactions, including interactions such as salt bridges and water bridges. Specific binding members describe members of a pair of molecules that have binding specificity to each other. The members of a specific binding pair can be naturally derived or produced in whole or in part synthetically. One member of the molecular pair has an area, or cavity, on its surface that specifically binds to and is therefore complementary to the specific spatial and polar organization of the other member of the molecular pair. Therefore, the members of the pair have the property of specifically binding to each other. Examples of specific binding member pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. The specific binding members of the binding pair exhibit high affinity and binding specificity for binding to each other. Typically, the affinity between a pair of specific binding members is characterized by K d (dissociation constant) is 10 -6 M or lower, e.g. 10 -7 M or lower, including 10 -8 M or lower, e.g. 10 -9 M or lower, 10 -10 M or lower, 10 -11 M or lower, 10 -12 M or lower, 10 -13 M or lower, 10 -14 M or lower, including 10 -15 M or lower. "Affinity" refers to the strength of binding, and increased binding affinity is associated with a lower KD. In one embodiment, affinity is determined by surface plasmon resonance (SPR), for example, as used by a Biacore system. The affinity of one molecule for another molecule is determined by measuring the binding kinetics of the interaction, for example, at 25°C. "Affinity" refers to the strength of binding, and increased binding affinity is associated with a lower KD. In one embodiment, affinity is determined by surface plasmon resonance (SPR), for example, as used by a Biacore system. The affinity of one molecule for another molecule is determined by measuring the binding kinetics of the interaction, for example, at 25°C.

[0024] Specific binding members can be proteinaceous. As used herein, the term "proteinaceous" refers to a structural portion consisting of amino acid residues. A proteinaceous structural portion can be a polypeptide. In some cases, a proteinaceous specific binding member is an antibody. In certain embodiments, a proteinaceous specific binding member is an antibody fragment, such as an antibody binding fragment that specifically binds to a polymer dye. As used herein, the terms "antibody" and "antibody molecule" are used interchangeably and refer to a protein consisting of one or more polypeptides that are substantially encoded by all or part of recognized immunoglobulin genes. Recognized immunoglobulin genes (e.g., in humans) include κ (k), λ (l) and heavy chain loci, which together constitute countless 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. An immunoglobulin light chain or heavy chain variable region is composed of a "framework" region (FR) interrupted by three hypervariable regions (also referred to as "complementarity determining regions" or "CDRs"). The scope of the framework 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 numbering of all antibody amino acid sequences discussed herein conforms to the Kabat system. The sequences of the framework regions of different light chains or heavy chains are relatively conserved within a species. The framework region of an antibody, that is, the combined framework region of the light chain and the heavy chain, is used to position and arrange the CDR. The CDR is primarily responsible for binding to the antigenic epitope. The term "antibody" is intended to include full-length antibodies and may refer to natural antibodies, engineered antibodies, or recombinantly produced antibodies from any organism for experimental, therapeutic or other purposes, as further defined below.

[0025] Antibody fragments of interest include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv or other antigen-binding subsequences of antibodies, which are either produced by modifying the entire antibody or synthesized de novo using recombinant DNA technology. Antibodies can be monoclonal or polyclonal and can have other specific activities on cells (e.g., antagonistic antibodies, agonistic antibodies, neutralizing antibodies, inhibitory antibodies or stimulating antibodies). It is understood that antibodies can have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other antibody functions.

[0026] In certain embodiments, the specific binding member is a Fab fragment, a F(ab')2 fragment, a scFv, a diabody or a triabody. In certain embodiments, the specific binding member is an antibody. In some cases, the specific binding member is a murine antibody or a binding fragment thereof. In some cases, the specific binding member is a recombinant antibody or a binding fragment thereof.

[0027] Method described herein may include a plurality of steps. As required, each step may be performed after a predetermined amount of time has passed between steps. Therefore, the time for performing each step may be 1 second or longer, 10 seconds or longer, 30 seconds or longer, 60 seconds or longer, 5 minutes or longer, 10 minutes or longer, 60 minutes or longer and may include 5 hours or longer. In certain embodiments, each subsequent step may be performed immediately after the previous step is completed. In other embodiments, a certain step may be performed after the incubation or waiting time after the previous step is completed, and the incubation or waiting time may be, for example, a waiting time of several minutes to overnight.

[0028] As used herein, the terms "assess," "determine," "measure," "evaluate," and "measure" are used interchangeably and include both quantitative and qualitative determinations.

[0029] As used herein, the term "separation" refers to the physical separation of two elements (eg, by size or affinity, etc.) as well as the degradation of one element while leaving the other element intact.

[0030] The term "linker" or "connector" refers to a connecting structure portion that connects two groups and has a skeleton of 100 atoms or less atoms in length. A linker or connector can be a covalent bond or a chain of 1 to 100 atoms connecting two groups, for example, a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more carbon atoms in length, wherein the linker can be linear, branched, cyclic or monatomic. In some cases, the linker is a branched linker, which refers to a connecting structure portion that connects three or more groups. In some cases, one, two, three, four or five or more carbon atoms of the linker skeleton can be optionally substituted by sulfur, nitrogen or oxygen heteroatoms. In some cases, the linker skeleton includes a connecting functional group, such as ether, thioether, amino, amide, sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester or imines. The bonds between the backbone atoms may be saturated or unsaturated, and in some cases, no more than one, two or three unsaturated bonds are present in the linker backbone. The linker may include one or more substituents, such as alkyl, aryl or alkenyl groups. The linker may include, but is not limited to, polyethylene glycol; ethers, thioethers, tertiary amines, alkyls, which may 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 backbone may include cyclic groups, such as aryl, heterocyclic or cycloalkyl, wherein 2 or more atoms of the cyclic group, such as 2, 3 or 4 atoms are included in the backbone. The linker may be cleavable or non-cleavable.

[0031] 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 solvated in an aqueous environment, e.g., under physiological conditions, and confers improved water solubility to the molecule to which it is attached. WSG can increase the solubility of a tandem dye or a component thereof (e.g., a donor or acceptor fluorophore) in a predominantly aqueous solution compared to a control tandem dye or a component thereof lacking WSG. The water-soluble group can be any convenient hydrophilic group that is well solvated in an aqueous environment.

[0032] Various water-soluble polymer groups can be applicable to the WSG of theme dyestuff.Any convenient water-soluble group (WSG) can be included in dyestuff described herein, to provide increased water solubility.Although the increase of solubility may be different, in some cases, the increase (compared with the compound without WSG) is 2 times or more, for example 5 times, 10 times, 25 times, 50 times, 100 times or more.In some cases, hydrophilic water-soluble group is charged, for example, positively charged or negatively charged.In some cases, hydrophilic water-soluble group is neutral hydrophilic group.In some embodiments, WSG is branched (for example, as described herein).In some cases, WSG is linear.In some embodiments, WSG is hydrophilic polymer, for example, polyethylene glycol, modified PEG, peptide sequence, peptoid, carbohydrate, oxazoline, polyol, dendritic polymer, dendritic polyglycerol, cellulose, chitosan or its derivatives. Water-soluble groups of interest include, but are not limited to, carboxylates, phosphonates, phosphates, sulfonates, sulfates, sulfinates, sulfoniums, esters, polyethylene glycols (PEGs) and modified PEGs, hydroxyls, amines, amino acids, ammonium, guanidino, pyridinium, polyamines and sulfoniums, polyols, linear or cyclic sugars, primary, secondary, tertiary or quaternary amines and polyamines, phosphonate groups, phosphinate groups, ascorbate groups, glycols including polyethers, -COOM', -SO3M', -PO3M', -NR3 + 、Y′、(CH2CH2O) p R and mixtures thereof, wherein Y' can be any halogen, sulfate, sulfonate or oxyanion, p can be 1 to 500, each R can independently be 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--, glycol and polyethylene glycol, wherein yy is selected from 1 to 1000, and X is selected from O, S and NR ZZ , R ZZ and R YY Independently selected from H and C 1-3 In some cases, WSG is (CH2) x (OCH2CH2) y OCH3, wherein each x is independently an integer from 0 to 20, and each y is independently an integer from 0 to 50. In some cases, the water-soluble group includes a nonionic polymer (eg, a PEG polymer) substituted at the terminal end with an ionic group (eg, a sulfonate group).

[0033] In some embodiments of the above formula, the pendant groups of interest include those selected from (CH2)x (OCH2CH2) y OCH3 substituents, wherein each x is independently an integer from 0 to 20, each y is independently an integer from 0 to 50; the benzyl group is optionally substituted by one or more halogen, hydroxyl, C1-C 12 Alkoxy or (OCH2CH2) z OCH3 substitution, wherein each z is independently an integer from 0 to 50. In some cases, the substituent is (CH2)3(OCH2CH2) 11 In some embodiments, one or more substituents are benzyl substituted with at least one WSG group (e.g., one or two WSG groups) selected from (CH2) x (OCH2CH2) y OCH3, wherein each x is independently an integer from 0 to 20, and each y is independently an integer from 0 to 50.

[0034] Multiple WSG can be included in the single position of the theme dye by a branched linking group. In certain embodiments, the branched linking group is an aralkyl substituent further substituted by a water-soluble group. Therefore, in some cases, the branched linking group group is a substituent of dye, which connects the dye to two or more water-soluble groups. In certain embodiments, the branched linking group is an amino acid, for example, a lysine amino acid connected to three groups by an amino group and a carboxylic acid group. In some cases, multiple WSGs are mixed by a branched linking group to give the desired solubility of the dye. In some cases, WSG is a water-soluble nonionic side chain group that can give more than 50mg / mL. In some cases, WSG is a water-soluble nonionic side chain group that can give more than 100mg / mL. In some embodiments, dye includes a substituent selected from alkyl, aralkyl and heterocyclic groups, and each group is further replaced by a hydrophilic polymer group including a water-soluble group, such as polyethylene glycol (PEG) (for example, a PEG group with 6-24 units).

[0035] Interesting water-soluble polymers that can be used in WSG include polyethylene glycol (PEG) groups or modified PEG groups. Interesting water-soluble polymers include, but are not limited to, polyalkylene oxide-based polymers, such as polyethylene glycol "PEG" (see, for example, "Poly (ethylene glycol) Chemistry: Biotechnical and Biomedical Applications", JM Harris, Ed., Plenum Press, New York, NY (1992); and "Poly (ethylene glycol) Chemistry and Biological Applications", JM Harris and S. Zalipsky, Eds., ACS (1997); and International Patent Applications: WO 90 / 13540, WO 92 / 00748, WO 92 / 16555, WO 94 / 04193, WO94 / 14758, WO 94 / 17039, WO 94 / 18247, WO 94 / 28937, WO 95 / 11924, WO 96 / 00080, WO 96 / 23794, WO 98 / 07713, WO 98 / 41562, WO 98 / 48837, WO 99 / 30727, WO 99 / 32134, WO 99 / 33483, WO 99 / 53951, WO 01 / 26692, WO 95 / 13312, WO 96 / 21469, WO 97 / 03106, WO 99 / 45964 and U.S. Patent Nos. 4,179,337; 5,075,046; 5,089,261; 5,100,992; 5,134,192; 5,166,309; 5,171,264; 5,213,891; 5,219,564; 5,275,838; 5,281,698; 5,298,643; 5,312,808; 5,321,095; 5,324,844; 5,349,001; 5,352,756; 5,405,877; 5,455,027; 5,446,090; 5,470,829; 5,478,805; 5,567,422; 5,605,976; 5,612,460; 5,614,549; 5,618,528; 5,672,662; 5,637,749; 5,643,575; 5,650,388; 5,681,567; 5,686,110; 5,730,990; 5,739,208; 5,756,593; 5,808,096; 5,824,778; 5,824,784; 5,840,900; 5,874,500; 5,880,131; 5,900,461; 5,902,588; 5,919,442; 5,919,455; 5,932,462; 5,965,119; 5,965,566; 5,985,263; 5,990,237; 6,011,042; 6,013,283; 6,077,939; 6,113,906; 6,127,355; 6,177,087; 6,180,095; 6,194,580; 6,214,966).

[0036] Examples of water-soluble polymers of interest include, but are not limited to, those containing polyalkylene oxides, polyamide alkylene oxides, or derivatives thereof, including polyalkylene oxides and polyamide alkylene oxides containing ethylene oxide repeating units of the formula -(CH2-CH2-O)-. Other examples of polymers of interest include those having the formula -[C(O)-XC(O)-NH-Y-NH] n -or-[NH-Y-NH-C(O)-XC(O)] n -a polyamide having a molecular weight greater than 1,000 Daltons, wherein X and Y are divalent groups which may be the same or different and which may be branched or linear, and n is a discrete integer from 2 to 100, such as from 2 to 50, wherein one or both of X and Y comprise a biocompatible, substantially non-antigenic, water-soluble repeating unit which may be linear or branched. Other examples of water-soluble repeating units include ethylene oxide of the formula -(CH2-CH2-O)- or -(O-CH2-CH2)-. The number of such water-soluble repeating units may vary significantly, with the number of such units being from 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, 6-100, such as from 2 to 50 or 6 to 50. An example of one embodiment is an example in which one or both of X and Y are selected from: -((CH2) n1 -(CH2-CH2-O) n2 -(CH2)- or -((CH2) n1 -(O-CH2-CH2) n2 -(CH2) n-1 -), wherein n1 is 1 to 6, 1 to 5, 1 to 4 or 1 to 3, wherein n2 is 2 to 50, 2 to 25, 2 to 15, 2 to 10, 2 to 8 or 2 to 5. A further example of one embodiment is that wherein X is -(CH2-CH2)-, wherein Y is -(CH2-(CH2-CH2-O)3-CH2-CH2-CH2)- or -(CH2-CH2-CH2-(O-CH2-CH2)3-CH2)-.

[0037] The term "modified polymer", such as modified PEG, refers to a water-soluble polymer that is modified or derivatized at either or both ends to, for example, include a terminal substituent (e.g., a terminal alkyl, substituted alkyl, alkoxy or substituted alkoxy, etc.) and / or a terminal connecting functional group (e.g., an amino or carboxylic acid group suitable for attachment via amide bond formation) suitable for connecting the polymer to a molecule of interest (e.g., attached to a light-collecting chromophore via a branching group). The subject water-soluble polymers can be modified to include any convenient linking group. It will be appreciated that in some cases, the water-soluble polymers may include some dispersity with respect to polymer length, depending on the preparation and / or purification methods of the polymer starting material. In some cases, the water-soluble polymers are monodisperse.

[0038] The water-soluble polymer may include one or more spacers or linkers. Examples of spacers or linkers include linear or branched moieties including 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 moieties including alkyl, aryl, heteroalkyl, heteroaryl, alkoxy, etc., which may include, for example, up to 18 carbon atoms or even additional polymer chains.

[0039] The water-soluble polymer structure part or one or more spacers or linkers of the polymer structure part (if present) can include a biostable or biodegradable polymer chain or unit. For example, polymers with repeating linkers have different degrees of stability under physiological conditions, depending on the instability of the bond. Polymers with such bonds can be classified according to their relative hydrolysis rates based on the known hydrolysis rates of low molecular weight analogs under physiological conditions, for example, from less stable to more stable, for example, polyurethane (-NH-C (O)-O)> polyorthoester (-OC ((OR) (R'))-O-)> polyamide (-C (O)-NH). Similarly, the connection system attaching the water-soluble polymer to the target molecule can be biostable or biodegradable, for example, from less stable to more stable: carbonate (-OC (O)-O)> ester (-C (O)-O)> carbamate (-NH-C (O)-O-)> orthoester (-OC ((OR) (R'))-O-)> amide (-C (O)-NH-). In general, sulfated polysaccharides may need to be avoided due to the instability of the sulfate groups. In addition, the use of polycarbonates and polyesters may be less desirable. These bonds are provided as examples only and are not intended to limit the types of bonds that may be employed in the polymer chains or linking systems of the water-soluble polymers that may be used in the WSG disclosed herein.

[0040] Water-soluble group (WSG) can give theme dye or polymer tandem dye the water solubility that surpasses 10mg / mL, for example surpasses 20mg / mL, surpasses 30mg / mL, surpasses 40mg / mL, surpasses 50mg / mL, surpasses 60mg / mL, surpasses 70mg / mL, surpasses 80mg / mL, surpasses 90mg / mL or surpasses 100mg / mL.In some cases, branched nonionic water-soluble group (WSG) can give theme dye or polymer tandem dye (for example, aqueous buffer) 20mg / mL or larger water solubility, for example 30mg / mL or larger, 40mg / mL or larger, 50mg / mL or larger, 60mg / mL or larger, 70mg / mL or larger, 80mg / mL or larger, 90mg / mL or larger, 100mg / mL or larger, or even larger.It is appreciated that the water-soluble dye based on dipyrromethene forms discrete water solvation nano particle in aqueous system under certain conditions. In some cases, water-solvated nanoparticles are able to resist aggregation and can be used in a variety of biological assays.

[0041] The terms "polyethylene oxide", "PEO", "polyethylene glycol" and "PEG" are used interchangeably and refer to a polyethylene glycol having the formula --(CH2----O--) n -described chain polymer group or its derivative. 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 have any convenient length and can include a variety of terminal groups and / or additional substituents, including but not limited to alkyl, aryl, hydroxy, amino, acyl, acyloxy and amido terminal groups and / or substituents. PEG groups that may be suitable for use in the subject multichromophores include those described by S. Zalipsky in “Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates”, Bioconjugate Chemistry 1995, 6(2), 150-165 and by Zhu et al. in “Water-Soluble Conjugated Polymers for Imaging, Diagnosis, and Therapy”, Chem. Rev., 2012, 112(8), pp 4687-4735.

[0042] The term "alkyl" by itself or as part of another substituent refers to a saturated branched or straight-chain monovalent hydrocarbon group derived by removing a hydrogen atom from a single carbon atom of a parent alkane. Interested alkyl groups include, but are not limited to, methyl; ethyl, propyl such as propane-1-yl or propane-2-yl; and butyl such as butane-1-yl, butane-2-yl, 2-methyl-propane-1-yl or 2-methyl-propane-2-yl. In some embodiments, the alkyl group comprises 1 to 20 carbon atoms. In some embodiments, the alkyl group comprises 1 to 10 carbon atoms. In certain embodiments, the low alkyl group comprises 1 to 6 carbon atoms, such as 1 to 4 carbon atoms. For example, the term includes linear and branched hydrocarbon groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), tert-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0043] The term "substituted alkyl" refers to an alkyl group, as defined herein, substituted with from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxaaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NR a R b , wherein R' and R" may be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocycle.

[0044] "Alkoxy" refers to the group -O-alkyl, where alkyl is as defined herein. For example, alkoxy includes methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, and the like. The term "alkoxy" also refers to the groups alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0045] The term "substituted alkoxy" refers to substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- groups, wherein substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0046] "Alkenyl" refers to a monovalent, branched or linear, cyclic or acyclic hydrocarbon radical comprising a carbon-carbon double bond. Exemplary alkenyl groups include vinyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosene and tetracosene. In some cases, the alkenyl group comprises 1 to 24 carbon atoms, such as 1 to 18 carbon atoms or 1 to 12 carbon atoms. The term "lower alkenyl" refers to an alkyl group with 1 to 6 carbon atoms.

[0047] "Alkynyl" or "alkyne" refers to a straight chain or branched monovalent hydrocarbon radical having 2 to 6 carbon atoms, preferably 2 to 3 carbon atoms, and having at least 1 and preferably 1 to 2 sites of triple bond unsaturation. Examples of such alkynyl groups include ethynyl (-C≡CH) and propargyl (-CH2C≡CH).

[0048] The term "substituted alkynyl" or "substituted alkyne" refers to an alkynyl group as defined herein having from 1 to 5 substituents, or from 1 to 3 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxaaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0049] "Heterocyclyl" refers to a monoradical cyclic group that contains heteroatoms (e.g., O, S, N) as ring atoms and is not aromatic (ie, to distinguish heterocyclyl from heteroaryl). Exemplary heterocyclyl groups include piperidinyl, tetrahydrofuranyl, dihydrofuranyl, and thiocyanato.

[0050] "Amino" refers to the group -NH2. The term "substituted amino" refers to the group -NRR, wherein each R is independently selected from hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl, provided that at least one R is not hydrogen.

[0051] "Aryl" by itself or as part of another substituent refers to a monovalent aromatic hydrocarbon radical derived by removing one hydrogen atom from a single carbon atom of an aromatic ring system. Aryl groups of interest include, but are not limited to, those derived from aceanthrene, acenaphthene, acephenanthrylene, anthracene, azulene, benzene, In some embodiments, the aryl group comprises 6 to 20 carbon atoms. In some embodiments, the aryl group comprises 6 to 12 carbon atoms. Examples of aryl groups are phenyl and naphthyl.

[0052] "Substituted aryl" refers to an aryl group substituted with from 1 to 5 substituents or from 1 to 3 substituents (unless the definition of aryl substituents is otherwise limited) selected from the group consisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxyalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl.

[0053] "Heteroaryl" by itself or as part of another substituent refers to a monovalent heteroaryl radical derived by the removal of one hydrogen atom from a single atom of a heteroaromatic ring system. Heteroaryl groups of interest include, but are not limited to, those derived from acridine, arsindene, carbazole, β-carboline, chroman, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, In some embodiments, the heteroaryl group is derived from a 5-20 yuan heteroaryl. In some embodiments, the heteroaryl group is derived from a 5-10 yuan heteroaryl. In some embodiments, the heteroaryl group is derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole and pyrazine.

[0054] "Heterocycle", "heterocyclic", "heterocycloalkyl" and "heterocyclyl" refer to a saturated or unsaturated group having a single ring or multiple fused rings (including fused bridged rings and spiro ring systems) and having 3 to 20 ring atoms (including 1 to 10 heteroatoms). The ring atoms are selected from nitrogen, sulfur or oxygen, wherein, in a fused ring system, one or more rings can be cycloalkyl, aryl or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atoms of the heterocyclic group are optionally oxidized to provide an N-oxide, -S(O)- or -SO2- structural moiety.

[0055] Examples of heterocyclic and heteroaryl groups include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indoline, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenanthroline, Thiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also known as thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

[0056] "Substituted heteroaryl" refers to a heteroaryl group substituted with 1 to 5 substituents or 1 to 3 substituents (unless the definition of substituents is otherwise limited) selected from the group consisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, amido, alkaryl, aryl, aryloxy, azido, carboxyl, carboxyalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyamido, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl and trihalomethyl.

[0057] The term "alkaryl" or "aralkyl" refers to the groups -alkylene-aryl and substituted alkylene-aryl wherein alkylene, substituted alkylene and aryl are as defined herein.

[0058] "Alkylene" refers to a divalent aliphatic hydrocarbon group preferably having 1 to 6 and more preferably 1 to 3 carbon atoms, which is straight chain or branched and optionally substituted with one or more selected from -O-, -NR- 10 -、-NR 10 C(O)-、-C(O)NR 10 -, etc. By way of example, the term includes methylene (-CH2-), ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), isopropylene (-CH2CH(CH3)-), (-C(CH3)2CH2CH2-), (-C(CH3)2CH2C(O)-), (-C(CH3)2CH2C(O)NH-), (-CH(CH3)CH2-), and the like. "Substituted alkylene" refers to an alkylene group having from 1 to 3 hydrogens replaced by substituents as described for the carbon in the definition of "substituted" below.

[0059] "Substituted" refers to a group in which one or more hydrogen atoms are independently replaced by the same or different substituents. Substituents of interest include, but are not limited to, alkylenedioxy (such as methylenedioxy), -M, -R 60 , -O - ,=O,-OR 60 、-SR 60 , -S - , =S, -NR 60 R 61 , =NR 60, -CF3, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -S(O)2O - 、-S(O)2OH、-S(O)2R 60 、-OS(O)2O - 、-OS(O)2R 60 、-P(O)(O - )2、-P(O)(OR 60 )(O - )、-OP(O)(OR 60 )(OR 61 )、-C(O)R 60 、-C(S)R 60 、-C(O)OR 60 、-C(O)NR 60 R 61 ,-C(O)O - 、-C(S)OR 60 、-NR 62 C(O)NR 60 R 61 、-NR 62 C(S)NR 60 R 61 、-NR 62 C(NR 63 )NR 60 R 61 and -C(NR 62 )NR 60 R 61 , wherein M is halogen; R 60 , R 61 , R 62 and R 63 are independently hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, or optionally, R 60 and R 61 Together with the nitrogen atom to which they are bound, they form a cycloheteroalkyl or substituted cycloheteroalkyl ring; R 64 and R 65 R is independently hydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, or optionally R 64 and R 65 Together with the nitrogen atom to which they are bound, they form a cycloheteroalkyl or substituted cycloheteroalkyl ring. In certain embodiments, the substituents include -M, -R 60 ,=O,-OR 60 、-SR60 , -S - , =S, -NR 60 R 61 , =NR 60 , -CF3, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -S(O)2R 60 、-OS(O)2O - 、-OS(O)2R 60 、-P(O)(O - )2、-P(O)(OR 60 )(O - )、-OP(O)(OR 60 )(OR 61 )、-C(O)R 60 、-C(S)R 60 、-C(O)OR 60 、-C(O)NR 60 R 61 、-C(O)O - 、-NR 62 C(O)NR 60 R 61 In certain embodiments, the substituents include -M, -R 60 ,=O,-OR 60 、-SR 60 、-NR 60 R 61 , -CF3, -CN, -NO2, -S(O)2R 60 、-P(O)(OR 60 )(O - )、-OP(O)(OR 60 )(OR 61 )、-C(O)R 60 、-C(O)OR 60 、-C(O)NR 60 R 61 ,-C(O)O - In certain embodiments, the substituents include -M, -R 60 ,=O,-OR 60 、-SR 60 、-NR 60 R 61 , -CF3, -CN, -NO2, -S(O)2R 60 、-OP(O)(OR 60 )(OR 61 )、-C(O)R 60 、-C(O)OR 60 、-C(O)O -, where R 60 , R 61 and R 62 As defined above. For example, a substituted group may carry a methylenedioxy substituent or one, two or three substituents selected from halogen atoms, (1-4C)alkyl groups and (1-4C)alkoxy groups. When the substituted group is an aryl or heteroaryl group, the substituent (e.g., as described herein) may be referred to as an "aryl substituent".

[0060] "Ester" refers to a group of formula -C(O)(OH).

[0061] "Substituted ester" refers to a radical of the formula -C(O)(O)(R') where R' is selected from alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl, cycloalkyl, and substituted cycloalkyl.

[0062] It is understood that, in all of the substituents defined above, polymers obtained by defining a substituent as itself having further substituents (e.g., a substituted aryl having a substituted aryl as a substituent, which itself is substituted with a substituted aryl, which is further substituted with a substituted aryl, etc.) are not intended to be included herein. In such cases, the maximum number of such substitutions is three. For example, sequential substitution of substituted aryl groups specifically contemplated herein is limited to substituted aryl-(substituted aryl)-substituted aryl.

[0063] "Acyl" refers to a group of the formula -C(O)R, where R is alkyl, alkenyl, or alkynyl. For example, an acetyl group has the formula -C(O)CH3.

[0064] "Halo" and "halogen" refer to chloro, bromo, fluoro and iodo radicals.

[0065] "Carboxy," "carboxyl," and "carboxylate" refer to the -CO2H group and its salts.

[0066] "Imine" means =NR X A group in which R X is any substituent, for example, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl.

[0067] "Sulfonyl" refers to the group -SO2R, where R is alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, and substituted forms thereof. Exemplary sulfonyl groups include -SO2CH3 and -SO2(C6H5).

[0068] Unless otherwise specified, a reference to an atom is intended to include all isotopes of that atom. For example, a reference to H is intended to include 1H. 2 H (i.e. D) and 3 H (i.e. T), reference to C means including 12 C and all isotopes of carbon (e.g. 13 C) In addition, any group described includes all stereoisomers of that group.

[0069] Unless otherwise indicated, nomenclature for substituents not specifically defined herein is derived by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent "arylalkoxycarbonyl" refers to the group (aryl)-(alkyl)-OC(O)-.

[0070] For any group containing one or more substituents disclosed herein, it is of course understood that such groups do not contain any sterically impractical and / or synthetically unfeasible substitutions or substitution patterns. In addition, the subject compounds include all stereochemical isomers resulting from the substitutions of these compounds.

[0071] Green excitable tandem dyes are provided. The tandem dyes of embodiments of the present invention include: a non-conjugated polymer backbone; one or more green excitable donor fluorophores connected to the non-conjugated polymer backbone; and one or more acceptor fluorophores connected to the non-conjugated polymer backbone; wherein the donor fluorophore and the acceptor fluorophore are in an energy transfer relationship. Also provided are methods of preparing and using the tandem dyes, and kits that include the dyes and can be used in embodiments of the methods.

[0072] Before describing exemplary embodiments in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used in the specification.

[0073] 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 the invention belongs. Nevertheless, for clarity and ease of reference, certain terms are defined below. In addition, representative illustrative methods and materials are described herein, although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention.

[0074] Where a numerical range is provided, it is understood that each intermediate value between the upper and lower limits of the range (unless the context clearly indicates otherwise, up to one tenth of the lower limit unit) and any other stated or intermediate values ​​in the stated range are encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also encompassed within the present invention, subject to any specifically excluded limitations in the stated range. When the stated range includes one or two limits, ranges excluding one or both of the included limits are also encompassed within the present invention.

[0075] Certain ranges of values ​​given herein are preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that follows it, as well as numbers that are close to or approximately the number that follows the term. In determining whether a number is close to or approximately a specifically recited number, the close or approximate unrecited number may be a number that, in the context presented, provides a substantial equivalent to the specifically recited number.

[0076] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with the publication citation. Citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0077] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims can be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only," and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0078] As will be apparent to those skilled in the art after reading this disclosure, each of the various embodiments described and illustrated herein has discrete components and features that can be easily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any described method can be executed in the order of events described or in any other order that is logically possible.

[0079] The apparatus and method have been or will be described for grammatical fluency and functional explanation, but it should be clearly understood that unless expressly stated in accordance with 35 U.S.C. §112, the claims should not be interpreted as being necessarily limited in any way to the interpretation of "means" or "step" limitations, but should be given the full scope of the meaning and equivalents provided by the claims under the doctrine of equivalents, and the case where the claims are expressly stated in accordance with 35 U.S.C. §112 should be given all legal equivalents under 35 U.S.C. §112.

[0080] In further describing various embodiments of the invention, green excitable tandem dyes are first reviewed in more detail, followed by methods of using and making the dyes, and kits including the conjugates.

[0081] Green excitable tandem dye

[0082] As described above, the present disclosure provides green excitable fluorescent tandem dye conjugates. Green excitable tandem dyes are dyes that can be excited by green light (e.g., green light provided by a green laser). In some cases, green excitable tandem dyes can be excited by light with a wavelength range of 500 to 565nm. The excitation maximum of the green excitable tandem dyes of the present invention can vary, and in some embodiments, ranges from 500 to 565nm, such as 510 to 555nm, 520nm to 545nm, 525nm to 540nm, 530nm to 535nm, or 527nm to 537nm. Therefore, the green excitable tandem dyes of the present invention can be excited by a laser that produces light with a wavelength range of 500 to 565nm, such as 510 to 555nm, 520nm to 545nm, 525nm to 540nm, 530nm to 535nm, or 527nm to 537nm. Examples of such lasers include, but are not limited to, 514nm Ar + Laser, 515-520nm GaN laser, 532nm Nd:YAG (frequency doubled) laser, 543nm He-Ne laser and 539nm Xe laser.

[0083] As described above, the green excitable tandem dye of an embodiment of the present invention comprises: a non-conjugated polymer backbone; one or more green excitable donor fluorophores connected to the non-conjugated polymer backbone; and one or more acceptor fluorophores connected to the non-conjugated polymer backbone; wherein the donor fluorophore and the acceptor fluorophore are in an energy transfer relationship. Each component of the green excitable dye will now be reviewed in more detail.

[0084] Green excitable donor fluorophore

[0085] As described above, the tandem dyes of the present invention include one or more green excitable donor fluorophores. The green excitable donor fluorophore is a fluorophore that absorbs light in a green wavelength range (e.g., from 500nm to 565nm, such as 510 to 555nm, 520nm to 545nm, 525nm to 540nm, or 530nm to 535nm). In some cases, the maximum absorption range of the green excitable donor fluorophore is 500nm to 565nm, such as 510 to 555nm, 520nm to 545nm, 525nm to 540nm, 530nm to 535nm, or 527nm to 537nm. In certain embodiments, the green excitable donor fluorophore exhibits a narrow absorption spectrum. A narrow absorption spectrum refers to a narrow absorption spectrum with a full width at half maximum (FWHM) of the maximum absorption peak of 35 nm or less, such as 25 nm or less, 20 nm or less, 15 nm or less, 12 nm or less, or 10 nm or less, such as measured in ethanol or phosphate buffered saline (PBS) or deionized water. The donor green excitable fluorophore of embodiments of the present invention can vary. The types of donor green excitable fluorophores that can be found in the tandem dyes of the present invention include, but are not limited to, dipyrromethene-based fluorophores, fluorescein fluorophores, and rhodamine fluorophores.

[0086] In some cases, the donor green excitable fluorophore is a dipyrromethene-based fluorophore. The term "dipyrromethene-based dye" refers to a dye that includes the following core structure:

[0087]

[0088] In some cases, the extinction coefficient of the dipyrromethene-based dye is 5 x 10 5 cm -1 M -1 or larger, such as 6x 10 5 cm -1 M -1 or larger, 7x 10 5 cm -1 M -1 or larger, 8x 10 5 cm -1 M -1 or larger, 9x 10 5 cm -1 M -1 or larger, such as 1x 10 6 cm -1 M -1 or larger, 1.5x 10 6 cm -1 M -1 or larger, 2x 10 6 cm-1 M -1 or larger, 2.5x 10 6 cm -1 M -1 or larger, 3x 10 6 cm -1 M -1 or larger, 4x 10 6 cm -1 M -1 or larger, 5x 10 6 cm -1 M -1 or larger, 6x 10 6 cm -1 M -1 or larger, 7x10 6 cm -1 M -1 or larger or 8x 10 6 cm -1 M -1 In some embodiments, the molar extinction coefficient of the polymer tandem dye is 5 x 10 5 M -1 cm -1 In certain embodiments, the molar extinction coefficient of the tandem dye is 1 x 10 6 M -1 cm -1 or larger.

[0089] In some cases, the dipyrromethene-based dye is a dipyrromethene-boron-based dye (e.g., a BODIPY-type dye). In some cases, the water-soluble dipyrromethene-based dye has formula (I):

[0090]

[0091] in:

[0092] R 1 -R 7 Each is independently selected from H, alkyl, halogen, substituted alkyl, alkoxy, substituted alkoxy, amino, substituted amino, amide, substituted amide, thiol, substituted thiol, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG) and -T 1 -J 1 ,or

[0093] Optionally, selected from R 6 and R 7 , R 2 and R 3, R 5 and R 6 , R 3 and R 4 , R 2 and R 1 and R 7 and R 1 Any one or more pairs of substituents in the group together form a divalent group and are annularly linked and, together with the carbon atoms to which they are bonded, provide a 5- or 6-membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring (e.g., a 5- or 6-membered ring comprising carbon atoms and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, a water-soluble group (WSG) and -T 1 -J 1 Substituents are substituted;

[0094] T 1 is an optional linker;

[0095] J 1 is a repeating unit of the compound of formula (I); and

[0096] Y 1 and Y 2 is independently selected from F, OH, H, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and a water-soluble group (WSG), or

[0097] Y 1 and Y 2 Together with the boron atom to which they are bound, they form a 5- or 6-membered ring (e.g., a 5- or 6-membered ring comprising a boron atom, a carbon atom, and 0-3 heteroatoms selected from O, S, and N), which ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, a water-soluble group (WSG), and -T 1 -Z 1 substituted by a substituent.

[0098] In some cases, the green excitable donor group of formula (I) is 1 The groups are attached to the polymer backbone.

[0099] In some cases, Y 1 and Y 2 Each is independently selected from substituted alkynyl. 1 and Y2 Can have the formula -(alkynylene)-(optional linker)-(polyethylene glycol group). For example, Y 1 or Y 2 The group may be -(alkynylene)-CH2-O-(CH2CH2O) n CH3, wherein n is an integer ranging from 0 to 20. In some cases, Y 1 and Y 2 Has the following structure:

[0100]

[0101] In some cases, R 4 and R 5 At least one of R is selected from substituted alkenyl, aryl and substituted aryl. 4 and R 5 When the group is a substituted alkenyl group, the alkenyl group may be substituted by an aryl group. 4 and R 5 At least one of the following structures is selected:

[0102]

[0103] The aryl group is optionally further substituted, for example by a polyethylene glycol group or a cyano group, or is unsubstituted.

[0104] In some cases, R 5 and R 6 Together they form a divalent group and are linked in a ring and together with the carbon atoms to which they are bonded provide a 5 or 6 membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring.

[0105] In some cases, R 2 and R 3 Together they form a divalent group and are linked in a ring and, together with the carbon atoms to which they are bound, provide a 5- or 6-membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring. In some cases, the divalent group and the carbon atom to which they are bound form a fused aromatic ring.

[0106] Exemplary dipyrromethene-based green excitable fluorophores are described in International Patent Application PCT / US2022 / 021137, which is incorporated herein by reference.

[0107] In some embodiments, one or more green excitable donor fluorophores are xanthene-based fluorophores. Xanthene-based fluorophores are fluorophores that include a xanthene core and can be xanthene or a derivative thereof. Xanthene has the formula:

[0108]

[0109] In some cases, one or more xanthene-based fluorophores have Formula (II) or are salts thereof:

[0110]

[0111] in:

[0112] Each R 21 and R 22 independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carbonyl, carboxyl, cyano, ether, halogen, hydroxyl, imine, and nitro;

[0113] R 23 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carboxyl, cyano, ether, halogen, hydroxyl, and nitro; and

[0114] a and b are each independently an integer of 0 to 4.

[0115] In some cases, R 23 is aryl, substituted aryl, heteroaryl, or substituted heteroaryl. In some cases, R 23 is an aryl group substituted with a carboxyl group. In some cases, there is at least one ether or hydroxyl R 21 In some cases, there is at least one oxo group R 22 In some embodiments, there is at least one amino group R 21 In some embodiments, there is at least one R 22 Imine group. In some embodiments, each green excitable donor fluorophore is a xanthene-based fluorophore.

[0116] In some embodiments, one or more xanthene-based fluorophores are fluorescein-based fluorophores. In other words, in some embodiments, one or more donor green excitable fluorophores are fluorescein-based fluorophores. Fluorescein-based fluorophores are fluorescein-based fluorophores that include a fluorescein core and can be fluorescein or a derivative thereof. Fluorescein has the following structure:

[0117]

[0118] In some cases, the fluorescein-based fluorophore has formula (III) or is a salt thereof:

[0119]

[0120] in:

[0121] Each R 31 , R 32 and R 33 independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carboxyl, cyano, ether, halogen, hydroxyl, and nitro;

[0122] R 34 and R 35 each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

[0123] c and d are each independently an integer from 0 to 3;

[0124] e is an integer from 0 to 4.

[0125] In some cases, each R 31 , R 32 and R 33 is independently selected from halo (e.g., F, Cl, Br, I) and nitro (i.e., NO2). 34 is H. In some cases, R 35 is H. In some embodiments, the fluorescein-based fluorophore is a salt of formula (II), for example, wherein OR 34 OR 35 At least one of O - Na + , O - K + and O - Li + In some embodiments, each green excitable donor fluorophore is a fluorescein-based fluorophore.

[0126] Exemplary fluorescein-based fluorophores comprising a group of formula (III) include Eosin B, Eosin Y, Erythrosin B, and Rose Bengal, the structures of which are shown below. Approximate maximum absorption wavelengths are: Eosin B at 522 nm, Eosin Y at 517 nm, Erythrosin B at 527 nm, and Rose Bengal at 551 nm.

[0127]

[0128] In some embodiments, one or more xanthene-based fluorophores are rhodamine-based fluorophores. In other words, in some embodiments, one or more green excitable donor fluorophores are rhodamine-based fluorophores. Rhodamine-based fluorophores are fluorophores that include a rhodamine (which may be rhodamine or a derivative thereof) core. The chloride salt of rhodamine has the following structure:

[0129]

[0130] In some cases, the rhodamine-based fluorophore has formula (IV) or is a salt thereof:

[0131]

[0132] in:

[0133] Each R 41 , R 42 and R 43 independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carboxyl, cyano, ether, halogen, hydroxyl, and nitro;

[0134] Each R 44 and R 45 independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carboxyl, cyano, ether, halogen, hydroxyl, and nitro;

[0135] X - It is an anion;

[0136] f and g are each independently an integer from 0 to 3;

[0137] h is an integer from 0 to 5.

[0138] In some cases, at least one R 44 is an alkyl group, for example, C 1-6 Alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl and tert-butyl. In some cases, at least one R 44 is H. In some embodiments, at least one R 45 is an alkyl group, for example, C 1-6 Alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl and tert-butyl. In some cases, at least one R 45is H. In some cases, h is 1, 2, 3, 4, or 5, and at least one R 43 is a substituted ester, for example, -C(O)-O-(alkyl), -C(O)-O-(aryl), or -C(O)-O-(heteroaryl). In some cases, h is 1, 2, 3, 4, or 5, and at least one R 43 In some embodiments, X - Is selected from F - , Cl - Br - ,I - , acetate, and 1 / 2 (sulfate) anions. In some embodiments, each green excitable donor fluorophore is a rhodamine-based fluorophore.

[0139] For example, the rhodamine fluorophore of formula (IV) may be rhodamine 6G or rhodamine B, which have the following chemical structures.

[0140] The maximum absorption wavelength of rhodamine 6G is 530nm, and the maximum absorption wavelength of rhodamine B is 541nm.

[0141]

[0142] Acceptor Fluorophore

[0143] As mentioned above, the tandem dye of the present invention includes one or more acceptor fluorophores. In some cases, the tandem dye includes a single acceptor fluorophore. In other cases, the tandem dye includes two or more acceptor fluorophores. Any convenient fluorescent dye can be used as an acceptor fluorophore in the polymer tandem dye. The acceptor fluorophore (e.g., each A) can be a small molecule fluorophore. The acceptor fluorophore (e.g., each A) can be a dye molecule selected from rhodamine, perylene, diimide, coumarin, xanthene, cyanine, polymethine (polymethine), pyrene, thiazine, acridine, dipyrromethene difluoride boron, naphthylimide, phycobiliprotein, dinoflagellate chlorophyll protein (peridinum chlorophyll protein), its conjugate and combination thereof. In certain embodiments, the acceptor fluorophore (A) is a cyanine dye, a xanthene dye, a coumarin dye, a thiazine dye or an acridine dye. In some cases, the acceptor fluorophore (A) is selected from DY 431, DY 485XL, DY 610, DY 640, DY 654, DY 682, DY700, DY 701, DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 778, DY 782, DY 800, DY831, and diethylaminocoumarin. Fluorescent dyes of interest include, but are not limited to, Cascade Blue, Cascade Yellow, Coumarin, Cy5, Cy5.5, Cy-Chrome, Phycoerythrin, PerCP (peridin chlorophyll-a protein), PerCP-Cy5.5, Lucifer Yellow, Marina Blue, Oregon Green 488, Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, 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 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY R6G, BODIPY TMR, BODIPY TR, their conjugates and combinations thereof. Lanthanide chelates of interest include, but are not limited to, europium chelates, terbium chelates and samarium chelates. In some embodiments, the polymer tandem dye comprises a multichromophore linked to an acceptor fluorophore selected from Cy5, Cy5.5, Cy7, Alexa488, Alexa 647 and Alexa700.In certain embodiments, the polymer tandem dye comprises a multichromophore linked to an acceptor fluorophore selected from the group consisting of Dyomics dyes (e.g., DY 431, DY 610, DY 633, DY 640, DY 651, DY 654, DY 682, DY 700, DY 701, DY 704, DY730, DY 731, DY 732, DY 734, DY 752, DY 754, DY 778, DY 782, DY 800, or DY 831) and diethylaminocoumarin. In certain instances, the acceptor fluorophore (A) is selected from the group consisting of Texas Red, California Red, iFluor594, Cascade Blue, Cascade Yellow, coumarin,. Cy-Chrome, DyLight350, DyLight 405, DyLight 488, DyLight 594, DyLight633, DyLight 649, DyLight 680, DyLight 750, DyLight 800, Phycoerythrin, PerCP (Peridinophyton Chlorophyll-a Protein), PerCP-Cy5.5, Lucifer Yellow, Marina Blue, Oregon Green 488, Alexa 350. Alexa 430. Alexa 488. Alexa 594. Alexa 633. Alexa 647、Alexa 660、Alexa 680, 7-amino-4-methylcoumarin-3-acetic acid, 576 / 589, 581 / 591, 630 / 650, 650 / 665, R6G, TMR, TR, their conjugates and combinations thereof.

[0144] If desired, the acceptor fluorophore present in the tandem dyes of embodiments of the invention may be substituted with one or more water-soluble groups (WSG), for example as defined above.

[0145] Polymer backbone

[0146] As described above, the tandem fluorescent dye of the embodiment of the present invention includes a polymer backbone. In some cases, the polymer backbone is composed of non-conjugated repeating units with any convenient configuration (e.g., linear, branched or dendritic polymer configuration). The polymer backbone can be a linear polymer. The polymer backbone can be branched. In some cases, the dye conjugate includes a plurality of pendant donor chromophore groups, each pendant donor chromophore group is independently connected to the non-conjugated repeating unit of the polymer backbone. The configuration of the pendant group can be realized during or after the polymer backbone synthesis. The incorporation of the pendant group can be realized in a sequence-specific manner by random configuration, block configuration or by stepwise synthesis, depending on the specific synthesis method used.

[0147] The term "unit" refers to a structural subunit of a polymer. The term "unit" is intended to include monomers, comonomers, coblocks, repeating units, etc. "Repeat unit" is a subunit of a polymer, which is defined by the unique structural features of the minimum number of units required for the unit to be considered as a monomer, so that when the unit is repeated n times, the resulting structure describes a polymer or its block. In some cases, a polymer may include two or more different repeating units, for example, when the polymer is a multi-block polymer, a random arrangement of units, or a defined sequence, each block may define a different repeating unit. It should be understood that the arrangement of repeating units or blocks is varied, and in the illustrated formula of the polymer backbone described herein, any convenient linear arrangement of various lengths may be included in the structure of the overall polymer. It should be understood that a polymer may also be represented by a formula represented by the mol% value of each unit in the polymer, and such a formula may represent a variety of arrangements of repeating units, such as random or multi-block polymers or defined residue sequences. In some cases, the repeating unit of a polymer includes a single monomer group. In some cases, the repeating unit of a polymer includes two or more monomer groups, i.e., comonomer groups, such as two, three, four or more comonomer groups. The term "comonomer" or "comonomer group" refers to a structural unit of a polymer, which itself can be part of a repeating unit of the polymer.

[0148] The backbone of tandem dyes can have any convenient length. In some cases, the specific number of monomer repeat units or fragments of the chromophore can fall in the range of 2 to 500,000, such as 2 to 100,000, 2 to 30,000, 2 to 10,000, 2 to 3,000 or 2 to 1,000 units or fragments, or such as 5 to 100,000, 10 to 100,000, 100 to 100,000, 200 to 100,000 or 500 to 50,000 units or fragments. In some cases, the specific number of monomer repeat units or fragments of the backbone can fall in the range of 2 to 1,000, such as 2 to 500, 2 to 100, 3 to 100, 4 to 100, 5 to 100, 6 to 100, 7 to 100, 8 to 100, 9 to 100 or 10 to 100 units or fragments. In some cases, the specific number of monomer repeating units or fragments of the backbone may fall within the range of 2 to 500, such as 2 to 400, 2 to 300, 2 to 200, or 2 to 100 units or fragments. In some cases, the specific number of monomer repeating units or fragments of the backbone may fall within the range of 2 to 100 repeating monomer units, such as 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, or 2 to 30 units or fragments.

[0149] The polymer backbone may have a random configuration of non-conjugated repeat units. The polymer backbone may include a block or co-block configuration of non-conjugated repeat units. Alternatively, the polymer backbone may include a specific defined sequence of non-conjugated repeat units, such as the amino acid residues of a polypeptide sequence. These configurations may be characterized by polymer segments of repeat units (e.g., as described herein), which themselves may be repeated throughout the modular scaffold.

[0150] "Non-conjugated" means that at least a portion of the repeat units include saturated backbone groups (e.g., groups having two or more consecutive single covalent bonds) that exclude π conjugation or a delocalized electronic structure extending from one repeat unit to the next along the polymer backbone. It should be understood that even though a repeat unit may not be conjugated to an adjacent repeat unit, such a repeat unit may also include one or more separate unsaturated groups, including unsaturated bonds (e.g., alkenyl or alkynyl unsaturated bonds) and / or aryl or heteroaryl groups, which may be part of the backbone. In some cases, each repeat unit of the polymer backbone includes a side chain that includes a pendant group attached or a chemoselective tag for attachment to a pendant group.

[0151] In certain embodiments of the tandem dye, the polymer backbone is a linear polymer. In some cases, the linear polymer is selected from a peptide, a peptoid, a hydrocarbon polymer, and a PEG polymer. In some cases, the linear polymer is a peptide. In some cases, the linear polymer is a peptoid. In some cases, the polymer is a hydrocarbon polymer. In some other cases, the polymer is a PEG polymer. More details about polymer backbones that can be used in embodiments of the present invention can be found in PCT Application Serial No. PCT / US2019 / 024662 (published as WO2019 / 191482) and PCT Application Serial No. PCT / US2020 / 019510 (published as WO2020 / 222894); the disclosures of these applications are incorporated herein by reference.

[0152] In some cases, the tandem dye includes a linear peptide backbone with 2 to 100 amino acids (e.g., 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, or 2 to 30 amino acids). In some cases, the linear peptide backbone includes 2 or more amino acids, e.g., 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, up to a maximum of 100 amino acids. In some cases, the tandem dye includes a linear peptide backbone with 5 to 30 amino acids (e.g., 5 to 25, 5 to 20, 5 to 15, or 5 to 10 amino acids).

[0153] If desired, the polymer backbone present in the tandem dyes of embodiments of the invention may be substituted with one or more water solubilizing groups (WSG) (eg as defined above).

[0154] Other aspects of green excitable tandem dyes

[0155] The green excitable polymer fluorescent tandem dye of the embodiment of the present invention can be varied. In some cases, the polymer fluorescent tandem dye of the embodiment of the present invention includes: a polymer backbone; a plurality of green excitable pendant donor fluorophores, each of which is independently connected to a repeating unit of the polymer backbone; and one or more pendant acceptor fluorophores, which are connected to a repeating unit of the polymer backbone, wherein the pendant donor fluorophore and the acceptor fluorophore are in an energy transfer relationship. Therefore, the polymer tandem dye of the embodiment of the present invention includes one or more pendant donor fluorophores and one or more pendant acceptor fluorophores (which are configured to be close to one or more pendant donor fluorophores to receive energy), for example, both are connected to a common polymer backbone. In some embodiments, a plurality of pendant donor fluorophores exist and are configured to be close to the pendant acceptor fluorophore for energy transfer, wherein in some cases, the plurality of pendant donor fluorophores are 2 to 20, for example, 2 to 15, for example, 2 to 10. The term "pendant group" refers to a side chain group that is connected to the backbone but is not a part of the backbone itself. In the embodiment of the polymer tandem dye, the donor fluorophore is able to transfer energy to the connected acceptor fluorophore. Therefore, the subject tandem dye includes a connected acceptor signaling fluorophore, which is close to the donor fluorophore system to receive energy, that is, close to at least one connected donor fluorophore to receive energy. The specific configuration of the pendant group can be determined and controlled by the arrangement of the repeating units of the lower polymer backbone (also referred to as "modular scaffold" herein), and the pendant group is attached to the backbone. The tandem dye can include a plurality of water-soluble groups, which are attached to any convenient position of the support and / or pendant group, to provide a water-soluble polymer dye. The polymer backbone, i.e., the modular scaffold, can be composed of repeating units, which form a polymer backbone with side chain groups, and the pendant group can be attached to the side chain group. The repeating unit can be arranged into various configurations, to provide a tandem dye with desired spectral characteristics. The distance and arrangement between the covalent attachment sites of the pendant donor fluorophore and the acceptor fluorophore (when present) can be controlled to provide the desired energy transfer process.

[0156] As mentioned above, if necessary, the polymer backbone and / or the pendant fluorophore (i.e., the donor fluorophore and the acceptor fluorophore) may include one or more water-soluble groups (WSG). In some cases, WSG is a pendant group directly connected to the polymer backbone, such as a side chain of the polymer backbone. In some cases, WSG is a substituent group attached to a pendant donor fluorophore or a pendant acceptor fluorophore. In some cases, each pendant donor fluorophore group is substituted by one or more WSG. 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 solvated in an aqueous environment, such as under physiological conditions, and imparts improved water solubility to the molecule to which it is attached. Compared with a control dye lacking WSG, WSG can increase the solubility of a given polymer tandem dye in a primary aqueous solution. The water-soluble group can be any convenient hydrophilic group that is well solvated in an aqueous environment. The solubility that the water-soluble tandem dye of the present disclosure has under aqueous conditions makes it particularly suitable for application in a variety of biological assays. Various water-soluble polymer groups can be applicable to the WSG of the subject dye. Any convenient water solubilizing group (WSG) may be included in the dyes described herein to provide increased water solubility, for example as described above.

[0157] As described above, the polymer fluorescent tandem dye of the embodiment of the present invention includes a plurality of pendant donor fluorophores and one or more pendant acceptor fluorophores. In some cases, the number of donor fluorophores exceeds the number of acceptor fluorophores. In certain embodiments of the tandem dye of the present invention, the ratio of donor fluorophores to acceptor fluorophores is selected from 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1 and 20: 2. In some cases, the ratio of donor fluorophores to acceptor fluorophores is 5: 1. In some cases, the ratio of donor fluorophores to acceptor fluorophores is 6: 1. In some cases, the ratio of donor fluorophores to acceptor fluorophores is 7: 1. In some cases, the ratio of donor fluorophores to acceptor fluorophores is 8: 1. In some cases, the ratio of donor fluorophores to acceptor fluorophores is 9: 1. In some cases, the ratio of donor fluorophores to acceptor fluorophores is 10: 1.

[0158] As described above, in the polymeric fluorescent tandem dyes of the present invention, the pendant donor fluorophore and the acceptor fluorophore are in an energy transfer relationship. Thus, in embodiments of the present invention, excitation of the donor can result in energy transfer to and emission of energy from the covalently attached acceptor signaling fluorophore. Energy transfer mechanisms between the donor chromophore and the linked acceptor signaling fluorophore include, for example, resonance energy transfer (e.g., (or fluorescence) resonance energy transfer, FRET), quantum charge exchange (Dexter energy transfer), etc. These energy transfer mechanisms can be relatively short distances; that is, the proximity of the chromophores of the light-collecting multi-chromophore system to each other and / or to the acceptor fluorophore provides efficient energy transfer. Under conditions of efficient energy transfer, when the incident light ("pump light") is at a wavelength absorbed by the chromophore of the light-collecting chromophore and transferred from it, the emission of the luminescent acceptor fluorophore is more intense than when the luminescent acceptor fluorophore is directly excited by the pump light, and the emission of the acceptor fluorophore can be amplified. "Effective" energy transfer means that 10% or more (e.g., 20% or more or 30% or more, 40% or more, 50% or more) of the energy collected by the donor chromophore is transferred to the acceptor. "Amplification" means that when excited by energy transfer from the donor light-collecting chromophore system, the signal from the acceptor fluorophore is 1.5 times or more than that of directly exciting the acceptor fluorophore with incident light of equal intensity. The signal can be measured using any convenient method. In some cases, a signal of 1.5 times or more refers to the intensity of the emitted light. In some cases, a signal of 1.5 times or greater refers to an increased signal-to-noise ratio. In certain embodiments of tandem dyes, the acceptor fluorophore emission when excited by the chromophore is 1.5 times or greater, e.g., 2 times or greater, 3 times or greater, 4 times or greater, 5 times or greater, 6 times or greater, 8 times or greater, 10 times or greater, 20 times or greater, 50 times or greater, 100 times or greater, or even greater, compared to direct excitation of the acceptor fluorophore with incident light.

[0159] The tandem dye of the present invention embodiment can have any convenient molecular weight (MW). In some cases, the MW of the tandem dye can be expressed as mean molecular weight. In some cases, the mean molecular weight of the tandem dye is in the range of 500 to 500,000, for example 1,000 to 100,000, 2,000 to 100,000, 10,000 to 100,000 or even mean molecular weight in the range of 50,000 to 100,000 dalton. In some cases, the molecular weight of the polymer fluorescent dye is in the range of 5 to 75kDa, for example 10 to 50kDa, for example 15 to 45kDa.

[0160] As mentioned above, the tandem dye of the embodiment of the present invention is a green excitable tandem dye. Therefore, the green excitable tandem dye is a dye that can be excited by green light (for example, the green light provided by a green laser). In some cases, the green excitable tandem dye can be excited by light with a wavelength range of 500nm to 565nm, for example 510 to 555nm, 520nm to 545nm, 525nm to 540nm or 530nm to 535nm. The excitation maximum of the green excitable tandem dye of the present invention can vary, and in some embodiments, ranges from 500 to 565nm, for example 510 to 555nm, 520nm to 545nm, 525nm to 540nm or 530nm to 535nm.

[0161] In some cases, the tandem dye exhibits an effective Stokes shift in the range of 25 nm to 300 nm, such as 50 nm to 250 nm or 75 nm to 200 nm. In some cases, when the light harvesting chromophore is directly excited with 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, 250 nm or greater.

[0162] The emission of the polymer tandem dye can have a quantum yield of 0.03 or more, e.g., 0.04 or more, 0.05 or more, 0.06 or more, 0.07 or more, 0.08 or more, 0.09 or more, 0.1 or more, 0.15 or more, 0.2 or more, 0.3 or more, or even higher. In some cases, the extinction coefficient of the polymer tandem dye is 5x10 5 cm -1 M -1 or larger, such as 6x 10 5 cm -1 M -1 or larger, 7x 10 5 cm -1 M -1 or larger, 8x 10 5 cm -1 M -1 or larger, 9x10 5 cm -1 M -1 or larger, such as 1x 10 6 cm -1 M -1 or larger, 1.5x 10 6 cm-1 M -1 or larger, 2x 10 6 cm -1 M -1 or larger, 2.5x 10 6 cm -1 M -1 or larger, 3x 10 6 cm -1 M -1 or larger, 4x 10 6 cm -1 M -1 or larger, 5x 10 6 cm -1 M -1 or larger, 6x10 6 cm -1 M -1 or larger, 7x 10 6 cm -1 M -1 or larger or 8x 10 6 cm -1 M -1 In some embodiments, the molar extinction coefficient of the polymer tandem dye is 5 x 10 5 M -1 cm -1 In certain embodiments, the molar extinction coefficient of the tandem dye is 1 x 10 6 M -1 cm -1 or larger.

[0163] In certain embodiments, the subject tandem dye provides a fluorescent emission of the acceptor fluorophore that is brighter than the fluorescence that such fluorescent dye could emit alone. The brightness of the emission of the polymer tandem dye can be 50 mM -1 cm -1 or higher, e.g. 60 mM -1 cm -1 or higher, 70mM -1 cm -1 or higher, 80mM -1 cm -1 or higher, 90mM -1 cm -1 or higher, 100mM -1 cm -1 or higher, 150mM -1 cm -1 or higher, 200mM -1 cm -1 or higher, 250mM -1 cm -1or higher, 300mM -1 cm -1 In some cases, the brightness of the emission of the tandem dye is at least 5 times higher than the brightness of the directly excited acceptor fluorophore, such as at least 10 times higher, at least 20 times higher, at least 30 times higher, at least 50 times higher, at least 100 times higher, at least 300 times higher, or even higher than the brightness of the directly excited acceptor fluorophore.

[0164] In addition to the above-mentioned properties, the tandem dyes of embodiments of the present invention may also have one or more additional desired spectral characteristics, such as a specific emission maximum wavelength, extinction coefficient, quantum yield, and the like.

[0165] Various emission spectra depend on various factors, such as the selected comonomers, linking groups, substituents and attached acceptor fluorophores contained in the tandem dye. In some embodiments, the maximum emission wavelength of the tandem dye is in the range of 570nm to 900nm, such as in the range of 580nm to 875nm, 590nm to 850nm or 600nm to 825nm.

[0166] In some embodiments, a polymeric tandem dye may include a segment of formula (Ia):

[0167]

[0168] in:

[0169] Each M 1 and M 2 are independently unsaturated comonomers (e.g., amino acid residues);

[0170] Each S 1 and S 2 are independently an optional non-conjugated spacer unit;

[0171] Each D 1 Independently for M 1 a linked pendant green excitable donor fluorophore (e.g., as described herein);

[0172] Each A 1 Independently for M 2 the attached acceptor fluorophore;

[0173] x is 75 mol% or greater; and

[0174] y is 25 mol% or less.

[0175] The first repeating unit (M 1 -S 1 ) and the second repeating unit (M 2 -S 2) can be arranged in a random or co-block configuration. In certain cases of Formula (Ia), D of the first repeating unit 1 The pendant group includes two or more (e.g., two or three) different types of pendant light absorbing chromophores that together provide a light harvesting multichromophore system. In certain instances of Formula (Ia), D of the first repeating unit 1 The pendant groups are all identical.

[0176] In some cases of Formula (Ia), x is 80 mol% or greater, e.g., 85 mol% or greater, 90 mol% or greater, 95 mol% or greater, 96 mol% or greater, 97 mol% or greater, 98 mol% or greater, or 99 mol% or greater. In some cases of Formula (Ia), y is 20 mol% or less, e.g., 15 mol% or less, 10 mol% or less, 5 mol% or less, 4 mol% or less, 3 mol% or less, 2 mol% or less, 1 mol% or less.

[0177] In some cases, the polymer tandem dye comprises a segment of formula (IIa):

[0178]

[0179] in:

[0180] The polymer backbone of the non-conjugated repeating unit comprises SM 1 , SM 2 and SM 3 comonomers, each of which is independently a non-conjugated comonomer;

[0181] Each D 1 Independently for SM 1 a linked pendant green excitable donor fluorophore (e.g., as described herein);

[0182] Each A 1 Independently for SM 2 the attached acceptor fluorophore;

[0183] Each Z 2 Independently for SM 3 attached optional side chain groups;

[0184] x is 50 mol% or greater; and

[0185] y+z is 50 mol% or less.

[0186] Z 2may be absent or any convenient side chain group, such as a light absorbing chromophore, a chemoselective tag, a linker, a linked biomolecule, an acceptor fluorophore, WSG (e.g., as described in PCT Application Serial No. PCT / US2019 / 024662 (published as WO 2019 / 191482), the disclosure of which is incorporated herein by reference), etc. In certain cases of Formula (IIa), when Z 2 When it does not exist, SM 3 is a spacer comonomer. In certain cases of formula (IIa), SM 3 It contains Z 2 The Z 2 The group is a second pendant light absorbing chromophore, wherein each D 1 and each Z 2 Together they provide a light harvesting multichromophore system. In some cases, the SM 3 is a second chemoselective tag (Z 2 ) comonomer, for example, with Z 1 Orthogonal tags or protected functional groups that provide for selective installation of moieties of interest.

[0187] In some cases of formula (IIa), x is 60mol% or more, for example 65mol% or more, 70mol% or more, 75mol% or more, 80mol% or more, 85mol% or more, 90mol% or more, 95mol% or more, or even more. In some cases of formula (IIa), y+z is 40mol% or less, for example 30mol% or less, 25mol% or less, 20mol% or less, 15mol% or less, 10mol% or less, 5mol% or less, or even less. In some cases of formula (IIa), y is at least 1mol% and is 25mol% or less, for example 20mol% or less, 15mol% or less, 10mol% or less, 5mol% or less, or even less. In some cases of formula (IIa), z is at least 1mol% and is 10mol% or less, for example 5mol% or less, or even less.

[0188] In some cases, the polymer tandem dye comprises a segment of formula (IIIa):

[0189]

[0190] in:

[0191] The polymer backbone of the non-conjugated repeating unit comprises SM 1 and SM 2 comonomers, each of which is independently a non-conjugated comonomer;

[0192] Each D 1 Independently for SM 1 a linked pendant green excitable donor fluorophore (e.g., as described herein);

[0193] Each A 1 Independently for SM 2 the attached acceptor fluorophore;

[0194] x is 75 mol% or greater; and

[0195] y is 25 mol% or less.

[0196] In certain embodiments of Formula (IIIa), SM 1 and SM 2 Each is independently a saturated non-conjugated comonomer, for example, a comonomer that provides only a single covalent CC bond. In some embodiments of Formula (IIIa), SM 1 and SM 2 Each is independently a partially saturated non-conjugated comonomer, for example, a comonomer that provides a single C=C covalent double bond in the saturated covalent bond backbone. The first and second repeating units (SM 1 and SM 2 ) can be arranged in a random configuration, a block or co-block configuration or in a specific order. In certain cases of formula (IIIa), SM 1 D 1 The pendant group includes two or more (e.g., two or three) different types of pendant light absorbing chromophores that together provide a light harvesting multichromophore system. In certain instances of formula (IIIa), D of the first repeating unit 1 The pendant groups are all identical.

[0197] In some cases of formula (IIIa), x is 80 mol% or greater, e.g., 85 mol% or greater, 90 mol% or greater, 95 mol% or greater, 96 mol% or greater, 97 mol% or greater, 98 mol% or greater, or 99 mol% or greater. In some cases of formula (IIIa), y is 20 mol% or less, e.g., 15 mol% or less, 10 mol% or less, 5 mol% or less, 4 mol% or less, 3 mol% or less, 2 mol% or less, 1 mol% or less.

[0198] In some cases, the polymeric tandem dye has formula (IVa):

[0199]

[0200] in:

[0201] Each D 1 independently a pendant green excitable donor fluorophore (e.g., as described herein);

[0202] Each A 1 independently for the acceptor fluorophore;

[0203] Each L 1 and L 2 are independently linkers;

[0204] p1 and q1 are independently 0 or 1, where p1+q1≤1;

[0205] p2 and q2 are independently 0 or 1, where p1+q1≤1;

[0206] x is 75 mol% or more;

[0207] y is 25 mol% or less; and

[0208] G 1 and G 2 Each is independently selected from a terminal group, a polymer segment, a light absorbing (eg, donor) chromophore group, an acceptor fluorophore, a linker, and a linker with a specific binding member, such as described above.

[0209] In some embodiments of Formula (IVa), p1 and p2 are each 0, q1 and q2 are each 1 (e.g., a β3-amino acid residue). In some embodiments of Formula (IVa), p1 and p2 are each 1, q1 and q2 are each 0 (e.g., a β2-amino acid residue). In some cases, p1, p2, q1, and q2 are each 0, and the polymer tandem dye has Formula (Va):

[0210]

[0211] in:

[0212] Each D 1 independently a pendant green excitable donor fluorophore (e.g., as described herein);

[0213] Each A 1 independently for the acceptor fluorophore;

[0214] L 1 and L 2 Each is independently a linker;

[0215] x is 75 mol% or greater;

[0216] y is 25 mol% or less; and

[0217] G1 and G 2 Each is independently selected from an end group, a polymer segment, a light absorbing (e.g., donor) chromophore group, a linker, and a linker with a specific binding member, such as described above. It will be appreciated that the tandem dye described by formula (Va) includes any convenient arrangement of comonomers in a defined linear sequence, these monomers having a total of defined x and y mol%. In some cases, the A-containing 1 The comonomers of D1 are spaced throughout the polymer backbone sequence so that there are always one or more comonomers containing D1 on both sides.

[0218] In certain instances of Formula (Va), the polymeric tandem dye comprises a segment of Formula (VIa):

[0219]

[0220] in:

[0221] Each D 1 is independently a pendant green excitable donor fluorophore (eg, as described herein); each A 1 are independently acceptor chromophores; each L 1 and L 2 is independently a linking group; n and p are each independently an integer from 1 to 20, wherein n+p≥2; and m is 1 or 2.

[0222] In some instances of Formula (VIa), n and p are each independently 1 to 10, e.g., 2 to 20, 3 to 10, or 3 to 6. In some instances of Formula (VIa), n+p is an integer from 2 to 20, e.g., 3 to 20, 4 to 20, 5 to 20, 5 to 15, or 5 to 12. In certain embodiments of Formula (VIa), m is 1.

[0223] The subject polymer tandem dye may include multiple segments of formula (VIa), wherein each segment includes two D-containing 1 A single comonomer block containing A 1 In some cases, the multichromophore includes two or more segments of formula (VIa) adjacent to each other to provide a copolymer containing 2-20 D 1 The blocks of comonomers (e.g., 3 to 20, 4 to 20, 5 to 20, 5 to 15 or 5 to 12 containing D 1 Two separate A-containing blocks separated by 1 Comonomer. Thus, in certain embodiments, the polymer tandem dye comprises q segments of a block copolymer and has formula (VIIa):

[0224]

[0225] Where: Each (n) q and each (p) q are independently integers from 1 to 20, wherein for each of the q segments, (n) q +(p) q ≥3; and q is an integer from 1 to 100.

[0226] In certain embodiments, the polymer tandem dye has formula (VIIIa):

[0227]

[0228] in

[0229] Each D 1 independently a pendant green excitable donor fluorophore (e.g., as described herein);

[0230] Each A 1 independently for the acceptor chromophore;

[0231] Each L 1 and L 2 are independently linkers;

[0232] x is 75 mol% or greater;

[0233] y is 25 mol% or less; and

[0234] G 1 and G 2 Each is independently selected from a terminal group, a polymer segment, a donor chromophore group, a linker, and a linker with a specific binding member, for example as described above.

[0235] In certain embodiments, the polymeric tandem dye has Formula (IXa):

[0236]

[0237] in:

[0238] Each D 1 independently a pendant green excitable donor fluorophore (e.g., as described herein);

[0239] Each A 1 independently for the acceptor fluorophore;

[0240] Each L 1 and L 2 are independently linkers;

[0241] x is 75 mol% or greater;

[0242] y is 25 mol% or less; and

[0243] G 1 and G 2 Each is independently selected from a terminal group, a polymer segment, a donor chromophore group, an acceptor fluorophore group, a linker group, and a linker group with a specific binding member, such as described above.

[0244] In some cases of Formula (IVa), (Va), (XIIIa), and (IXa), x is 80 mol% or greater, e.g., 85 mol% or greater, 90 mol% or greater, 95 mol% or greater, 96 mol% or greater, 97 mol% or greater, 98 mol% or greater, or 99 mol% or greater. In some cases of Formula (IV), (V), (XIII), and (IX), y is 20 mol% or less, e.g., 15 mol% or less, 10 mol% or less, 5 mol% or less, 4 mol% or less, 3 mol% or less, 2 mol% or less, 1 mol% or less.

[0245] In certain embodiments, the polymeric tandem dye has formula (Xa):

[0246]

[0247] in:

[0248] Each D 1 independently a pendant green excitable donor fluorophore (e.g., as described herein);

[0249] Each A 1 independently for the acceptor fluorophore;

[0250] Each L 1 , L 2 and L 3 are independently linkers;

[0251] a, b and c are the mol% values ​​of each comonomer;

[0252] d represents the overall degree of polymerization or average length of the polymer (e.g., d is 2-1000, such as 2-500, 2-200, 2-100 or 2-50);

[0253] WSG is a water-soluble group (e.g., as described above and in PCT Application Serial No. PCT / US2019 / 024662 (published as WO 2019 / 191482), the disclosure of which is incorporated herein by reference); and

[0254] G 1 and G 2Each is independently selected from a terminal group, a polymer segment, a donor chromophore group, an acceptor fluorophore group, a linker group, and a linker group with a specific binding member, such as described above.

[0255] In some cases of formula (Xa), c=0. In some cases of formula (Xa), a>0 and b>0. In some cases of formula (Xa), a is 80mol% or more, such as 85mol% or more, 90mol% or more, 95mol% or more, 96mol% or more, 97mol% or more, 98mol% or more, or 99mol% or more. In some cases of formula (Xa), b is 20mol% or less, such as 15mol% or less, 10mol% or less, 5mol% or less, 4mol% or less, 3mol% or less, 2mol% or less, 1mol% or less. In some cases of formula (Xa), a is 65-95mol%, b is 5-35mol% and c is 0-30mol%, wherein a+b+c=100%.

[0256] Any convenient end group (e.g., G 1 and G 2 ) can be used at the end of the tandem fluorescent dye. As used herein, the terms "end group" and "terminal group" are used interchangeably to refer to a group located at the end of a tandem dye polymer structure, such as described herein. 1 and G 2 Groups include, but are not limited to, H, chemoselective groups, linkers (which may be activated or inactivated), and linkers conjugated to specific binding members, such as described above. It is understood that the G located at the polymer backbone 1 and / or G 2 The linker at the position of G can be selected to be orthogonal to any other linker that may be present on the tandem dye side chain, including a chemoselective tag (e.g., as described herein). 1 and / or G 2 In certain embodiments, G 1 and / or G 2 The moiety includes a carboxylic acid functional group or a derivative thereof.

[0257] In some embodiments of the formulae described herein, G 1 and G 2 At least one of them is -L 6 -Z 4 , where L 6 is a linker (eg, as described herein), and Z 4 is a specific binding member (e.g., as described herein). In some embodiments of the formulae described herein, G 1and G 2 At least one of them is -L 6 -Z 3 , where L 6 is a linker (eg, as described herein), and Z 3 is a chemoselective tag (e.g., as described herein). Any convenient chemoselective tag and conjugation chemistry may be suitable for use with the subject light-harvesting chromophore. Chemoselective tags of interest include, but are not limited to, amines, active esters, maleimides, thiols, sulfur (VI) fluoride exchange chemistry (SuFEX), sulfonyl fluorides, Diers Alder cycloaddition click reagents and click chemistry, tetrazines, trans-cyclooctenes, aldehydes, alkoxyamines, alkynes, cyclooctynes, azides, and the like. In some cases, Z 3 is selected from carboxylic acids, active esters (e.g., N-hydroxysuccinimide esters (NHS) or sulfo-NHS), amino groups, maleimides, iodoacetyl groups, and thiols. In certain embodiments of the formulae described herein, G 1 and G 2 At least one of is described by the following structure:

[0258] *-Ar-LZ

[0259] wherein Ar is a π-conjugated aromatic group, L is a linker, and Z is a chemoselective tag or specific binding member. In some cases, the LZ group can be directly attached to a terminal comonomer. In certain embodiments of the formula described herein, G 1 and G 2 At least one of is described by the following structure:

[0260]

[0261] in:

[0262] q is 0 or an integer from 1 to 12;

[0263] L is an optional linker; and

[0264] Z is a chemoselective tag or a specific binding member. More detailed information about the WSG group that can be found in the tandem dyes of embodiments of the present invention can be found in PCT application serial number PCT / US2019 / 024662 (published as WO2019 / 191482), the disclosure of which is incorporated herein by reference.

[0265] method

[0266] Aspects of the present invention include methods for assessing the presence of target analytes in samples. Aspects of the method include contacting samples with marker specific binding members (which specifically bind to target analytes) to produce a determination composition comprising a sample contacted with marker specific binding members. The marker specific binding members used in each embodiment of the inventive method include specific binding members conjugated with green excitable polymer fluorescent tandem dyes, such as described above. In the following sections, the target analyte can be a target molecule of interest or a reagent (such as an antibody) bound to a target molecule, depending on whether the marker specific binding member is used as the first label or the second label. Any convenient method can be used to contact samples with marker specific binding members that specifically bind to target analytes to produce a determination composition. In some cases, samples are contacted with marker specific binding members under conditions where marker specific binding members specifically bind to target analytes (if present). In order to specifically bind marker specific binding members to target analytes, suitable media can be used to maintain the biological activity of sample components and single domain antibodies. The culture medium can be a balanced salt solution, such as physiological saline, PBS, Hank's balanced salt solution, etc., which is conveniently supplemented with fetal bovine serum, human platelet lysate or other factors, combined with an acceptable buffer at a low concentration such as 5-25mM. Convenient buffers include HEPES, phosphate buffer, lactate buffer, etc. Various media are commercially available and can be used according to the properties of the target analyte, including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., supplemented with fetal bovine serum or human platelet lysate in some cases. The final component of the culture medium (which can be a solution) can be selected according to the components contained in the sample. The temperature at which the label specific binding member and the target analyte specifically bind can vary, and in some cases can be within the range of 5°C to 50°C, such as 10°C to 40°C, 15°C to 40°C, 20°C to 40°C, such as 20°C, 25°C, 30°C, 35°C or 37°C (for example, as described above). In some cases, the temperature at which specific binding occurs is selected to be compatible with the biological activity of specific binding members and / or target analytes. In some cases, 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 convenient incubation time for specific binding can be selected to allow the formation of a desired amount of binding complex, in some cases, 1 minute (min) or longer, such as 2min or longer, 10min or longer, 30min or longer, 1 hour or longer, 2 hours or longer, or even 6 hours or longer.

[0267] Any convenient specific binding member can be used in the labeling specific binding member adopted in the inventive method. Interested specific binding members include but are not limited to those specific binding members of cell surface proteins that specifically bind to a variety of cell types, and the cell type includes but is not limited to stem cells (e.g., pluripotent stem cells, hematopoietic stem cells), T cells, T regulatory cells, dendritic cells, B cells (e.g., memory B cells, 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 erythrocytes. Interested target cells include cells with convenient cell surface markers or antigens that can be captured by convenient specific binding member conjugates. In some embodiments, target cells are selected from cells containing HIV, Treg cells, antigen-specific T cell colonies, tumor cells, or hematopoietic progenitor cells (CD34+) from whole blood, bone marrow or umbilical cord blood. Any convenient cell surface protein or cell marker can be targeted and specifically bound to the conjugate used in the subject method. In some embodiments, target cells include cell surface markers selected from cell receptors and cell surface antigens. In some cases, the target cells can include 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, disialylganglioside GD2, and CD71.

[0268] Any convenient target can be selected to utilize the subject method for assessment. Interested 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 phosphorylation, glycosylation, ubiquitination, SUMOylation 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. Target protein can be any protein of interest, such as treatment or diagnosis targets, including but not limited to: hormones, growth factors, transcription factors, receptors, enzymes, cytokines, osteoinductive factors, colony stimulating factors and immunoglobulins. The term "target protein" is intended to include recombinant molecules and synthetic molecules, which can be prepared using any convenient recombinant expression method or using any convenient synthesis method, or commercial purchase. In some embodiments, the polymer dye conjugate includes an antibody or an antibody fragment. In the subject method, any convenient target analyte that can be targeted specifically combined with an antibody of interest or an antibody fragment.

[0269] In some embodiments, the target analyte is associated with a cell. In some cases, the target analyte is a cell surface marker of a cell. In some cases, the cell surface marker is selected from a cell receptor and a cell surface antigen. In some cases, the target analyte is an intracellular target, and the method further includes processing cells so that the marker specific binding members can be close to the intracellular target, such as by permeabilizing or lysing the cells. Therefore, the marker specific binding members used in the method of the present invention can target cell surface or intracellular antigens. Alternatively, the marker specific binding members used in the method of the present invention can target an antibody, and the antibody specifically binds to the target cell surface or intracellular antigen.

[0270] In some embodiments, the sample may include a heterogeneous cell population from which a target cell is isolated. In some cases, the sample includes peripheral whole blood, peripheral whole blood in which red blood cells have been lysed before cell separation, cord blood, bone marrow, density gradient purified peripheral blood mononuclear cells or homogenized tissue. In some cases, the sample includes hematopoietic progenitor cells (e.g., CD34+ cells) in whole blood, bone marrow or cord blood. In certain embodiments, the sample includes tumor cells in peripheral blood. In some cases, the sample includes (or is suspected of including) viral cells (e.g., HIV).

[0271] Mark specific binding members can be used in subject methods, such as labeling target cells, particles, targets or analytes with polymer series fluorescent dyes.For example, mark specific binding members can be used for labeling cells to be processed (for example, detection, analysis and / or sorting) in flow cytometer.Mark specific binding members can include specific binding members, such as antibodies or their binding fragments, which specifically bind to cell surface proteins (for example, as described herein) of various cell types.Mark specific binding members can be used for studying various biological (such as cell) characteristics or processes, such as cell cycle, cell proliferation, cell differentiation, DNA repair, T cell signaling, apoptosis, cell surface protein expression and / or presentation, etc.Mark specific binding members can be used for including (or can include) any application of antibody-mediated labeled cells, particles or analytes.

[0272] Aspects of the method include determining whether there is a marker specific binding member-target analyte binding complex in the assay composition (i.e., the sample contacted by the marker specific binding member) to assess whether the target analyte is present in the sample. Once the sample contacts the marker specific binding member, any convenient method can be used to determine the produced assay composition to determine the presence of the marker specific binding member-target analyte binding complex. The marker specific binding member-target analyte binding complex is a binding complex produced when the marker specific binding member and the target analyte (or the main binding member for the target antigen, for example, an antibody, depending on the embodiment) are specifically bound, if there is a target analyte. Determining the assay composition may include detecting a fluorescent signal from the binding complex, if there is a binding complex. In some cases, the assay includes a separation step, wherein the target analyte (if present) is separated from the sample. A variety of methods can be used to separate the target analyte from the sample, such as by being fixed on a support. Interested assay methods include but are not limited to any convenient methods and assay forms wherein specific binding members are used such as avidin-biotin or hapten-anti-hapten antibodies. Methods and assay formats of interest that may be suitable for use with the subject compositions include, but are not limited to, flow cytometry methods, in situ hybridization methods, enzyme-linked immunosorbent assays (ELISAs), Western blot analysis, magnetic cell separation assays, and fluorescent dye purification chromatography.

[0273] In certain embodiments, the method also includes contacting the sample with the second specific binding members that specifically binds the target analyte. In some cases, the second specific binding members is supported. The component (for example, the second specific binding members) of the subject method can be fixed using any convenient support. In some cases, the support is a particle, such as a magnetic particle. In some cases, the second specific binding members and the polymer dye conjugate produce a sandwich complex, and any convenient method can be used to separate and detect the sandwich complex (if present). In some embodiments, the method also includes flow cytometry analysis of polymer dye conjugate-target analyte binding complex, i.e., fluorescently labeled target analyte. Determination of the presence of marker specific binding members-target analyte binding complex can provide a determination result (for example, qualitative or quantitative determination data), which can be used to assess whether there is a target analyte in a sample.

[0274] In the subject method, any convenient support can be used to fix any convenient component of the method, such as labeling specific binding members, targets, second specific binding members, etc. Interested support includes but is not limited to: solid matrix, wherein matrix can have a variety of structures, such as sheets, beads or other structures such as plates with holes; beads, polymers, particles, fiber nets, hydrogels, porous matrixes, pins, microarray surfaces, chromatographic supports, etc. In some cases, support is selected from particles, planar solid matrixes, fiber nets, hydrogels, porous matrixes, pins, microarray surfaces and chromatographic supports. Support can be incorporated into the system, which provides cell separation assisted by any convenient method (such as manually operated syringes, centrifuges or automatic liquid handling systems). In some cases, support can be used for automated liquid handling systems, to achieve high-throughput separation of cells, such as flow cytometers.

[0275] In some embodiments of the method, the separation step includes applying an external magnetic field to fix the magnetic particles. Any convenient magnet can be used as an external magnetic field (e.g., magnetic field gradient) source. In some cases, the external magnetic field is produced by a magnetic source, such as produced by a permanent magnet or an electromagnet. In some cases, fixing the magnetic particles means that the magnetic particles are gathered near the surface closest to the magnetic field gradient source (i.e., magnet).

[0276] Separation can also include one or more optional washing steps to remove unbound material of the sample from the support. Any convenient washing method can be used, for example, washing the immobilized support with a biocompatible buffer that retains the specific binding interactions of the polymeric dye and the specific binding members. The unbound material of the sample separated and optionally cleaned from the support provides an enriched target cell population, in which unwanted cells and materials can be removed.

[0277] In certain embodiments, the method includes detecting a labeled target analyte. Detecting a labeled target analyte can include exciting a polymer fluorescent tandem dye with one or more lasers, and then using one or more optical detectors to detect the fluorescence emission from the polymer fluorescent tandem dye. The detection of the labeled target can be carried out using any convenient instrument and method, including but not limited to flow cytometry, FACS system, fluorescence microscopy; using a microplate reader for fluorescence, cold light (luminescence), ultraviolet and / or visible light detection; high performance liquid chromatography (HPLC); and mass spectrometry. When using fluorescently labeled components in the methods and compositions of the present disclosure, it is recognized that different types of fluorescence detection systems can be used to practice the subject method. In some cases, high throughput screening can be performed, such as a system using 96 wells or larger microtiter plates. A variety of methods for measuring fluorescent materials can be used, such as those described in Lakowicz, JR, Principles of Fluorescent Spectroscopy, New York: Plenum Press (1983); Herman, B., Resonance energy transfer microscopy, in: Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed. Taylor, 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.

[0278] Fluorescence in a sample can be measured using a fluorometer. In some cases, excitation radiation from an excitation source having a first wavelength passes through an excitation optical device. The excitation optical device causes the excitation radiation to excite the sample. In response, the fluorescently labeled target in the sample emits radiation having a wavelength different from the excitation wavelength. The collection optical device then collects the emitted light from the sample. The device may include a temperature controller to keep the sample at a specific temperature while scanning the sample. In some cases, a multi-axis translation stage moves a microtiter plate holding multiple samples in order to locate different wells to be exposed. The multi-axis translation stage, temperature controller, autofocus function, and electronic devices associated with imaging and data collection can be managed by a properly programmed digital computer. The computer can also convert the data collected during the assay into another format for presentation.

[0279] In some embodiments, the method for assessing the presence of a target analyte in a sample further comprises detecting fluorescence in a flow cytometer. In some embodiments, the method for assessing the presence of a target analyte in a sample further comprises imaging the sample contacted with the labeling composition using fluorescence microscopy. Fluorescence microscopy imaging can be used to identify polymer dye conjugate-target analyte binding complexes in the contacted sample to assess whether the target analyte exists. Microscopic methods of interest that can be used for the subject method include laser scanning confocal microscopy.

[0280] Also provided is a method for producing green excitable polymer fluorescent tandem dyes, for example as described herein. In some embodiments, the method includes: contacting a specific binding member with a green excitable polymer fluorescent tandem dye (for example as described herein) to produce a specific binding member / polymer fluorescent tandem dye conjugate, wherein the polymer fluorescent tandem dye includes a conjugated label for covalently linking the dye to a specific binding member. The term "conjugated label" refers to a group including a chemoselective functional group (for example, as described herein), which can be covalently linked to a compatible functional group of a specific binding member after optional activation and / or deprotection. Any convenient conjugated label can be used in the subject polymer dye so that the polymer fluorescent tandem dye is conjugated to a specific binding member (for example, an antibody or fragment thereof of interest). In some embodiments, the conjugated label includes a terminal functional group selected from amino, carboxylic acid or derivatives thereof, thiol, hydroxyl, hydrazine, hydrazide, azide, alkyne and protein reactive groups (for example, amino reactive, thiol reactive, hydroxy reactive, imidazole reactive or guanidinyl reactive). Any convenient method and reagent can be applied to the subject method so that the conjugated label is covalently connected to the heterosexual binding member. The method of interest for marking the target includes but is not limited to those methods and reagents described by Hermanson, Bioconjugate Techniques, Third edition, Academic Press, 2013. The contact step can be carried out in an aqueous solution. In some cases, the conjugated label includes an amino functional group and the target molecule includes an activated ester functional group, such as NHS ester or sulfo-NHS ester, or vice versa. In some cases, the conjugated label includes a maleimide functional group and the target molecule includes a thiol functional group, or vice versa. In some cases, the conjugated label includes an alkyne (e.g., cyclooctyne group) functional group and the target molecule includes an azide functional group, or vice versa, which can be conjugated by click chemistry. In some cases, the method includes a separation step, wherein the specific binding members of the product label are separated from the reaction mixture (e.g., excess reagent or unlabeled specific binding members). A variety of methods can be used to separate the target from the sample, such as by immobilization on a support, precipitation, chromatography, and the like.

[0281] In some cases, the method also includes the specific binding members of detection and / or analysis product labeling. In some cases, the method also includes fluorescence detection labeling specific binding members. Any convenient method can be used in conjunction with subject method and composition to detect and / or analyze specific binding members. The method for analyzing the target of interest that can be used for subject method includes but is not limited to flow cytometry, fluorescence microscopy, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assay and fluorescent dye purification chromatography. Interested 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.

[0282] Detection can be directly realized by polymer tandem dye, or indirectly realized by auxiliary detection system. The latter can be based on any one or several different principle combinations, including but not limited to the immunological or non-immunological bridging and signal amplification systems (such as biotin-streptavidin technology, protein A and protein G mediated technology, or nucleic acid probe / anti-nucleic acid probe, etc.) of the anti-species antibody of antibody labeling and other forms. Suitable reporter molecules can be those molecules known in immunocytochemistry, molecular biology, light, fluorescence and electron microscopy, cell immune phenotype, cell sorting, flow cytometry, cell visualization, detection, counting and / or signal output quantitative fields. More than one specific and / or non-specific antibodies can be labeled and used simultaneously or sequentially to enhance target detection, differentiation and / or analysis.

[0283] system

[0284] Aspects of the present invention further include systems for practicing subject methods and compositions. The sample analysis system may include a sample field of view or a flow channel containing a sample and a marker-specific binding member of the present invention, such as described above. In some embodiments, the system is a flow cytometry system, including: a flow cytometer comprising a flow path; a composition in the flow path, wherein the composition comprises: a sample and a marker-specific binding member (e.g., as described herein). In some embodiments, the system for analyzing a sample is a fluorescence microscopy system, including: a fluorescence microscope comprising a sample field of view; and a composition disposed in the sample field of view, wherein the composition comprises a sample; and a marker-specific binding member (e.g., as described herein).

[0285] In certain embodiments of the system, the composition further comprises a second specific binding member that is support-bound and specifically binds to the target analyte. In some cases, the support comprises magnetic particles. Thus, in some cases, the system may further comprise a controllable external paramagnetic field configured to be applied to the assay region of the flow channel.

[0286] The sample may comprise cells. In some cases, the sample is a biological sample containing cells. In some cases, the sample comprises a labeled specific binding member that specifically binds to the target cell. In some cases, the target analyte specifically bound by the specific binding member is a cell surface marker of the cell. In some cases, the cell surface marker is selected from a cell receptor and a cell surface antigen.

[0287] In some cases, the system may further include a light source configured to direct light to the assay region or sample field of view of the flow channel. The system may include a detector configured to receive a signal from the assay region or sample field of view of the flow channel, wherein the signal is provided by the fluorescent composition. Optionally, the sample analysis system may include one or more additional detectors and / or light sources for detecting one or more additional signals.

[0288] In some aspects, the system may further include a computer-based system configured to detect the presence of a fluorescent signal. "Computer-based system" refers to hardware devices, software devices, and data storage devices that are used to analyze the information of the present invention. The minimum hardware of the computer-based system of the present invention includes a central processing unit (CPU), an input device, an output device, and a data storage device. The technician can easily recognize that any currently available computer-based system is suitable for use in the subject system. The data storage device can include any product containing any record of the information of the present invention as described above, or a storage accessor that can access this product.

[0289] "Recording" data, programming, or other information on a computer readable medium refers to the process of storing the information using any such method known in the art. Any convenient data storage structure may be selected based on the means used to access the stored information. Storage may be performed using a variety of data processor programs and formats, such as word processing text files, database formats, etc.

[0290] "Processor" refers to any hardware and / or software combination that performs its required functions. For example, any processor herein may be a programmable digital microprocessor, such as available in the form of an electronic controller, mainframe, server, or personal computer (desktop or portable). Where the processor is programmable, suitable programming may be transmitted to the processor from a remote location, or may be pre-stored in a computer program product (such as 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 a program and may be read by a suitable reader that communicates with each processor at its corresponding site.

[0291] In addition to the sensor device and signal processing module (for example as described above), the system of the present invention may include many additional components, such as data output devices, such as displays and / or speakers, data input devices (such as interface ports, keyboards, etc.), fluid processing components, power supplies, etc.

[0292] In certain aspects, the system comprises a flow cytometer. Suitable flow cytometry systems may include, but are not limited to, Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan; 49 (pt 1): 17-28; Linden, et al., Semin Throm Hemost. 2004 Oct; 30 (5): 502-11; Alison, et al. J Pathol, 2010 Dec; 222 (4): 335-344; and Herbig, et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255; the disclosure of which is incorporated herein by reference. In some cases, flow cytometry systems of interest include BD Biosciences FACSCanto TM Flow cytometer, BD Biosciences 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 TMFlow cytometer, BD Biosciences LSRFortessa TM X-20 flow cytometer, BD Biosciences FACSPresto TM Flow cytometer, BD Biosciences FACSVia TM Flow cytometer and BD Biosciences FACSCalibur TM Cell sorter, BD Biosciences FACSCount TM Cell sorter, BD Biosciences FACSLyric TM Cell sorter, BD Biosciences Via TM Cell sorter, BD Biosciences Influx TM Cell sorter, BD Biosciences Jazz TM Cell sorter, BD Biosciences Aria TM Cell sorter, BD Biosciences FACSAria TM II cell sorter, BD Biosciences FACSAria TM III cell sorter, BD Biosciences FACSAria TM Fusion Cell Sorter and BD Biosciences FACSMelody TM Cell sorter, BD Biosciences FACSymphony TM S6 cell sorter, etc.

[0293] In some embodiments, the subject system is a flow cytometry system, such as U.S. Pat. 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, 9,947,834, and 9,113,967. those described in, for example, 4,084,975,595, 4,095,494, 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, and 4,498,766, the disclosures of which are incorporated herein by reference in their entireties.

[0294] In some cases, the flow cytometry systems of the present invention are configured to image particles in a flowing stream by fluorescence imaging using radio frequency labeled emission (FIRE), such as described in Diebold, et al. Nature Photonics Vol. 7(10); 806-810 (2013) and in U.S. Pat. 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, 10,40 8,758, 10,451,538, 10,620,111 and those described in U.S. Patent Publication Nos. 2017 / 0133857, 2017 / 0328826, 2017 / 0350803, 2018 / 0275042, 2019 / 0376895 and 2019 / 0376894, the disclosures of which are incorporated herein by reference.

[0295] Other systems can also be used to practice the subject method. In some aspects, the system can be a fluorometer or microscope loaded with a sample of the fluorescent composition of any embodiment discussed herein. The fluorometer or microscope can include a light source configured to direct light to the assay area or sample field of view of the flow channel. The fluorometer or microscope can also include a detector configured to receive a signal from the assay area or field of view of the flow channel, wherein the signal is provided by the fluorescent composition.

[0296] Reagent test kit

[0297] Aspects of the present invention further include kits for practicing subject methods and compositions. Compositions of the present invention can be included in kits as reagents or as starting materials, or are provided for, for example, in the above-mentioned methods. Kits can include labeling specific binding members (e.g., as described above) and containers. Any convenient container can be used, such as holes, boxes, bags, heat-insulated containers, etc. in tubes, bottles, or porous strips or plates. The subject kit can also include one or more components and instructions for use selected from primer-specific binding members, support binding specific binding members, cells, support, biocompatible aqueous elution buffer, controls (positive and / or negative) etc. for given target analytes as required. A given kit can include reagents suitable for detecting a single target analyte, or multiple reagents suitable for detecting two or more different target analytes, for example, wherein a given kit is configured to be used for multiple detection applications.

[0298] In certain embodiments, the kit is used to assess the presence of a target analyte (such as an intracellular target) in a sample. Therefore, in some cases, the kit comprises one or more components suitable for permeabilization or lysis of cells. The one or more additional components of the kit can be provided in a separate container (e.g., a separate tube, bottle, or a hole in a porous strip or plate).

[0299] In some cases, the kit further comprises a reagent for performing flow cytometry. Reagents of interest include, but are not limited to, buffer for reconstructing and diluting, buffer for contacting cell samples with chromophores, wash buffer, control cells, control beads, fluorescent beads for flow cytometry calibration, and combinations thereof. The kit may also include one or more cell fixatives, such as paraformaldehyde, glutaraldehyde, methanol, acetone, formalin, or any combination thereof or buffer. In addition, the kit may include a cell permeabilization reagent, such as methanol, acetone, or detergent (e.g., triton, NP-40, saponin, tween 20, digitonin, leucoperm, or any combination thereof or buffer. Other protein transport inhibitors, cell fixation reagents, and cell permeabilization reagents familiar to those skilled in the art are within the scope of the subject kit.

[0300] The composition of the kit can be provided as a liquid composition, such as any suitable buffer. Alternatively, the composition of the kit can be provided as a dry composition (e.g., it can be lyophilized), and the kit can optionally include one or more buffers for reconstructing the dry composition. In some aspects, the kit can include aliquots of the composition, which are provided in separate containers (e.g., separate tubes, bottles, or holes in porous strips or plates).

[0301] In addition, one or more components can be combined into a single container such as a glass or plastic vial, tube or bottle. In some cases, the test kit may further include a container (e.g., a box, bag, insulated container, bottle, tube, etc.) in which all components (and their individual containers) are present. The test kit may further include a package that is separate from or attached to the test kit container and has information about the test kit, components and / or instructions for use of the test kit printed on the package.

[0302] In addition to the above-mentioned components, the subject test kit can further include instructions for practicing the subject method. These instructions can be present in the subject test kit in various forms, one or more of which can be present in the test kit. One of the forms in which these instructions can exist is as packaging, package inserts, etc. of printed information, test kit on suitable medium or substrate (for example, one or more sheets of paper printed with information). Another means can be a computer-readable medium, for example, a floppy disk, CD, DVD, portable flash drive, etc., with information recorded. Another means that can exist is a website address, which can be used to access the information at the removed site by the Internet. Any convenient means can be present in the test kit.

[0303] use

[0304] Green excitable tandem dyes as described herein and marker-specific binding members, compositions, methods and systems comprising the dye can be used for a variety of applications, including diagnostic and research applications, where it is necessary to label, detect and / or analyze a target of interest. Such applications include methods such as cell counting, microscopy, immunoassays (e.g., competitive or non-competitive), assessment of free analytes, assessment of receptor binding ligands. Compositions, systems and methods described herein can be used for analyzing any of a variety of samples, including but not limited to biological fluids, cell culture samples and tissue samples. In some aspects, compositions, systems and methods described herein can be used for methods where analytes (if present) in samples are detected using fluorescent markers, such as fluorescence activated cell sorting or analysis, immunoassays, immunostaining, etc. In some cases, compositions and methods are used for applications where it is interesting to assess the presence of target analytes in samples. In some aspects, green excitable tandem dyes can be excited by a green laser with a wavelength of, for example, 532nm.

[0305] In some cases, the methods and compositions are used in any assay format where there is interest in detecting and / or analyzing a target from a sample, including but not limited to flow cytometry, fluorescence microscopy, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assays, and fluorescent dye purification chromatography. In some cases, the methods and compositions are used in any application where there is interest in fluorescently labeling a target molecule. The subject compositions can be adapted for any convenient application using specific binding member pairs such as biotin-streptavidin and hapten-anti-hapten antibodies.

[0306] The following examples are offered by way of illustration and not limitation.

[0307] Example

[0308] Example 1: Synthesis of tandem dyes

[0309] A summary of some different methods for synthesizing tandem dyes with green excitable donor fluorophores can be found in Steward et al., "Solid Phase Peptide Synthesis", WH Freeman Co., San Francisco, 1969; Bodanszky et al., "Peptide Synthesis", John Wiley & Sons, Second Edition, 1976 and Meienhofer, "Hormonal Proteins and Peptides", Vol. 2, p. 46, Academic Press (New York), 1983; and in Kent, Ann. Rev. Biochem., 57, 957, 1988 for solid phase peptide synthesis and Schroder et al., "The Peptides", Vol. 1, Academic Press (New York), 1965 for solution synthesis. Any convenient protecting group strategy may be used, such as, but not limited to, Fmoc solid phase peptide synthesis and Boc solid phase peptide synthesis strategies. In Boc solid phase peptide synthesis, Boc-amino protecting group is used at amino terminal, and benzyl or benzyl-based or other convenient protecting groups can be used to protect side chain functional groups. In Fmoc solid phase peptide synthesis, Fmoc-amino protecting group is used at amino terminal, and tert-butyl or benzyl-based or other convenient protecting groups can be used to protect side chain functional groups. Convenient protecting groups that can be used for such synthetic methods are described in the above-mentioned references and McOmic "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. Tandem dyes with a green excitable donor fluorophore and different acceptor fluorophores were prepared, namely Tandem 570 (DMX6) (with an acceptor providing 570 nm emission), Tandem 600 (DMX5) (with an acceptor providing 600 nm emission), Tandem 650 (DMX4) (with an acceptor providing 650 nm emission), Tandem 712 (DMX3) (with an acceptor providing 712 nm emission), Tandem 750 (DMX2) (with an acceptor providing 750 nm emission), Tandem 780 (DMX1) (with an acceptor providing 780 nm emission) and Tandem 850 (with an acceptor providing 850 nm emission).

[0310] The tandem dyes of the structures shown below were experimentally synthesized. Such tandem dyes have a non-conjugated polypeptide backbone and a BODIPY-type green excitable donor chromophore. Different acceptor chromophores are attached to obtain embodiments referred to herein as DMX-1 to DMX-6. Polyethylene glycol (PEG) groups can increase water solubility. Antibodies are also attached to the tandem dyes.

[0311]

[0312] wherein n and m are integers, for example 1 to 20, such as 2 to 15, for example 3 to 10, and may be the same or different as desired.

[0313] Implementation Acceptor chromophore DMX-1 Cy7 DMX-2 ATTO725 DMX-3 Cy5.5 DMX-4 Cy5 DMX-5 CF594 DMX-6 Cy3

[0314] Example 2: UV-Visible Absorption Spectra of Tandem Dyes

[0315] UV-2600 spectrophotometer (dual beam, single monochromator Shimadzu Scientific Instruments) is set to collect data from 900nm to 300nm with a slit width of 1.0nm in 1nm increments. The stock solution of each tandem dye is dissolved in 1x phosphate buffered saline (PBS), and 2mL is transferred to the sample cuvette [1cm polymethyl methacrylate cuvette]. Then 2mL of 1x PBS is transferred to the second cuvette. The cuvette containing 1x PBS is placed in the blank cuvette position in the UV-2600, and the tandem dye sample sample cuvette is placed in the sample cuvette position. The instrument absorbance is automatically zeroed at 900nm, and the UV-visible spectrum from 900nm to 300nm is collected.

[0316] Figure 1 The UV-visible absorption spectra of the tandem dyes DMX-1 to DMX-6 are shown. Each tandem dye has a maximum absorption of about 530 nm. For DMX-1, -2, -3, -4 and -5, smaller absorption peaks are also observed at about 770 nm, 740 nm, 690 nm, 650 nm and 610 nm, respectively.

[0317] Example 3: Fluorescence of polymer tandem dyes

[0318] A small amount of stock solution of polymer tandem dye in 1x PBS was diluted to 25 mA.U. in a total volume of 2 mL of 1x PBS. The solution was transferred to a sample cuvette [1 cm polymethyl methacrylate cuvette] and placed in the sample chamber of the fluorometer. The sample was excited with a 532 nm green laser and the emission scan range was set to 500 nm to 900 nm. For signal detection, both the fluorescence signal (S1) and the instrument noise (R1) were recorded. The correction file applied to the raw output signal converts the S1 and R1 signals into corrected fluorescence (S1c) and corrected noise (R1c). The corrected fluorescence S1c was then normalized according to the corrected noise to obtain the final reported fluorescence emission of the sample.

[0319] Figure 2 Normalized emission spectra of polymer tandem dyes DMX-1 to DMX-6 when excited by a 532 nm green laser are shown. For embodiments DMX-1, -2, -3, -4, -5, and -6, the maximum emission is approximately 780 nm, 740 nm, 700 nm, 665 nm, 625 nm, and 560 nm, respectively.

[0320] Figure 3 Shown are overlays of the normalized absorption (ie, excitation) spectra of samples DMX-1 to DMX-6 and their corresponding normalized emission spectra.

[0321] Example 4: Fluorescence of polymer tandem dyes

[0322] Additional polymer tandem dyes were constructed and tested as described above. Figure 4A and 4B Results are presented for tandem dyes with donors and acceptors: a BODIPY-type donor with green light absorption and acceptors with various different emission spectra. Figure 5A and 5B Results are presented for tandem dyes with the following donors and acceptors: a rhodamine-type donor with green light absorption and acceptors with various different emission spectra. Figures 6A to 6G Results are presented for tandem dyes with various donors that absorb green light and acceptors with various different emission spectra.

[0323] Notwithstanding the appended claims, the present disclosure is also defined by the following clauses:

[0324] 1. A tandem dye comprising:

[0325] Non-conjugated polymer backbone;

[0326] one or more green excitable donor fluorophores attached to the non-conjugated polymer backbone; and

[0327] one or more acceptor fluorophores attached to the non-conjugated polymer backbone,

[0328] Wherein the donor fluorophore and the acceptor fluorophore are in an energy transfer relationship.

[0329] 2. The tandem dye according to item 1, wherein the one or more green excitable donor fluorophores have an absorption maximum between 500 nm and 565 nm.

[0330] 3. The tandem dye according to item 2, wherein the one or more green excitable donor fluorophores have an absorption maximum between 520 nm and 545 nm.

[0331] 4. The tandem dye according to any one of items 1 to 3, wherein the one or more green excitable donor fluorophores are dipyrromethene-based fluorophores.

[0332] 5. The tandem dye according to any one of items 1 to 4, wherein the dipyrromethene-based dye is a dipyrromethene-boron-based fluorophore.

[0333] 6. The tandem dye according to item 5, wherein the dipyrromethene-boron based fluorophore has formula (I):

[0334]

[0335] in:

[0336] R 1 -R 7 Each is independently selected from H, alkyl, halogen, substituted alkyl, alkoxy, substituted alkoxy, amino, substituted amino, amide, substituted amide, thiol, substituted thiol, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG) and -T 1 -J 1 ,or

[0337] Optionally, selected from R 6 and R 7 , R 2 and R 3 , R 5 and R 6 , R 3 and R 4 , R 2 and R 1 and R 7 and R 1Any one or more pairs of substituents in the group together form a divalent group and are annularly linked and, together with the carbon atoms to which they are bound, provide a 5- or 6-membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring (e.g., a 5- or 6-membered ring comprising carbon atoms and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, a water-soluble group (WSG) and -T 1 -J 1 Substituents are substituted;

[0338] T 1 is an optional linker;

[0339] J 1 is a repeating unit of the compound of formula (I); and

[0340] Y 1 and Y 2 is independently selected from F, OH, H, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and a water-soluble group (WSG), or

[0341] Y 1 and Y 2 Together with the boron atom to which they are bound, they form a 5- or 6-membered ring (e.g., a 5- or 6-membered ring comprising a boron atom, a carbon atom, and 0-3 heteroatoms selected from O, S, and N), which ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, a water-soluble group (WSG), and -T 1 -Z 1 substituted by a substituent.

[0342] 7. The tandem dye according to any one of items 1 to 6, wherein the one or more green excitable donor fluorophores are xanthene-based fluorophores.

[0343] 8. The tandem dye according to item 7, wherein one or more xanthene-based fluorophores have formula (II) or are salts thereof:

[0344]

[0345] in:

[0346] Each R 21 and R 22independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carbonyl, carboxyl, cyano, ether, halogen, hydroxyl, imine, and nitro;

[0347] R 23 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carboxyl, cyano, ether, halogen, hydroxyl, and nitro; and

[0348] a and b are each independently an integer of 0 to 4.

[0349] 9. The tandem dye according to any one of items 7-8, wherein one or more xanthene-based fluorophores are fluorescein-based fluorophores.

[0350] 10. The tandem dye according to item 9, wherein one or more fluorescein-based fluorophores have formula (III) or are salts thereof:

[0351]

[0352] in:

[0353] Each R 31 , R 32 and R 33 independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carboxyl, cyano, ether, halogen, hydroxyl, and nitro;

[0354] R 34 and R 35 each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

[0355] c and d are each independently an integer from 0 to 3;

[0356] e is an integer from 0 to 4.

[0357] 11. The tandem dye according to any one of items 7 to 10, wherein one or more xanthene-based fluorophores is a rhodamine-based fluorophore.

[0358] 12. The tandem dye according to item 11, wherein the one or more rhodamine-based fluorophores have formula (IV) or are salts thereof:

[0359]

[0360] in:

[0361] Each R 41 , R 42 and R 43 independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carboxyl, cyano, ether, halogen, hydroxyl, and nitro;

[0362] Each R 44 and R 45 independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carboxyl, cyano, ether, halogen, hydroxyl, and nitro;

[0363] X - It is an anion;

[0364] f and g are each independently an integer from 0 to 3;

[0365] h is an integer from 0 to 5.

[0366] 13. The tandem dye according to any one of items 1 to 12, wherein the one or more green excitable donor fluorophores each independently comprise a water-soluble group.

[0367] 14. The tandem dye according to item 13, wherein the water-soluble group comprises a non-ionic water-soluble group.

[0368] 15. The tandem dye according to item 14, wherein the non-ionic water-soluble group comprises a polyalkylene glycol.

[0369] 16. The tandem dye according to item 15, wherein the polyalkylene glycol comprises polyethylene glycol.

[0370] 17. The tandem dye according to any one of items 1 to 16, wherein the one or more acceptor fluorophores have an emission maximum between 550 and 850 nm.

[0371] 18. The tandem dye according to any one of items 1 to 17, wherein the one or more acceptor fluorophores are selected from coumarin dyes, cyanine dyes, polymethines, naphthylimides, thiazine dyes and acridine dyes.

[0372] 19. The tandem dye according to any one of items 1 to 18, wherein the number of donor fluorophores exceeds the number of acceptor fluorophores.

[0373] 20. The tandem dye according to any one of items 1 to 19, wherein the number of donor fluorophores ranges from 2 to 20.

[0374] 21. The tandem dye according to any one of items 1 to 20, wherein the tandem dye comprises a single acceptor fluorophore.

[0375] 22. The tandem dye according to any one of items 1 to 21, wherein the tandem dye is linked to a biomolecule.

[0376] 23. The tandem dye according to item 22, wherein the biomolecule is selected from the group consisting of carbohydrates, lipids, nucleic acids and proteins.

[0377] 24. The tandem dye according to any one of items 22-23, wherein the biomolecule is a specific binding member.

[0378] 25. The tandem dye according to item 24, wherein the specific binding member is an antibody or a binding fragment or derivative thereof.

[0379] 26. The tandem dye according to any one of items 1 to 25, wherein the tandem dye is linked to a chemoselective group.

[0380] 27. The tandem dye according to any one of items 1 to 26, wherein the non-conjugated polymer backbone comprises a peptide.

[0381] 28. The tandem dye according to item 27, wherein the peptide comprises 5 to 100 amino acid residues.

[0382] 29. A method of assessing the presence of a target analyte in a sample, the method comprising:

[0383] (a) contacting the sample with a labeled specific binding member that specifically binds to the target analyte to produce a labeled sample, wherein the labeled specific binding member comprises a specific binding member conjugated to a tandem dye, the tandem dye comprising:

[0384] Non-conjugated polymer backbone;

[0385] one or more green excitable donor fluorophores attached to the non-conjugated polymer backbone; and

[0386] one or more acceptor fluorophores attached to the non-conjugated polymer backbone,

[0387] wherein the donor fluorophore and the acceptor fluorophore are in an energy transfer relationship; and

[0388] (b) assaying the labeled composition for the presence of a labeled specific binding member-target analyte binding complex to assess the presence or absence of the target analyte in the sample.

[0389] 30. The method according to item 29, wherein the one or more green excitable donor fluorophores have an absorption maximum between 500 nm and 565 nm.

[0390] 31. The method according to item 30, wherein the one or more green excitable donor fluorophores have an absorption maximum between 520 nm and 545 nm.

[0391] 32. The method according to any one of items 29 to 31, wherein the one or more green excitable donor fluorophores are dipyrromethene-based fluorophores.

[0392] 33. The method according to item 32, wherein the dipyrromethene-based dye is a dipyrromethene-boron-based fluorophore.

[0393] 34. The method according to item 33, wherein the dipyrromethene-boron based fluorophore has formula (I):

[0394]

[0395] in:

[0396] R 1 -R 7 Each is independently selected from H, alkyl, halogen, substituted alkyl, alkoxy, substituted alkoxy, amino, substituted amino, amide, substituted amide, thiol, substituted thiol, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG) and -T 1 -J 1 ,or

[0397] Optionally, selected from R 6 and R 7 , R 2 and R 3 , R 5 and R 6 , R 3 and R 4 , R 2 and R1 and R 7 and R 1 Any one or more pairs of substituents in the group together form a divalent group and are annularly linked and, together with the carbon atoms to which they are bound, provide a 5- or 6-membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring (e.g., a 5- or 6-membered ring comprising carbon atoms and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, a water-soluble group (WSG) and -T 1 -J 1 Substituents are substituted;

[0398] T 1 is an optional linker;

[0399] J 1 is a repeating unit of the compound of formula (I); and

[0400] Y 1 and Y 2 is independently selected from F, OH, H, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and a water-soluble group (WSG), or

[0401] Y 1 and Y 2 Together with the boron atom to which they are bound, they form a 5- or 6-membered ring (e.g., a 5- or 6-membered ring comprising a boron atom, a carbon atom, and 0-3 heteroatoms selected from O, S, and N), which ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, a water-soluble group (WSG), and -T 1 -Z 1 substituted by a substituent.

[0402] 35. The method according to any one of items 29 to 34, wherein the one or more green excitable donor fluorophores are xanthene-based fluorophores.

[0403] 36. The method according to item 35, wherein the one or more xanthene-based fluorophores have formula (II) or are salts thereof:

[0404]

[0405] in:

[0406] Each R 21 and R22 independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carbonyl, carboxyl, cyano, ether, halogen, hydroxyl, imine, and nitro;

[0407] R 23 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carboxyl, cyano, ether, halogen, hydroxyl, and nitro; and

[0408] a and b are each independently an integer of 0 to 4.

[0409] 37. The method according to any one of items 35-36, wherein the one or more xanthene-based fluorophores are fluorescein-based fluorophores.

[0410] 38. The method according to item 37, wherein the one or more fluorescein-based fluorophores have formula (III) or a salt thereof:

[0411]

[0412] in:

[0413] Each R 31 , R 32 and R 33 independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carboxyl, cyano, ether, halogen, hydroxyl, and nitro;

[0414] R 34 and R 35 each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

[0415] c and d are each independently an integer from 0 to 3;

[0416] e is an integer from 0 to 4.

[0417] 39. The method according to any one of items 35 to 38, wherein the one or more xanthene-based fluorophores are rhodamine-based fluorophores.

[0418] 40. The method according to item 39, wherein the one or more rhodamine-based fluorophores have formula (IV) or are salts thereof:

[0419]

[0420] in:

[0421] Each R 41 , R 42 and R 43 independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carboxyl, cyano, ether, halogen, hydroxyl, and nitro;

[0422] Each R 44 and R 45 independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carboxyl, cyano, ether, halogen, hydroxyl, and nitro;

[0423] X - It is an anion;

[0424] f and g are each independently an integer from 0 to 3;

[0425] h is an integer from 0 to 5.

[0426] 41. The method according to any one of items 29-40, wherein the one or more green excitable donor fluorophores each independently comprise a water soluble group.

[0427] 42. The method according to item 41, wherein the water-soluble group comprises a non-ionic water-soluble group.

[0428] 43. The method according to item 42, wherein the non-ionic water-soluble group comprises a polyalkylene glycol.

[0429] 44. The method according to item 43, wherein the polyalkylene glycol comprises polyethylene glycol.

[0430] 45. The method according to any one of items 29-44, wherein the one or more acceptor fluorophores have an emission maximum between 550 and 850 nm.

[0431] 46. ​​The method according to any one of items 29 to 45, wherein the one or more acceptor fluorophores are selected from coumarin dyes, cyanine dyes, polymethines, naphthylimides, thiazine dyes and acridine dyes.

[0432] 47. The method according to any one of items 29 to 46, wherein the number of donor fluorophores exceeds the number of acceptor fluorophores.

[0433] 48. The method according to any one of items 29-47, wherein the number of donor fluorophores ranges from 2 to 20.

[0434] 49. The method according to any one of items 29 to 48, wherein the tandem dye comprises a single acceptor fluorophore.

[0435] 50. The method according to any one of items 29 to 49, wherein the tandem dye is linked to a biomolecule.

[0436] 51. The method according to item 50, wherein the biomolecule is selected from the group consisting of carbohydrates, lipids, nucleic acids and proteins.

[0437] 52. The method according to any of items 50-51, wherein the biomolecule is a specific binding member.

[0438] 53. The method according to item 52, wherein the specific binding member is an antibody or a binding fragment or derivative thereof.

[0439] 54. The method according to any one of items 29 to 53, wherein the tandem dye is linked to a chemoselective group.

[0440] 55. The method according to any one of items 29 to 54, wherein the non-conjugated polymer backbone comprises a peptide.

[0441] 56. The method according to item 55, wherein the peptide comprises 5 to 100 amino acid residues.

[0442] 57. The method according to any one of items 29 to 56, further comprising contacting the sample with a second specific binding member that is support-bound and specifically binds to the target analyte.

[0443] 58. The method according to item 57, wherein the support comprises magnetic particles.

[0444] 59. The method according to any one of items 57-58, wherein the target analyte is associated with a cell.

[0445] 60. The method according to item 59, wherein said target analyte is a cell surface marker of said cell.

[0446] 61. The method according to item 60, wherein said cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen.

[0447] 62. The method according to any one of items 29-59, wherein the target analyte is an intracellular target.

[0448] 63. The method according to item 62, wherein the method further comprises permeabilizing or lysing the cells.

[0449] 64. The method according to any one of items 29 to 63, wherein said determining comprises analyzing said labeled sample by flow cytometry.

[0450] 65. A method for synthesizing a fluorescently labeled tandem dye, the method comprising:

[0451] preparing a precursor non-conjugated polymer backbone comprising a first type of residue, a second type of residue, and a third type of residue having first, second, and third orthogonal reactive side chains, respectively; and

[0452] conjugating a green excitable donor fluorophore to any first reactive side chain of the precursor non-conjugated polymer backbone, conjugating an acceptor fluorophore to any second reactive side chain of the precursor non-conjugated polymer backbone, and conjugating a biomolecule to any third reactive side chain of the precursor non-conjugated polymer backbone;

[0453] To produce the fluorescently labeled polymer tandem dye.

[0454] 66. The method according to item 65, wherein the one or more green excitable donor fluorophores have an absorption maximum between 500 nm and 565 nm.

[0455] 67. The method according to item 66, wherein the one or more green excitable donor fluorophores have an absorption maximum between 520 nm and 545 nm.

[0456] 68. The method according to any one of items 65-67, wherein the one or more green excitable donor fluorophores are dipyrromethene-based fluorophores.

[0457] 69. The method according to item 68, wherein the dipyrromethene-based dye is a dipyrromethene-boron-based fluorophore.

[0458] 70. The method according to item 69, wherein the dipyrromethene-boron based fluorophore has formula (I):

[0459]

[0460] in:

[0461] R 1 -R 7 Each is independently selected from H, alkyl, halogen, substituted alkyl, alkoxy, substituted alkoxy, amino, substituted amino, amide, substituted amide, thiol, substituted thiol, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG) and -T 1 -J 1 ,or

[0462] Optionally, selected from R 6 and R 7 , R 2 and R 3 , R 5 and R 6 , R 3 and R 4 , R 2 and R 1 and R 7 and R 1 Any one or more pairs of substituents in the group together form a divalent group and are annularly linked and, together with the carbon atoms to which they are bound, provide a 5- or 6-membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring (e.g., a 5- or 6-membered ring comprising carbon atoms and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, a water-soluble group (WSG) and -T 1 -J 1 Substituents are substituted;

[0463] T 1 is an optional linker;

[0464] J 1 is a repeating unit of the compound of formula (I); and

[0465] Y 1 and Y 2 is independently selected from F, OH, H, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and a water-soluble group (WSG), or

[0466] Y 1 and Y2 Together with the boron atom to which they are bound, they form a 5- or 6-membered ring (e.g., a 5- or 6-membered ring comprising a boron atom, a carbon atom, and 0-3 heteroatoms selected from O, S, and N), which ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, a water-soluble group (WSG), and -T 1 -Z 1 substituted by a substituent.

[0467] 71. The method according to any one of items 65-70, wherein the one or more green excitable donor fluorophores are xanthene-based fluorophores.

[0468] 72. The method according to item 71, wherein the one or more xanthene-based fluorophores have formula (II) or are salts thereof:

[0469]

[0470] in:

[0471] Each R 21 and R 22 independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carbonyl, carboxyl, cyano, ether, halogen, hydroxyl, imine, and nitro;

[0472] R 23 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carboxyl, cyano, ether, halogen, hydroxyl, and nitro; and

[0473] a and b are each independently an integer of 0 to 4.

[0474] 73. The method according to any one of items 71-72, wherein the one or more xanthene-based fluorophores are fluorescein-based fluorophores.

[0475] 74. The method according to item 73, wherein the one or more fluorescein-based fluorophores have formula (III) or a salt thereof:

[0476]

[0477] in:

[0478] Each R 31 , R 32 and R 33 independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carboxyl, cyano, ether, halogen, hydroxyl, and nitro;

[0479] R 34 and R 35 each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

[0480] c and d are each independently an integer from 0 to 3;

[0481] e is an integer from 0 to 4.

[0482] 75. The method according to any one of items 71 to 74, wherein the one or more xanthene-based fluorophores are rhodamine-based fluorophores.

[0483] 76. The method according to item 75, wherein the one or more rhodamine-based fluorophores have formula (IV) or are salts thereof:

[0484]

[0485] in:

[0486] Each R 41 , R 42 and R 43 independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carboxyl, cyano, ether, halogen, hydroxyl, and nitro;

[0487] Each R 44 and R 45 independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carboxyl, cyano, ether, halogen, hydroxyl, and nitro;

[0488] X - It is an anion;

[0489] f and g are each independently an integer from 0 to 3;

[0490] h is an integer from 0 to 5.

[0491] 77. The method according to any one of items 65-76, wherein the one or more green excitable donor fluorophores each independently comprise a water soluble group.

[0492] 78. The method according to item 77, wherein the water-soluble groups comprise non-ionic water-soluble groups.

[0493] 79. The method according to item 78, wherein the non-ionic water-soluble group comprises a polyalkylene glycol.

[0494] 80. The method according to item 79, wherein the polyalkylene glycol comprises polyethylene glycol.

[0495] 81. The method according to any one of items 65-80, wherein the one or more acceptor fluorophores have an emission maximum between 550 and 850 nm.

[0496] 82. The method according to any one of items 65 to 81, wherein the one or more acceptor fluorophores are selected from coumarin dyes, cyanine dyes, polymethines, naphthylimides, thiazine dyes and acridine dyes.

[0497] 83. The method according to any one of items 65-82, wherein the number of donor fluorophores exceeds the number of acceptor fluorophores.

[0498] 84. The method according to any one of items 65-83, wherein the number of donor fluorophores ranges from 2 to 20.

[0499] 85. The method according to any one of items 65 to 84, wherein the tandem dye comprises a single acceptor fluorophore.

[0500] 86. The method according to any one of items 65 to 85, wherein the tandem dye is linked to a biomolecule.

[0501] 87. The method according to item 86, wherein the biomolecule is selected from the group consisting of carbohydrates, lipids, nucleic acids and proteins.

[0502] 88. Method according to any of items 86-87, wherein said biomolecule is a specific binding member.

[0503] 89. The method according to item 88, wherein the specific binding member is an antibody or a binding fragment or derivative thereof.

[0504] 90. The method according to any one of items 65 to 89, wherein the tandem dye is linked to a chemoselective group.

[0505] 91. The method according to any one of items 65 to 90, wherein the non-conjugated polymer backbone comprises a peptide.

[0506] 92. The method according to item 91, wherein the peptide comprises 5 to 100 amino acid residues.

[0507] 93. A kit comprising:

[0508] A tandem dye, the tandem dye comprising:

[0509] Non-conjugated polymer backbone;

[0510] one or more green excitable donor fluorophores attached to the non-conjugated polymer backbone; and one or more acceptor fluorophores attached to the non-conjugated polymer backbone,

[0511] Wherein the donor fluorophore and the acceptor fluorophore are in an energy transfer relationship.

[0512] 94. The kit according to item 93, wherein the one or more green excitable donor fluorophores have an absorption maximum between 500 nm and 565 nm.

[0513] 95. The kit according to item 94, wherein the one or more green excitable donor fluorophores have an absorption maximum between 520 nm and 545 nm.

[0514] 96. The kit according to any one of items 93 to 95, wherein the one or more green excitable donor fluorophores are dipyrromethene-based fluorophores.

[0515] 97. The kit according to item 96, wherein the dipyrromethene-based dye is a dipyrromethene-boron-based fluorophore.

[0516] 98. The kit according to item 97, wherein the dipyrromethene-boron based fluorophore has formula (I):

[0517]

[0518] in:

[0519] R 1 -R 7Each is independently selected from H, alkyl, halogen, substituted alkyl, alkoxy, substituted alkoxy, amino, substituted amino, amide, substituted amide, thiol, substituted thiol, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG) and -T 1 -J 1 ,or

[0520] Optionally, selected from R 6 and R 7 , R 2 and R 3 , R 5 and R 6 , R 3 and R 4 , R 2 and R 1 and R 7 and R 1 Any one or more pairs of substituents in the group together form a divalent group and are annularly linked and, together with the carbon atoms to which they are bound, provide a 5- or 6-membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring (e.g., a 5- or 6-membered ring comprising carbon atoms and 0-3 heteroatoms selected from O, S and N), which ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, a water-soluble group (WSG) and -T 1 -J 1 Substituents are substituted;

[0521] T 1 is an optional linker;

[0522] J 1 is a repeating unit of the compound of formula (I); and

[0523] Y 1 and Y 2 is independently selected from F, OH, H, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and a water-soluble group (WSG), or

[0524] Y 1 and Y 2Together with the boron atom to which they are bound, they form a 5- or 6-membered ring (e.g., a 5- or 6-membered ring comprising a boron atom, a carbon atom, and 0-3 heteroatoms selected from O, S, and N), which ring may be unsubstituted or further independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, a water-soluble group (WSG), and -T 1 -Z 1 substituted by a substituent.

[0525] 99. The kit according to any one of items 93 to 98, wherein the one or more green excitable donor fluorophores are xanthene-based fluorophores.

[0526] 100. The kit according to item 99, wherein the one or more xanthene-based fluorophores have formula (II) or are salts thereof:

[0527]

[0528] in:

[0529] Each R 21 and R 22 independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carbonyl, carboxyl, cyano, ether, halogen, hydroxyl, imine, and nitro;

[0530] R 23 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carboxyl, cyano, ether, halogen, hydroxyl, and nitro; and

[0531] a and b are each independently an integer from 0 to 4.

[0532] 101. The kit according to any of items 99 to 100, wherein the one or more xanthene-based fluorophores are fluorescein-based fluorophores.

[0533] 102. The kit according to item 101, wherein the one or more fluorescein-based fluorophores have formula (III) or are salts thereof:

[0534]

[0535] in:

[0536] Each R 31 , R 32 and R 33 independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carboxyl, cyano, ether, halogen, hydroxyl, and nitro;

[0537] R 34 and R 35 each independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl;

[0538] c and d are each independently an integer from 0 to 3;

[0539] e is an integer from 0 to 4.

[0540] 103. The kit according to any one of items 99 to 102, wherein the one or more xanthene-based fluorophores are rhodamine-based fluorophores.

[0541] 104. The kit according to item 103, wherein the one or more rhodamine-based fluorophores have formula (IV) or are salts thereof:

[0542]

[0543] in:

[0544] Each R 41 , R 42 and R 43 independently selected from alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carboxyl, cyano, ether, halogen, hydroxyl, and nitro;

[0545] Each R 44 and R 45 independently selected from H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, substituted heterocyclyl, acyl, alkoxy, amino, azido, carboxyl, cyano, ether, halogen, hydroxyl, and nitro;

[0546] X - It is an anion;

[0547] f and g are each independently an integer from 0 to 3;

[0548] h is an integer from 0 to 5.

[0549] 105. The kit according to any one of items 93 to 104, wherein the one or more green excitable donor fluorophores each independently comprise a water soluble group.

[0550] 106. The kit according to item 105, wherein the water-soluble group comprises a non-ionic water-soluble group.

[0551] 107. The kit according to item 106, wherein the non-ionic water-soluble group comprises a polyalkylene glycol.

[0552] 108. The kit according to item 107, wherein the polyalkylene glycol comprises polyethylene glycol.

[0553] 109. The kit according to any of items 93 to 108, wherein the one or more acceptor fluorophores have an emission maximum between 550 and 850 nm.

[0554] 110. The kit according to any of items 93 to 109, wherein the one or more acceptor fluorophores are selected from the group consisting of coumarin dyes, cyanine dyes, polymethines, naphthylimides, thiazine dyes and acridine dyes.

[0555] 111. The kit according to any of items 93 to 110, wherein the number of donor fluorophores exceeds the number of acceptor fluorophores.

[0556] 112. The kit according to any of items 93 to 111, wherein the number of donor fluorophores ranges from 2 to 20.

[0557] 113. The kit according to any one of items 93 to 112, wherein the tandem dye comprises a single acceptor fluorophore.

[0558] 114. The kit according to any of items 93 to 113, wherein the tandem dye is linked to a biomolecule.

[0559] 115. The kit according to item 114, wherein the biomolecule is selected from the group consisting of carbohydrates, lipids, nucleic acids and proteins.

[0560] 116. The kit according to any of items 113-114, wherein the biomolecule is a specific binding member.

[0561] 117. The kit according to item 116, wherein the specific binding member is an antibody or a binding fragment or derivative thereof.

[0562] 118. The kit according to any one of items 93 to 117, wherein the tandem dye is linked to a chemoselective group.

[0563] 119. The kit according to any one of items 93 to 118, wherein said non-conjugated polymer backbone comprises a peptide.

[0564] 120. The kit according to item 119, wherein said peptide comprises 5 to 100 amino acid residues.

[0565] In at least some previously described embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, unless such replacement is technically infeasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the above methods and structures without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter defined by the appended claims.

[0566] Those skilled in the art will appreciate that, in general, the terms used herein, and particularly in the appended claims (e.g., the bodies of the appended claims), are generally intended to be "open" terms (e.g., the term "including" should be interpreted as "including but not limited to", the term "having" should be interpreted as "having at least", the term "comprising" should be interpreted as "including but not limited to", etc.). Those skilled in the art will also appreciate that if a specific number of claim recitations is intended to be introduced, such an intent will be explicitly recited in the claim, and in the absence of such a recitation, such an intent is not present. For example, to aid understanding, the following appended claims may contain the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be interpreted as implying that the introduction of a claim recitation by the indefinite article "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"); the same applies to the use of definite articles to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, one skilled in the art will recognize that such recitation should be interpreted as indicating at least the recited number (e.g., the mere recitation "two recitations" without other modifiers means at least two recitations, or two or more recitations). In addition, in those cases where a convention similar to "at least one of A, B, and C, etc." is used, generally, such construction is intended that one skilled in the art will understand the meaning of the convention (e.g., "a system having at least one of A, B, and C" will include, but is not limited to, systems including A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In addition, in those cases where a convention similar to "at least one of A, B, or C, etc." is used, generally, such construction is intended that one skilled in the art will understand the meaning of the convention (e.g., "a system having at least one of A, B, or C" will include, but is not limited to, systems including A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art will also appreciate that virtually any transitional word and / or phrase presenting two or more alternative terms, whether in the specification, claims or drawings, should be understood to contemplate the possibility of including one, either or both terms. For example, the phrase "A or B" will be understood to include the possibility of "A" or "B" or "A and B".

[0567] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0568] As will be understood by those skilled in the art, for any and all purposes, for example, in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily considered to be fully described and enables the same range to be decomposed into at least equal halves, one-third, one-quarter, one-fifth, one-tenth, etc. As a non-limiting example, each range discussed herein can be easily decomposed into a lower third, a middle third, and an upper third, etc. As will also be understood by those skilled in the art, all such as "until", "at least", "greater than", "less than" and similar languages ​​include the numbers listed and refer to the ranges that can be subsequently decomposed into sub-ranges as described above. Finally, as will be understood by those skilled in the art, the range includes each individual member. Therefore, for example, a group with 1-3 articles refers to a group with 1, 2 or 3 articles. Similarly, a group with 1-5 articles refers to a group with 1, 2, 3, 4 or 5 articles, and so on.

[0569] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to one skilled in the art in light of the teachings of this invention that certain changes and modifications may be made without departing from the spirit or scope of the appended claims.

[0570] Therefore, the foregoing only illustrates the principles of the present invention.It should be understood that those skilled in the art will be able to think of various arrangements, although not explicitly described or shown herein, these arrangements embody the principles of the present invention and are included in its spirit and scope.In addition, all examples and conditional language recorded herein are mainly intended to help readers understand the principles of the present invention and the concepts contributed by the inventors to promote this area, and should be interpreted as not being limited to such specific examples and conditions of recording.In addition, all statements of the principles, aspects and embodiments of the present invention and their specific examples recorded herein are intended to cover their structural and functional equivalents.In addition, such equivalents are intended to include currently known equivalents and equivalents developed in the future, that is, no matter how the structure, any element of the developed performance of the same function.In addition, any content disclosed herein is not intended to be dedicated to the public, regardless of whether such disclosures are clearly recorded in the claims.

[0571] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Instead, the scope and spirit of the present invention are embodied by the appended claims. In the claims, 35 U.S.C. § 112 (f) or 35 U.S.C. § 112 (6) is expressly defined as being cited for such a limitation in a claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of a certain limitation in a claim; if such exact phrase is not used in the limitation in a claim, 35 U.S.C. § 112 (f) or 35 U.S.C. § 112 (6) is not cited.

Claims

1. A tandem dye comprising: Non-conjugated polymer backbone; one or more green excitable donor fluorophores attached to the non-conjugated polymer backbone; and one or more acceptor fluorophores attached to the non-conjugated polymer backbone, Wherein the donor fluorophore and the acceptor fluorophore are in an energy transfer relationship.

2. The tandem dye of claim 1, wherein the one or more green excitable donor fluorophores are dipyrromethene-based fluorophores, xanthene-based fluorophores, or rhodamine-based fluorophores.

3. The tandem dye according to any one of claims 1-2, wherein the one or more green excitable donor fluorophores each independently comprise a water-soluble group.

4. The tandem dye according to any one of claims 1 to 3, wherein the one or more acceptor fluorophores have an emission maximum between 550 and 850 nm.

5. The tandem dye according to any one of claims 1 to 4, wherein the one or more acceptor fluorophores are selected from the group consisting of coumarin dyes, cyanine dyes, polymethines, naphthylimides, thiazine dyes and acridine dyes.

6. The tandem dye according to any one of claims 1 to 5, wherein the number of donor fluorophores exceeds the number of acceptor fluorophores.

7. The tandem dye according to any one of claims 1 to 6, wherein the number of donor fluorophores ranges from 2 to 20.

8. The tandem dye of any one of claims 1 to 7, wherein the tandem dye comprises a single acceptor fluorophore.

9. The tandem dye according to any one of claims 1 to 8, wherein the tandem dye is linked to a biomolecule.

10. The tandem dye of claim 9, wherein the biomolecule is a specific binding member.

11. The tandem dye of claim 10, wherein the specific binding member is an antibody or a binding fragment or derivative thereof.

12. The tandem dye according to any one of claims 1 to 11, wherein the non-conjugated polymer backbone comprises a peptide.

13. A method of assessing the presence of a target analyte in a sample, the method comprising: (a) contacting the sample with a labeled specific binding member that specifically binds to the target analyte to produce a labeled sample, wherein the labeled specific binding member comprises a specific binding member conjugated to a tandem dye, the tandem dye comprising: Non-conjugated polymer backbone; one or more green excitable donor fluorophores attached to the non-conjugated polymer backbone; and one or more acceptor fluorophores attached to the non-conjugated polymer backbone, wherein the donor fluorophore and the acceptor fluorophore are in an energy transfer relationship; as well as (b) assaying the labeled composition for the presence of a labeled specific binding member-target analyte binding complex to assess the presence or absence of the target analyte in the sample.

14. A method for synthesizing a fluorescently labeled tandem dye, the method comprising: preparing a precursor non-conjugated polymer backbone comprising a first type of residue, a second type of residue, and a third type of residue having first, second, and third orthogonal reactive side chains, respectively; and conjugating a green excitable donor fluorophore to any first reactive side chain of the precursor non-conjugated polymer backbone, conjugating an acceptor fluorophore to any second reactive side chain of the precursor non-conjugated polymer backbone, and conjugating a biomolecule to any third reactive side chain of the precursor non-conjugated polymer backbone; To generate fluorescently labeled polymer tandem dyes.

15. A kit comprising: A tandem dye, the tandem dye comprising: Non-conjugated polymer backbone; one or more green excitable donor fluorophores attached to the non-conjugated polymer backbone; and one or more acceptor fluorophores attached to the non-conjugated polymer backbone, Wherein the donor fluorophore and the acceptor fluorophore are in an energy transfer relationship.

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