Water-soluble polymer dyes

Abstract

Water soluble polymeric dyes and polymeric tandem dyes are provided. The present invention relates to a polymer dye comprising a hydrosolvated light harvesting multichromophore having a conjugated segment of an aryl or heteroaryl comonomer, the aryl or heteroaryl comonomer comprising a branched nonionic water soluble group (WSG), the branched nonionic water soluble group (WSG) comprising two or more water soluble polymers. Specific binding members comprising aggregation resistant labels of the subject water soluble polymer dyes are also provided. Also provided are methods of assessing a sample for the presence of a target analyte and methods of labeling a target molecule using the subject polymeric dyes. Systems and kits for practicing the subject methods are also provided.

Description

[0001] This application is a divisional application of the Chinese patent application with the application date of December 11, 2017, application number 201780068448.9, and invention name “Water-soluble polymer dye”. Technical Field

[0002] The present application relates to the field of polymer dyes, in particular to the field of water-soluble polymer dyes and polymer tandem dyes. Background Art

[0003] Fluorescent dyes are compounds that emit (usually) different wavelengths of light when illuminated by the wavelengths they absorb. Fluorescent dyes can be used for multiple applications in biochemistry, biology and medicine, for example, for diagnostic kits, microscopy or drug screening. Fluorescent dyes are characterized by allowing users to select many parameters of suitable dyes according to the desired purpose. Parameters of interest include excitation wavelength maximum, emission wavelength maximum, Stokes shift, extinction coefficient, fluorescence quantum yield and fluorescence lifetime. Dyes can be selected according to applications of interest, so as to, for example, allow excitation radiation to penetrate into biological samples, minimize background fluorescence and / or reach a 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. Summary of the invention

[0005] Water-soluble polymer dyes and polymer tandem dyes are provided. The polymer dye includes a water-solvated light-harvesting multichromophore, the water-solvated light-harvesting multichromophore has a conjugated segment of an aryl or heteroaryl comonomer, the aryl or heteroaryl comonomer includes a branched nonionic water-soluble group (WSG), and the branched nonionic water-soluble group (WSG) includes two or more water-soluble polymers. In some cases, the branched nonionic water-soluble group (WSG) of the present disclosure can give the multichromophore a solubility in water exceeding 50 mg / mL. The polymer tandem dye also includes a signal chromophore covalently linked to the multichromophore, and the multichromophore is in energy receiving proximity to the signal chromophore. A specific binding member comprising a label that resists aggregation of the subject water-soluble polymer dye is also provided. A method for evaluating a sample for the presence of a target analyte and a method for labeling a target molecule using a subject polymer dye are also provided. A system and a kit for practicing the subject method are also provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] It should be understood that the drawings described below are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0007] Figure 1 Several exemplary comonomers of the present disclosure are shown, including exemplary branched nonionic water-solubilizing groups (WSGs).

[0008] Figure 2 The structural formula of an exemplary tandem polymeric dye of the present disclosure including a fluorene comonomer with an exemplary branched nonionic water-soluble group (WSG) is shown.

[0009] Figure 3 The structural formula of an exemplary tandem polymeric dye of the present disclosure including a fluorene comonomer with an exemplary branched nonionic water-soluble group (WSG) is shown, wherein x, y, and z represent the mole % values ​​of the comonomer in the polymeric dye.

[0010] definition

[0011] 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.

[0012] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. In addition, for clarity and ease of reference, certain terms are defined below.

[0013] It must 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 indicates otherwise. For example, the term "a primer" refers to one or more primers, i.e., a single primer and multiple primers. It should be further noted that the claims may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a prerequisite basis for using exclusive terminology such as "only," "only," etc., or using a "negative" limitation in conjunction with the recitation of claim elements.

[0014] As used herein, the term "sample" refers to a material or material mixture containing one or more analytes of interest, in some cases in liquid form. In some embodiments, the term, used in its broadest sense, refers to any plant, animal or bacterial material containing cells or producing cell metabolites, such as, for example, tissues or liquids (including but not limited to plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) separated from individuals or tissues or liquids separated from in vitro cell culture components, and samples from the environment. The term "sample" may also refer to a "biological sample". As used herein, the term "biological sample" refers to a subset of a whole organism or its tissues, cells or components (e.g., body fluids, including but not limited to blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and semen). "Biological sample" may also refer to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or components, or a fraction or portion thereof, 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 from an animal or plant. The biological sample may include a cell. The term "cell" is used in its conventional sense to refer to the basic structural unit of a eukaryotic organism and a prokaryotic organism having at least a nucleus and a cell membrane. In certain embodiments, the cell includes a prokaryotic cell, such as a cell from a bacteria. In other embodiments, the cell includes a eukaryotic cell, such as a cell obtained from a biological sample of an animal, a plant or a fungus.

[0015] As used herein, the terms "carrier binding" and "connected to a carrier" are used interchangeably and refer to a moiety (e.g., a specific binding member) that is covalently or non-covalently connected to a carrier of interest. Covalent connection can involve a chemical reaction of two compatible functional groups (e.g., two chemoselective functional groups, an electrophile and a nucleophile, etc.) to form a covalent bond between the two moieties of interest (e.g., a carrier and a specific binding member). In some cases, non-covalent connection can involve specific binding between two moieties of interest (e.g., two affinity moieties, such as a hapten and an antibody or a biotin moiety and streptavidin, etc.). In some cases, non-covalent connection can involve absorption of a substrate.

[0016] As used herein, 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 polymers of coded and non-coded amino acids, chemically or biochemically modified or derived amino acids, and polypeptides with modified peptide backbones, wherein the conventional backbone has been replaced by a non-naturally occurring or synthetic backbone. The polypeptide can have any suitable 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 peptide length is between 5 and 30 amino acids.

[0017] As used herein, the term "isolated" means that the portion of interest is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free, and even at least 99% free from other components with which the portion was associated prior to purification.

[0018] A "plurality" includes at least 2 members. In some cases, a plurality can have 10 or more, such as 100 or more, 1000 or more, 10,000 or more, 10 ... 6 or more, 10 7 or more, 10 8 or more or 10 9 or more members.

[0019] Numerical ranges are inclusive of the numbers defining the range.

[0020] As used herein, the term "specific binding" refers to the ability of a capture agent (or a first member of a specific binding pair) to preferentially bind to a specific analyte (or a second member of a specific binding pair) present, for example, in a homogeneous mixture of different analytes. In some instances, the specific binding interaction will discriminate between a desired analyte and an undesired analyte in a sample with a specificity for the desired analyte that exceeds that for an undesired analyte by a factor of 10 or more, such as 100 or more, or 1000 or more. In some cases, the affinity between the capture agent and the analyte when they are specifically bound in the capture agent / analyte complex is at least 10 -8 M. At least 10 -9 M, such as up to 10 -10 M.

[0021] As used herein, the terms "affinity" and "avidity" have the same meaning and are used interchangeably herein."Affinity" refers to the strength of binding, with increased binding affinity correlating to a lower Kd.

[0022] The method described herein comprises a plurality of steps. Each step can be performed after a predetermined amount of time has passed between steps as required. Therefore, the time between each step can 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 include 5 hours or longer. In certain embodiments, each subsequent step is performed immediately after the previous step is completed. In other embodiments, the step can be performed after incubation or waiting time (e.g., a few minutes to overnight waiting time) after the previous step is completed.

[0023] As used herein, the terms "assess," "determine," "measure," and "assess," and "determine" are used interchangeably and include both quantitative and qualitative determinations.

[0024] 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, leaving the other element intact.

[0025] As used herein, the term "linker" or "linking key" refers to a connecting portion that connects two groups and has a main chain of 100 atoms or less in length. A linker or linking key can be a covalent bond connecting two groups or a chain of 1 to 100 atoms in length, such as a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more carbon atoms in length, wherein the chain can be straight, branched, cyclic or a single atom. In some cases, the linker is a branched linker, which refers to a connecting portion that connects three or more groups. In some cases, one, two, three, four or five or more carbon atoms of the linker main chain may be optionally substituted by sulfur, nitrogen or oxygen heteroatoms. In some cases, the linker main chain includes a connecting functional group such as ether, thioether, amino, amide, sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester or imine. The bonds between the backbone atoms may be saturated or unsaturated, and in some cases, there are no more than one, two or three unsaturated bonds in the backbone of the linker. The linker may include one or more substituents, such as alkyl, aryl or alkenyl. The linker may include, but is not limited to, polyethylene glycol; ether; thioether; tertiary amine; alkyl, 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 backbone of the linker may include a cyclic group, such as an aryl, heterocyclic or cycloalkyl group, wherein the backbone includes 2 or more atoms of the cyclic group, such as 2, 3 or 4 atoms. The linker may be cleavable or non-cleavable.

[0026] As used herein, the terms "polyethylene oxide", "PEO", "polyethylene glycol" and "PEG" are used interchangeably and refer to a polyethylene glycol having the formula --(CH2--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 suitable length and can include multiple terminal groups and / or additional substituents, including but not limited to alkyl, aryl, hydroxyl, amino, acyl, acyloxy and amido terminal groups and / or substituents. PEG groups that may be adapted 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.

[0027] As used herein, 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. Alkyl groups of interest include, but are not limited to, methyl; ethyl; propyl, such as prop-1-yl or prop-2-yl; and butyl, such as but-1-yl, but-2-yl, 2-methyl-prop-1-yl or 2-methyl-prop-2-yl. In some embodiments, the alkyl group includes 1 to 20 carbon atoms. In some embodiments, the alkyl group includes 1 to 10 carbon atoms. In certain embodiments, the alkyl group includes 1 to 6 carbon atoms, such as 1 to 4 carbon atoms. For example, the term includes straight and branched chain 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-).

[0028] The term "substituted alkyl" refers to an alkyl group as defined herein in which one or more of the carbon atoms in the alkyl chain has been optionally replaced by a radical such as -O-, -N-, -S-, -S(O)-n -(wherein n is 0 to 2), -NR-(wherein R is hydrogen or alkyl), and has 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, oxyaminoacyl, 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 heterocyclyl.

[0029] "Alkoxy" refers to an -O-alkyl group, wherein 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 alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O- groups, wherein alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0030] 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.

[0031] "Amino" refers to a -NH2 group.

[0032] The term "substituted amino" refers to a -NRR group where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl, with the proviso that at least one R is not hydrogen.

[0033] "Aryl" by itself or as part of another substituent refers to a monovalent aromatic hydrocarbon radical derived by the removal of one hydrogen atom from a single carbon atom of an aromatic ring system. Aryl radicals of interest include, but are not limited to, radicals derived from aceanthrene, acenaphthylene, acephenanthrene, anthracene, azulene, benzene, In some embodiments, the aryl group includes 6 to 20 carbon atoms. In some embodiments, the aryl group includes 6 to 12 carbon atoms. Examples of aryl groups are phenyl and naphthyl.

[0034] "Heteroaryl" by itself or as part of another substituent refers to a monovalent heteroaromatic ring radical derived by removing one hydrogen atom from a single atom of a heteroaromatic ring system. Heteroaryl groups of interest include, but are not limited to, groups 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, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, triazole, benzotriazole, thiophene, triazole, xanthene, benzodioxole, and the like. In certain embodiments, heteroaryl is a 5-20 membered heteroaryl. In certain embodiments, heteroaryl is a 5-10 membered heteroaryl. In certain embodiments, heteroaryl is those groups derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole and pyrazine.

[0035] "Heterocycle", "heterocyclic", "heterocycloalkyl" and "heterocyclyl" refer to a saturated or unsaturated group having a monocyclic or multiple fused rings (including fused bridged ring systems and spirocyclic ring systems) and having 3 to 20 ring atoms, including 1 to 10 heteroatoms. These ring atoms are selected from the group consisting of nitrogen, sulfur or oxygen, wherein in the fused ring system, one or more of the 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 N-oxides, -S(O)- or -SO2- moieties.

[0036] 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, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, dihydroindoline, 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.

[0037] Unless otherwise limited by the definition of heterocyclic substituents, such heterocyclic groups may be optionally substituted with 1 to 5 or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, 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, -SO2-heteroaryl, and fused heterocycle.

[0038] The term "alkaryl" or "aralkyl" refers to -alkylene-aryl and substituted alkylene-aryl groups wherein alkylene, substituted alkylene and aryl are as defined herein.

[0039] "Alkylene" refers to a straight or branched divalent aliphatic hydrocarbon group, preferably having 1 to 6, more preferably 1 to 3 carbon atoms, and optionally interrupted by one or more groups selected from: -O-, -NR 10 -、-NR 10 C(O)-、-C(O)NR 10-, etc. For 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-), etc. "Substituted alkylene" refers to an alkylene group in which 1 to 3 hydrogens are replaced as described for a carbon in the definition of "substituted" below.

[0040] "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, where M is halogen; R 60 , R 61 , R 62 and R 63 R is 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; and 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 optional 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 、-SR 60 , -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, the substituent may carry a methylenedioxy substituent or one, two or three substituents selected from halogen atoms, (1-4C) alkyl and (1-4C) alkoxy. When the substituted group is an aryl or heteroaryl group, one or more substituents (e.g., as described herein) may be referred to as "one or more aryl substituents".

[0041] Additional definitions of terms may appear throughout the specification. DETAILED DESCRIPTION

[0042] As outlined above, water-soluble polymer dyes and polymer tandem dyes are provided. The polymer dye includes a water-solvated light-harvesting multichromophore, the water-solvated light-harvesting multichromophore having a conjugated segment of an aryl or heteroaryl comonomer, the aryl or heteroaryl comonomer including a branched nonionic water-soluble group (WSG), the branched nonionic water-soluble group (WSG) comprising two or more water-soluble polymers. In some cases, the branched nonionic water-soluble group (WSG) of the present disclosure can impart a solubility in water exceeding 50 mg / mL to the multichromophore. The polymer tandem dye also includes a signal chromophore covalently linked to the multichromophore, the multichromophore being in energy-receiving proximity to the signal chromophore. A specific binding member comprising a label that resists aggregation of the subject water-soluble polymer dye is also provided. A method for evaluating a sample for the presence of a target analyte and a method for labeling a target molecule using the subject polymer dye are also provided. A system and a kit for practicing the subject method are also provided.

[0043] Before describing various embodiments in more detail, it should be understood that the teachings of the present disclosure are not limited to the specific embodiments described, and therefore can certainly be varied. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments, and is not intended to be limiting, because the scope of this teaching will be limited only by the appended claims.

[0044] The paragraph headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described in any way. Although the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents as will be understood by those skilled in the art.

[0045] 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 present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, exemplary methods and materials are now described.

[0046] The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present claims are not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates that may be independently confirmed.

[0047] It will be apparent to those skilled in the art upon reading this disclosure that each individual embodiment described and illustrated herein has discrete components and features that can be readily separated or combined with the features of any other several embodiments without departing from the scope or spirit of the present teachings. Any recited method can be performed in the order of events recited or in any other order that is logically possible.

[0048] All patents and publications mentioned herein, including all sequences disclosed within such patents and publications, are expressly incorporated herein by reference.

[0049] In further describing the present invention, water-soluble polymeric dyes comprising light-harvesting multichromophores and related polymeric tandem dyes further comprising signal chromophores are first described in more detail. Next, labeled specific binding members comprising the subject polymeric dyes are described. Then, methods of interest using the subject polymeric dyes are reviewed. Systems and kits useful for practicing the methods of the present disclosure are also described.

[0050] Polymer Dyes

[0051] As outlined above, the present disclosure provides water-soluble polymer dyes. The subject water-soluble polymer dyes include light-harvesting multichromophores, and the light-harvesting multichromophores have conjugated segments of aryl and / or heteroaryl comonomers, and the aryl and / or heteroaryl comonomers include branched nonionic water-soluble groups (WSG). Branched nonionic water-soluble groups can be connected to comonomers at any suitable position to provide side chain groups protruding away from the conjugated polymer backbone, thereby providing high solvation in an aqueous environment. In some cases, branched nonionic water-soluble groups (WSG) include two or more water-soluble polymers, and each polymer has 6-100, such as 6-50, 6-40 or 6-30 monomer units. As used herein, the terms "water-solubilizing group", "water-soluble group" and WSG are used interchangeably, and refer to groups or substituents that are well solvated in an aqueous environment, for example, under physiological conditions and impart improved water solubility to the molecules connected thereto. In some embodiments, WSG increases the solubility of the multichromophore in a predominantly aqueous solution compared to a control multichromophore lacking WSG.The water-solubilizing group can be any suitable hydrophilic group that is well solvated in an aqueous environment.

[0052] The water-soluble polymer dyes disclosed herein have solubility under aqueous conditions, making them particularly suitable for application to a variety of biological assays. The subject water-soluble polymers and their conjugates can resist undesirable aggregation, which provides favorable fluorescence and spectral properties in various biological assays. Aggregation of the dye is undesirable because it can lead to a reduction in the fluorescent signal, for example, through aggregation-induced quenching of the dye fluorescence. The subject water-soluble conjugated polymer dyes can be used as fluorescent reporters for a variety of biosensors and provide signals with excellent brightness and a wide range of excitation and emission wavelength options for applications such as flow cytometry and imaging.

[0053] In some instances, the solubility of the water-soluble polymer dye in water or buffer is 1mg / mL or higher, such as 3mg / mL or higher, 10mg / mL or higher, 20mg / mL or higher, 30mg / mL or higher, 40mg / mL or higher, 50mg / mL or higher, 60mg / mL or higher, 70mg / mL or higher, 80mg / mL or higher, 90mg / mL or higher, 100mg / mL or higher or even higher.Should be appreciated that under certain conditions, the water-soluble polymer dye can form discrete water-solvated nanoparticle in aqueous systems.In some cases, the water-solvated nanoparticle resists gathering and can be used for multiple biological assays.

[0054] As used herein, the terms "light-harvesting multichromophore," "polymer dye," and "conjugated polymer" are used interchangeably and refer to a conjugated polymer having a structure capable of capturing light of a specific absorption maximum wavelength and converting it into emitted light of a longer emission maximum wavelength. In some cases, the light-harvesting multichromophore itself is fluorescent. Conjugated polymers (CPs) are characterized by a delocalized electronic structure and can have an effective conjugation length that is significantly shorter than the polymer chain length because the backbone can contain a large number of conjugated segments in close proximity. In some cases, conjugated polymers are effective for light harvesting and provide optical amplification through Forster energy transfer to an acceptor.

[0055] As used herein, the term "unit" refers to a structural subunit of a polymer. The term unit is intended to include monomers, comonomers, coblocks, conjugated segments, repeating units, etc. "Repeat unit" is a subunit of a polymer, which is defined by the minimum number of different structural features required for a 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 a polymer is a random arrangement of a multi-block polymer or unit, each block may define different repeating units, such as n-blocks and m-blocks. It should be understood that a variety of arrangements of n and / or m repeating units or blocks are possible, and in the illustrated formula of the subject multi-chromophore described herein, any suitable linear arrangement of n and m coblocks of various lengths is included in the structure of the overall polymer. It should be understood that a polymer may also be represented by a formula according to the molar % value of each unit in a polymer, and the formula may represent a variety of arrangements of repeating units, such as random or multi-block polymers. In some cases, the repeating unit of a polymer includes a single monomer group. In some instances, 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. As used herein, the term "comonomer" or "comonomer group" refers to a structural unit of a polymer, which itself can be a part of a repeating unit of a polymer. In some embodiments, a conjugated polymer includes a block copolymer consisting of a polymerized monomer block. In such cases, the block copolymer can be described as having different repeating units, each repeating unit corresponding to a different co-block of the polymer. In some cases, the polymer is a diblock copolymer containing two different co-blocks. In such cases, the polymer can be described as including a co-block, wherein each co-block can be composed of a co-monomer, such as one, two, three or more co-monomers.

[0056] The multichromophore may have any suitable length. In some cases, the specific number of monomer repeat units or segments of the multichromophore may be 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 segments, 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 segments. In some examples, the specific number of monomer repeating units or segments of the multichromophore can range from 2 to 1,000, such as 2 to 500, 2 to 100, 3 to 100, 4 to 100, or, 5 to 100, 6 to 100, 7 to 100, 8 to 100, 9 to 100, or 10 to 100 units or segments.

[0057] The multichromophore can have any suitable molecular weight (MW). In some cases, the MW of the multichromophore can be expressed as an average molecular weight. In some instances, the average molecular weight of the polymeric dye is 500-500,000, such as 1,000 to 100,000, 2,000 to 100,000, 10,000 to 100,000, or even an average molecular weight in the range of 50,000 to 100,000.

[0058] In some embodiments, the multichromophore includes a specific aryl or heteroaryl comonomer that constitutes 5% or more (e.g., 5 mol%) of the multichromophore by molar concentration, such as 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 45% or more, 50% or more, 60% or more, 70% or more, or even more by molar concentration of the multichromophore. In such cases, the multichromophore may include 5 or more repeating units, such as 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, 200 or more, 500 or more, 1000 or more, 10,000 or more, or even more repeating units. In such cases, the multichromophore may include 5 or more comonomer units, such as 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, 200 or more, 500 or more, 1000 or more, 10,000 or more, or even more comonomer units. In certain embodiments, the aromatic or heteroaromatic comonomer of interest constitutes 25% or more on a molar basis, such as 30% or more, 40% or more, 45% or more, 50% or more, or even more on a molar basis, of a multichromophore comprising 5 or more repeating units, such as 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 repeating units.

[0059] In some embodiments, the multichromophore includes a plurality of first optically active units forming a conjugated system having an absorption wavelength at which the first optically active units absorb light to form an excited state (e.g., as described herein). In certain instances, the multichromophore includes a conjugated polymer segment or oligomeric structure including n-conjugated repeating units that reduce the band gap.

[0060] The subject multi-chromophore can have one or more desired spectral characteristics, such as specific absorption maximum wavelength, specific emission maximum wavelength, extinction coefficient, quantum yield etc. The subject polymer dye and polymer tandem dye provide a variety of absorption and emission characteristics, and these characteristics depend on a variety of factors, such as selected comonomer, linking group, substituent and the signal chromophore of the optional connection of composition polymer. In some cases, the absorption maximum wavelength of polymer dye ranges from 300 to 600nm, such as 350nm to 560nm or 400nm to 560nm. In certain embodiments, the absorption maximum wavelength of multi-chromophore is 600nm or less, such as 575nm or less, 550nm or less, 525nm or less, 500nm or less, 475nm or less, 450nm or less, 440nm or less, 430nm or less, 420nm or less, 410nm or less, 400nm or less or even less. In certain embodiments, multi-chromophore only absorbs UV light. In some instances, the absorption maximum wavelength of the multichromophore ranges from 300nm to 400nm. In some instances, the absorption maximum wavelength of the multichromophore ranges from 300nm to 350nm. In some instances, the absorption maximum wavelength of the multichromophore ranges from 350nm to 400nm. In some instances, the absorption maximum wavelength of the multichromophore ranges from 400nm to 600nm, such as 400nm to 560nm. In some instances, the absorption maximum wavelength of the multichromophore ranges from 400nm to 450nm. In some instances, the absorption maximum wavelength of the multichromophore ranges from 450nm to 500nm. In some instances, the absorption maximum wavelength of the multichromophore ranges from 500nm to 550nm. In some instances, the absorption maximum wavelength of the multichromophore ranges from 550nm to 600nm.

[0061] In some embodiments, the multichromophore has an emission maximum wavelength in the range of 300 to 900 nm, such as 350 to 850 nm, 350 to 600 nm, 360 to 500 nm, 370 to 500 nm, 380 to 500 nm, 390 to 500 nm, or 400 to 500 nm, wherein specific examples of emission maxima of interest include, but are not limited to, 395 nm ± 5 nm, 460 nm ± 5 nm, 490 nm ± 5 nm, 550 nm ± 5 nm, 560 nm ± 5 nm, 605 nm ± 5 nm, 650 nm ± 5 nm, 680 nm ± 5 nm, 700 nm ± 5 nm, 805 nm ± 5 nm. In certain instances, the multichromophore has an emission maximum wavelength selected from the group consisting of 395 nm, 460 nm, 490 nm, 550 nm, 560 nm, 605 nm, 650 nm, 680 nm, 700 nm, and 805 nm. In some instances, the multichromophore has an emission maximum wavelength of 395 nm ± 5 nm. In some instances, the multichromophore itself has an emission maximum wavelength in the range of 375 to 900 nm (such as in the range of 380 to 900 nm, 390 to 900 nm, or 400 to 900 nm).

[0062] In some embodiments, the extinction coefficient of the multichromophore 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, 3x10 6 cm -1 M -1 or larger, 4x 106 cm -1 M -1 or larger, 5x 10 6 cm -1 M -1 or larger, 6x 10 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 such cases, the multichromophore may have 5 or more repeating units, such as 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or even more repeating units. In some embodiments, the molar extinction coefficient of the multichromophore is 5 x 10 5 M -1 cm -1 In certain embodiments, the molar extinction coefficient of the multichromophore is 1 x 10 6 M -1 cm -1 or larger.

[0063] In some embodiments, the multichromophore has an extinction coefficient of 40,000 cm -1 M -1 / repeating unit or larger, such as 45,000cm -1 M -1 / Repeat unit or larger, 50,000cm -1 M -1 / Repeat unit or larger, 55,000cm -1 M -1 / Repeat unit or larger, 60,000cm -1 M -1 / Repeat unit or larger, 70,000cm -1 M -1 / Repeating unit or larger, 80,000cm -1 M -1 / Repeat unit or larger, 90,000cm -1 M -1 / Repeating unit or larger, 100,000cm -1 M -1 / repeat unit or more, or even more. In some examples, the 40,000 cm -1 M -1 / repeat unit or greater is the average extinction coefficient. In some examples, the repeating unit of the multichromophore may include a single monomer, two comonomers, or three or more comonomers. In some examples, the extinction coefficient of the multichromophore is 40,000 cm -1 M -1 / comonomer or greater, such as 45,000cm -1 M -1 / comonomer or larger, 50,000cm -1 M -1 / comonomer or larger, 55,000cm -1 M -1 / comonomer or larger, 60,000cm -1 M -1 / comonomer or larger, 70,000cm -1 M -1 / comonomer or larger, 80,000cm -1 M -1 / comonomer or greater, 90,000cm -1 M -1 / comonomer or larger, 100,000cm -1 M -1 / comonomer or greater, or even greater. In some examples, the 40,000 cm -1 M -1 / comonomer or greater is the average extinction coefficient.

[0064] It should be understood that in some cases, the subject multi-chromophore may include a random arrangement of co-blocks (e.g., n and m co-blocks) or n and m repeating units. The subject multi-chromophore may include any suitable linear arrangement of n and m co-blocks with various lengths within the structure of the overall polymer. In some cases, the linear arrangement of the comonomers shown in the structure herein is random. In addition, the multi-chromophore may include any suitable co-monomer arrangement within these n and / or m co-blocks. Unless otherwise indicated, all possible co-monomer arrangements are intended to be included in the polymer dyes described herein. A variety of polymer synthesis methods can be utilized to prepare co-monomers and co-blocks of interest in the preparation of the subject multi-chromophore. It should be understood that in some cases, a polymerization method can produce a composition comprising a conjugated polymer population, the composition comprising some changes in a specific length and / or end group (i.e., end group) present in each conjugated polymer in the population. The formula shown herein may refer to a population or subpopulation of a single compound or polymeric compound. As used herein, * indicates a site for covalent attachment to the unsaturated backbone of a conjugated polymer. It is understood that the formula of the subject polymers shown herein is one representation of the structure, and other representations, such as those indicating the mole % ratio of a particular comonomer in a conjugated polymer segment, may also be used.

[0065] In some examples, the multichromophore includes a conjugated segment having the structure of Formula (I):

[0066]

[0067] in:

[0068] F 1 is a fused tricyclic comonomer substituted with a water solubilizing group (WSG) (e.g., as described herein);

[0069] M 1 is an aromatic or heteroaromatic comonomer;

[0070] n is an integer from 1 to 100,000; and

[0071] * indicates the site for covalent attachment to the unsaturated backbone or terminal group of the conjugated polymer;

[0072] Among them, F 1 and M 1At least one of the M is substituted by a branched nonionic water-soluble group (WSG) comprising two or more water-soluble polymers, each of which has 6 or more monomer units. In some cases, the branched nonionic water-soluble group (WSG) can impart a solubility in water of 10 mg / mL or higher (e.g., more than 10 mg / mL, more than 20 mg / mL, more than 30 mg / mL, more than 40 mg / mL, or more than 50 mg / mL) to the multichromophore. In some embodiments of formula (I), M 1 is substituted with a branched nonionic water-soluble group (WSG) comprising two or more water-soluble polymers, each of which has 6 or more monomer units; and F 1 is substituted with one or more (e.g., two) linear nonionic water-soluble groups (WSG) comprising a linear water-soluble polymer having 6 or more monomer units. In certain embodiments of formula (I), F 1 It is a fused three-ring comonomer.

[0073] In some embodiments of Formula (I), the multichromophore includes a conjugated segment having the structure of Formula (I):

[0074]

[0075] in:

[0076] F 1 is a fused 6-5-6 tricyclic comonomer substituted with a branched nonionic water solubilizing group (WSG) comprising two or more water soluble polymers, each of which has 6 or more monomer units;

[0077] M 1 is an aromatic or heteroaromatic comonomer;

[0078] n is an integer from 1 to 100,000; and

[0079] * indicates the site for covalent attachment to the unsaturated backbone or terminal group of the conjugated polymer;

[0080] The branched nonionic water-soluble group (WSG) can impart a solubility of 10 mg / mL or higher (eg, more than 10 mg / mL) in water to the multichromophore.

[0081] In some examples of formula (I), the two or more water-soluble polymers have 7 or more monomeric units, such as 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, or even more monomeric units; and in some cases, up to 100 units, such as up to 50 monomeric units, up to 40 or up to 30 monomeric units. In some examples of formula (I), the two or more water-soluble polymers each independently have 6-100 monomeric units, such as 6-50, 6-40, 6-30, 10-30, 10-20 units, 12-20 or 12-16 monomeric units. In some cases of formula (I), F 1 It is a substituted fluorene comonomer.

[0082] In some cases, the branched nonionic water-soluble groups (WSG) can impart a solubility in water (e.g., aqueous buffer) of 20 mg / mL or more to the multichromophore, such as 30 mg / mL or more, 40 mg / mL or more, 50 mg / mL or more, 60 mg / mL or more, 70 mg / mL or more, 80 mg / mL or more, 90 mg / mL or more, 100 mg / mL or more, or even more. In some cases, the branched nonionic water-soluble groups (WSG) can impart a solubility in water (e.g., aqueous buffer) of more than 20 mg / mL to the multichromophore, such as more than 30 mg / mL, more than 40 mg / mL, more than 50 mg / mL, more than 60 mg / mL, more than 70 mg / mL, more than 80 mg / mL, more than 90 mg / mL, or more than 100 mg / mL.

[0083] The aromatic or heteroaromatic comonomers of interest include, but are not limited to, fused tricyclic comonomers, such as fluorene comonomers, carbazole comonomers, thiole comonomers or bridged biphenyl comonomers. A fused tricyclic comonomer is a comonomer comprising a tricyclic aromatic group having three fused rings, and its configuration is that two aromatic or heteroaryl 6-membered rings are fused to a central 5-membered or 6-membered carbocyclic or heterocyclic ring. In some cases, the fused tricyclic comonomer comprises two benzo or pyrido rings fused to a central 5-membered or 6-membered carbocyclic or heterocyclic ring. The fused tricyclic comonomer can be π-conjugated with the adjacent comonomer of the polymer backbone by any suitable ring atom of the fused ring. The central 5-membered or 6-membered ring can be an aromatic or partially saturated carbocyclic or heterocyclic ring, and may also include a side chain substituent, such as a linker for WSG and / or a chemically selective tag. A bridged biphenyl comonomer is a fused tricyclic comonomer with a biphenyl group, wherein the two benzene rings are further interconnected by a central 6-membered ring. In certain examples, the fused tricyclic comonomer is described by the following structure:

[0084]

[0085] in:

[0086] Y is C(R 3 )2、-C(R 3 )2C(R 3 )2-、-C(R 3 )2Si(R 3 )2-、NR 3 、Si(R 3 )2 or Se;

[0087] Each Z is independently CH, CR or N;

[0088] Each R 3 independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido, aralkyl, substituted aralkyl, PEG moiety, -L 11 -Z 1 , where L 11 is a linker and Z 1 is a chemoselective tag (e.g., a tag comprising a chemoselective functional group) and WSG; and

[0089] Each R is independently H or one or more aryl or heteroaryl substituents, and any two suitable R groups are optionally cyclically connected. In some cases, each R refers to one or two ring substituents independently selected from halogen, sulfonate, alkoxy, substituted alkoxy, alkyl and substituted alkyl, and any two suitable R groups are optionally cyclically connected. In some instances, at least two of Z in each ring are CH or CR. In some instances, one and only one of Z in each ring is N.

[0090] In certain examples, the fused tricyclic comonomer is described by one of the following structures:

[0091]

[0092] in:

[0093] Y is C(R 3 )2、-C(R 3 )2C(R 3 )2-、-C(R 3 )2Si(R 3 )2-、NR 3 、Si(R 3 )2 or Se;

[0094] Each R 3independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido, aralkyl, substituted aralkyl, PEG moiety, -L 11 -Z 1 , where L 11 is a linker and Z 1 is a chemoselective tag (e.g., a tag comprising a chemoselective functional group) and WSG; and

[0095] Each R is independently H, R 3 Or one or more aryl or heteroaryl substituents, and any two suitable R groups are optionally cyclically connected. In some cases, each R refers to one or two ring substituents independently selected from halogen, sulfonate, alkoxy, substituted alkoxy, alkyl and substituted alkyl, and any two suitable R groups are optionally cyclically connected. The symbol "*" represents the site of the unsaturated main chain for covalent connection to the conjugated polymer, such as π conjugated segments, end groups, linking groups and connected specific binding members. It should be understood that for any formula of the fused tricyclic comonomer including 2,7-π-conjugation described herein, similar formulas including similar 3,6-π-conjugated comonomers can also be shown. In some cases, the fused tricyclic comonomer is a fluorene comonomer, wherein Y is C(R 3 )2. In some cases, the fused tricyclic comonomer is a carbazole comonomer, wherein Y is NR 3 In some cases, the fused tricyclic comonomer is a silole comonomer, wherein Y is Si(R 3 )2. In some cases, the fused tricyclic comonomer is a bridged biphenyl comonomer, wherein Y is -C(R 3 )2C(R 3 )2-. In some cases, the fused tricyclic comonomer is a bridged biphenyl comonomer, wherein Y is -CHR 3 CHR 3 -. In certain examples of any fused tricyclic comonomer described herein, each R is independently selected from H, halogen, alkoxy, substituted alkoxy, alkyl, and substituted alkyl. In certain cases, each R is independently selected from H, fluorine, chlorine, methoxy, substituted alkoxy, alkyl, and substituted alkyl.

[0096] In certain embodiments of the fused tricyclic comonomer, the comonomer includes two R substituents cyclically linked to provide an optionally further substituted carbocyclic or heterocyclic ring A:

[0097]

[0098] Where Y is C(R 3)2、-C(R 3 )2C(R 3 )2-、-C(R 3 )2Si(R 3 )2-、NR 3 、Si(R 3 )2 or Se; and each R 3 independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido, aralkyl, substituted aralkyl, PEG moiety, -L 11 -Z 1 , where L 11 is a linker and Z 1 is a chemoselective tag (e.g., a tag comprising a chemoselective functional group) and WSG. In certain examples, the fused tricyclic comonomer has the following structure:

[0099]

[0100] Each R 8 -R 9 independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido, aralkyl, substituted aralkyl, PEG moiety, -L 11 -Z 1 , where L 11 is a linker and Z 1 is a chemoselective tag (e.g., a tag comprising a chemoselective functional group) and WSG. In some cases of the comonomer, Y is C(R 3 )2.

[0101] In certain examples, the fused tricyclic comonomer is described by one of the following structures:

[0102]

[0103] in:

[0104] Each R 3 independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido, aralkyl, substituted aralkyl, PEG moiety, -L 11 -Z 1 , where L 11 is a linker and Z 1is a chemoselective tag (e.g., a tag comprising a chemoselective functional group) and WSG (e.g., as described herein); and

[0105] Each R is independently H, R 3 or one or more aryl or heteroaryl substituents. In some cases, each R refers to one or two ring substituents independently selected from halogen, sulfonate, alkoxy, substituted alkoxy, alkyl, and substituted alkyl. In some cases, each R is fluoro or methoxy. In some cases, at least one R 3 is WSG. In some cases, at least one R 3 For -L 11 -Z 1 .

[0106] In certain examples, the fused tricyclic comonomer is described by the following structure:

[0107]

[0108] Wherein each WSG is a branched or linear water-soluble group, and each R 3 is H, alkyl, substituted alkyl. In some cases, each WSG is a linear WSG (e.g., as described herein). In some examples, the fused three-ring comonomer is described by the following structure:

[0109]

[0110] Where T 5 is an optional linking group; each s is independently an integer from 6 to 100 (e.g., 6 to 50); and each R and R 11 are independently hydrogen, alkyl or substituted alkyl.

[0111] A fused 6-5-6 tricyclic comonomer is a comonomer comprising a tricyclic aromatic group having three fused rings, the configuration of which is 6-5-6, i.e., two benzo rings are fused to a central 5-membered ring. The 5-membered ring may be a carbocyclic or heterocyclic ring and may also include side chain substituents on the ring atoms that are not fused to the benzo ring. In certain instances, the fused 6-5-6 tricyclic comonomer is described by the following structure:

[0112]

[0113] Where: Z is -C(R 1 )2-、Si(R 1 )2-or-N(R 1 )-; each R is independently H or one or more aryl substituents; and each R 1independently selected from branched nonionic water-soluble groups (WSG), alkyl groups, substituted alkyl groups, aralkyl groups, substituted aralkyl groups, PEG moieties, and -L 11 -Z 1 , where L 11 is a linker and Z 1 is a chemoselective tag (e.g., a tag comprising a chemoselective functional group) or WSG. As used in any formula described herein, * represents a site for covalent attachment to an unsaturated backbone or terminal group of a conjugated polymer. In some embodiments, when Z is -N(R 1 )-, the fused 6-5-6 tricyclic comonomer is a carbazole comonomer. Any suitable carbazole comonomer may be utilized in the subject multichromophore. In some embodiments, when Z is -C(R 1 )2-, the fused 6-5-6 tricyclic comonomer is a fluorene comonomer. Any suitable fluorene comonomer may be utilized in the subject multichromophore. In certain embodiments of the fused 6-5-6 tricyclic comonomer, each R 1 is selected from benzyl substituted with one, two or more PEG moieties or alkyl substituted with two or more PEG moieties. In some embodiments, Z is -Si(R 1 )2-. It is to be understood that for any fluorene comonomer described herein, also included in the present disclosure is the corresponding comonomer wherein the C atom of Z is replaced by Si.

[0114] Fluorene comonomers are comonomers comprising aromatic groups having a 9H-fluorene core structure, which is substituted at position 9 by one or more any suitable side chain substituents. In some cases, the fluorene comonomer is a 9,9-disubstituted fluorene. The fluorene comonomer is conjugated to the adjacent polymer backbone groups through any suitable position of the fluorene core structure, such as any two positions in positions 1-8 (see numbering scheme below). In some embodiments, the fluorene core structure is connected to the adjacent polymer backbone groups through positions 2 and 7. In certain embodiments, the fluorene comonomer is described by the following structure:

[0115]

[0116] Among them: Each R 1 independently selected from branched nonionic water-soluble groups (WSG), alkyl groups, substituted alkyl groups, aralkyl groups, substituted aralkyl groups, PEG moieties, and -L 11 -Z 1 , where L 11 is a linker and Z 1 is a chemoselective tag (e.g., a tag comprising a chemoselective functional group) or WSG. In certain embodiments of the fluorene comonomer, each R 1is a branched nonionic water-soluble group (WSG). In certain examples of the fluorene comonomer, each R 1 is selected from benzyl substituted with one, two or more PEG moieties or alkyl substituted with two or more PEG moieties. 1 Functional groups can be used to covalently link a multichromophore to an acceptor chromophore (e.g., as described herein). 1 Includes an amino group for covalent attachment to an acceptor chromophore. In certain embodiments, Z 1 Includes a carboxylic acid group or a derivative thereof for covalent attachment to an acceptor chromophore. In certain embodiments, L 11 To connect to two or more Z 1 In some examples, the fluorene comonomer is further connected by one, two or more R groups selected from the group consisting of 1, 3, 4, 5, 6 and 8. 5 and / or R 6 Substituent substitution, where R 5 and R 6 are independently selected from a water solubilizing group (WSG) and an aryl substituent (eg, as described herein).

[0117] In certain examples, the fluorene comonomer is described by the following structure:

[0118]

[0119] Among them: Each R 1 As defined above; and R 5 and R 6 and R is independently selected from H, a water solubilizing group, or an aryl substituent (eg, as described herein).

[0120] In some examples, the fluorene comonomer is described by the following structure:

[0121]

[0122] Each R 2 is an alkyl group substituted with a water solubilizing group or a branched linker connected to two or more water solubilizing groups (e.g., PEG-disubstituted benzyl or PEG-substituted alkyl). In certain examples of the fluorene comonomer, each R 2 is substituted with one, two or three PEG moieties (e.g. -O(CH2CH2O) n R', wherein R' is H or alkyl, and n is 1-20, for example 3-16, such as n is 8-16) substituted benzyl. In certain examples of the fluorene comonomer, each R 2 For a -O(CH2CH2O) nR' groups (eg, at positions 2, 3, or 4) are substituted benzyl, wherein R' is H or alkyl, and n is 1-20, such as 3-16, such as 8-16. In certain examples of fluorene comonomers, each R 2 For two -O (CH2CH2O) n R' groups (e.g., at the 2,4-, 3,4-, or 3,5-positions) substituted benzyl, wherein each R' is independently H or alkyl, and each n is independently 1-20, such as 3-16, such as n is 8-16. In certain examples of fluorene comonomers, each R 2 For three -O (CH2CH2O) n R' groups (e.g., at the 2,4,6-, 2,4,5-, or 3,4,5-positions) substituted benzyl, wherein each R' is independently H or alkyl, and each n is independently 1-20, such as 3-16, such as n is 8-16. In certain examples of fluorene comonomers, each R 2 is a lower alkyl substituted with a trivalent branched group, each of which is substituted with two PEG moieties (e.g., -CO-NR"2 or -O(CH2R")2 trivalent branched groups, wherein each R" is independently a PEG moiety (e.g., -O(CH2CH2O) n R', wherein R' is H or alkyl, and n is 1-20, for example 3-16, such as n is 8-16). In certain examples of the fluorene comonomer, each R 2 is a branched nonionic water-solubilizing group (WSG) (eg, as described herein).

[0123] In certain embodiments, the fluorene comonomer is described by the following structure:

[0124]

[0125] Where R 3 is an alkyl group substituted with a water solubilizing group (e.g., an alkyl group substituted with PEG) or a branched nonionic water solubilizing group (WSG) (e.g., as described herein), and R 4 For L 2 -Z 2 , where L 2 is a linker and Z 2 is a chemoselective tag (e.g., for conjugation with an acceptor chromophore). In some examples, the fluorene comonomer is described by the following structure:

[0126]

[0127] Where: R 3is a substituent comprising a water solubilizing group (e.g., as described herein) or a branched nonionic water solubilizing group (WSG) (e.g., as described herein); R 4 For L 2 -Z 2 , where L 2 is a linker and Z 2 is a chemoselective tag (eg, for conjugation to a receptor chromophore); and R 5 and R 6 is independently selected from H, a water solubilizing group, and an aromatic substituent (e.g., an alkyl, a substituted alkyl, an alkoxy, a substituted alkoxy, a halogen, or a nitro group). In certain examples of the fluorene comonomer, R 3 is a lower alkyl substituted with a trivalent branched group, each of which is substituted with two PEG moieties (e.g., -CO-NR"2 or -O(CH2R")2 trivalent branched groups, wherein each R" is a PEG moiety (e.g., -O(CH2CH2O) n R', wherein R' is H or alkyl, and n is 6-20, for example 8-16, such as n is 12-16). In certain examples of the fluorene comonomer, R 3 is a branched nonionic water-solubilizing group (WSG) (eg, as described herein).

[0128] Any fluorene comonomer described herein can be used in the subject multichromophores, such as the multichromophores of formula (I)-(IV). In some cases, the multichromophore includes one of the following structures as part of the polymer backbone:

[0129]

[0130] Each R 3 is independently a water solubilizing group connected through an optional linker or an optionally substituted alkyl, aralkyl or aryl group; Ar is an optionally substituted aryl or heteroaryl group; n is an integer from 1 to 100,000; and the terminal is a site for covalent attachment to an unsaturated backbone or terminal group of a conjugated polymer. In certain embodiments, each R 3 is independently substituted alkyl. In certain embodiments, each R 3 is independently substituted aralkyl. In some cases, each R 3 and each Ar is independently substituted (through an optional linker) with a water solubilizing group, an acceptor chromophore, a chemoselective functional group, or a specific binding moiety.

[0131] In certain embodiments of Formula (I), F 1 Has the following structure:

[0132]

[0133] Where: Z is -C(R 1 )(R 2 )-、-Si(R 1 )(R 2 )-or-N(R 1 )-; and R 1 and R 2 and independently a nonionic WSG, such as a linear or branched nonionic WSG.

[0134] In certain embodiments of Formula (I), F 1 Has the following structure:

[0135]

[0136] Where: Z is -C(R 1 )(R 2 )-、-Si(R 1 )(R 2 )-or-N(R 1 )-; and R 1 and R 2 Independently is a branched nonionic WSG comprising two or more water-soluble polymers, each of which has, for example, 6 to 30 monomer units.

[0137] Branched nonionic water-soluble group

[0138] Branched nonionic water-soluble group (WSG) comprises the branched group connected with the comonomer connected thereto, and provides further connection with two, three or more nonionic water-soluble polymers.In some instances, the two or more water-soluble polymers utilized in WSG are polyethylene glycol (PEG) groups or modified PEG groups.Any suitable branched group can be utilized, which provides 3 or more attachment points (for example, 4 or more), such as one with the attachment point of the comonomer of the conjugated polymer main chain and two or more (for example, 2,3,4 or more) with the attachment point of the nonionic water-soluble polymer.Branched group can utilize any suitable connection functional group and connection chemical substance to be connected with comonomer and nonionic water-soluble polymer by optional linking base.In some instances, branched group is non-aromatic.In some instances, branched group is cyclic. In some cases, the branched group is a substituted aryl or heteroaryl ring, for example a trisubstituted or tetrasubstituted aryl ring, such as a trisubstituted or tetrasubstituted benzene ring; or a trisubstituted or tetrasubstituted heteroaryl ring, such as a trisubstituted or tetrasubstituted pyridine ring. In some instances, the branched group is acyclic. In some instances, the branched group is an atom, such as C, Si, N. In some instances, the branched group is a connecting functional group, such as an acylamino or sulfonamide group. In some instances, the branched group is an amino acid residue or a branched linker, such as glycerol or amino-glycerol.

[0139] Water-soluble polymers of interest include, but are not limited to, polyalkylene oxide based polymers such as polyethylene glycol "PEG" (see, e.g., "Poly (ethylene glycol) Chemistry: Biotechnical and Biomedical Applications", J. M. Harris, ed., Plenum Press, New York, NY (1992); and "Poly (ethylene glycol) Chemistry and Biological Applications", J. M. Harris and S.Zalipsky, ed., ACS (1997); Japanese international applications: WO 90 / 13540, WO 92 / 00748, WO 92 / 16555, WO94 / 04193, WO 94 / 14758, WO 94 / 17039, WO 94 / 18247, WO 94 / 28937, WO 95 / 11924, WO 96 / 00080, WO 96 / 23794, WO 98 / 07713, WO 98 / 41562, WO 98 / 48837, WO 99 / 30727, WO 99 / 32134, WO 99 / 33483, WO 99 / 53951, WO 01 / 26692, WO 95 / 13312, WO 96 / 21469, WO 97 / 03106, WO 99 / 45964; USPTO 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; 5,6 43,575; 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; 5,919,4 42; 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).

[0140] Examples of water-soluble polymers of interest include, but are not limited to, polymers 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 polymers of the formula -[C(O)-XC(O)-NH-Y-NH]n- or -[NH-Y-NH-C(O)-XC(O)] having a molecular weight greater than 1,000 Daltons. n -polyamide, wherein X and Y are divalent groups that may be the same or different and may be branched or linear, and n is a discrete integer from 2 to 100 (such as 2 to 50), and wherein either or both of X and Y comprise biocompatible, substantially non-antigenic water-soluble repeating units that 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 these water-soluble repeating units may vary significantly, with the number of these units being 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, 6-100, such as 2 to 50 or 6 to 50. An example of an embodiment is one in which one or both of X and Y are selected from the following embodiments: -((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. Another example of an embodiment is one in which X is -(CH2-CH2)-, and in which Y is -(CH2-(CH2-CH2-O)3-CH2-CH2-CH2)- or -(CH2-CH2-CH2-(O-CH2-CH2)3-CH2)-.

[0141] As used herein, modified polymers, such as modified PEG, refer to water-soluble polymers that are modified or derivatized at one or both ends, for example to include terminal substituents (e.g., terminal alkyl, substituted alkyl, alkoxy or substituted alkoxy, etc.) and / or terminal connecting functional groups (e.g., amino acids or carboxylic acid groups suitable for connecting via amide bond formation) suitable for connecting the polymer to a multichromophore (e.g., via a branching group). The subject water-soluble polymers can be adapted to include any suitable connecting group.

[0142] It should be understood that in some cases, the water-soluble polymers may include some dispersity relative to the polymer length, depending on the preparation and / or purification methods of the polymer starting material. In some instances, the subject water-soluble polymers are monodisperse. In some instances, the subject water-soluble polymers are substantially monodisperse, e.g., containing 20 wt % or less of non-target material, such as 15 wt % or less, 10 wt % or less, 5 wt % or less, 2 wt % or less, or 1 wt % or less.

[0143] The water-soluble polymer may contain one or more spacers or linkers. Examples of spacers or linkers include linear or branched moieties comprising one or more repeating units employed 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 contain, for example, up to 18 carbon atoms or even additional polymer chains.

[0144] When present, the water-soluble polymer portion, or one or more spacers or linkers of the polymer portion, may include a biostable or biodegradable polymer chain or unit. For example, polymers with repeated linking bonds have varying degrees of stability under physiological conditions, depending on bond instability. Polymers with such bonds can be classified according to their relative hydrolysis rates under physiological conditions based on the hydrolysis rates of known low molecular weight analogs, 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 linking bond system connecting 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-). Generally, it may be desirable to avoid the use of sulfated polysaccharides, depending on the instability of the sulfate groups. In addition, the use of polycarbonates and polyesters may not be desirable. These bonds are provided by way of example and are not intended to limit the types of bonds that may be employed in the polymer chains or linker systems of the water-soluble polymers that may be used for the modified aldehyde-labeled polypeptides disclosed herein.

[0145] In some examples, the branched nonionic WSG is independently selected from: -Y 1 -(CH2CH2O) r -R'; -Y 1 -O-CH[(CH2) q -O-(CH2CH2O) r-R']2; and -Y 1 -CH2-Ph(Y 1 -(CH2CH2O) r -R') s ; where Y 1 Selected from covalent bonds, -O-, -CONH-, -NHCO-, NHSO2-, -SO2-NH-, -CONR-, -NRCO-, NRSO2-, -SO2-NR- (e.g., wherein R is alkyl or substituted alkyl), -(CH2) q -SO2-NH-, -(CH2) q -CONH- and -(CH2) q -O-; q and r are each independently an integer from 1 to 50; s is 1, 2 or 3; and each R' is independently H, an alkyl group (eg, methyl) or a substituted alkyl group.

[0146] In some examples, the branched nonionic WSG has one of the following formulas:

[0147]

[0148] in:

[0149] Each B 1 and B 2 are independently branching groups;

[0150] Each W 1 independently a nonionic water-soluble polymer, for example comprising 6 or more monomer units;

[0151] T 3 is an optional linker to the fused 6-5-6 tricyclic comonomer; and

[0152] Each p and q is independently 0 or 1, where each T, if present 1 and each T 2 In some embodiments, each W 1 W is independently PEG or a modified PEG polymer. In certain instances, each W 1 W is independently selected from substituted alkyl, PEG or modified PEG groups and WSG. In certain instances, each W 1 Independently PEG or modified PEG polymers having 6-30 monomer units, such as 6-24 or 10-30, 10-24 or 10-20, 12-24, 12-20, 12-16 or 16-20 monomer units.

[0153] In certain embodiments, the branched nonionic WSG has the formula:

[0154]

[0155] in:

[0156] Each B 1 is a branching group;

[0157] Each W 1 independently a nonionic water-soluble polymer, for example comprising 6 or more monomer units;

[0158] T 3 is an optional linker to the fused 6-5-6 tricyclic comonomer; and

[0159] Each p is independently 0 or 1, where each T, if present 1 In some embodiments, each W 1 W is independently PEG or a modified PEG polymer. In certain instances, each W 1 W is independently selected from substituted alkyl, PEG or modified PEG groups and WSG. In certain instances, each W 1 is independently PEG or a modified PEG polymer having 6-30 monomer units, such as 6-24 or 10-30, 10-24 or 10-20, 12-24, 12-20, 12-16 or 16-20 monomer units. In some embodiments of the branched nonionic WSG, B 1 is a tetra-substituted aryl group (eg, 1,3,4,5-phenyl).

[0160] In some embodiments of the branched nonionic WSG (eg, as shown in the above formula), B 1 is selected from CH, N, C(=O)N, SO2N, trisubstituted aryl (e.g., 1,3,5-phenyl), tetrasubstituted aryl, and trisubstituted heteroaryl. In some embodiments of the branched nonionic WSG, each p is 0. In some embodiments of the branched nonionic WSG, p is 1, and each T 1 Selected from -(CH2) n -O-, -O-(CH2) n -、-(CH2) n - and -O-, wherein n is 1 to 12, such as 1 to 6. In some embodiments of the branched nonionic WSG, each T 2 and / or T 3 Independently a C1-C12-alkyl linker, such as a C1-C6-alkyl linker, wherein one or more backbone atoms are optionally substituted by heteroatoms.

[0161] In some embodiments, the branched nonionic WSG is selected from one of the following structures:

[0162]

[0163]

[0164] in:

[0165] T 5 is an optional linker to a comonomer (e.g., a fused 6-5-6 tricyclic comonomer);

[0166] T 6 is a linking group;

[0167] Each s is independently an integer from 6 to 100 (e.g., 6 to 50); and

[0168] Each R 11 are independently hydrogen, alkyl or substituted alkyl.

[0169] In certain instances, each s is independently 6 to 30, such as 6 to 24, 6 to 20, 11 to 20, 12 to 20, 12 to 18, or 12 to 16. In certain instances, each s is independently 6 to 30, such as 6 to 24, 8 to 24, 10 to 24, 12 to 24, 13 to 24, 14 to 24, 15 to 22, or 16 to 20. In some cases, each s is independently 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. In some embodiments, each s is independently 7 or greater, such as 8, 9 or greater, 10 or greater, 11 or greater, 12 or greater, 13 or greater, 14 or greater, 15 or greater, or even greater; and in some cases, has up to 50 monomer units, such as up to 40, up to 30, or up to 24 monomer units. In some examples, each s is independently 6-30 monomer units, such as 6-24 or 10-30, 10-24 or 10-20, 12-24, 12-20, 12-16, or 16-20 monomer units. In some cases, each s is the same. In some embodiments of branched nonionic WSG, T 5 and / or T 6 is a C1-C12-alkyl linker, such as a C1-C6-alkyl linker, wherein one or more backbone atoms are optionally substituted by a heteroatom (e.g., —O—). In some embodiments of the branched nonionic WSG, each R 11 is H. In some embodiments of the branched nonionic WSG, each R 11 It is methyl.

[0170] In some embodiments of Formula (I), F 1is a fluorene comonomer substituted with two branched nonionic WSG groups (e.g., as described herein) (e.g., disubstituted at the 9 position). In some embodiments of Formula (I), F 1 is a fluorene comonomer substituted with two linear nonionic WSG groups (eg, as described herein) (eg, disubstituted at the 9 position).

[0171] In some embodiments of Formula (I), F 1 Has the following structure:

[0172]

[0173] Where s is 6-60;

[0174] t is 0-10 (e.g., 0-6, such as 0, 1, 2 or 3);

[0175] R 11 is H, alkyl or substituted alkyl. In certain cases, each R 11 It is methyl.

[0176] In some embodiments of Formula (I), F 1 One of the following comonomers:

[0177]

[0178] Any suitable water solubilizing group (WSG) may be included in the multichromophores described herein (e.g., multichromophores of formula (I)-(IV)) to provide increased water solubility. Although the increase in solubility can vary, in some instances, the increase (compared to compounds without WSG) is 2 times or more, e.g., 5 times, 10 times, 25 times, 50 times, 100 times or more. In some cases, the hydrophilic water solubilizing group is charged, e.g., positively charged or negatively charged. In some cases, the hydrophilic water solubilizing group is a neutral hydrophilic group. In some embodiments, WSG is branched (e.g., as described herein). In some instances, WSG is linear. In some embodiments, WSG is a hydrophilic polymer, e.g., polyethylene glycol, modified PEG, peptide sequence, peptoid, carbohydrate, oxazoline, polyol, dendritic macromolecule, dendritic polyglycerol, cellulose, chitosan, or derivatives thereof. Water solubilizing 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, guanidines, pyridines, 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 from 1 to 500; each R can independently be H or an alkyl group (such as 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 1-1000, and X is selected from O, S and NR ZZ And R ZZ and R YY Independently selected from H and C 1-3 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 solubilizing group includes a nonionic polymer (eg, a PEG polymer) substituted at the terminal end with an ionic group (eg, a sulfonate).

[0179] In some embodiments of the formula, the comonomer includes a substituent selected from: (CH2) x (OCH2CH2) y OCH3, wherein each x is independently an integer from 0 to 20, each y is independently an integer from 0 to 50; and phenyl optionally substituted by one or more halogen, hydroxyl, C1-C 12 Alkoxy or (OCH2CH2) z OCH3, wherein each z is independently an integer from 0 to 50. In some examples, the substituent is (CH2)3(OCH2CH2) 11 In some embodiments, one or more of the substituents is a benzyl group substituted with at least one WSG group (eg, 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.

[0180] Multiple WSG can be included in a single position in the subject multi-chromophore through a branched linker. In certain embodiments, the branched linker is an aralkyl substituent further substituted by a water-solubilizing group. Therefore, in some cases, the branched linker group is a substituent of a multi-chromophore that connects the multi-chromophore to two or more water-solubilizing groups. In certain embodiments, the branched linker is an amino acid, such as a lysine amino acid connected to three groups by an amino group and a carboxylic acid group. In some cases, multiple WSGs are incorporated into the branched linker to give the desired solubility of the multi-chromophore. In some instances, WSG is a nonionic side chain group that can give a solubility in water exceeding 50 mg / mL. In some instances, WSG is a nonionic side chain group that can give a solubility in water exceeding 100 mg / mL. In some embodiments, the multi-chromophore includes a substituent selected from the group consisting of: an alkyl, an aralkyl, and a heterocyclic group, each of which is further replaced by a hydrophilic polymer group including a water-solubilizing group such as polyethylene glycol (PEG) (e.g., a PEG group of 2-20 units).

[0181] In some examples of any formula described herein, one or more of the comonomers are replaced by WSG and / or a connected signal chromophore. Any suitable WSG can be adapted to be included in the comonomer of the subject polymer dye. In some examples of any formula described herein, one or more of the comonomers are replaced by WSG, and the WSG is independently selected from one of the following structures:

[0182]

[0183]

[0184] Among them: Each R 11 is H, alkyl (eg, methyl) or substituted alkyl; T 5 is an optional linking group; T 6is a linking group; and each s is independently an integer from 1 to 100 (e.g., 1 to 50, or 6 to 50). In some instances, each s is independently 1 to 50, such as 3 to 40, 3 to 30, 3 to 20, 3 to 15, 3 to 12, or 6 to 12. In some instances, each s is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, each s is 3. In some cases, each s is 4. In some cases, each s is 5. In some cases, each s is 6. In some cases, each s is 7. In some cases, each s is 8. In some cases, each s is 9. In some cases, each s is 10. In some cases, each s is 11. In some cases, each s is 12. In some cases, each s is 14. In some cases, each s is 16. In some embodiments, T 5 is an alkyl linker, such as a C1-C12 or C1-C6 alkyl linker. 5 is a substituted alkyl linker. In some embodiments, T 5 is an alkoxy linker (eg, -O-alkyl-). In some embodiments, T 5 It is understood that hydroxyl-terminated PEG chains can be used in place of methoxy-terminated PEG chains in any WSG group described herein. In some embodiments, each R 11 is H. In some embodiments, each R 11 It is methyl.

[0185] In certain examples of any of the formulas described herein, one or more of the comonomers is substituted with WSG independently selected from one of the following structures:

[0186]

[0187] Where: T 5 is an optional linking group; and each s is an integer from 1 to 50. In certain embodiments of WSG, T 5does not exist. In some instances, each s of WSG is independently 1 to 20, such as 3 to 20, 3 to 15, 3 to 12, or 6 to 12. In some cases, each s of WSG is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some cases, each s of WSG is 3. In some cases, each s of WSG is 4. In some cases, each s of WSG is 5. In some cases, each s of WSG is 6. In some cases, each s of WSG is 7. In some cases, each s of WSG is 8. In some cases, each s of WSG is 9. In some cases, each s of WSG is 10. In some cases, each s of WSG is 11. In some cases, each s of WSG is 12. In some cases, each s of WSG is 14. In some cases, each s of WSG is 16. In some embodiments, T 5 is an alkyl linker, such as a C1-C12 or C1-C6 alkyl linker. 5 is a substituted alkyl linker. In some embodiments, T 5 is an alkoxy linker (eg, -O-alkyl-). In some embodiments, T 5 is a substituted alkoxy. It should be understood that a hydroxyl-terminated PEG chain can be used in place of a methoxy-terminated PEG chain in any of the WSG groups described above. In certain examples of any of the formulas described herein, one or more of the comonomers is replaced by WSG, which is a dendrimer independently selected from one of the following structures:

[0188]

[0189] In certain examples of any of the formulas described herein, one or more of the comonomers is substituted with WSG, which is a polyol selected from one of the following structures:

[0190]

[0191] In certain examples of any of the formulas described herein, one or more of the comonomers is substituted with WSG, which is an oxazoline of the structure:

[0192]

[0193] In certain examples of any of the formulas described herein, one or more of the comonomers is replaced by WSG, which is a peptoid selected from one of the following structures:

[0194]

[0195] In some embodiments of formula (I), n is 5 or greater, such as 10 or greater, 30 or greater, 100 or greater, 300 or greater, 1000 or greater, 3000 or greater, or even greater. It should be understood that the conjugated polymer population can be represented by a thematic formula that includes some variations with respect to a specific length. In some cases, the conjugated segment can constitute 25 mol% or more of the multichromophore, such as 30 mol% or more, 40 mol% or more, 50 mol% or more, 60 mol% or more, 70 mol% or more, 80 mol% or more, or 90 mol% or more.

[0196] In some embodiments of Formula (I), the multichromophore has the structure of Formula (II):

[0197]

[0198] in:

[0199] M 1 、M 2 and L 1 are independently aryl or heteroaryl comonomers;

[0200] Z 1 is a linked chemoselective functional group or a linked acceptor chromophore;

[0201] x and y represent the molar concentration % of the unit in the multichromophore; and

[0202] G 1 and G 2 Each independently selected from end groups, π conjugated segments, linking groups and connected specific binding members. In some examples of formula (II), x is 5% or more (e.g., 5 mol%), such as 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or even more, accounting for the molar concentration of the multichromophore, wherein x+y is 100%. In some examples of Formula (II), y is 5% or more (e.g., 5 mol%), such as 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or even more, based on the molar concentration of the multichromophore.

[0203] In some embodiments of Formulas (I)-(II), the polymeric dye has the structure of Formula (IIa):

[0204]

[0205] where F 1 is a fused tricyclic comonomer substituted with a water solubilizing group (WSG) (e.g., as described herein); M 1 、M 2 and L 1 is independently an aryl or heteroaryl comonomer (eg, as described herein); Z 1 is a linked chemical selective functional group or a linked acceptor chromophore; G 1 and G 2Each is independently selected from an end group, a π-conjugated segment, a linker, and a linked specific binding member; and w, x, y, and z represent the molar concentration % of the unit in the multichromophore. In some examples of Formula (IIa), w is 5% or more (e.g., 5 mol%), such as 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or even more, by molar concentration of the multichromophore, wherein w+x+y+z is 100%. In some examples of Formula (IIa), x is 5% or more (e.g., 5 mol%), such as 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or even more, based on the molar concentration of the multichromophore, wherein w+x+y+z is 100%. In some examples of Formula (IIa), y is 5% or more (e.g., 5 mol%), such as 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or even more, based on the molar concentration of the multichromophore. In some examples of Formula (IIa), z is 5% or more (e.g., 5 mol%), such as 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or even more, based on the molar concentration of the multichromophore, wherein w+x+y+z is 100%.

[0206] In some embodiments, the polymeric dye has the structure of Formula (IIb):

[0207]

[0208] wherein u, v, w, x, y and z represent the mole % values ​​of each comonomer in the multichromophore; Y 1-3 Each Z group is independently a fused 6-5-6 tricyclic comonomer (eg, as described herein); M 1 、M2 and M 3 Each is an aryl or heteroaryl comonomer; and G 1 and G 2 Each independently selected from end group, π conjugated chain segment, linking group and connected specific binding members.It should be understood that any polymer dye described herein can be represented by formula (IIb).In some examples of formula (IIb), u+w+y constitutes 5% or more (e.g., 5 mol %) of multichromophore by molar concentration, such as 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more or even more.In some instances, z is 0. In some examples of Formula (IIb), v+x+z constitutes 80% or less (e.g., 80 mol%) of the multichromophore on a molar basis, such as 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or even less.

[0209] In some examples of formula (IIb), u is 5% or more, such as 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or even more. In some examples of formula (IIb), w is 5% or more, such as 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or even more. In some examples of formula (IIb), y is 1% or more, such as 2% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or even more. In some examples of formula (IIb), v is 80% or less, such as 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or even less. In some examples of formula (IIb), x is 80% or less, such as 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or even less. In some examples of formula (IIb), z is 80% or less, such as 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or even less.

[0210] In some embodiments of Formula (I), the multichromophore has the structure of Formula (III):

[0211]

[0212] in:

[0213] F1 It is a fused 6-5-6 tricyclic comonomer;

[0214] M 1 、M 2 and L 1 are independently aryl or heteroaryl comonomers;

[0215] Z 1 is a linked chemoselective functional group or a linked acceptor chromophore;

[0216] Each n is independently an integer from 1 to 10,000;

[0217] each m is 0 or independently an integer from 1 to 10,000;

[0218] p is an integer from 1 to 100,000; and

[0219] G 1 and G 2 Each is independently selected from a terminal group, a π-conjugated segment, a linker, and a linked specific binding member.

[0220] In certain embodiments of Formula (III), the multichromophore has the structure of Formula (IV):

[0221]

[0222] In some examples of Formulas (II)-(IV), L 1 To be Z 1 and a branched nonionic water-soluble group (WSG) (e.g., as described herein) substituted fused 6-5-6 tricyclic comonomer. In certain examples of formulas (II)-(IV), L 1 Has the following structure:

[0223]

[0224] Where: R 3 is a branched nonionic water-soluble group (WSG) (e.g., as described herein); and R 4 For L 2 -Z 2 , where L 2 is a linker and Z 2 is a chemically selective functional group or an acceptor chromophore. 1 In certain embodiments, R 3 Select one of the following structures:

[0225]

[0226] In L 1 In some cases, T5 is a C1-C6 linker. In some cases, each s is 6 to -30, such as 6 to 20, 11 to 20, 12 to 20, or 12 to 16. In some cases, each s is independently 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, each s is 11. In some cases, each s is 12. In some cases, each s is 13. In some cases, each s is 14. In some cases, each s is 15. In some cases, each s is 16. In some cases, each s is 17. In some cases, each s is 18. In some cases, each s is 19. In some cases, each s is 20. In L 1 In certain embodiments, R 4 For L 2 -Z 2 , where L 2 is a linker (e.g., a C1-C20 linker that optionally includes one or more heteroatoms or linking functional groups (e.g., -CONH- or SO2NH-) in the linker backbone; and Z 2 is a chemoselective functional group (e.g., an amino group or a protected amino group, or a carboxylic acid or its active ester, or a protected carboxylic acid). 1 Has one of the following structures:

[0227]

[0228] In some embodiments of Formulas (I)-(IV), the polymeric dye has the structure of Formula (V) or (VI):

[0229]

[0230]

[0231] wherein each WSG is independently a branched or linear WSG (e.g., as described herein); Z 1 is a linked chemical selective functional group or a linked acceptor chromophore; G 1 and G 2 Each is independently selected from a terminal group, a π-conjugated segment, a linker, and a linked specific binding member; and x, y, and z represent the molar concentration % of the unit in the multichromophore. In some cases of formula (V)

[0232] In some embodiments of Formulas (I)-(VI), the polymeric dye has the structure of Formula (VII or (VIII):

[0233]

[0234]

[0235] wherein each s is independently an integer from 6 to 50, such as 6-30, 6 to 20, 11 to 20, 12 to 20 or 12 to 16; each R 11 are independently hydrogen, alkyl or substituted alkyl; DYE is an attached acceptor chromophore (eg, fluorophore); r is 0-6 (eg, 0, 1, 2, 3 or 4); and q is 1-12, such as 1-6 (eg, 1, 2, 3 or 4).

[0236] Any suitable comonomer may be adapted to include side chain substituents that provide for conjugation of the polymeric dye to a moiety of interest, such as a signaling chromophore or a biomolecule.Any comonomer may include a substituent that imparts increased water solubility to the polymeric dye.

[0237] In some embodiments of any formula described herein, n, m and p are selected so that the multichromophore includes 2 to 100,000 repeating units (i.e., monomer repeating units) in total, wherein the multichromophore may include a plurality of different monomer repeating units. In some instances, when m is 0, p is 1 and n is 2 to 100,000. It should be understood that the conjugated polymers of the formula described herein may also be represented by a formula providing a mole % value of each comonomer in the polymer. It should also be understood that in some cases, the polymerization process may produce a composition comprising a conjugated polymer population, the composition comprising some changes in a specific length and / or end group (i.e., terminal group) present in each conjugated polymer in the population. The formula shown herein may refer to a population or subpopulation of a single compound or a polymeric compound.

[0238] Aromatic or heteroaromatic comonomers

[0239] As outlined above, the subject polymeric dye comprises the conjugated segments of the aryl or heteroaryl comonomers connected by covalent bonds, vinylidene or ethynylene groups. In addition to the comonomers described in the above exemplary formulas, a variety of aryl and heteroaryl comonomers can be used in the subject polymeric dye. Any suitable aryl and heteroaryl comonomers can be used in the subject multichromophore, for example, in the band gap modified unit, and can be evenly or randomly distributed along the conjugated polymer as described herein.

[0240] In some embodiments of the formulae described herein (eg, formulae (I)-(IV)), the aromatic or heteroaromatic comonomer (eg, L 1 、M 1 、M 2 and M 3 ) are independently selected from one of formula (XXIII)-(XXVI):

[0241]

[0242] in

[0243] Cy 2-7 refers to an aryl or heteroaryl group containing 2 to 7 fused and / or unfused rings;

[0244] Y 2 , Y 3 and Y 4 Independently selected from -CR 3 -、NR 3 , N, O, S and -C(=O)- and together form a 5- or 6-membered fused aryl or heteroaryl ring;

[0245] Each R 3 is one or more independently selected from H, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano, alkoxy substituted alkoxy and -T 1 -Z 1 a ring substituent of

[0246] R 1 and R 2 independently selected from H, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano, alkoxy, substituted alkoxy, and -T 1 -Z 1 , or R 1 and R 2 together form an optionally substituted 5- or 6-membered fused aryl, heteroaryl, cycloalkyl or heterocyclic ring;

[0247] Y 5 N or CR 5 And Y 7 N or CR 7 ;

[0248] R 4 -R 7 independently selected from H, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano, alkoxy, substituted alkoxy, and -T 1 -Z 1 ;

[0249] Z 1 is a chemoselective functional group or an attached signaling chromophore; and

[0250] T 1 For the connection base.

[0251] Cy 2-7is an aryl or heteroaryl group that may include 2, 3, 4, 5, 6, or 7 rings, which may be fused together to form a fused ring system or may be unfused (i.e., linked together by a single covalent bond). 2-7 In some embodiments, Cy 2-7 It is composed of 5-membered and / or 6-membered carbocyclic and / or heterocyclic rings. 2-7 is a bicyclic, tricyclic or tetracyclic aromatic or heteroaromatic group, such as naphthalene, anthracene, acridine or quinoline.

[0252] In certain embodiments of Formulas (XXIII)-(XXVI), the comonomer (e.g., M 1 、M 2 and M 3 ) is independently selected from one of the following structures (a) to (x):

[0253]

[0254]

[0255]

[0256] in:

[0257] Each Y 8 Independently C(R 3 )2、-C(R 3 )2C(R 3 )2-、-C(R 3 )2Si(R 3 )2-、NR 3 or Si(R 3 )2;

[0258] X is S or O; each R 3 are independently H, a water solubilizing group (e.g., as described herein), amino, substituted amino, halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano, alkoxy, substituted alkoxy, and -T. 1 -Z 1 ;

[0259] R 1 and R 2 independently selected from H, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano, alkoxy, substituted alkoxy, and -T 1 -Z 1 , or R 1 and R2 Together they form may be optionally substituted (e.g. by R 3 a 5-membered or 6-membered fused aryl ring, heteroaryl ring, cycloalkyl ring or heterocyclic ring substituted with a cycloalkyl group;

[0260] Z 1 is a chemoselective functional group or an attached signaling chromophore; and

[0261] T 1 For the connection base.

[0262] In some instances, Y 8 C(R 3 )2. In some instances, Y 8 -C(R 3 )2C(R 3 )2-. In certain instances, Y 8 NR 3 In some instances, Y 8 Si(R 3 )2. In certain embodiments of formula (a)-(x), when the comonomer is a linking comonomer, R 1 -R 7 At least one of them is -T 1 -Z 1 In certain embodiments of formulae (a)-(x), the comonomer includes a water-soluble group (e.g., as described herein). In certain embodiments of formulae (these), R 3 is H, halogen (e.g., F), or alkoxy (e.g., methoxy). It is understood that in any of the structures of (a)-(x) described herein, a N heteroatom may be included to convert a phenyl or fused benzo ring into a pyridyl or fused pyrido ring. Such heteroatom substituted forms of formula (a)-(x) are intended to be included in the present disclosure. In some examples of formula (XXIII)-(XXVI), one or more of the comonomers has a structure of one of the formulas (these) in which the benzo ring is replaced by a pyrido ring:

[0263]

[0264] where Y 8 Independently C(R 4 )2、-C(R 4 )2C(R 4 )2-、NR 4 or Si(R 4 )2.

[0265] In some examples of formulas (XXIII)-(XXVI) and (a)-(x), the comonomer (M 2 、M 3 and M4 ) is independently selected from one of the following structures (ba) to (cd):

[0266]

[0267]

[0268]

[0269] in:

[0270] Y 9 C(R 4 )2、-C(R 4 )2C(R 4 )2- or Si(R 4 )2;

[0271] X is S or O;

[0272] Each R 4 are independently H, a water solubilizing group (e.g., as described herein), alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano, alkoxy, substituted alkoxy, and -T. 1 -Z 1 ;and

[0273] R 1 and R 2 independently selected from H, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano, alkoxy, substituted alkoxy, and -T 1 -Z 1 , or R 1 and R 2 together to form a 5-membered or 6-membered fused aryl or heteroaryl ring which may be optionally substituted; in certain embodiments of formulas (ba)-(cd), when the comonomer is a linking comonomer, R 4 At least one of them is -T 1 -Z 1 In certain embodiments of Formulas (ba)-(cd), R 4 A water solubilizing group is included (eg, as described herein).

[0274] The aryl and heteroaryl comonomers described herein (e.g., comonomers of formula (I)-(IV), (a)-(x), and (ba)-(cd)) may include a water solubilizing group WSG (e.g., as described herein). In some embodiments, the aryl and heteroaryl comonomers include a branched nonionic WSG selected from one of the following structures:

[0275]

[0276] Where: T 5 is an optional linking group to a comonomer; each s is independently an integer from 6 to 50 or 6-30, such as from 6 to 20, 11 to 20, 12 to 20 or 12 to 16; and each R 11 In some cases, each s is independently hydrogen, alkyl, or substituted alkyl. In some cases, each s is independently 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, each s is 11. In some cases, each s is 12. In some cases, each s is 13. In some cases, each s is 14. In some cases, each s is 15. In some cases, each s is 16. In some cases, each s is 17. In some cases, each s is 18. In some cases, each s is 19. In some cases, each s is 20. In some embodiments, T 5 is an alkyl linker, such as a C1-C6 alkyl linker. 5 is a substituted alkyl linker. In some embodiments, T 5 is an alkoxy linker (eg, -O-alkyl-). In some embodiments, T 5 is a substituted alkoxy group.

[0277] In certain examples of comonomers, the aromatic and heteroaromatic comonomers include WSG selected from one of the following structures (e.g., R 3 and / or R 4 ):

[0278]

[0279] Where: T 5 is an optional linker; and each s is an integer from 1 to 50. In certain instances, each s is independently 1 to 20, such as 3 to 20, 3 to 15, 3 to 12, or 6 to 20 or 6 to 12 or 12-16. In certain cases, each s is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In some cases, each s is 3. In some cases, each s is 4. In some cases, each s is 5. In some cases, each s is 6. In some cases, each s is 7. In some cases, each s is 8. In some cases, each s is 9. In some cases, each s is 10. In some cases, each s is 11. In some embodiments, T 5 is an alkyl linker, such as a C1-C6 alkyl linker. 5is a substituted alkyl linker. In some embodiments, T 5 is an alkoxy linker (eg, -O-alkyl-). In some embodiments, T 5 is a substituted alkoxy group.

[0280] In certain embodiments of the formulae described herein (eg, formulae (I)-(IV)), the comonomer (eg, M 1 、M 2 and M 3 ) are independently selected from one of the following structures:

[0281]

[0282]

[0283]

[0284]

[0285] wherein each s is independently 1-30, such as 6-30, such as 6 to 20, 11 to 20, 12 to 20, or 12 to 16. In certain cases, each s is independently 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, each s is 4. In some cases, each s is 5. In some cases, each s is 6. In some cases, each s is 7. In some cases, each s is 8. In some cases, each s is 9. In some cases, each s is 10. In some cases, each s is 11. In some cases, each s is 12. In some cases, each s is 13. In some cases, each s is 14. In some cases, each s is 15. In some cases, each s is 16. In some cases, each s is 17. In some cases, each s is 18. In some cases, each s is 19. In some cases, each s is 20.

[0286] In some examples, a multichromophore (eg, a multichromophore of Formula (I)-(IV)) includes a comonomer (eg, L 1 ):

[0287]

[0288] Wherein WSG is an optional water-soluble group; T 2 is a linker; and Dye is a signal chromophore.

[0289] In certain embodiments, the aryl or heteroaryl comonomer is an optionally substituted comonomer selected from 2,1,3-benzothiadiazole, 2,1,3-benzoxadiazole, benzoxidazole, benzoselenadiazole, benzotellurodiazole, naphthoselenadiazole, 4,7-di(thiophen-2-yl)-2,1,3-benzothiadiazole, squarylium dyes, quinoxaline, perylene, perylene diimide, diketopyrrolopyrrole, thienopyrazine low bandgap commercial dyes, olefins and cyano-substituted olefins, and isomers thereof. In some examples, the aryl and heteroaryl comonomers useful for the subject multichromophore are selected from a'-m' having the following structures:

[0290]

[0291] Where * = site for covalent attachment to the unsaturated backbone, and each R is independently H, a nonionic side group capable of imparting solubility in water (e.g., WSG), or -L 2 -Z 2 , where L 2 is a linker and Z 2 is a chemoselective tag or a linked metal complex. In certain examples of a'-m', each R is H or a water solubilizing group (e.g., as described herein). In some cases, each R is a (CH2) x (OCH2CH2) y OCH3 substituted alkyl or benzyl, wherein each x is independently an integer from 0 to 20, and each y is independently an integer from 0 to 50. In certain examples of a'-m', each R is (CH2)3(OCH2CH2) 11 OCH3. In certain examples of a'-m', each R is H or a nonionic branched WSG (eg, as described herein).

[0292] In some embodiments, the multichromophore includes an absorbance-modulating comonomer having the following structure:

[0293]

[0294] Where X is O or S, R 41 and R 42 Each is independently H, halogen, WSG, alkyl, substituted alkyl, alkoxy, and substituted alkoxy. In some instances, X is O. In some instances, X is S. In some embodiments, X is O and R 41 and R 42 Each is H. In certain embodiments, X is S and R41 and R 42 Each is H.

[0295] In some examples, the aryl or heteroaryl comonomer is a substituted or unsubstituted phenyl, biphenyl or pyridyl comonomer. In certain embodiments, the aryl or heteroaryl comonomer is selected from substituted or unsubstituted 1,4-phenyl, substituted or unsubstituted 1,3-phenyl, substituted or unsubstituted 4,4'-biphenyl, substituted or unsubstituted 2,5-pyridyl and substituted or unsubstituted 2,6-pyridyl. In certain examples, the absorbance adjustment comonomer is an optionally substituted aryl or heteroaryl comonomer selected from one of the following structures:

[0296]

[0297] Where Z 2 -Z 5 Each independently represents CR or N, wherein at least one Z 2 -Z 5 is N; and each R and each R 11 -R 16 is independently selected from the group consisting of hydrogen, a water solubilizing group, a halogen, a cyano group, an alkoxy group, a substituted alkoxy group, an alkyl group, and a substituted alkyl group. 2 -Z 5 One and only one of is N. In certain embodiments, Z 2 -Z 5 Two and only two of are N. In some instances, R 11 , R 12 and R 14 Each is H. In some examples, R 12 and R 14 Each is H. In some examples, R 11 and R 13 Each is H. In some cases, R 15 and R 16 Each is H. In some instances, the halogen is fluorine.

[0298] In some cases, the aryl or heteroaryl comonomer is selected from one of the following:

[0299]

[0300] wherein n is 1-20 and R' is H or lower alkyl. In some embodiments of the aryl or heteroaryl comonomer structure, n is an integer from 6 to 20, such as 12-20 or 12-16 or 16-20.

[0301] In some embodiments, the multichromophore includes a substituted aromatic comonomer described by the following structure:

[0302]

[0303]

[0304]

[0305]

[0306] In some embodiments, the multichromophore includes a substituted aromatic comonomer described by the following structure:

[0307]

[0308] wherein n is 1-20 and R' is H or a lower alkyl. In certain examples, n is 3 to 12. In some embodiments, the multichromophore includes a substituted aromatic comonomer described by the following structure:

[0309]

[0310] Wherein each n is independently 1-20, and each R' is independently H or lower alkyl. In certain embodiments of the substituted aryl or heteroaryl comonomer structures shown above, n is 3. In some examples, n is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In certain examples, R' is methyl. In certain examples, R' is hydrogen. In some embodiments, the multichromophore includes a substituted aryl comonomer described by the following structure:

[0311]

[0312] In some embodiments, the multichromophore includes a substituted aromatic comonomer described by the following structure:

[0313]

[0314] In some embodiments of any of the formulas described herein, the multichromophore includes a substituted aromatic comonomer (M1) having the structure:

[0315]

[0316] wherein s is 6-50 (e.g., 6-40, 6-30, or 6-20); and each R 11 are independently H, alkyl or substituted alkyl. In some embodiments, the multichromophore includes a substituted aryl comonomer comprising a branched nonionic WSG described by one of the following structures:

[0317]

[0318] Any of the absorbance-modulating comonomers described above may be used in the subject multichromophores, such as those of Formulae (I)-(IV).

[0319] Polymer tandem dyes

[0320] The water-soluble light-harvesting multichromophore itself can be fluorescent and capable of transferring energy to a connected acceptor signal chromophore. Therefore, the polymer tandem dye of the present invention also includes a covalently linked acceptor signal chromophore that is in energy-receiving proximity to the donor water-solvated light-harvesting multichromophore. Therefore, excitation of the multichromophore donor causes energy transfer to and emission from the covalently linked acceptor signal chromophore. The number of signal chromophore acceptor units connected to the donor water-solvated light-harvesting multichromophore can vary, wherein in some instances, the number ranges from 1 mol % to 50 mol %, such as 5 mol % to 25 mol % or 10 mol % to 25 mol %.

[0321] Energy transfer mechanisms from the fluorescent water-solvated light-harvesting multichromophore donor to the linked acceptor signal chromophore include, for example, resonance energy transfer (e.g., (or fluorescence) resonance energy transfer, FRET), quantum charge exchange (Dexter energy transfer), etc. In some instances, the range of these energy transfer mechanisms is relatively short; that is, the close proximity of the light-harvesting multichromophore system to the acceptor provides efficient energy transfer. In some instances, under conditions of efficient energy transfer, the emission from the luminescent signal chromophore is more intense when the incident light ("pump light") is at a wavelength absorbed by the light-harvesting multichromophore than when the luminescent signal chromophore is directly excited by the pump light, in which case the emission from the acceptor is amplified.

[0322] "Effective" energy transfer means that 10% or more of the energy harvested by the donor is transferred to the acceptor, such as 20% or more or 30% or more. "Amplification" means that when excited by energy transfer from the donor light-harvesting multichromophore, the signal from the signal chromophore is 1.5x or greater than that directly excited by incident light of equivalent intensity. Any suitable method can be used to measure the signal. In some cases, a signal of 1.5x or greater refers to the intensity of the emitted light. In some cases, a signal of 1.5x or greater refers to an increased signal-to-noise ratio. In certain embodiments of polymer tandem dyes, compared with incident light directly exciting the signal chromophore, the signal chromophore emission when excited by the multichromophore is 1.5 times or greater, such as 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 than that directly exciting the signal chromophore by the incident light.

[0323] The linked luminescent signal chromophore emission of the polymer tandem dye may have a quantum yield of 0.03 or greater, such as 0.04 or greater, 0.05 or greater, 0.06 or greater, 0.07 or greater, 0.08 or greater, 0.09 or greater, 0.1 or greater, 0.15 or greater, 0.2 or greater, 0.3 or greater, or even greater. In some examples, the polymer tandem dye has a quantum yield of 5 x 10 5 cm -1 M -1 or larger extinction coefficient, such as 6x 10 5 cm -1 M -1 or larger, 7x 10 5 cm -1 M -1 or larger, 8x10 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, 4x10 6 cm -1 M -1 or larger, 5x10 6 cm -1 M -1 or larger, 6x 10 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 polymer tandem dye has 5 x 10 5 M -1 cm -1In certain embodiments, the polymer tandem dye has a molar extinction coefficient of 1 x 10 6 M -1 cm -1 or greater molar extinction coefficient.

[0324] The subject polymer tandem dyes provide fluorescent emission from the luminescent signal chromophore dyes that is brighter than the emission that these luminescent dyes could provide individually. The emission of the linked luminescent signal chromophore of the polymer tandem dye can have a 50 mM -1 cm -1 or greater brightness, such as 60mM -1 cm -1 or greater, 70mM -1 cm -1 or greater, 80mM -1 cm -1 or greater, 90mM -1 cm -1 or greater, 100mM -1 cm -1 or greater, 150mM -1 cm -1 or greater, 200mM -1 cm -1 or greater, 250mM -1 cm -1 or larger, 300mM -1 cm -1 In some examples, the brightness of the emission of the linked signal chromophore of the polymer tandem dye is at least 5 times the brightness of the directly excited luminescent dye, such as at least 10 times, at least 20 times, at least 30 times, at least 50 times, at least 100 times, at least 300 times, or even greater than the brightness of the directly excited luminescent dye.

[0325] In some embodiments, the polymer tandem dye includes: a water-soluble light-harvesting multichromophore comprising a conjugated segment having a structure of formula (I):

[0326]

[0327] in:

[0328] F 1 It is a fused three-ring comonomer substituted with a water-soluble group (WSG);

[0329] M 1 is an aromatic or heteroaromatic comonomer;

[0330] n is an integer from 1 to 100,000; and

[0331] * indicates the site for covalent attachment to the unsaturated backbone or terminal group of the conjugated polymer;

[0332] Among them, F 1 and M 1 At least one of the water-soluble polymers is substituted with a branched nonionic water-soluble group (WSG) comprising two or more water-soluble polymers, each of which has 6 or more monomer units; and

[0333] A signal chromophore is covalently attached to the multichromophore, the multichromophore being in energy-accepting proximity to the signal chromophore.

[0334] In some embodiments, the polymer tandem dye includes: a water-soluble light-harvesting multichromophore comprising a conjugated segment having a structure of formula (I):

[0335]

[0336] in:

[0337] F 1 is a fused 6-5-6 tricyclic comonomer substituted with a branched nonionic water solubilizing group (WSG) comprising two or more water soluble polymers, each of which has 6-30 monomer units (e.g., 10-20 units, 12-16 units, etc.);

[0338] M 1 is an aromatic or heteroaromatic comonomer;

[0339] n is an integer from 1 to 100,000; and

[0340] * indicates the site for covalent attachment to the unsaturated backbone or terminal group of the conjugated polymer; and

[0341] The signal chromophore covalently linked to the multichromophore, the multichromophore is in energy receiving proximity with the signal chromophore. Side chain nonionic water-soluble groups (WSG) can give polymer tandem dyes solubility in water exceeding 10mg / mL, such as exceeding 20mg / mL, exceeding 30mg / mL, exceeding 40mg / mL, exceeding 50mg / mL, exceeding 60mg / mL, exceeding 70mg / mL, exceeding 80mg / mL, exceeding 90mg / mL or exceeding 100mg / mL. In some cases, side chain nonionic water-soluble groups (WSG) can give polymer tandem dyes solubility in water (for example, aqueous buffer) 20mg / mL or higher, such as 30mg / mL or higher, 40mg / mL or higher, 50mg / mL or higher, 60mg / mL or higher, 70mg / mL or higher, 80mg / mL or higher, 90mg / mL or higher, 100mg / mL or higher or even higher.

[0342] In certain embodiments or formulae described herein (e.g., formulae (I)-(IV) and (a)-(w) and (ba)-(cb)), a signal chromophore (e.g., as described herein) is included as a comonomer substituent. Any suitable signal chromophore can be attached to any suitable polymeric dye described herein via coupling of compatible chemoselective functional groups. The signal chromophore can be selected to provide a desired emission spectrum and emission maximum wavelength.

[0343] As used herein, the terms "chemoselective functional group" and "chemoselective tag" are used interchangeably and refer to a functional group that can selectively react with another compatible functional group to form a covalent bond, in some cases after one of the functional groups is optionally activated. 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 by click chemistry (e.g., azides and alkyne groups (e.g., cyclooctyne groups), as well as hydroxyl groups, hydrazide groups, hydrazine groups, aldehydes, ketones, azides, alkynes, phosphines, epoxides, and the like.

[0344] Any suitable linking comonomer may be incorporated into the subject multichromophore to provide a linking group to which any suitable moiety of interest (e.g., a linked signal chromophore) may be attached. Linking comonomers of interest include, but are not limited to, those described in the formula herein, as well as fluorene comonomers, phenylene vinylene comonomers, phenylene ethynylene comonomers, carbazole comonomers, C2-C 12Alkyne comonomers, arylene-ethynylene comonomers, heteroarylene-ethynylene comonomers, arylene comonomers and heteroarylene comonomers. As used herein, the terms aryl or heteroaryl comonomer and arylene or heteroarylene comonomer are used interchangeably. In some cases, the connecting comonomer is a substituted aryl comonomer. In some cases, the connecting comonomer is a substituted heteroaryl comonomer. In some cases, the connecting comonomer is a substituted or unsubstituted 1,4-phenyl, a substituted or unsubstituted 1,3-phenyl, a substituted or unsubstituted 4,4'-biphenyl, a substituted or unsubstituted 2,5-pyridyl and a substituted or unsubstituted 2,6-pyridyl.

[0345] In some examples of any of the formulas described herein, the signal chromophore is linked to a comonomer that comprises 1% to 50% by mole of the polychromophore, such as 1% to 20%, 1% to 10%, or 11% to 20% by mole. In some cases, the polychromophore is a conjugated polymer comprising 5 or more repeating units.

[0346] Any suitable chemoselective functional groups may be included in the subject polychromophores (e.g., including -Z 1 , -Z 2 and / or G 1 or G 2 The terminal groups include, but are not limited to, carboxylic acid, active esters (e.g., NHS or sulfo-NHS esters), amino, hydroxyl, thiol, maleimide, iodoacetyl, hydrazide, hydrazine, aldehyde, ketone, azide, alkyne, phosphine, and epoxide. It should be understood that in the polymer tandem dye structures described herein, in some cases, the group Z 1 and Z 2 Appearing at equivalent positions in the structure, where these groups are used interchangeably, refer to linked signal chromophores or chemoselective functional groups that can subsequently be conjugated to a suitable chromophore precursor to generate a linked signal chromophore.

[0347] In some cases, the signal chromophore is a fluorophore. In some cases, the signal chromophore is a quencher. Any suitable fluorescent dye can be used as the acceptor chromophore in the polymer tandem dye. The terms "fluorescent dye" and "fluorophore" are used interchangeably herein. 1 ) can be a dye molecule selected from the group consisting of rhodamine, coumarin, xanthene, cyanine, polymethine, pyrene, boron dipyrrole fluoride, naphthylimide, phycobiliprotein, peridinin chlorophyll protein, conjugates thereof, and combinations thereof. In certain embodiments, the signal chromophore (Z 1 ) are cyanine dyes, xanthene dyes, coumarin dyes, thiazine dyes and acridine dyes. In some examples, the signal chromophore (Z 1) is selected from DY 431, DY 485XL, DY500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 778, DY 782, DY 800, DY 831, Biotium CF 555, Cy 3.5 and diethylaminocoumarin. In some embodiments, the acceptor chromophore is a cyanine dye, a xanthene dye, a coumarin dye, a thiazine dye and or an acridine dye. Fluorescent dyes of interest include, but are not limited to, fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine 110, CascadeBlue, Cascade Yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP (peridinyl chlorophyll a protein), PerCP-Cy5.5, JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, OregonGreen 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 514, 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY FL, BODIPY FL-Br2, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY R6G, BODIPY TMR, BODIPY TR, their conjugates and their combinations. 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 polymer dye linked to an acceptor fluorophore selected from the group consisting of Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Alexa488, Alexa 647, and Alexa700. In certain embodiments, the polymer tandem dye comprises a polymer dye linked to an acceptor fluorophore selected from the group consisting of: Dyomics dyes (such as DY 431, DY 485XL, DY 500XL, DY530, DY 610, DY 633, DY 640, DY 651, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY731, DY 732, DY 734, DY 752, DY 754, DY 778, DY 782, DY 800, or DY 831), Biotium CF 555, Cy 3.5, and diethylaminocoumarin.

[0348] In some embodiments, the emission maximum wavelength of the selected signal chromophore is in the range of 300 to 900 nm, such as 350 to 850 nm, 350 to 600 nm, 360 to 500 nm, 370 to 500 nm, 380 to 500 nm, 390 to 500 nm, or 400 to 500 nm, wherein specific examples of emission maxima of the signal chromophore of interest include, but are not limited to, 395 nm ± 5 nm, 420 nm ± 5 nm, 430 nm ± 5 nm, 440 nm ± 5 nm, 450 nm ± 5 nm, 460 nm ± 5 nm, 470 nm ± 5 nm, 480 nm ± 5 nm, 490 nm ± 5 nm, 500 nm ± 5 nm, 510 nm ± 5 nm, 520 nm ± 5 nm, 530 nm ± 5 nm, 540 nm ± 5 nm, 550 nm ± 5 nm, 560 nm ± 5 nm, 570 nm ± 5 nm, 580 nm ± 5 nm, 590 nm ± 5 nm, 600 nm ± 5 nm, 610 nm ± 610 nm 460nm±5nm, 470nm±5nm, 480nm±5nm, 490nm±5nm, 500nm±5nm, 510nm±5nm, 520nm±5nm, 530nm±5nm, 540nm±5nm, 550nm±5nm, 560nm±5nm, 570nm±5nm, 580nm±5nm, 590nm±5nm, 605nm±5nm, 650nm±5nm, 680nm±5nm, 700nm±5nm, 805nm±5nm.

[0349] Terminal Group

[0350] Any suitable terminal group (e.g., G 1 and G 2 ) can be used at the end of the subject multichromophore. As used herein, the terms "terminal group" and "terminal group" are used interchangeably to refer to a group located at the end of the polymeric structure of a multichromophore (e.g., as described herein). G of interest 1 and G 2The group includes, but is not limited to, a terminal capping group, a π-conjugated segment, a linking group, and a linked specific binding member. In some embodiments, the terminal capping group is a monovalent group that is conjugated to the main chain of the multichromophore after polymerization. In some instances, the terminal capping group is an aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, or substituted alkyl. In some cases, the terminal comonomer is directed to be connected to a chemically selective tag or linking group. In some cases, the terminal capping group is derived from a monomer used in the polymerization method, such as an end group such as a halogen (e.g., Br), boric acid, or borate that can be further conjugated. In some instances, G 1 and / or G 2 As used herein, a π-conjugated segment refers to any suitable segment of a conjugated polymer that can be conjugated to a multichromophore, i.e., allows π electrons to be delocalized across adjacent units. In certain embodiments, G 1 and / or G 2 is a linker, such as a linker comprising a functional group suitable for conjugation with a specific binding moiety. It will be appreciated that G can be selected to be located at the polychromophore. 1 and / or G 2 The linker at the position to allow for attachment to the side chain that may be present in the multi-chromophore (e.g., Z 2 In some embodiments, the amino functional group or its derivative is included in G. 1 and / or G 2 and a carboxylic acid functional group or a derivative thereof is included in Z 2 In certain embodiments, the carboxylic acid functional group or its derivative is included in G 1 and / or G 2 and an amino functional group or a derivative thereof is included in Z 2 Place.

[0351] In some embodiments of the formulae described herein, G 1 and G 2 At least one of them is -L 3 -Z 4 , where L 3 is a linker (e.g., 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 1 and G 2 At least one of them is -L 3 -Z 3 , where L 3 is a linker (e.g., as described herein) and Z 3is a chemoselective tag (e.g., as described herein). Any suitable chemoselective tag and conjugation chemistry can be adapted for use with the subject multichromophore. 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 examples, Z 3 is selected from the group consisting of a carboxylic acid, an active ester (e.g., N-hydroxysuccinimide ester (NHS) or sulfo-NHS), an amino group, a maleimide, an iodoacetyl group, and a thiol group. In certain embodiments of the formulae described herein, G 1 and G 2 At least one of is described by the following structure:

[0352] *-Ar-LZ

[0353] 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 direct attachment 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:

[0354]

[0355] in:

[0356] q is 0 or an integer from 1 to 12;

[0357] L is an optional linker; and

[0358] Z is a chemoselective tag or a specific binding member.

[0359] In certain embodiments, Z is a biomolecule. The biomolecule of interest includes, but is not limited to, polypeptides, polynucleotides, carbohydrates, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs thereof, and combinations thereof. In certain instances, Z is an antibody. In some instances, Z is an antibody fragment or a binding derivative thereof. In some cases, an antibody fragment or a binding derivative thereof is selected from the group consisting of: a Fab fragment, a F(ab')2 fragment, a scFv, a diabody, and a triabody.

[0360] It is understood that for any of the structures and formulae shown herein, in some instances of the subject multichromophores, the terminal group shown may be located at the opposite end to the terminal group shown, e.g., terminal group G 1In some embodiments of the multichromophores described herein (eg, Formulas (II) to (VI)), G 1 and G 2 At least one of the following structures 1-33 is selected:

[0361]

[0362]

[0363]

[0364] * = site for covalent attachment to the unsaturated backbone;

[0365] Wherein R' is independently H, halogen, C1-C 12 Alkyl, (C1-C 12 Alkyl)NH2, C2-C 12 Olefins, C2-C 12 Alkynes, C3-C 12 Cycloalkyl, C1-C 12 Halogenated alkyl, C2-C 18 (Hetero)aryl, C2-C 18 (Hetero)arylamino, -[CH2-CH2] r′ -Z 1 , or (C1-C 12 )alkoxy-X 1 ; and where Z 1 is -OH or -COOH; X 1 -NH2, -NHCOOH, -NHCOOC(CH3)3, -NHCO(C3-C12)cycloalkyl(C1-C4)alkyl-N-maleimide; or -NHCO[CH2-CH2-O] s′ (CH2) s′ NH2; r' is an integer from 1 to 20; and each s' is independently an integer from 1 to 20; (CH2)3(OCH2CH2) x″ OCH3, wherein x" is independently an integer from 0 to 50; or optionally substituted by one or more halogen, hydroxyl, C1-C 12 Alkoxy, or (OCH2CH2) y″ CH3-substituted benzyl, wherein each y″ is independently an integer from 0 to 50 and R′ is different from R;

[0366] Where k is 2, 4, 8, 12 or 24;

[0367] Where R 15 Selected from the group consisting of lu having the following structure:

[0368]

[0369] * = site for covalent attachment to the backbone.

[0370] In some embodiments of the multichromophores described herein (eg, Formulas (V)-(XVI)), at least one terminal group (eg, T 2 -Z 2 , G 1 , G 2 , -LZ, -L 3 -Z 3 ) or a side chain group is selected from one of the following structures:

[0371]

[0372] wherein r is 0 or an integer from 1 to 50; k is 0 or an integer from 1 to 50 (e.g., 1 to 20); R 1 As defined for any fluorene comonomer described herein; and R 16 Selected from H, OH, NH2, -NH(CH2)r-NH2 and -NH(CH2) r COOH. In some instances, r is 1 to 20, such as 3 to 20, 3 to 15, 3 to 12, or 6 to 12. In some cases, r is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some cases, r is 3. In some cases, r is 4. In some cases, r is 5. In some cases, r is 6. In some cases, r is 7. In some cases, r is 8. In some cases, r is 9. In some cases, r is 10. In some cases, r is 11.

[0373] In some embodiments, the multichromophore includes one or more of the following groups, for example as terminal or pendant groups for conjugation with a compatible functional group on another molecule:

[0374]

[0375]

[0376] Labeled specific binding member

[0377] Aspects of the present disclosure include specific binding members of labels. The specific binding members of labels are conjugates of theme polymer dyes (e.g., as described herein) and specific binding members. Any polymer dye or polymer tandem dye described herein can be conjugated with specific binding members. Specific binding members and polymer dyes can be conjugated to each other (e.g., covalently linked) at any suitable position of two molecules by an optional linking group.

[0378] In some embodiments, the labeled specific binding members are resistant to aggregation. As used herein, "resistance to aggregation" means that the labeled specific binding members are able to form a uniform aqueous composition in an aqueous buffer of interest without aggregation precipitates, and the labeled specific binding members are at a concentration of 1 mg / ml or higher, such as 2 mg / ml or higher, 3 mg / ml or higher, 4 mg / ml or higher, 5 mg / ml or higher, 6 mg / ml or higher, 7 mg / ml or higher, 8 mg / ml or higher, 9 mg / ml or higher, 10 mg / mL or higher or even higher. In certain embodiments, the labeled specific binding members that resist aggregation include: a water-soluble light-harvesting multichromophore, the water-soluble light-harvesting multichromophore comprising a conjugated segment having a structure of formula (I):

[0379]

[0380] in:

[0381] F 1 It is a fused three-ring comonomer substituted with a water-soluble group (WSG);

[0382] M 1 is an aromatic or heteroaromatic comonomer;

[0383] n is an integer from 1 to 100,000; and

[0384] * indicates the site for covalent attachment to the unsaturated backbone or terminal group of the conjugated polymer;

[0385] where F 1 and M 1 At least one of the water-soluble polymers is substituted with a branched nonionic water-soluble group (WSG) comprising two or more water-soluble polymers, each of which has 6 or more monomer units; and

[0386] A specific binding member is covalently linked to the multichromophore.

[0387] In certain embodiments, the aggregation-resistant labeled specific binding member comprises: a water-soluble light-harvesting multichromophore comprising a conjugated segment having the structure of formula (I):

[0388]

[0389] in:

[0390] F 1is a fused 6-5-6 tricyclic comonomer substituted with a branched nonionic water solubilizing group (WSG) comprising two or more water soluble polymers, each of which has 6-30 monomer units (e.g., 10-20 units, 12-16 units, etc.);

[0391] M 1 is an aromatic or heteroaromatic comonomer;

[0392] n is an integer from 1 to 100,000; and

[0393] * indicates the site for covalent attachment to the unsaturated backbone or terminal group of the conjugated polymer; and

[0394] A specific binding member is covalently linked to the multichromophore.

[0395] The branched nonionic water-soluble group (WSG) can give the specific binding members of the labeling solubility in water exceeding 1mg / mL, such as exceeding 2mg / mL, exceeding 3mg / mL, exceeding 4mg / mL, exceeding 5mg / mL, exceeding 6mg / mL, exceeding 7mg / mL, exceeding 8mg / mL, exceeding 9mg / mL or exceeding 10mg / mL. In some cases, the branched nonionic water-soluble group (WSG) can give solubility in water (for example, aqueous buffer) of polymeric dye 10mg / mL or higher, such as giving polymeric dye 20mg / mL or higher, 30mg / mL or higher, 40mg / mL or higher, 50mg / mL or higher, 60mg / mL or higher, 70mg / mL or higher, 80mg / mL or higher, 90mg / mL or higher, 100mg / mL or higher or even higher solubility.

[0396] As used herein, the term "specific binding member" refers to one member of a pair of molecules that have binding specificity for each other. One member of the pair may have an area or cavity on its surface that specifically binds to an area or cavity on the surface of the other member of the pair. Thus, the members of the pair have the property of specifically binding to each other to produce a binding complex. In some embodiments, the affinity between the specific binding members in the binding complex is characterized by a K d (dissociation constant) is 10 -6 M or smaller, such as 10 -7 M or smaller, including 10 -8 M or smaller, e.g. 10 -9 M or smaller, 10 -10 M or smaller, 10 -11 M or smaller, 10 -12 M or smaller, 10 -13M or smaller, 10 -14 M or smaller, including 10 -15 M or less. In some embodiments, the specific binding member specifically binds with high affinity. High affinity means that the binding member specifically binds with an apparent affinity characterized by: an apparent K d 10x 10 -9 M or smaller, such as 1x 10 -9 M or smaller, 3x 10 -10 M or smaller, 1x 10 -10 M or smaller, 3x 10 -11 M or smaller, 1x 10 -11 M or smaller, 3x 10 -12 M or smaller or 1x 10 -12 M or smaller.

[0397] Specific binding members can be protein. As used herein, the term "protein" refers to a portion consisting of amino acid residues. The protein portion can be a polypeptide. In some cases, the protein specific binding member is an antibody. In certain embodiments, the protein specific binding member is an antibody fragment, such as a binding fragment of an antibody 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 a recognized immunoglobulin gene. Recognized immunoglobulin genes, such as in humans, include κ (k), λ (I) and heavy chain genetic loci, which together constitute countless variable region genes, and constant region genes μ (u), δ (d), γ (g), σ (e) and α (a) encoding IgM, IgD, IgG, IgE and IgA isotypes, respectively. The 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 extent of the framework region and CDR has been precisely defined (see "Sequences of Proteins of Immunological Interest," E. Kabat et al., US Department of Health and Human Services, (1991)). The numbering of all antibody amino acid sequences discussed herein conforms to the Kabat system. The sequences of the framework regions of different light or heavy chains are relatively conserved within species. The framework region of an antibody, i.e., the combined framework region of the constitutive light and heavy chains, is used to position and align the CDR. The CDR is primarily responsible for binding to the epitope of the antigen. 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 use in experiments, treatments, or other purposes as further defined below.

[0398] Antibody fragments of interest include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv or other antigen-binding subsequences of antibodies, produced by modifying whole antibodies or synthesized de novo using recombinant DNA technology. Antibodies can be monoclonal or polyclonal, and can have other specific activities on cells (e.g., antagonists, agonists, neutralizing antibodies, inhibitory antibodies or stimulating antibodies). It should be understood that antibodies can have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other antibody functions.

[0399] 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 certain instances, the specific binding member is a recombinant antibody or a binding fragment thereof.

[0400] In some embodiments, the labeled specific binding member comprises: a water-solvated light-harvesting multichromophore (e.g., as described herein); and a signal chromophore covalently linked to the multichromophore, the signal chromophore being in energy-receiving proximity to the multichromophore (e.g., as described herein); and a specific binding member covalently linked to the multichromophore. In some examples of labeled specific binding members, a multichromophore of any formula described herein (e.g., formulas (V) and (VII)-(VIII)), wherein: G 1 and G 2 Each is independently selected from the group consisting of a terminal group (eg, a terminal group), a π-conjugated segment, a linker, and a linked specific binding member, wherein G 1 and G 2 At least one of the is a linked specific binding member.

[0401] method

[0402] As described above, aspects of the present invention include methods for evaluating a sample for the presence of a target analyte. Aspects of the method include contacting the sample with a polymer dye conjugate that specifically binds to the target analyte to produce a sample contacted with a labeled composition. As used herein, the terms "polymer dye conjugate" and "labeled specific binding member" are used interchangeably. Therefore, the polymer dye conjugate may include: (i) a water-solvated polymer dye (e.g., as described herein); and (ii) a specific binding member (e.g., as described herein). In some instances, the polymer dye conjugate also includes a signal chromophore covalently linked to a multichromophore of the polymer dye, the multichromophore being in energy-receiving proximity to the signal chromophore.

[0403] Any suitable method can be used to contact the sample with a polymer dye conjugate, which specifically binds the target analyte to produce a sample contacted by the labeling composition. In some instances, the sample is contacted with a polymer dye conjugate under the condition that the specific binding members specifically bind the target analyte (if present). For the specific binding of the specific binding members of the conjugate to the target analyte, an appropriate solution that keeps the biological activity of the sample components and the specific binding members can be used. The solution can be a balanced salt solution, such as normal saline, PBS, Hank's balanced salt solution, etc., suitably supplemented with fetal calf serum, human platelet lysate or other factors, combined with acceptable low concentration buffers (such as 5-25mM). Suitable buffer includes HEPES, phosphate buffer, lactate buffer, etc. Various culture media are commercially available, and can be used according to the properties of the target analyte, including dMEM, HBSS, dPBS, RPMI, Iscove's culture media, etc., supplemented with fetal calf serum or human platelet lysate in some cases. The final component of the solution can be selected according to the sample components included.

[0404] The temperature at which the specific binding member of the conjugate specifically binds to the target analyte can vary, and in some cases can be 5°C to 50°C, such as 10°C to 40°C, 15°C to 40°C, 20°C to 40°C, for example 20°C, 25°C, 30°C, 35°C or 37°C (e.g., as described herein). In some cases, the temperature at which specific binding occurs is selected to be compatible with the biological activity of the specific binding member and / or the target analyte. In some instances, the temperature is 25°C, 30°C, 35°C or 37°C. In some cases, the specific binding member is an antibody or a fragment thereof, and the temperature at which specific binding occurs is room temperature (e.g., 25°C), 30°C, 35°C or 37°C. Any suitable incubation time for specific binding can be selected to allow the formation of a desired amount of binding complex, and in some instances, can be 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.

[0405] Any suitable specific binding member can be utilized in the conjugate. Interested specific binding members include but are not limited to those agents that specifically bind to the cell surface proteins of various cell types, and the cell types include but are 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 suitable cell surface markers or antigens, and the cells can be captured by suitable specific binding member conjugates. In some embodiments, target cells are selected from cells containing HIV, Treg cells, antigen-specific T cell groups, tumor cells, or hematopoietic progenitor cells (CD34+) from whole blood, bone marrow, or umbilical cord blood. In the subject method, any suitable cell surface protein or cell marker can be targeted to specifically bind to polymer dye conjugates. In some embodiments, target cells include cell surface markers selected from cell receptors and cell surface antigens. In some cases, the target cell may 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-terminal), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71.

[0406] Any suitable target can be selected for evaluation using the subject method. Targets of interest include, but are not limited to, nucleic acids, such as RNA, DNA, PNA, CNA, HNA, LNA or ANA molecules; proteins, such as fusion proteins; modified proteins, such as phosphorylated proteins, glycosylated proteins, ubiquitinated proteins, SUMO-ylated proteins or acetylated proteins; or antibodies, peptides, aggregated biomolecules, cells, small molecules, vitamins and drug molecules. As used herein, the term "target protein" refers to all members of the target family and fragments thereof. The target protein can be any protein of interest, such as a therapeutic or diagnostic target, including but not limited to: hormones, growth factors, receptors, enzymes, cytokines, osteoinductive factors, colony stimulating factors and immunoglobulins. The term "target protein" is intended to include recombinant molecules and synthetic molecules that can be prepared using any suitable recombinant expression method or using any suitable synthetic method, or commercially available. In some embodiments, the polymer dye conjugate includes an antibody or an antibody fragment. In the subject method, any suitable target analyte that can be targeted specifically to bind an antibody or an antibody fragment of interest can be used.

[0407] In some embodiments, the target analyte is associated with a cell. In some instances, the target analyte is a cell surface marker of a cell. In some cases, the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen. In some instances, the target analyte is an intracellular target, and the method further comprises lysing the cell.

[0408] In some embodiments, the sample may include a heterogeneous cell population from which the target cell can be separated. In some instances, the sample includes peripheral whole blood, peripheral whole blood that has cracked red blood cells 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 certain instances, the sample is a sample including (or suspected to include) viral cells (e.g., HIV).

[0409] The specific binding members of labeling can be used for subject method, for example, for labeling target cells, particles, targets or analytes with polymer dyes or polymer tandem dyes.For example, the specific binding members of labeling can be used for labeling cells to be processed in flow cytometers (for example, detection, analysis and / or sorting).The specific binding members of labeling can include antibodies (for example, as described herein) specifically binding to cell surface proteins of, for example, various cell types.The specific binding members of labeling can be used for studying various biological (for example, 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.The specific binding members of labeling can be used for any application of antibody-mediated labeling including (or can include) cells, particles or analytes.

[0410] In some examples of the methods, the labeled specific binding member comprises a multichromophore as described herein (eg, according to any one of formulas (I)-(IV)). 1 and G 2 Each is independently selected from the group consisting of a terminal group, a π-conjugated segment, a linker, and a linked specific binding member, wherein G 1 and G 2 At least one of the is a linked specific binding member.

[0411] Aspects of the method include measuring the sample contacted with the labeling composition for the presence of the polymer dye conjugate-target analyte binding complex to assess whether there is a target analyte in the sample. Once the sample is contacted with the polymer dye conjugate, any suitable method can be used to measure the sample contacted with the labeling composition for the presence of the polymer dye conjugate-target analyte binding complex. The polymer dye conjugate-target analyte binding complex is a binding complex produced when the specific binding member of the conjugate specifically binds to the target analyte (if present). Determining the sample contacted with the labeling composition can include detecting the fluorescent signal from the binding complex (if present). In some cases, the determination 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, for example, by being fixed on a carrier. The assay method of interest includes but is not limited to any suitable method and assay format in which the use of the specific binding member such as avidin-biotin or hapten-anti-hapten antibodies is of interest. Methods and assay formats of interest that may be adapted for use with the subject compositions include, but are not limited to, flow cytometry methods, in situ hybridization methods, enzyme-linked immunosorbent assays (ELISA), Western blot analysis, magnetic cell separation assays, and fluorescent dye purification chromatography.

[0412] In certain embodiments, the method also includes contacting the sample with a second specific binding member specifically binding to a target analyte. In some instances, the second specific binding member is carrier-bound. Any suitable carrier can be used to fix the component (e.g., the second specific binding member) of the subject method. In some instances, the carrier is a particle, such as a magnetic particle. In some instances, the second specific binding member and the polymer dye conjugate produce a sandwich complex, which can be separated and detected (if present) using any suitable method. In some embodiments, the method also includes analyzing polymer dye conjugate-target analyte binding complexes, i.e., fluorescently labeled target analytes, by flow cytometry. Measured for the presence of polymer dye conjugate-target analyte binding complexes can provide measurement results (e.g., qualitative or quantitative measurement data), which can be used to assess whether there is a target analyte in a sample.

[0413] Any suitable carrier can be used to fix any suitable component of the method in the subject method, such as a labeled specific binding member, a target, a second specific binding member, etc. The carrier of interest includes, but is not limited to: a solid matrix, wherein the matrix can have a variety of configurations, such as a sheet, a bead or other structure, such as a plate with holes; beads, polymers, particles, fiber meshes, hydrogels, porous matrices, needles, microarray surfaces, chromatographic carriers, etc. In some cases, the carrier is selected from the group consisting of particles, planar solid matrices, fiber meshes, hydrogels, porous matrices, needles, microarray surfaces, and chromatographic carriers. The carrier can be incorporated into a system that is assisted by any suitable method to achieve cell separation, such as a manually operated syringe, a centrifuge, or an automated liquid handling system. In some cases, the carrier can be used for an automated liquid handling system for high-throughput separation of cells, such as a flow cytometer.

[0414] In some embodiments of the method, the separation step includes applying an external magnetic field to fix the magnetic particles. Any suitable magnet can be used as the source of the external magnetic field (e.g., magnetic field gradient). In some cases, the external magnetic field is produced by a magnetic source, such as a permanent magnet or an electromagnet. In some cases, fixing the magnetic particles means that the magnetic particles accumulate near the surface of the magnetic field gradient source, i.e., the magnet.

[0415] The separation may also include one or more optional washing steps to remove unbound material of the sample from the carrier. Any suitable washing method may be used, such as washing the fixed carrier with a biocompatible buffer that retains the specific binding interaction of the polymeric dye and the specific binding member. Separating and optionally washing away unbound material of the sample from the carrier provides an enriched target cell population in which unwanted cells and material can be removed.

[0416] In certain embodiments, the method also includes detecting a labeled target. Detecting a labeled target may include exciting a multichromophore with one or more lasers, followed by detecting fluorescence emission from a polymer tandem dye using one or more optical detectors. Any suitable instrument and method can be used to detect the labeled target, including but not limited to flow cytometry, FACS systems, fluorescence microscopy; fluorescence, luminescence, ultraviolet and / or visible light detection using a plate reader; high performance liquid chromatography (HPLC); and mass spectrometry. When using fluorescently labeled components in the methods and compositions of the present disclosure, it is recognized that different types of fluorescence detection systems can be used to practice the subject method. In some cases, high throughput screening can be performed, for example, using a system of 96 wells or larger microtiter plates. A variety of methods for measuring fluorescent materials can be used, such as those described in, for example, Lakowicz, JR, Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983); Herman, B., Resonance energy transfer microscopy, In: Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, Vol. 30, 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.

[0417] 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 emission from the sample. The device may include a temperature controller to keep the sample at a specific temperature while the sample is scanned. In some instances, a multi-axis translation stage moves a microtiter plate holding multiple samples so as to locate different wells to be exposed. The multi-axis translation stage, temperature controller, autofocus feature, and electronics 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.

[0418] In some embodiments, the method for evaluating a sample for the presence of a target analyte further comprises detecting fluorescence in a flow cytometer. In some embodiments, the method for evaluating a sample for the presence of a target analyte further comprises imaging the sample contacted with the labeled 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. Microscopy methods of interest that can be used for the subject method include laser scanning confocal microscopy.

[0419] Also provided is a method for labeling a target molecule. The subject polymer dye can be used for a variety of labeling, separation, detection and / or analysis methods. In some embodiments, the method comprises: contacting the target molecule with a polymer dye (e.g., as described herein) to produce a labeled target molecule, wherein the polymer dye comprises a conjugated tag covalently linked to the target molecule. In some cases, the polymer dye also includes a signal chromophore covalently linked to the multichromophore of the polymer dye, and the multichromophore is in energy receiving proximity to the signal chromophore. In some examples of the method, the polymer dye member includes a multichromophore according to any one of formulas (I)-(IV) (e.g., as described herein), wherein G 1 and G 2 One of them is a terminal group, G 1 and G 2 The other one in is a conjugated label.

[0420] As used herein, the term "conjugated tag" refers to a group comprising a chemoselective functional group (e.g., as described herein) that can be covalently linked to a compatible functional group of a target molecule after optional activation and / or deprotection. Any suitable conjugated tag can be utilized in the subject polymer dye to conjugate the dye to the target molecule of interest. In some embodiments, the conjugated tag includes a terminal functional group selected from the group consisting of an amino group, a carboxylic acid or a derivative thereof, a thiol, a hydroxyl group, a hydrazine, a hydrazide, an alkyne, and a protein reactive group (e.g., amino-reactive, thiol-reactive, hydroxyl-reactive, imidazole-reactive, or guanidino-reactive).

[0421] Any suitable method and reagent can be adapted for the subject labeling method, so that the conjugated label is covalently linked to the target molecule. The method of interest for labeling the target includes but is not limited to those methods and reagents described by Hermanson, Bioconjugate Techniques, 3rd edition, Academic Press, 2013. The contacting step can be carried out in an aqueous solution. In some instances, the conjugated label includes an amino functional group, and the target molecule includes an activated ester functional group, such as an NHS ester or a sulfo-NHS ester, or vice versa. In some instances, the conjugated label includes a maleimide functional group, and the target molecule includes a thiol functional group, or vice versa. In some instances, the conjugated label includes an alkyne (e.g., a cyclooctyne group) functional group, and the target molecule includes an azide functional group, or vice versa, which can be conjugated by click chemistry.

[0422] Any suitable target molecule can be selected for labeling using the subject method. Target molecules of interest include, but are not limited to, nucleic acids, such as RNA, DNA, PNA, CNA, HNA, LNA or ANA molecules; proteins, such as fusion proteins; modified proteins, such as phosphorylated proteins, glycosylated proteins, ubiquitinated proteins, SUMO-ylated proteins or acetylated proteins; or antibodies, peptides, aggregated biomolecules, cells, small molecules, vitamins and drug molecules. As used herein, the term "target protein" refers to all members of the target family and fragments thereof. The target protein can be any protein of interest, such as a therapeutic or diagnostic target, including but not limited to: hormones, growth factors, receptors, enzymes, cytokines, osteoinductive factors, colony stimulating factors and immunoglobulins. The term "target protein" is intended to include recombinant molecules and synthetic molecules that can be prepared using any suitable recombinant expression method or using any suitable synthesis method, or commercially available. In some embodiments, the target molecule is a specific binding member (e.g., as described herein). In some instances, the specific binding member is an antibody. In some instances, the specific binding member is an antibody fragment or its binding derivative. In some cases, the antibody fragment or binding derivative thereof is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a scFv, a diabody, and a triabody.

[0423] In some cases, the method includes a separation step, wherein the labeled target molecule is separated from the reaction mixture (e.g., excess reagent or unlabeled target). The target can be separated from the sample using a variety of methods, such as by immobilization on a carrier, precipitation, chromatography, etc.

[0424] In some instances, the method also includes detecting and / or analyzing the target molecule of the label. In some instances, the method also includes carrying out fluorescence detection to the target molecule of the label. In conjunction with the subject method and composition, any suitable method can be used to detect and / or analyze the target molecule of the label. The method of the target of interest that can be used for the analysis of the 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.

[0425] Detection can be achieved directly by polymer dyes or polymer tandem dyes, or indirectly by a secondary detection system. The latter can be based on any one or combination of several different principles, including but not limited to antibody-labeled anti-species antibodies and other forms of immunological or non-immunological bridging and signal amplification systems (e.g., biotin-streptavidin technology, protein A and protein G-mediated technology, or nucleic acid probes / anti-nucleic acid probes, etc.). Suitable reporter molecules can be those known in the fields of immunocytochemistry, molecular biology, light, fluorescence and electron microscopy, cell immunophenotyping, cell sorting, flow cytometry, cell visualization, detection, counting and / or signal output quantification. More than one antibody with specific and / or nonspecific properties can be labeled and used simultaneously or sequentially to enhance target detection, identification and / or analysis.

[0426] system

[0427] Aspects of the present invention also include systems for practicing subject methods and compositions. Sample analysis systems may include sample fields of view or flow channels loaded with sample and labeled specific binding members. In some embodiments, the system is a flow cytometry system, comprising: a flow cytometer comprising a flow path; a composition in the flow path, wherein the composition comprises sample and labeled specific binding members (e.g., as described herein). In some embodiments, the system for analyzing a sample is a fluorescence microscopy system, comprising: a fluorescence microscope comprising a sample field of view; and a composition arranged in the sample field of view, wherein the composition comprises a sample; and labeled specific binding members (e.g., as described herein).

[0428] In some examples of the system, the labeled specific binding member includes: a water-solvated light-harvesting multichromophore (e.g., as described herein) and a specific binding member that specifically binds to a target analyte covalently linked to the multichromophore. In some cases, the labeled specific binding member further comprises a signal chromophore covalently linked to the multichromophore of the polymeric dye, the multichromophore being in energy-receiving proximity to the signal chromophore. In some examples of the subject system, the labeled specific binding member, the multichromophore is described by any one of formulas (I)-(IV) (e.g., as described herein), wherein: G 1 and G 2 Each is independently selected from the group consisting of a terminal group, a π-conjugated segment, a linker, and a linked specific binding member, wherein G 1 and G 2 At least one of the is a linked specific binding member.

[0429] In certain embodiments of the system, the composition further comprises a second specific binding member that is carrier-bound and specifically binds to the target analyte. In some cases, the carrier comprises magnetic particles. Therefore, in certain instances, the system may further comprise a controllable external paramagnetic field that is configured to be applied to the assay region of the flow channel.

[0430] The sample may include cells. In some instances, the sample is a biological sample containing cells. In some instances, the sample comprises a specific binding member that specifically binds to a label of a target cell. In some instances, the target analyte specifically bound by the specific binding member is a cell surface marker of a cell. In some cases, the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen.

[0431] In certain aspects, 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.

[0432] In some aspects, the system may also include a computer-based system configured to detect the presence of a fluorescent signal. "Computer-based system" refers to a hardware device, software device, and data storage device for analyzing 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 understand that any currently available computer-based system is suitable for the subject system. The data storage device can include any manufacture that includes a record of the information of the present invention as described above, or a memory access device that can access such a manufacture.

[0433] "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 suitable data storage structure may be selected based on the means used to access the stored information. A variety of data processor programs and formats may be used for storage, such as word processing text files, database formats, etc.

[0434] "Processor" refers to any hardware and / or software combination that will perform its required functions. For example, any processor herein may be a programmable digital microprocessor, such as may be provided 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 previously stored in a computer program product (such as a portable or fixed computer-readable storage medium, whether magnetically, optically, or solid-state device-based). For example, a magnetic medium or optical disc may carry the programming, and may be read by a suitable reader that communicates with each processor at its respective station.

[0435] In addition to the sensor devices and signal processing modules (e.g., as described above), the system of the present invention may include a number of additional components, such as data output devices (e.g., monitors and / or speakers), data input devices (e.g., interface ports, keyboards, etc.), fluid handling components, power supplies, etc.

[0436] In certain aspects, the system comprises a flow cytometer. Flow cytometers of interest include, but are not limited to, those described in U.S. Pat. Nos. 4,704,891; 4,727,029; 4,745,285; 4,867,908; 5,342,790; 5,620,842; 5,627,037; 5,701,012; 5,895,922; and 6,287,791; the disclosures of these patents are incorporated herein by reference.

[0437] Other systems can be used to practice the subject method. In some aspects, the system can be a fluorometer or microscope equipped 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 an assay area or sample field of view of a flow channel. The fluorometer or microscope can also include a detector configured to receive a signal from an assay area or sample field of view of a flow channel, wherein the signal is provided by the fluorescent composition.

[0438] Reagent test kit

[0439] Aspects of the invention also include kits for practicing the subject methods and compositions.The compositions of the invention can be included in a kit as reagents, as starting materials or provided for use, for example, in the methods described above.

[0440] The kit may include: a polymer dye and a container comprising a water-solvated light-harvesting multichromophore (e.g., as described herein). Any suitable container may be used, such as a tube, a bottle, or a hole in a porous strip or plate, a box, a bag, an insulated container, etc. The subject kit may also include one or more components selected from the following: a polymer tandem dye, a fluorophore, a specific binding member, a specific binding member conjugate, a carrier-bound specific binding member, a cell, a carrier, a biocompatible aqueous elution buffer; and instructions for use. In some embodiments of the kit, the multichromophore is covalently linked to the specific binding member. In some instances, the specific binding member is an antibody. In certain instances, the specific binding member is an antibody fragment or a binding derivative thereof. In some cases, the antibody fragment or its binding derivative is selected from the group consisting of: a Fab fragment, a F(ab')2 fragment, a scFv, a double antibody, and a three-antibody.

[0441] In certain embodiments, the kit can be used to evaluate a sample for the presence of a target analyte (such as an intracellular target). Thus, in some instances, the kit includes one or more components suitable for lysing cells. One or more additional components of the kit can be provided in a separate container (e.g., a separate tube, bottle, or separate well in a porous strip or plate).

[0442] In some aspects, the kit also includes 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 multiple chromophores, wash buffer, control cells, control beads, fluorescent beads for flow cytometer calibration, and combinations thereof. The kit may also include one or more cell fixation reagents, such as paraformaldehyde, glutaraldehyde, methanol, acetone, formalin, or any combination or buffer thereof. In addition, the kit may include a cell permeabilization reagent, such as methanol, acetone, or a detergent (e.g., triton, NP-40, saponin, Tween 20, digitonin, leucoperm, or any combination or buffer thereof. Other protein transport inhibitors, cell fixation reagents, and cell permeabilization reagents familiar to the technician are within the scope of the subject kit.

[0443] 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 provided in separate containers (e.g., separate tubes, bottles, or separate holes in porous strips or plates).

[0444] In addition, one or more components can be incorporated into a single container, such as a glass or plastic vial, tube or bottle. In some instances, the kit may also include a container (e.g., such as a box, bag, insulated container, bottle, tube, etc.) in which all components (and their individual containers) are present. The kit may also include packaging that is separate from or attached to the kit container and has printed thereon information about the kit, the components of the kit, and / or the instructions for use of the kit.

[0445] In addition to the above-mentioned components, the subject test kit may also include instructions for practicing the subject method. These instructions may be present in the subject test kit in various forms, wherein one or more may be present in the test kit. A form in which these instructions may exist is to be printed on suitable medium or substrate (for example, one or more sheets of paper for printing information thereon), printed in the packaging of the test kit, printed on the information in the packaging insert etc. And another mode will be a computer-readable medium for recording information thereon, for example a floppy disk, CD, DVD, portable flash drive etc. Another possible mode is the website address that can be used by the Internet, with access information at a remote site. Any suitable device may all be present in the test kit.

[0446] Practicality

[0447] Polymer dyes, compositions, methods and systems as described herein can be used for a variety of applications in which it is desired to mark, detect and / or analyze a target of interest, including diagnostic and research applications. Such applications include methodologies such as cytometry, microscopy, immunoassays (e.g., competitive or non-competitive), the assessment of free analytes, the assessment of receptor binding ligands, etc. Compositions, systems and methods as described herein can be used for 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 as described herein can be used for methods such as in fluorescent activated cell sorting or analysis, immunoassays, immunostaining, etc., using fluorescent markers to detect analytes in samples (if present). In some instances, the compositions and methods can be used for applications in which it is of interest to evaluate samples for the presence of target analytes.

[0448] In some cases, the methods and compositions can be used for any assay format in which it is of interest to detect and / or analyze a target in a sample, including but not limited to flow cytometry, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assays, and fluorescent dye purification chromatography. In certain instances, the methods and compositions can be used for any application in which it is of interest to fluorescently label a target molecule. The subject composition can be adapted for any suitable application in which specific binding member pairs (such as biotin-streptavidin and hapten-anti-hapten antibodies) can be used.

[0449] Example

[0450] Example 1: Design of water-soluble conjugated polymers

[0451] Comonomers for water-soluble conjugated polymer dyes have been developed that provide excellent aqueous solubility, processability, and improved performance of such dyes in flow cytometry.

[0452] Various strategies were employed to increase the amount of water-soluble side chains and reduce the number of hydrophobic groups to provide high purity and high reactivity of these monomers in polymerization reactions to produce conjugated polymers. Additionally, for some monomers, synthetic methodologies were employed to improve the purity of the final product.

[0453] The subject water-soluble conjugated polymer dyes are used as fluorescent reporters for a variety of biosensors and exhibit excellent brightness and a wide selection of excitation and emission wavelengths for flow cytometry, imaging, and other applications.

[0454] Conjugated polymer dyes can be very hydrophobic and tend to adopt planar stackable conformations. The key step in realizing conjugated polymers as dyes in biosensing is to bring these structures into aqueous buffer solutions suitable for biological targets of interest. The subject conjugated polymers obtain water solubility by chemically modifying the monomers that damage the polymer, thereby achieving high water solubility and improved water solubility. The subject water-soluble groups are covalently linked to the comonomers that form the polymer backbone. Due to the high water solubility of one or more comonomers incorporated into the polymer, the resulting polymer is soluble. Due to the increased length of the water-soluble part, the improved purification technology that produces pure highly reactive monomers and / or the synthetic strategy that allows the key monomers to be obtained in high purity, these higher solubility monomers are possible.

[0455] The water solubility requirements for conjugated polymer dyes used for biosensing are high solubility in buffer solutions and low nonspecific binding in the assay of interest. Typically, the solubility of the conjugate of the polymer dye and the biosensing group (e.g., antibody) is lower than the solubility of the dye itself. This occurs when there is more than one binding site on the conjugated polymer or biosensing biomolecule, resulting in higher molecular weight cross-linked species with lower solubility and specificity in some cases, which may be different from the intended synthetic target but cannot be separated from the desired material.

[0456] Aggregation of the conjugate can occur when the solubility of the dye-biosensor conjugate becomes negligible. This aggregation can manifest itself as precipitation during the conjugation reaction, precipitation of the product upon standing, and the presence of ultimately unwanted material in the bioassay, which results in degraded data quality. Aggregation of polymerized material once initiated can become a persistent problem, and even if such precipitate is purified from the product, the resulting conjugate may not be suitable for downstream processing. The subject comonomers (e.g., Figure 1 ) can provide polymer dye conjugates that avoid such aggregation problems. They are the product of several strategic elements. First, the length of the water-soluble oligoethylene glycol chain provides high solubility. In some cases, the chain length exceeds 11 repeating units per chain, such as 12-16 repeating units. Secondly, in some structures, the hydrophobic phenyl used for high WSG side chain branches is replaced by hydrophilic branched elements (such as amides, sulfonamides and branched ethylene glycol connecting bonds). With regard to amide bonds, there are other strategic elements.

[0457] Utilizing amide bond formation to attach large water soluble groups provides improved purification of the aforementioned synthetic steps. In particular, the formation of boronate groups (for polymerization) can result in product mixtures that are difficult to properly purify. This becomes even more difficult when large polar water soluble groups are installed first. By moving the attachment of large water soluble groups to a simple, high yield reaction at the end of the protocol, the boronate precursor can be thoroughly purified in a straightforward manner. This contrasts with previous monomers that required attachment of ethylene glycol oligomers earlier in the synthesis. The resulting material has a higher solubility and is more reactive in polymerization reactions for producing water soluble conjugated polymer dyes. When these higher solubility monomers are incorporated into conjugated polymers, the solubility characteristics of the resulting antibody conjugates are improved.

[0458] The advantage of this design is the high solubility of the gained polymeric dye.Due to the improvement of dissolving strategy, the polymeric dye is obviously more soluble, has low viscosity and is easier to process.The conjugation yield is much higher, and the gained data is also improved.By increasing the amount of water-soluble side chains and changing the connection strategy with the monomeric part that becomes a polymer backbone part, the significant improvement of solubility and gained dye characteristics can be obtained.The antibody conjugate of theme polymeric dye (for example, comprising long oligoethylene glycol side chains and comprising the dyestuff of non-aromatic side chain groups (such as alkyl ether link, amide link or sulfonamide link)) is especially resisting aggregation.

[0459] Example 2: Synthesis of conjugated polymers via CH bond arylation

[0460] The synthesis of the subject polymer dyes can be achieved by a technique known as CH bond arylation (Scheme 1). Other methods such as the Suzuki coupling method (Scheme 2) or the Stille coupling method (Scheme 3) can also be utilized.

[0461] Scheme 1: CH bond arylation method

[0462]

[0463] Scheme 2: Suzuki coupling method

[0464]

[0465] Scheme 3: Stille coupling method

[0466]

[0467] Notwithstanding the appended claims, the disclosure set forth herein is also defined by the following terms:

[0468] Item 1. A water-soluble light-harvesting multichromophore comprising a conjugated segment having a structure of formula (I):

[0469]

[0470] in:

[0471] F 1 It is a fused three-ring comonomer substituted with a water-soluble group (WSG);

[0472] M 1 is an aromatic or heteroaromatic comonomer;

[0473] n is an integer from 1 to 100,000; and

[0474] * indicates the site for covalent attachment to the unsaturated backbone or terminal group of the conjugated polymer;

[0475] where F 1 and M 1 At least one of the water-soluble groups is substituted with a branched nonionic water-soluble group (WSG) comprising two or more water-soluble polymers, each of which has 6-50 (e.g., 6-40 or 6-30) monomer units.

[0476] Item 2. The water-soluble light-harvesting multichromophore according to Item 1, wherein

[0477] F 1 is a fused 6-5-6 tricyclic comonomer substituted with a branched nonionic water solubilizing group (WSG) comprising two or more water soluble polymers, each of which has 6 to 30 monomer units;

[0478] M 1 is an aromatic or heteroaromatic comonomer;

[0479] n is an integer from 1 to 100,000; and

[0480] * indicates the site for covalent attachment to the unsaturated backbone or terminal group of the conjugated polymer;

[0481] The branched nonionic water-soluble group (WSG) can endow the multichromophore with a solubility in water exceeding 50 mg / mL.

[0482] Clause 3. The water-soluble light-harvesting multichromophore according to clause 1 or 2, wherein the fused tricyclic comonomer is described by the following structure:

[0483]

[0484] in:

[0485] Y is C(R 3 )2、-C(R 3 )2C(R3 )2-、-C(R 3 )2Si(R 3 )2-、NR 3 、Si(R 3 )2 or Se;

[0486] each Z is independently CH, CR or N, wherein at least two of the Zs in each ring are CH or CR;

[0487] Each R 3 independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido, aralkyl, substituted aralkyl, PEG moiety, -L 1 -Z 1 , where L 1 is a linker and Z 1 is a chemoselective tag (e.g., a tag comprising a chemoselective functional group) and WSG; and

[0488] Each R is independently H or one or more aryl or heteroaryl substituents (eg, WSG, halogen, alkoxy, substituted alkoxy, alkyl, or substituted alkyl), and any two suitable R groups are optionally cyclically linked.

[0489] Item 4. The water-soluble light-harvesting multichromophore according to Item 3, wherein the fused tricyclic comonomer is described by one of the following structures:

[0490]

[0491] in:

[0492] Each R 3 independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido, aralkyl, substituted aralkyl, PEG moiety, -L 1 -Z 1 , where L 1 is a linker and Z 1 is a chemoselective tag (eg, a tag comprising a chemoselective functional group) and WSG (eg, as described herein).

[0493] Item 5. The water-soluble light-harvesting multichromophore according to Item 4, wherein at least one R 3 For WSG.

[0494] Item 6. The water-soluble light-harvesting multichromophore according to Item 4 or 5, wherein at least one R 3For -L 1 -Z 1 , where L 1 is a linker and Z 1 Chemically selective labeling.

[0495] Clause 7. The water-soluble light-harvesting multichromophore according to any one of clauses 1 to 6, wherein F 1 Has the following structure:

[0496]

[0497] in:

[0498] Z is -C(R 1 )(R 2 )-、-Si(R 1 )(R 2 )-or-N(R 1 )-;and

[0499] R 1 and R 2 Independently, a branched nonionic WSG comprising two or more water-soluble polymers, each of which has 6 to 30 monomer units.

[0500] Clause 8. The water-soluble light-harvesting multichromophore according to any one of clauses 1 to 7, wherein the branched nonionic WSG has one of the following formulae:

[0501]

[0502] in:

[0503] Each B 1 and B 2 are independently branching groups;

[0504] Each W 1 are independently water-soluble polymers comprising 6 to 24 monomer units;

[0505] T 3 is an optional linker to the fused 6-5-6 tricyclic comonomer; and

[0506] Each p and q is independently 0 or 1, where each T, if present 1 and each T 2 are independently linking groups.

[0507] Item 9. The water-soluble light-harvesting multichromophore according to Item 8, wherein:

[0508] B 1is selected from CH, N, C(═O)N, SO2N, trisubstituted aryl, tetrasubstituted aryl and trisubstituted heteroaryl;

[0509] Each p is 0 or 1, where each T, if present 1 Selected from -(CH2) n -O-, -(CH2) n - and -O-, wherein n is 1 to 6; and

[0510] T 2 is a C1-C6 alkyl linking group, one or more atoms of which are optionally substituted by heteroatoms.

[0511] Clause 10. The water-soluble light-harvesting multichromophore according to any one of clauses 1 to 9, wherein the branched nonionic WSG is selected from one of the following structures:

[0512]

[0513]

[0514] in:

[0515] T 5 is an optional linker to the fused 6-5-6 tricyclic comonomer;

[0516] T 6 is a linking group;

[0517] Each s is an integer from 6 to 24; and

[0518] Each R 11 are independently hydrogen, alkyl or substituted alkyl.

[0519] Clause 11. The water-soluble light-harvesting multichromophore according to any one of clauses 1 to 10, wherein F 1 One of the following comonomers:

[0520]

[0521]

[0522] Clause 12. The water-soluble light-harvesting multichromophore according to any one of clauses 1 to 11, wherein the multichromophore has a structure of formula (II):

[0523]

[0524] in:

[0525] M 1 、M 2 and L 1are independently aryl or heteroaryl comonomers;

[0526] Z 1 is a linked chemoselective functional group or a linked acceptor chromophore;

[0527] x and y represent the molar concentration % of said unit in said multichromophore; and

[0528] G 1 and G 2 Each is independently selected from a terminal group, a π-conjugated segment, a linker, and a linked specific binding member.

[0529] Clause 13. The water-soluble light-harvesting multichromophore according to any one of Clauses 1 to 12, wherein the multichromophore has a structure of formula (III):

[0530]

[0531] in:

[0532] F 1 It is a fused three-ring comonomer;

[0533] M 1 、M 2 and L 1 are independently aryl or heteroaryl comonomers;

[0534] Z 1 is a linked chemoselective functional group or a linked acceptor chromophore;

[0535] Each n is independently an integer from 1 to 10,000;

[0536] each m is 0 or independently an integer from 1 to 10,000;

[0537] p is an integer from 1 to 100,000; and

[0538] G 1 and G 2 Each is independently selected from a terminal group, a π-conjugated segment, a linker, and a linked specific binding member.

[0539] Item 14. The water-soluble light-harvesting multichromophore according to Item 13, wherein the multichromophore has a structure of formula (IV):

[0540]

[0541] Item 15. The water-soluble light-harvesting multichromophore according to Items 12-14, wherein L 1 To be Z 1 and a fused tricyclic comonomer substituted with a branched nonionic water-soluble group (WSG).

[0542] Item 16. The water-soluble light-harvesting multichromophore according to Item 15, wherein L 1 Has the following structure:

[0543]

[0544] in:

[0545] R 3 is a branched nonionic water-soluble group (WSG); and

[0546] R 4 For L 2 -Z 2 , where L 2 is a linker and Z 2 It is a chemically selective functional group or an acceptor chromophore.

[0547] Item 17. The water-soluble light-harvesting multichromophore according to Item 15, wherein L 1 Has the following structure:

[0548]

[0549] in:

[0550] R 3 It is a nonionic water-soluble group (WSG);

[0551] R 4 For L 2 -Z 2 , where L 2 is a linker and Z 2 is a chemoselective functional group or an acceptor chromophore; and

[0552] Each R is an optional substituent (eg, a substituted alkyl), and two R groups may be optionally cyclically linked to form a carbocyclic or heterocyclic ring (eg, a 7-membered heterocyclic ring).

[0553] Item 18. The water-soluble light-harvesting multichromophore according to Item 16 or 17, wherein L 1 Has one of the following structures:

[0554]

[0555] Clause 19. The water-soluble light-harvesting multichromophore according to any one of clauses 1 to 18, wherein the aromatic or heteroaromatic comonomer is independently selected from one of formulae (XXIII) to (XXVI):

[0556]

[0557] in

[0558] Cy 2-7 refers to an aryl or heteroaryl group containing 2 to 7 fused and / or unfused rings;

[0559] Y 2 , Y 3 and Y 4 Independently selected from -CR 3 -、NR 3 , N, O, S and -C(=O)- and together form a 5- or 6-membered fused aryl or heteroaryl ring;

[0560] Each R 3 is one or more independently selected from H, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano, alkoxy substituted alkoxy and -T 1 -Z 1 a ring substituent of

[0561] R 1 and R 2 independently selected from H, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano, alkoxy, substituted alkoxy, and -T 1 -Z 1 , or R 1 and R 2 together form an optionally substituted 5- or 6-membered fused aryl, heteroaryl, cycloalkyl or heterocyclic ring;

[0562] Y 5 N or CR 5 And Y 7 N or CR 7 ;

[0563] R 4 -R 7 independently selected from H, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano, alkoxy, substituted alkoxy, and -T 1 -Z 1 ;

[0564] Z 1 is a chemoselective functional group or a linked signaling chromophore; and

[0565] T 1 For the connection base.

[0566] Item 20. The water-soluble light-harvesting multichromophore according to Item 19, wherein the aromatic or heteroaromatic comonomer is independently selected from one of the following structures (a) to (x):

[0567]

[0568]

[0569]

[0570] in:

[0571] Y 8 C(R 3 )2、-C(R 3 )2C(R 3 )2-、-C(R 3 )2Si(R 3 )2-、NR 3 or Si(R 3 )2;

[0572] X is S or O; each R 3 are independently H, a water-solubilizing group, an amino group, a substituted amino group, a halogen group, a cyano group, an alkyl group, a substituted alkyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, an acyl group, a substituted acyl group, a sulfonic acid group, a cyano group, an alkoxy group, a substituted alkoxy group, and -T. 1 -Z 1 ;

[0573] R 1 and R 2 independently selected from H, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano, alkoxy, substituted alkoxy, and -T 1 -Z 1 , or R 1 and R 2 Together they form may be optionally substituted (e.g. by R 3 a 5-membered or 6-membered fused aryl ring, heteroaryl ring, cycloalkyl ring or heterocyclic ring substituted with a cycloalkyl group;

[0574] Z 1 is a chemoselective functional group or an attached signaling chromophore; and

[0575] T 1 For the connection base.

[0576] Clause 21. The water-soluble light-harvesting multichromophore according to any one of clauses 19-20, wherein the aromatic or heteroaromatic comonomer is independently selected from one of the following structures (ba) to (cd):

[0577]

[0578]

[0579] in:

[0580] X is S or O;

[0581] Each R 4 are independently H, a water-solubilizing group, an alkyl group, a substituted alkyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, an acyl group, a substituted acyl group, a sulfonic acid group, a cyano group, an alkoxy group, a substituted alkoxy group, and -T 1 -Z 1 ;and

[0582] R 1 and R 2 independently selected from H, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano, alkoxy, substituted alkoxy, and -T 1 -Z 1 , or R 1 and R 2 Together they form a 5- or 6-membered fused aryl or heteroaryl ring which may be optionally substituted.

[0583] Item 22. The water-soluble light-harvesting multichromophore according to any one of Items 19-20, wherein the aryl or heteroaryl comonomer is independently selected from substituted or unsubstituted 1,4-phenyl, substituted or unsubstituted 1,3-phenyl, substituted or unsubstituted 4,4'-biphenyl, substituted or unsubstituted 2,5-pyridyl and substituted or unsubstituted 2,6-pyridyl.

[0584] Item 23. The water-soluble light-harvesting multichromophore according to Item 22, wherein the aromatic or heteroaromatic comonomer is selected from one of the following structures:

[0585]

[0586] Where Z 2 -Z 5 Each independently represents CR or N, wherein at least one Z 2 -Z 5 is N; and each R and each R 11 -R 16 Independently selected from the group consisting of hydrogen, water solubilizing groups, halogen, cyano, alkoxy, substituted alkoxy, alkyl, and substituted alkyl.

[0587] Clause 24. A water-soluble light-harvesting multichromophore according to any one of Clauses 19-23, wherein each comonomer substituent (eg, R3 -R 47 , R and each R 11 -R 16 ) are independently selected from one of the following structures:

[0588]

[0589]

[0590] in:

[0591] T 5 is an optional linking group (eg, a lower alkyl linking group);

[0592] Each T 6 is a linking group (e.g., a lower alkyl linking group);

[0593] R 11 and R are independently H, alkyl or substituted alkyl; and

[0594] Each s is an integer from 1 to 50.

[0595] Clause 25. The water-soluble light-harvesting multichromophore according to any one of clauses 19 to 24, wherein the aromatic or heteroaromatic comonomer is independently selected from one of the following structures:

[0596]

[0597]

[0598]

[0599]

[0600] Clause 26. The water-soluble light-harvesting multichromophore according to any one of Clauses 1 to 25, wherein the multichromophore has a structure of Formula (V):

[0601]

[0602] wherein each WSG is independently a branched or linear WSG (e.g., as described herein); Z 1 is a linked chemical selective functional group or a linked acceptor chromophore; G 1 and G 2 Each is independently selected from a terminal group, a π-conjugated segment, a linker, and a linked specific binding member; and x, y, and z represent the molar concentration % of the unit in the multichromophore.

[0603] Clause 27. The water-soluble light-harvesting multichromophore according to Clause 26, wherein the multichromophore has a structure of formula (VI):

[0604]

[0605] wherein each s is independently 6 to 50 (such as an integer of 6-30, 6 to 20, 11 to 20, 12 to 20, or 12 to 16; each R 11 are independently hydrogen, alkyl or substituted alkyl; DYE is an attached acceptor chromophore (eg, fluorophore); r is 0-6 (eg, 0, 1, 2, 3 or 4); and q is 1-12, such as 1-6 (eg, 1, 2, 3 or 4).

[0606] Item 28. A polymer tandem dye comprising:

[0607] A water-soluble light-harvesting multichromophore, wherein the water-soluble light-harvesting multichromophore comprises a conjugated segment having a structure of formula (I):

[0608]

[0609] in:

[0610] F 1 It is a fused three-ring comonomer substituted with a water-soluble group (WSG);

[0611] M 1 is an aromatic or heteroaromatic comonomer;

[0612] n is an integer from 1 to 100,000; and

[0613] * indicates the site for covalent attachment to the unsaturated backbone or terminal group of the conjugated polymer;

[0614] Among them, F 1 and M 1 At least one of the water-soluble polymers is substituted with a branched nonionic water-soluble group (WSG) comprising two or more water-soluble polymers, each of which has 6 to 50 (e.g., 6 to 40 or 6 to 30) monomer units; and

[0615] A signal chromophore is covalently attached to the multichromophore, the multichromophore being in energy-accepting proximity to the signal chromophore.

[0616] Item 29. The polymer tandem dye according to Item 28, wherein the multichromophore has an absorption maximum wavelength of 300 nm to 400 nm.

[0617] Item 30. The polymer tandem dye of Item 28, wherein the signal chromophore emission has a quantum yield of 0.1 or greater.

[0618] Item 31. The polymer tandem dye according to Item 28, wherein the signal chromophore has an emission of 50 mM - 1 cm -1 or higher brightness.

[0619] Item 32. The polymer tandem dye according to any one of Items 28-31, wherein the signal chromophore is a fluorophore.

[0620] Item 33. The polymer tandem dye according to any one of items 28-31, wherein the signal chromophore is a quencher.

[0621] Item 34. A polymer tandem dye according to any one of items 28-33, wherein the signal chromophore is selected from rhodamine, coumarin, xanthene, cyanine, polymethine, pyrene, dipyrrometheneborondifluoride, naphthylimide, phycobiliprotein, peridinum chlorophyllprotein, their conjugates and their combinations.

[0622] Item 35. A polymer tandem dye according to any one of Items 28-34, wherein the signal chromophore is selected from fluorescein, 6-FAM, rhodamine, Texas Red, California Red, iFluor594, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxy-p-methylamino acid, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy-Chrome, DyLight350, DyLight 405, DyLight 488, DyLight 549, DyLight 594, DyLight 633, DyLight 649, DyLight 680, DyLight 750, DyLight 800, Phycoerythrin, PerCP (Peridinyl Chlorophyll-a Protein), PerCP-Cy5.5, JOE (6-Carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), NED, ROX (5-(and-6)-Carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green500, Oregon Green 514, Alexa 350. Alexa 430. Alexa 488. Alexa 532. Alexa 546. Alexa 568. Alexa 594. Alexa 633. Alexa 647、Alexa 660、Alexa 680, 7-amino-4-methylcoumarin-3-acetic acid, FL, FL-Br2, 530 / 550, 558 / 568, 564 / 570, 576 / 589, 581 / 591, 630 / 650, 650 / 665, R6G, TMR, TR, their conjugates, and combinations thereof.

[0623] Clause 36. The polymer tandem dye according to any one of clauses 28 to 35, wherein:

[0624] The signal chromophore is linked to a comonomer that constitutes 5% to 50% by mole of the multichromophore; and

[0625] The multichromophore is a conjugated polymer comprising 5 or more repeating units.

[0626] Clause 37. A polymer tandem dye according to any one of clauses 28 to 36, wherein the multichromophore comprises a terminal group -L 3 -Z, where L 3 is a linker and Z is a specific binding member.

[0627] Item 38. The polymer tandem dye according to Item 37, wherein the linker is selected from the group consisting of alkyl, substituted alkyl, alkyl-amido, alkyl-amido-alkyl, and PEG moieties.

[0628] Item 39. A polymer tandem dye according to Item 38, wherein -L 3 -Z is described by the following structure:

[0629]

[0630] in:

[0631] q is 0 or an integer from 1 to 12; and

[0632] Z is a specific binding member.

[0633] Item 40. The polymer tandem dye according to any one of items 37-39, wherein Z is a biomolecule.

[0634] Item 41. The polymer tandem dye according to any one of items 37-40, wherein Z is an antibody.

[0635] Item 42. The polymer tandem dye according to any one of items 37-41, wherein Z is an antibody fragment or a binding derivative thereof.

[0636] Item 43. The polymer tandem dye according to Item 42, wherein the antibody fragment or binding derivative thereof is selected from the group consisting of: a Fab fragment, a F(ab')2 fragment, a scFv, a diabody and a triabody.

[0637] Item 44. A labeled specific binding member that resists aggregation, comprising:

[0638] A water-soluble light-harvesting multichromophore, wherein the water-soluble light-harvesting multichromophore comprises a conjugated segment having a structure of formula (I):

[0639]

[0640] in:

[0641] F 1 It is a fused three-ring comonomer substituted with a water-soluble group (WSG);

[0642] M 1 is an aromatic or heteroaromatic comonomer;

[0643] n is an integer from 1 to 100,000; and

[0644] * indicates the site for covalent attachment to the unsaturated backbone or terminal group of the conjugated polymer;

[0645] where F 1 and M 1 At least one of the water-soluble polymers is substituted with a branched nonionic water-soluble group (WSG) comprising two or more water-soluble polymers, each of which has 6 to 50 (e.g., 6 to 40 or 6 to 30) monomer units; and

[0646] A specific binding member is covalently linked to the multichromophore.

[0647] Item 45. The labeled specific binding member of Item 44, further comprising a signal chromophore covalently linked to the multichromophore, the multichromophore being in energy-accepting proximity to the signal chromophore.

[0648] Clause 46. A labeled specific binding member according to any one of clauses 44-45, wherein the specific binding member is an antibody.

[0649] Clause 47. A labeled specific binding member according to any one of clauses 44 to 46, wherein the specific binding member is an antibody fragment or a binding derivative thereof.

[0650] Clause 48. A labeled specific binding member according to Clause 47, wherein the antibody fragment or binding derivative thereof is selected from the group consisting of: a Fab fragment, a F(ab')2 fragment, a scFv, a diabody and a triabody.

[0651] Item 49. A labeled specific binding member according to Item 45, wherein the signal chromophore is selected from rhodamine, coumarin, xanthene, cyanine, polymethine, pyrene, boron dipyrrole fluoride, naphthylimide, phycobiliprotein, peridinin chlorophyll protein, conjugates thereof and combinations thereof.

[0652] Clause 50. A method of evaluating a sample for the presence of a target analyte, the method comprising:

[0653] (a) contacting the sample with an aggregation-resistant polymer-dye conjugate that specifically binds to the target analyte to produce a labeled composition-contacted sample, wherein the polymer-dye conjugate comprises:

[0654] (i) a water-soluble light-harvesting multichromophore, wherein the water-soluble light-harvesting multichromophore comprises a conjugated segment having a structure of formula (I):

[0655]

[0656] in:

[0657] F 1 It is a fused three-ring comonomer substituted with a water-soluble group (WSG);

[0658] M 1 is an aromatic or heteroaromatic comonomer;

[0659] n is an integer from 1 to 100,000; and

[0660] * indicates the site for covalent attachment to the unsaturated backbone or terminal group of the conjugated polymer;

[0661] Among them, F 1 and M1 At least one of the water-soluble polymers is substituted with a branched nonionic water-soluble group (WSG) comprising two or more water-soluble polymers, each of which has 6 to 50 (e.g., 6 to 40 or 6 to 30) monomer units; and

[0662] (ii) a specific binding member; and

[0663] (b) assaying the sample contacted with the labeling composition for the presence of the polymer dye conjugate-target analyte binding complex to assess the presence or absence of the target analyte in the sample.

[0664] Clause 51. The method of Clause 50, wherein the polymer-dye conjugate further comprises a signal chromophore covalently linked to the multichromophore, the multichromophore being in energy-accepting proximity to the signal chromophore.

[0665] Clause 52. The method according to any one of clauses 50-51, further comprising contacting the sample with a second specific binding member, which is support-bound and specifically binds the target analyte.

[0666] Clause 53. The method according to Clause 52, wherein the carrier comprises magnetic particles.

[0667] Clause 54. The method according to any one of Clauses 50-53, wherein the target analyte is associated with a cell.

[0668] Clause 55. The method according to Clause 54, wherein the target analyte is a cell surface marker of the cell.

[0669] Clause 56. The method according to Clause 55, wherein the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen.

[0670] Clause 57. The method according to Clause 56, wherein the target analyte is an intracellular target, and the method further comprises lysing the cell.

[0671] Clause 58. The method according to any one of Clauses 50-57, wherein the method further comprises analyzing the fluorescently labeled target analyte by flow cytometry.

[0672] Clause 59. A method of labeling a target molecule, the method comprising:

[0673] contacting the target molecule with a polymeric dye to produce a labeled target molecule, wherein the polymeric dye comprises:

[0674] A water-soluble light-harvesting multichromophore, wherein the water-soluble light-harvesting multichromophore comprises a conjugated segment having a structure of formula (I):

[0675]

[0676] in:

[0677] F 1 It is a fused three-ring comonomer substituted with a water-soluble group (WSG);

[0678] M 1 is an aromatic or heteroaromatic comonomer;

[0679] n is an integer from 1 to 100,000; and

[0680] * indicates the site for covalent attachment to the unsaturated backbone or terminal group of the conjugated polymer;

[0681] where F 1 and M 1 At least one of the water-soluble polymers is substituted with a branched nonionic water-soluble group (WSG) comprising two or more water-soluble polymers, each of which has 6 to 50 (e.g., 6 to 40 or 6 to 30) monomer units; and

[0682] A conjugated label is covalently linked to the target molecule.

[0683] Clause 60. The method of Clause 59, wherein the polymeric dye further comprises a signal chromophore covalently linked to the multichromophore, the multichromophore being in energy-accepting proximity to the signal chromophore.

[0684] Clause 61. The method according to any one of clauses 59-60, further comprising fluorescently detecting the labeled target molecule.

[0685] Clause 62. A method according to any one of clauses 59-61, wherein the conjugated tag comprises a terminal functional group selected from the group consisting of amino, thiol, hydroxyl, hydrazine, hydrazide, azide, alkyne, maleimide, iodoacetyl, amine, active ester and protein reactive group.

[0686] Clause 63. The method according to any one of clauses 59-62, wherein the target molecule is a specific binding member.

[0687] Clause 64. The method according to Clause 63, wherein the specific binding member is an antibody.

[0688] Clause 65. The method according to Clause 63, wherein the specific binding member is an antibody fragment or a binding derivative thereof.

[0689] Clause 66. The method according to Clause 65, wherein the antibody fragment or binding derivative thereof is selected from the group consisting of: a Fab fragment, a F(ab')2 fragment, a scFv, a diabody and a triabody.

[0690] Clause 67. A flow cytometry system comprising:

[0691] A flow cytometer including a flow path;

[0692] A composition in the flow path, wherein the composition comprises:

[0693] samples; and

[0694] A labeled specific binding member that resists aggregation, comprising:

[0695] A water-soluble light-harvesting multichromophore, wherein the water-soluble light-harvesting multichromophore comprises a conjugated segment having a structure of formula (I):

[0696]

[0697] in:

[0698] F 1 It is a fused three-ring comonomer substituted with a water-soluble group (WSG);

[0699] M 1 is an aromatic or heteroaromatic comonomer;

[0700] n is an integer from 1 to 100,000; and

[0701] * indicates the site for covalent attachment to the unsaturated backbone or terminal group of the conjugated polymer;

[0702] where F 1 and M 1 At least one of the water-soluble polymers is substituted with a branched nonionic water-soluble group (WSG) comprising two or more water-soluble polymers, each of which has 6 to 50 (e.g., 6 to 40 or 6 to 30) monomer units; and

[0703] A specific binding member specifically binds the target analyte and is covalently linked to the multichromophore.

[0704] Clause 68. The system of Clause 67, wherein the labeled specific binding member further comprises a signal chromophore covalently linked to the multichromophore, the multichromophore being in energy receiving proximity to the signal chromophore.

[0705] Clause 69. The system according to any of Clauses 67-68, wherein the composition further comprises a second specific binding member which is carrier-bound and specifically binds to the target analyte.

[0706] Clause 70. The system of Clause 69, wherein the carrier comprises magnetic particles.

[0707] Clause 71. The system of any one of Clauses 67-70, wherein the sample comprises cells.

[0708] Clause 72. The system of Clause 71, wherein the target analyte is a cell surface marker of the cell.

[0709] Clause 73. The system according to Clause 72, wherein the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen.

[0710] Clause 74. A kit comprising:

[0711] A water-soluble light-harvesting multichromophore, wherein the water-soluble light-harvesting multichromophore comprises a conjugated segment having a structure of formula (I):

[0712]

[0713] in:

[0714] F 1 It is a fused three-ring comonomer substituted with a water-soluble group (WSG);

[0715] M 1 is an aromatic or heteroaromatic comonomer;

[0716] n is an integer from 1 to 100,000; and

[0717] * indicates the site for covalent attachment to the unsaturated backbone or terminal group of the conjugated polymer;

[0718] where F 1 and M 1 At least one of the water-soluble polymers is substituted with a branched nonionic water-soluble group (WSG) comprising two or more water-soluble polymers, each of which has 6 to 50 (e.g., 6 to 40 or 6 to 30) monomer units; and

[0719] container.

[0720] Clause 75. The kit according to clause 74, further comprising one or more components selected from the group consisting of: a polymer tandem dye, a fluorophore, a specific binding member, a specific binding member conjugate, cells, a carrier, a biocompatible aqueous elution buffer, and instructions for use.

[0721] Clause 76. The kit according to any one of clauses 74-75, wherein the multichromophore is as described in any one of clauses 1-27.

[0722] Clause 77. The kit according to any one of clauses 74-76, wherein the multichromophore is covalently linked to a specific binding member.

[0723] Clause 78. The kit according to Clause 77, wherein the specific binding member is an antibody.

[0724] Clause 79. The kit according to Clause 77, wherein the specific binding member is an antibody fragment or a binding derivative thereof.

[0725] Clause 80. The kit according to Clause 79, wherein the antibody fragment or binding derivative thereof is selected from the group consisting of: a Fab fragment, a F(ab')2 fragment, a scFv, a diabody and a triabody.

[0726] Clause 81. The kit according to any one of clauses 74-80, wherein the multichromophore further comprises an acceptor signaling chromophore covalently linked to the multichromophore, the multichromophore being in energy receiving proximity to the acceptor signaling chromophore.

[0727] Although the foregoing invention has been described in considerable detail by way of illustration and example for purposes of clarity of understanding, in light of the teachings of the present invention, one of ordinary skill in the art will readily appreciate that certain changes and modifications may be made to the present invention without departing from the spirit or scope of the appended claims.

[0728] Therefore, the above only illustrates the principle of the present invention.It should be understood that those skilled in the art will be able to design various arrangements, which, although not explicitly described or shown herein, embody the principle of the present invention and are included in its spirit and scope.In addition, all embodiments and conditional language described herein are mainly intended to help readers understand the principle of the present invention and the conception of promoting technology provided by the inventor, and should be interpreted as not limiting such examples and conditions of special narration.In addition, all statements describing the principles, aspects and embodiments of the present invention and its specific embodiments herein are intended to cover their structural equivalents and functional equivalents.In addition, it is intended that such equivalents include currently known equivalents and equivalents developed in the future, that is, any element developed to perform the same function regardless of the structure.Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein.On the contrary, the scope and spirit of the present invention are embodied by the following content.

Claims

1. A water-soluble light-harvesting multichromophore comprising a conjugated segment having a structure of formula (I): in: F 1 It is a fused three-ring comonomer substituted with a water-soluble group (WSG); M 1 is an aromatic or heteroaromatic comonomer; n is an integer from 1 to 100,000; and * indicates the site for covalent attachment to the unsaturated backbone or terminal group of the conjugated polymer; where F 1 and M 1 At least one of the polychromophores is substituted with a branched nonionic water-soluble group comprising two or more water-soluble polymers, each of which has 6-50 monomer units; wherein the branched nonionic water-soluble group is capable of conferring a solubility in water of more than 50 mg / mL to the polychromophore.

2. The water-soluble light-harvesting multichromophore according to claim 1, wherein the fused tricyclic comonomer is described by the following structure: in: Y is C(R 3 )2、-C(R 3 )2C(R 3 )2-、-C(R 3 )2Si(R 3 )2-、NR 3 、Si(R 3 )2 or Se; each Z is independently CH, CR or N, wherein at least two of the Zs in each ring are CH or CR; Each R 3 independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido, aralkyl, substituted aralkyl, PEG moiety, -L 1 -Z 1 , where L 1 is a linker and Z 1 are chemoselective tags and WSG; and Each R is independently selected from H, WSG, halogen, alkoxy, substituted alkoxy, alkyl and substituted alkyl, or wherein two R groups are optionally cyclically linked to provide a carbocyclic or heterocyclic ring.

3. The water-soluble light-harvesting multichromophore according to claim 2, wherein the fused tricyclic comonomer is described by one of the following structures: in: Each R 3 independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido, aralkyl, substituted aralkyl, PEG moiety, -L 1 -Z 1 , where L 1 is a linker and Z 1 are chemoselective tags and WSG; and Each R is independently selected from H, WSG, halogen, alkoxy, substituted alkoxy, alkyl and substituted alkyl, or wherein two R groups are optionally cyclically linked to provide a carbocyclic or heterocyclic ring.

4. The water-soluble light-harvesting multichromophore according to claim 1, wherein the branched nonionic WSG has one of the following formulae: in: Each B 1 and B 2 are independently branched groups; Each W 1 are independently water-soluble polymers comprising 6 to 24 monomer units; T 3 is an optional linker to the fused 6-5-6 tricyclic comonomer; and Each p and q is independently 0 or 1, where each T, if present 1 and each T 2 are independently linking groups.

5. The water-soluble light-harvesting multichromophore according to claim 4, wherein the branched nonionic WSG is selected from one of the following structures: in: T 5 is an optional linker to the fused 6-5-6 tricyclic comonomer; T 6 is a linking group; Each s is an integer from 6 to 24; and Each R 11 are independently hydrogen, alkyl or substituted alkyl.

6. The water-soluble light-harvesting multichromophore according to claim 1, wherein F 1 One of the following comonomers:

7. The water-soluble light-harvesting multichromophore according to claim 1, wherein the multichromophore has a structure of formula (III): in: F 1 It is a fused three-ring comonomer; M 1 、M 2 and L 1 are independently aryl or heteroaryl comonomers; Z 1 is a linked chemoselective functional group or a linked acceptor chromophore; Each n is independently an integer from 1 to 10,000; each m is 0 or independently an integer from 1 to 10,000; p is an integer from 1 to 100,000; and G 1 and G 2 Each is independently selected from a terminal group, a π-conjugated segment, a linker, and a linked specific binding member.

8. The water-soluble light-harvesting multichromophore according to claim 7, wherein the multichromophore has a structure of formula (IV):

9. The water-soluble light-harvesting multichromophore according to claim 8, wherein L 1 Has the following structure: in: R 3 It is a nonionic water-soluble group (WSG); R 4 For L 2 -Z 2 , where L 2 is a linker and Z 2 is a chemoselective functional group or an acceptor chromophore; and Each R is independently selected from H, WSG, halogen, alkoxy, substituted alkoxy, alkyl and substituted alkyl, or wherein two R groups are optionally cyclically linked to provide a carbocyclic or heterocyclic ring.

10. The water-soluble light-harvesting multichromophore according to claim 9, wherein L 1 Has one of the following structures:

11. The water-soluble light-harvesting multichromophore according to claim 1, wherein the aromatic or heteroaromatic comonomer is independently selected from one of formula (XXIII)-(XXVI): in Cy 2-7 is an aryl or heteroaryl group containing 2 to 7 fused and / or unfused rings; Y 2 , Y 3 and Y 4 Independently selected from -CR 3 -、NR 3 , N, O, S and -C(=O)- and together form a 5- or 6-membered fused aryl or heteroaryl ring; Each R 3 is one or more independently selected from H, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano, alkoxy substituted alkoxy and -T 1 -Z 1 a ring substituent of R 1 and R 2 independently selected from H, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano, alkoxy, substituted alkoxy, and -T 1 -Z 1 , or R 1 and R 2 together form an optionally substituted 5- or 6-membered fused aryl, heteroaryl, cycloalkyl or heterocyclic ring; Y 5 N or CR 5 And Y 7 N or CR 7 ; R 4 -R 7 independently selected from H, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano, alkoxy, substituted alkoxy, and -T 1 -Z 1 ; Z 1 is a chemoselective functional group or an attached signaling chromophore; and T 1 For the connection base.

12. The water-soluble light-harvesting multichromophore according to claim 11, wherein the aromatic or heteroaromatic comonomer is independently selected from one of the following structures (a) to (x): in: Y 8 C(R 3 )2、-C(R 3 )2C(R 3 )2-、-C(R 3 )2Si(R 3 )2-、NR 3 or Si(R 3 )2; X is S or O; Each R 3 are independently H, a water-soluble group, an amino group, a substituted amino group, a halogen group, a cyano group, an alkyl group, a substituted alkyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, an acyl group, a substituted acyl group, a sulfonic acid group, a cyano group, an alkoxy group, a substituted alkoxy group, and -T 1 -Z 1 ; R 1 and R 2 independently selected from H, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano, alkoxy, substituted alkoxy, and -T 1 -Z 1 , or R 1 and R 2 together form an optionally substituted 5- or 6-membered fused aryl, heteroaryl, cycloalkyl or heterocyclic ring; Z 1 is a chemoselective functional group or an attached signaling chromophore; and T 1 For the connection base.

13. The water-soluble light-harvesting multichromophore according to claim 12, wherein the aromatic or heteroaromatic comonomer is independently selected from one of the following structures (ba) to (cd): in: X is S or O; Each R 4 are independently H, a water-solubilizing group, an alkyl group, a substituted alkyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, an acyl group, a substituted acyl group, a sulfonic acid group, a cyano group, an alkoxy group, a substituted alkoxy group, and -T 1 -Z 1 ;and R 1 and R 2 independently selected from H, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano, alkoxy, substituted alkoxy, and -T 1 -Z 1 , or R 1 and R 2 Together they form a 5- or 6-membered fused aryl or heteroaryl ring which may be optionally substituted.

14. The water-soluble light-harvesting multichromophore according to claim 1, wherein the aryl or heteroaryl comonomer is independently selected from substituted or unsubstituted 1,4-phenyl, substituted or unsubstituted 1,3-phenyl, substituted or unsubstituted 4,4'-biphenyl, substituted or unsubstituted 2,5-pyridyl and substituted or unsubstituted 2,6-pyridyl.

15. The water-soluble light-harvesting multichromophore according to claim 14, wherein the aromatic or heteroaromatic comonomer is selected from one of the following structures: Where Z 2 -Z 5 Each independently represents CR or N, wherein at least one Z 2 -Z 5 is N; and each R and each R 11 -R 16 Independently selected from the group consisting of hydrogen, water solubilizing groups, halogen, cyano, alkoxy, substituted alkoxy, alkyl, and substituted alkyl.

16. The water-soluble light-harvesting multichromophore according to claim 1, wherein each comonomer is substituted with WSG independently selected from one of the following structures: in: T 5 is an optional linker; Each T 6 is a linking group; R 11 and R are independently H, alkyl or substituted alkyl; and Each s is an integer from 1 to 50.

17. The water-soluble light-harvesting multichromophore according to claim 1, wherein the aromatic or heteroaromatic comonomer is independently selected from one of the following structures:

18. A polymer tandem dye comprising: A water-soluble light-harvesting multichromophore, wherein the water-soluble light-harvesting multichromophore comprises a conjugated segment having a structure of formula (I): in: F 1 It is a fused three-ring comonomer substituted with a water-soluble group (WSG); M 1 is an aromatic or heteroaromatic comonomer; n is an integer from 1 to 100,000; and * indicates the site for covalent attachment to the unsaturated backbone or terminal group of the conjugated polymer; where F 1 and M 1 At least one of the water-soluble polymers is substituted with a branched nonionic water-soluble group (WSG) comprising two or more water-soluble polymers, each of which has 6 to 50 monomer units; as well as A signal chromophore is covalently attached to the multichromophore, the multichromophore being in energy-accepting proximity to the signal chromophore.

19. A specific binding member for a marker that resists aggregation, comprising: A water-soluble light-harvesting multichromophore, wherein the water-soluble light-harvesting multichromophore comprises a conjugated segment having a structure of formula (I): in: F 1 It is a fused three-ring comonomer substituted with a water-soluble group (WSG); M 1 is an aromatic or heteroaromatic comonomer; n is an integer from 1 to 100,000; and * indicates the site for covalent attachment to the unsaturated backbone or terminal group of the conjugated polymer; where F 1 and M 1 At least one of the water-soluble polymers is substituted with a branched nonionic water-soluble group (WSG) comprising two or more water-soluble polymers, each of which has 6 to 50 monomer units; and A specific binding member is covalently linked to the multichromophore.

20. The labeled specific binding member of claim 19, further comprising a signal chromophore covalently linked to the multichromophore, the multichromophore being in energy receiving proximity to the signal chromophore.

21. A method of evaluating a sample for the presence of a target analyte, the method comprising: (a) contacting the sample with an aggregation-resistant polymer-dye conjugate that specifically binds to the target analyte to produce a labeled composition-contacted sample, wherein the polymer-dye conjugate comprises: (i) a water-soluble light-harvesting multichromophore, wherein the water-soluble light-harvesting multichromophore comprises a conjugated segment having a structure of formula (I): in: F 1 It is a fused three-ring comonomer substituted with a water-soluble group (WSG); M 1 is an aromatic or heteroaromatic comonomer; n is an integer from 1 to 100,000; and * indicates the site for covalent attachment to the unsaturated backbone or terminal group of the conjugated polymer; where F 1 and M 1 At least one of the water-soluble polymers is substituted with a branched nonionic water-soluble group (WSG) comprising two or more water-soluble polymers, each of which has 6 to 50 (e.g., 6 to 40 or 6 to 30) monomer units; and (ii) a specific binding member; and (b) assaying the sample contacted with the labeling composition for the presence of the polymer dye conjugate-target analyte binding complex to assess the presence or absence of the target analyte in the sample.

22. A method for labeling a target molecule, the method comprising: contacting the target molecule with a polymeric dye to produce a labeled target molecule, wherein the polymeric dye comprises: A water-soluble light-harvesting multichromophore, wherein the water-soluble light-harvesting multichromophore comprises a conjugated segment having a structure of formula (I): in: F 1 It is a fused three-ring comonomer substituted with a water-soluble group (WSG); M 1 is an aromatic or heteroaromatic comonomer; n is an integer from 1 to 100,000; and * indicates the site for covalent attachment to the unsaturated backbone or terminal group of the conjugated polymer; where F 1 and M 1 At least one of the water-soluble polymers is substituted with a branched nonionic water-soluble group (WSG) comprising two or more water-soluble polymers, each of which has 6 to 50 (e.g., 6 to 40 or 6 to 30) monomer units; and A conjugated label is covalently linked to the target molecule.

23. A flow cytometry system comprising: A flow cytometer including a flow path; A composition in the flow path, wherein the composition comprises: sample; and A labeled specific binding member that resists aggregation, comprising: A water-soluble light-harvesting multichromophore, wherein the water-soluble light-harvesting multichromophore comprises a conjugated segment having a structure of formula (I): in: F 1 It is a fused three-ring comonomer substituted with a water-soluble group (WSG); M 1 is an aromatic or heteroaromatic comonomer; n is an integer from 1 to 100,000; and * indicates the site for covalent attachment to the unsaturated backbone or terminal group of the conjugated polymer; where F 1 and M 1 At least one of the water-soluble polymers is substituted with a branched nonionic water-soluble group (WSG) comprising two or more water-soluble polymers, each of which has 6 to 50 (e.g., 6 to 40 or 6 to 30) monomer units; and A specific binding member specifically binds the target analyte and is covalently linked to the multichromophore.

24. A kit comprising: A water-soluble light-harvesting multichromophore, wherein the water-soluble light-harvesting multichromophore comprises a conjugated segment having a structure of formula (I): in: F 1 It is a fused three-ring comonomer substituted with a water-soluble group (WSG); M 1 is an aromatic or heteroaromatic comonomer; n is an integer from 1 to 100,000; and * indicates the site for covalent attachment to the unsaturated backbone or terminal group of the conjugated polymer; where F 1 and M 1 At least one of the water-soluble polymers is substituted with a branched nonionic water-soluble group (WSG) comprising two or more water-soluble polymers, each of which has 6 to 50 (e.g., 6 to 40 or 6 to 30) monomer units; and container.

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