Deep ultraviolet excitable hydrosolvated polymer dyes

Abstract

A hydrosolvated polymeric dye having a deep ultraviolet excitation spectrum is provided. The subject polymeric dyes include a light-condensing multi-chromophore having conjugated modified repeat units incorporated into a polymer backbone to produce segments of a pi-conjugated comonomer with limited pi-conjugation between the segments. Polymeric tandem dyes are also provided that further comprise a signal chromophore covalently linked to a multi-chromophore that receives energy close to the multi-chromophore. Specific binding members comprising a label of the subject hydrosolvated polymeric dyes are also provided. Also provided are methods of evaluating the presence of a target analyte in a sample as well as methods of labeling a target molecule in which a subject polymeric dye can be used. Systems and kits for practicing the subject methods are also provided.

Description

[0001] This application is a divisional application of a Chinese patent application with an application date of December 19, 2018, application number 201880060262.3, and invention name “Deep UV-Excitable Water-Solvated Polymer Dye”. Technical Field

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

[0003] Fluorescent dyes are compounds that emit (usually) different wavelengths of light when irradiated with the light of the wavelength they absorb. Fluorescent dyes can be used in various applications in biochemistry, biology and medicine, for example, for diagnostic kits, microscopy or drug screening. Fluorescent dyes are characterized by allowing the user to select many parameters of suitable dyes according to the desired purpose. The parameters of interest include excitation wavelength maximum, emission wavelength maximum, Stokes shift, extinction coefficient, fluorescence quantum yield and fluorescence lifetime. Dyes can be selected according to the application of interest, so as to, for example, allow the excitation radiation to penetrate into the biological sample, 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] A water-solvated polymer dye with a deep ultraviolet excitation spectrum is provided. The subject polymer dye comprises a light-gathering multichromophore having a conjugated modified repeating unit, which is incorporated into a polymer backbone to produce segments of π-conjugated comonomers with limited π-conjugation between segments. A polymer tandem dye is also provided, which further comprises a signal chromophore covalently attached to the multichromophore close to the multichromophore energy receiving. A labeled specific binding member comprising the subject water-solvated polymer dye is also provided. A method for evaluating the presence of a target analyte in a sample and a method for labeling a target molecule in which the subject polymer dye can be used 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 following figures are for illustration purposes only. The figures are not intended to limit the scope of the present teachings in any way.

[0007] Figure 1Depicted are exemplary comonomers, conjugated modifying repeat units, and polymeric dyes of the present disclosure.

[0008] Figure 2 Depicted are exemplary pi-conjugated segments of exemplary polymeric dyes of the present disclosure.

[0009] Figure 3 Exemplary polymeric dyes of the present disclosure are depicted.

[0010] Figure 4 Exemplary polymeric dyes of the present disclosure are depicted.

[0011] Figure 5 Depicted are exemplary polymeric dyes of the present disclosure that include branched π-conjugated segments.

[0012] Figures 6A to 6C Exemplary polymeric dyes of the present disclosure are depicted.

[0013] Figure 7 Normalized absorption spectra of several exemplary polymer dyes having deep UV excitation spectra and absorption maxima at 350 nm or less are shown.

[0014] Figure 8 Shown are normalized emission spectra of several exemplary polymeric dyes of the present disclosure.

[0015] Fig. 9 Conjugated with various signal chromophores, with Figure 3 Normalized emission spectra of several exemplary polymer tandem dyes (linked dyes) of the polymer dye structure of Dye 1 are shown.

[0016] definition

[0017] 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.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. However, for clarity and ease of reference, certain terms are defined below.

[0019] It must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural indicators, except where the context clearly specifies otherwise. For example, the term "dye" refers to one or more dyes, i.e., a single dye and multiple dyes. It should be further noted that the claims can be written to exclude any optional elements. Therefore, this statement is intended to be used as a prerequisite for the use of such exclusive terms such as "solely", "only", etc., or the use of "negative" restrictions related to the enumeration of claim elements.

[0020] As used herein, the term "sample" refers to a material or mixture of materials containing one or more analytes of interest, in some cases in liquid form. In some embodiments, the term, used in its broadest sense, refers to any plant, animal or bacterial material containing cells or producing cell metabolites, such as tissues or fluids (including but not limited to plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) separated from individuals or tissues or fluids 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, skin, external parts of the respiratory tract, intestinal tract and urogenital tract, 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 living organism (eukaryotic and prokaryotic) having at least a nucleus and a cell membrane. In certain embodiments, the cell includes a prokaryotic cell, such as a prokaryotic cell from a bacterium. In other embodiments, the cell includes a eukaryotic cell, such as a cell obtained from a biological sample of an animal, plant or fungus.

[0021] As used herein, the terms "carrier-bound" and "carrier-attached" are used interchangeably and refer to a moiety (e.g., a specific binding member) that is covalently or non-covalently attached to a carrier of interest. Covalent attachment 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 attachment 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 attachment can involve absorption of a substrate.

[0022] 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 in which conventional backbones have been replaced by non-naturally occurring or synthetic backbones. 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.

[0023] 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 that accompanied the portion prior to purification.

[0024] A "plurality" includes at least 2 members. In some cases, a plurality can have 5 or more, such as 10 or more, 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.

[0025] Numerical ranges are inclusive of the numbers defining the range.

[0026] 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 examples, the specific binding interaction will distinguish between a desired analyte and an undesired analyte in a sample with a specificity that exceeds that for an undesired analyte by 10 times or more, such as 100 times or more, or 1000 times or more. In some cases, the affinity between a capture agent and an analyte when they are specifically bound in the form of a capture agent / analyte complex is at least 10 -8 M. At least 10 -9 M, such as up to 10 -10 M.

[0027] 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 being associated with a lower Kd.

[0028] Methods described herein include multiple steps. Each step can be performed between steps after a predetermined amount of time has passed, as needed. Therefore, the time between performing 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, a step can be performed after incubation or waiting time (e.g., a few minutes to overnight waiting time) after the previous step is completed.

[0029] As used herein, the terms "evaluate," "determine," "measure," "assess," and "measure" are used interchangeably and include both quantitative and qualitative determination.

[0030] As used herein, the term "isolated" refers to the physical separation of two components (eg, by size or affinity, etc.) as well as the degradation of one component while the other component remains intact.

[0031] As used herein, the term "joint" or "linker" refers to a connecting portion that connects two groups and has a skeleton of 100 atoms or less in length. A joint or linker can be a covalent bond connecting two groups or a chain of length between 1 and 100 atoms, such as a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more carbon atoms, wherein the joint can be linear, branched, cyclic or a single atom. In some cases, the joint is a branched joint, 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 joint skeleton can be optionally substituted by heteroatom sulfur, nitrogen or oxygen. In some cases, the joint skeleton includes a connecting functional group, such as ether, thioether, amino, amide, sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester or imine. The bond between the skeleton atoms can be saturated or unsaturated, and in some cases, there are no more than one, two or three unsaturated bonds in the joint skeleton. The joint may include one or more substituents, such as alkyl, aryl or alkenyl. The joint may include but is not limited to polyethylene glycol, ether, thioether, tertiary amine, alkyl, which may be a linear or branched alkyl, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl) etc. The joint skeleton may include a cyclic group, such as an aryl, a heterocycle or a cycloalkyl, wherein the skeleton includes 2 or more atoms of the cyclic group, such as 2, 3 or 4 atoms. The joint may be breakable or non-breakable.

[0032] 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 various end groups and / or further substituents, including but not limited to alkyl, aryl, hydroxyl, amino, acyl, acyloxy and amido end groups and / or substituents. PEG groups that can be modified 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.

[0033] As used herein, the term "alkyl" by itself or as part of another substituent refers to a saturated branched or straight-chain monovalent hydrocarbon radical obtained 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 groups such as prop-1-yl or prop-2-yl; and butyl groups 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, for example, 1 to 4 carbon atoms. The term includes, for example, linear and branched hydrocarbon groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), tert-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-) and neopentyl ((CH3)3CCH2-).

[0034] 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 heteroatom 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, amide, acyloxy, amino, aminoacyl, aminoacyloxy, oxaaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, mercapto, 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.

[0035] "Alkoxy" refers to the group -O-alkyl, wherein alkyl is as defined herein. Alkoxy includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, and the like. The term "alkoxy" also refers to the groups alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-, wherein alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0036] The term "substituted alkoxy" refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O-, wherein substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0037] "Alkynyl" refers to a straight or branched monovalent hydrocarbon group having 2 to 6 carbon atoms, preferably 2 to 3 carbon atoms, and having at least 1 and preferably 1 to 2 sites of triple bond unsaturation. Examples of such alkynyl groups include ethynyl (-C≡CH) and propargyl (-CH2C≡CH).

[0038] The term "substituted alkynyl" refers to an alkynyl group as defined herein having from 1 to 5 substituents or from 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amido, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxoaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, mercapto, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0039] "Amino" refers to the group -NH2. The term "substituted amino" refers to the group -NRR, wherein 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, provided that at least one R is not hydrogen.

[0040] "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 groups 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 include phenyl and naphthyl.

[0041] "Substituted aryl", unless otherwise limited by the definition of aryl substituents, refers to aryl substituted with 1 to 5 substituents, or 1 to 3 substituents, selected from acyloxy, hydroxy, mercapto, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, amido, alkaryl, aryl, aromatic 1-Hydroxyl, -2-oxo, -2-deg.- 1- alkyl ...

[0042] "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, radicals 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, benzodioxol, and the like. In certain embodiments, the heteroaryl is a 5-20 membered heteroaryl. In certain embodiments, the heteroaryl is a 5-10 membered heteroaryl. In certain embodiments, the heteroaryl is those derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole and pyrazine.

[0043] "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 rings can be cycloalkyl, aryl or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In certain embodiments, one or more nitrogen and / or sulfur atoms of the heterocyclyl are optionally oxidized to provide N-oxide, -S(O)- or -SO2- moieties.

[0044] 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 ... oxazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also known as thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

[0045] "Substituted heteroaryl", unless otherwise limited by the definition of substituents, refers to heteroaryl substituted with 1 to 5 substituents, or 1 to 3 substituents selected from acyloxy, hydroxy, mercapto, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, amido, alkaryl, aryl, Aryloxy, azido, carboxyl, carboxyalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyamido, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl and trihalomethyl.

[0046] The term "alkaryl" or "aralkyl" refers to the groups -alkylene-aryl and substituted alkylene-aryl wherein alkylene, substituted alkylene and aryl are as defined herein.

[0047] "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. The term includes, for example, 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 having from 1 to 3 hydrogen atoms replaced by substituents as described under the carbon atoms in the definition of "substituted" below.

[0048] "Substituted" refers to a group in which one or more hydrogen atoms are independently replaced by one or more substituents that are the same or different. Substituents of interest include, but are not limited to, alkylenedioxy (e.g., 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 R61 , where M is halogen; R 60 , R 61 , R 62 and R 63 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 is independently hydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, or optionally, R 64 and R 65 Together with the nitrogen atom to which they are bound, they form a cycloheteroalkyl or substituted cycloheteroalkyl ring. In certain embodiments, the substituents include -M, -R 60 ,=O,-OR 60 、-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)2R60 、-P(O)(OR 60 )(O - )、-OP(O)(OR 60 )(OR 61 )、-C(O)R 60 、-C(O)OR 60 、-C(O)NR 60 R 61 ,-C(O)O - In certain embodiments, the substituents include -M, -R 60 ,=O,-OR 60 、-SR 60 、-NR 60 R 61 , -CF3, -CN, -NO2, -S(O)2R 60 、-OP(O)(OR 60 )(OR 61 )、-C(O)R 60 、-C(O)OR 60 、-C(O)O - , where R 60 , R 61 and R 62 As defined above. For example, a substituted group may carry a methylenedioxy substituent or one, two or three substituents selected from halogen atoms, (1-4C)alkyl 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".

[0049] "Sulfonamido" refers to the group -NR 21 S02R 22 , where R 21 and R 22 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocycle, and substituted heterocycle, and wherein R 21 and R 22 and optionally joined together with the atoms to which they are attached to form a heterocycle or substituted heterocyclyl, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle and substituted heterocycle are as defined herein.

[0050] It should be understood that in all substituted groups defined above, it is not intended herein to include polymers formed by limiting a substituent to have further substituents on itself (e.g., a substituted aryl has a substituted aryl as a substituent, which itself is substituted with a substituted aryl, which is further substituted with a substituted aryl, etc.). In such cases, the maximum number of such substitutions is three. For example, the continuous substitution of a substituted aryl specifically contemplated herein is limited to substituted aryl-(substituted aryl)-substituted aryl.

[0051] Nomenclature of substituents not expressly defined herein is obtained by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment unless otherwise indicated. For example, the substituent "arylalkoxycarbonyl" refers to the group (aryl)-(alkyl)-OC(O)-.

[0052] For any group disclosed herein containing one or more substituents, it is of course understood that such groups do not contain any substitution or substitution pattern that is sterically impractical and / or synthetically infeasible. In addition, the subject compounds include all stereochemical isomers resulting from the substitution of these compounds.

[0053] Additional definitions for terms may appear throughout the specification. DETAILED DESCRIPTION

[0054] As outlined above, water-solvated polymer dyes with deep ultraviolet excitation spectra are provided. The subject polymer dye comprises a light-gathering multichromophore, and the light-gathering multichromophore has a conjugated modified repeating unit, and the conjugated modified repeating unit is incorporated into the polymer backbone to produce a segment of a π-conjugated comonomer with limited π-conjugation between segments. In some cases, the polymer dye comprises a plurality of π-conjugated segments separated by the conjugated modified repeating units. A polymer tandem dye is also provided, which further comprises a signal chromophore covalently attached to the polymer dye close to the energy reception of the polymer dye. A labeled specific binding member comprising the subject water-solvated polymer dye is also provided. A method for evaluating the presence of a target analyte in a sample and a method for labeling a target molecule in which the subject polymer dye can be used are also provided. A system and a kit for practicing the subject method are also provided.

[0055] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific embodiments described, as such embodiments may of course vary. 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 restrictive, as the scope of the present invention will be limited only by the appended claims.

[0056] If a range of values ​​is provided, it is understood that each intermediate value between the upper and lower limits of the range (up to one tenth of the unit of the lower limit, unless the context clearly indicates otherwise) and any other stated or intermediate values ​​within the stated range are included in the present invention. The upper and lower limits of these smaller ranges may be independently included in these smaller ranges and are also included in the present invention, but are subject to any explicitly excluded limitations within the stated range. If the stated range includes one or both of the stated limits, the range excluding one or both of these included limits is also included in the present invention.

[0057] Certain ranges provided herein contain values ​​preceded by the term "about". The term "about" is used herein to provide literal support for the exact number that follows it, as well as a number that is close to or approximately the number that follows the term. In determining whether a value is close to or approximately an explicitly recited value, a close or approximate unrecited value may be a value that, in the context in which it is presented, provides a substantial equivalent to the explicitly recited value.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative exemplary methods and materials are now described.

[0059] All publications and patents cited in this specification are incorporated herein by reference, just as if each individual publication or patent was specifically and individually indicated as being incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials involved when citing the publication. Reference to any publication is directed to its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate the publication by virtue of prior invention. In addition, the publication date provided may be different from the actual publication date, which may need to be independently confirmed.

[0060] It should be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims can be drafted to exclude any optional elements. Thus, this statement is intended to serve as an antecedent basis for the use of such exclusive terminology as "solely", "only", etc., or the use of a "negative" limitation in connection with the enumeration of claim elements.

[0061] It will be apparent to those skilled in the art after 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 invention. Any recited method can be performed in the order of events recited or in any other order that is logically possible.

[0062] Although the apparatus and method have been or will be described through functional descriptions for the sake of grammatical fluency, it should be expressly understood that unless expressly set forth under 35 U.S.C. §112, the claims should not be interpreted as being limited in any way to "means" or "step" limitations, but should be given the full scope of meaning and equivalents provided by the definition of the claims under the judicial doctrine of equivalents, and in the event that a claim is expressly set forth under 35 U.S.C. §112, it should be given full statutory equivalent rights under 35 U.S.C. §112.

[0063] In further describing the present invention, water-solvated polymer dyes including light-gathering multichromophores and related polymer tandem dyes further including signal chromophores are first described in more detail. Next, labeled specific binding members including the subject polymer dyes are described. Then, methods of interest in which the subject polymer dyes can be used are reviewed. Systems and kits that can be used to practice the methods disclosed herein are also described.

[0064] Polymer Dyes

[0065] As outlined above, the present disclosure provides water-solvated polymer dyes having deep ultraviolet excitation spectra and / or deep UV absorption maxima. Deep ultraviolet (deep UV or DUV) is the region of the electromagnetic spectrum from about 200 nm to about 350 nm, e.g., 250 nm to 350 nm. Deep UV includes ultraviolet B (UVB) and ultraviolet C (UVC) radiation, wherein the wavelength of UVB radiation is in the range of 290 to 320 nm, and the wavelength of UVC radiation is in the range of 200 nm to 290 nm.

[0066] The term "deep ultraviolet excitation spectrum" may refer to an absorption spectrum of a polymer dye whose full width at half maximum (FWHM) limits the wavelength range to the deep UV region of the electromagnetic spectrum. Likewise, the absorption spectrum of such polymer dyes is located in the deep UV region. Figure 7 Normalized absorption spectra of several exemplary polymeric dyes whose FWHMs limit the wavelength to the deep UV region are shown.The subject polymeric dyes can have strong absorption capabilities at wavelengths ranging from 250 nm to 350 nm.

[0067] The term "deep UV absorption maximum" refers to a maximum absorption wavelength of a polymeric dye that is in the deep UV region of the electromagnetic spectrum, such as a maximum absorption wavelength of 350 nm or less, such as a maximum absorption wavelength in the range of 200 nm to 350 nm, such as 200 nm to 250 nm, 250 nm to 290 nm, 290 to 320 nm, or 320 nm to 350 nm. In some embodiments, the "deep UV absorption maximum" is a maximum absorption wavelength of 340 nm or less, such as 330 nm or less, 320 nm or less, 310 nm or less, 300 nm or less, 290 nm or less, 280 nm or less, 270 nm or less, or even less. Figure 7 Shown are normalized absorption spectra of several exemplary polymeric dyes having deep UV absorption maxima of 350 nm or less and deep UV excitation spectra with FWHM in the range of 200 nm to 350 nm.

[0068] The subject water-solvated polymer dye can be used for various multiple applications in which a group of fluorescent dyes can be independently excited to provide emission for high parameter analysis. The polymer dye can be used directly as a fluorescent marker, or coupled with a small molecule receptor signal chromophore in a tandem dye to provide various emission wavelengths within the range of 320 to 1200nm. In multicolor applications, multiple excitation sources can be used to achieve multiplicity and a large number of parameters. In addition to the narrow peak width, minimizing the absorption at other common excitation sources is also important for experiments with higher parameters. The cross-source excitation degree of the subject polymer dye can be adjusted by selecting a specific structure, comonomer, and a mixture of coblocks.

[0069] The subject polymer dye comprises a light-gathering multichromophore of a segment with one or more π-conjugated comonomers and one or more conjugated modified repeating units. The light-gathering multichromophore of the subject polymer dye can provide a desired absorption spectrum in the deep UV range, and has no significant absorption at longer wavelengths. By incorporating a conjugated modified repeating unit in the main chain (or backbone) of the polymer dye, so as to connect adjacent π-conjugated segments by one or more covalent bonds, thereby achieving a desired deep UV absorption spectrum, the conjugated modified repeating unit can prevent the p-orbital overlap between the one or more covalent bonds or reduce the p-orbital overlap degree between the one or more covalent bonds (thereby limiting π-conjugation). The conjugated modified repeating unit can regulate (e.g., reduce) extended π-conjugation, otherwise there will be such extended π-conjugation along the polymer dye backbone of the conjugated comonomer lacking such repeating units. If there is no such conjugated modified repeating unit, the polymer dye consisting only of the remaining π-conjugated comonomers will have extended electronic delocalization and a narrow band gap. The conjugated modified repeating units of the subject polymer dyes directly connect and regulate the conjugation between adjacent segments of the pi-conjugated comonomer to provide the desired effective conjugation length. By limiting the effective conjugation length of the resulting light-collecting multichromophore, a wider electronic band gap can be obtained to provide an absorption spectrum of short wavelength light including in the deep UV range. Likewise, the subject polymer dyes expand the spectral space into the deep UV region within the spectral coverage provided by conventional dyes and instruments.

[0070] Conjugated modified repeating unit

[0071] As used herein, the term "conjugated modified repeat unit" refers to a divalent unit of a polymer backbone that is connected to adjacent segments of a pi-conjugated comonomer and includes one or more connecting covalent bonds within the unit, wherein the one or more connecting covalent bonds prevent or reduce p orbital overlap between one or more covalent bonds, thereby limiting the electronic delocalization between two adjacent pi-conjugated segments. In some cases, the connecting covalent bond can be defined by a divalent atom, group, or joint that is directly covalently connected to an adjacent unit of the polymer backbone. In some cases, the connecting covalent bond is defined by a conjugated modified repeat unit that includes two unsaturated groups (e.g., aryl, heteroaryl, or alkenyl) separated by a stereo-strained covalent single bond in the repeat unit.

[0072] The conjugated modified repeating unit can limit the electronic delocalization along the polymer backbone, and therefore limit the effective conjugation length. In some cases, the conjugated modified repeating unit incorporates atoms, functional groups or joints (Z) in the backbone of the polymer dye to form saturated or partially saturated covalent bonds that limit or prevent the pi-conjugation in those covalent bonds. In some cases, the conjugated modified repeating unit includes a strained covalent single bond (Z) that connects two aryl or heteroaryl groups but limits the p-orbital overlap in the bond. This covalent single bond can be due to the incorporation of adjacent sterically hindered substituents on aryl or heteroaryl groups, causing two aryl or heteroaryl groups to be distorted outside the plane and produce strain.

[0073] The subject polymer dye can include multiple π-conjugated segments of comonomers connected via conjugated modified repeating units.π-conjugated segments can include single comonomers, two or more comonomers or a greater number of comonomers.In some examples, the effective conjugated length of π-conjugated segments is 30 or less, for example 20 or less, 18 or less, 16 or less, 14 or less, 12 or less, 10 or less, 8 or less, 6 or less or 5 or less.Any convenient arrangement of π-conjugated segments and conjugated modified repeating units can be used to provide required effective conjugated length.In some cases, polymer dye is the alternating copolymer of specific π-conjugated segments and conjugated modified repeating units.In some cases, polymer dye is the random copolymer of comonomers, and wherein conjugated modified repeating units are incorporated in polymer with certain frequency or molar concentration %, to obtain the average π-conjugated segment size providing required effective conjugated length in polymer.

[0074] The conjugated modified repeat unit may comprise two aryl or heteroaryl groups covalently linked to each other via a conjugated modified atom, group or linker (Z) which prevents or limits conjugation between the two aryl or heteroaryl groups, for example, 1 -Z-Ar 2 -unit, where Ar 1 and Ar 2 are two aryl or heteroaryl groups, and Z is a conjugated modified atom, group or linker (Z). The two aryl or heteroaryl groups (e.g., Ar 1 and Ar 2 ) can be 1,4-linked or 1,3-linked to the polymeric dye backbone and can be unsubstituted or further substituted, for example, with a water solubilizing group (e.g., as described herein). The two aryl or heteroaryl groups (e.g., Ar 1 and Ar 2 ) can be monocyclic or bicyclic. In some cases, the two aryl or heteroaryl groups (e.g., Ar 1 and Ar 2) is independently selected from 1,4-phenyl, 1,3-phenyl, 2,5-pyridyl, 2,6-pyridyl and 3,5-pyridyl, which are optionally further substituted. In some cases, the conjugated modified repeat unit has the formula -Ar 1 -Z-Ar 2 -, where Ar 1 and Ar 2 Each is independently an optionally further substituted 1,4-phenyl or 2,5-pyridyl. In some examples, the conjugated modified repeating unit has a structure of formula (XI):

[0075]

[0076] in:

[0077] Z is selected from the group consisting of a covalent bond, a saturated atom, a saturated group, and a functional group, provided that when Z is a covalent bond, R 22 , R 23 , R 25 and R 28 At least two (e.g., 2, 3, or 4) of them are not H;

[0078] R 21 -R 28 are independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro or bromo), sulfonate / ester, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide and water solubilizing groups, and wherein R 22 , R 23 , R 25 and R 28 One or more of are optionally cyclically linked to Z to provide a benzo-fused carbocyclic or heterocyclic ring; and

[0079] Each * is a site of covalent attachment to the polymer dye backbone. In some examples of Formula (XI), when Z is a covalent bond, R 22 , R 23 , R 25 and R 28 None of them is H, so that the p orbital overlap on Z is reduced due to steric strain. Therefore, z can be a covalent single bond for steric strain in the conjugated modified repeating unit of formula (XI).

[0080] In certain embodiments of formula (XI), Z is selected from a saturated carbon atom, a saturated heteroatom, a functional group, and a saturated linker. The saturated carbon atom (Z) of formula (XI) can be covalently bonded to the adjacent phenyl group via two monosaturated covalent bonds, thereby preventing or limiting the orbital overlap between the two benzene rings. 5)(R 6 )-, where R 5 and R 6 are independently H, alkyl or substituted alkyl. Similarly, the saturated heteroatom (Z) of formula (XI) refers to a heteroatom such as oxygen, sulfur, nitrogen or silicon, which is covalently bonded to the adjacent phenyl group through two monosaturated covalent bonds, thereby preventing or limiting the orbital overlap between the two benzene rings. In some examples, Z is O, S or NR' or -Si(R 5 )(R 6 )-, where R', R 5 and R 6 Z is independently H, alkyl or substituted alkyl. Similarly, a saturated linker can provide a backbone of three or more saturated covalent bonds between two adjacent phenyl rings of formula (XI). Any convenient linker can be used for the repeating unit of formula (XI). Linkers of interest used as Z in formula (XI) include, but are not limited to, alkyl linkers such as C2-C 12 Substituted or unsubstituted alkyl linker or C2-C6 substituted or unsubstituted alkyl linker, polyethylene glycol linker (e.g. -O-(CH2CH2O) n -, wherein n is 1-6, such as 1, 2, 3, 4, 5 or 6), or a modified polyethylene glycol (PEG) linker (e.g., a substituted PEG or a terminally modified PEG, such as -Z--(CH2CH2O) n -).

[0081] The functional group (Z) of formula (XI) may refer to a heteroatom-containing atomic group with characteristic properties, which provides a link between two phenyl groups of formula (XI), but prevents or limits conjugation between phenyl groups. Functional groups of interest for conjugated modified repeating units include, but are not limited to, oxygen-, sulfur- and / or nitrogen-containing functional groups, such as sulfonyl, keto, amide, ester, carbonate, carbamate or urea. Exemplary functional groups include -S(O)-, -SO2-, -SO2NR'-, -S(O)O-, -C(O)-, -C(O)NR'-, -C(O)O-, -OC(O)NR'-, -OC(O)O-, -NR'C(O)NR'-, wherein R' is H, alkyl or substituted alkyl. Exemplary conjugated modified repeating units of interest include, but are not limited to:

[0082]

[0083] In some embodiments of Formula (XI), Z is a covalent bond, R 22 and R 23 At least one of them is not H, and R 25 and R 28 At least one of is not H. In some cases, R 22 , R 23, R 25 and R 28 In some examples, three of R 22 , R 23 , R 25 and R 28 are not H. In some examples, R 22 , R 23 , R 25 and R 28 In some examples, two or more of R 23 and R 25 In this case, by 22 , R 23 , R 25 and / or R 28 The substituents are selected to induce torsional strain in the Z bond of the conjugated modified repeating unit, thereby reducing the planarity and orbital overlap between two adjacent benzene rings. In such cases, the conjugated modified repeating unit can regulate (e.g., limit) the electron delocalization between the pendant π-conjugated segments or comonomers conjugated to the two 1,4-benzene rings of formula (XI).

[0084] In some embodiments of Formula (XI), Z is a covalent bond, and the conjugated modified repeating unit comprises the following structure:

[0085]

[0086] in:

[0087] R 1 To R 4 are independently selected from hydrogen, alkyl, substituted alkyl, halogen (e.g., fluoro), cyano,

[0088] Where X is O, S, CONH or NH, provided that R 1 and R 2 Not hydrogen; each R 5 independently -(CH2CH2O) p R', wherein p is an integer from 0 to 50, R' is H, alkyl or substituted alkyl; and m is an integer from 0 to 10. In certain examples of formula (XIII), R 1 To R 4 At least two of the following are selected from:

[0089] Where X is O, S, CONH or NH, and each R 5 independently -(CH2CH2O) pR', wherein p is an integer from 0 to 50, and R' is H, alkyl or substituted alkyl. In certain examples of formula (XIII), R 1 and R 2 Selected from:

[0090] Where X is O, S, CONH or NH, and each R 5 independently -(CH2CH2O) p R', wherein each p is independently an integer from 0 to 50 (eg, 6-50, 6-40, 6-30, 8-30, 10-30, or 10-20, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and R' is H, alkyl, or substituted alkyl.

[0091] Exemplary conjugated modified repeating units of interest include, but are not limited to:

[0092]

[0093] Each R 5 independently -(CH2CH2O) p R', wherein p is an integer from 0 to 50 (e.g., from 0 to 30, from 0 to 20, or from 10 to 20), and R' is H, lower alkyl, or substituted lower alkyl. 5 In the water-soluble polymer, p is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; and R' is methyl. In certain cases, each p is 10, 11 or 12.

[0094] In some examples of Formula (XI), Z is C(R 5 )(R 6 )-, where R 5 and R 6 Each is cyclically linked to an adjacent phenyl ring of formula (XI), for example, to R 22 or R 23 Position ring connection, or with R 25 or R 28 In some embodiments of Formula (XI), Z is -C(R 5 )(R 6 )-, where R 5 With R 22 The ring is connected to provide a partially saturated carbocyclic ring (eg, a 5- or 6-membered carbocyclic ring) which is optionally further substituted, and R 6 With R 28The cyclic connection provides a partially saturated carbocyclic ring (e.g., a 5- or 6-membered carbocyclic ring) that is optionally further substituted. In certain embodiments of formula (XI), the conjugated modified repeating unit has a structure of formula (XII):

[0095]

[0096] in:

[0097] n 1 、n 2 、m 1 and m 2 independently 0 to 3, wherein n 1 +m 1 and n 2 +m 2 each independently 2 to 3;

[0098] R 16 and R 17 Each is independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro or bromo), sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide and a water solubilizing group; and

[0099] Each * is a site for covalent attachment to the polymer dye backbone. In certain examples of formula (XII), n 1 +m 1 and n 2 +m 2 In some examples of formula (XII), n 1 +m 1 and n 2 +m 2 In certain examples of formula (XII), each R 16 and R 17 are H. In certain examples of formula (XII), m 2 and m 2 are all 1, and R 16 and R 17 At least one of them is not H. In certain examples of formula (XII), m 2 and m 2 are all 1, and R 16 and R 17 At least one or two of the groups are water solubilizing groups.

[0100] Exemplary conjugated modified repeating units of interest include, but are not limited to:

[0101]

[0102] As used herein, the terms "light-collecting multichromophore," "polymer dye," and "conjugated polymer" are used interchangeably and refer to a conjugated polymer having a structure capable of capturing light having a specific absorption maximum wavelength and converting it into emitted light at a longer emission maximum wavelength. In some cases, the light-collecting 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 to an acceptor through Forster energy transfer.

[0103] 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 as the minimum number of different structural features required to be considered as a unit of 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, such as when the polymer is a multi-block polymer or a random arrangement of each unit, each block may define different repeating units, such as n-blocks and m-blocks. It should be understood that various 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 coblocks of n and m different 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 mol% value of each unit in the 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 examples, the repeating unit of the 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 the structural unit of a polymer, which itself can be a part of the repeating unit of a polymer. In some embodiments, the 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 co-blocks, wherein each co-block can be composed of co-monomers such as one, two, three or more co-monomers.

[0104] The multichromophore can have any suitable length. In some cases, the specific number of monomer repeat units or segments of the multichromophore can fall within the range of 2 to 500,000, such as 2 to 100,000, 2 to 30,000, 2 to 10,000, 2 to 3,000, or 2 to 1,000 units or segments, or such as 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 fall within the range of 2 to 1,000, such as 2 to 500, 2 to 100, 3 to 100, 4 to 100, 5 to 100, 6 to 100, 7 to 100, 8 to 100, 9 to 100, or 10 to 100 units or segments.

[0105] The polychromophore can have any suitable molecular weight (MW). In some cases, the MW of the polychromophore can be expressed as an average molecular weight. In some examples, the polymeric dye has an average molecular weight in the range of 500 to 500,000, such as 1,000 to 100,000, 2,000 to 100,000, 10,000 to 100,000, or even in the range of 50,000 to 100,000.

[0106] In some embodiments, the multichromophore includes a particular comonomer (e.g., an aromatic or heteroaryl comonomer) that comprises 5% or more (e.g., 5 mol%) on a molar basis in the multichromophore, 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 an even higher percentage on a molar basis in the multichromophore. In such cases, the polychromophore 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 higher repeating units. In such cases, the multichromophore can 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 higher comonomer units. In certain embodiments, the comonomer of interest (e.g., an aromatic or heteroaryl comonomer) comprises 25% or more on a molar basis in a polychromophore, such as 30% or more, 40% or more, 45% or more, 50% or more, or an even higher percentage on a molar basis in a polychromophore 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.

[0107] In some embodiments, the multichromophore includes a plurality of first optically active units forming a conjugated system or conjugated segment system (e.g., as described herein) having an absorption wavelength at which the first optically active unit absorbs light to form an excited state (e.g., as described herein). The subject multichromophore may have one or more desired spectral properties, such as a specific deep UV absorption spectrum or deep UV maximum wavelength (e.g., as described herein), a specific emission maximum wavelength, an extinction coefficient, a quantum yield, etc. The subject polymer dyes and polymer tandem dyes provide deep UV absorption spectra and a variety of emission spectra, depending on a variety of factors, such as the selected comonomers, linking groups, substituents, and optionally linked signal chromophores that make up the polymer.

[0108] In some embodiments, the multichromophore itself has an emission maximum wavelength in the range of 300 to 900 nm, e.g., 300 to 850 nm, 300 to 600 nm, 300 to 500 nm, 300 to 500 nm, 300 to 500 nm, 350 to 500 nm, or 400 to 500 nm, where specific examples of emission maxima of interest include, but are not limited to, 340 nm ± 5 nm, 350 nm ± 5 nm, 360 nm ± 5 nm, 370 nm ± 5 nm, 380 nm ± 5 nm, 390 nm ± 5 nm, 400 nm ± 5 nm, 410 nm ± 5 nm, 420 nm ± 5 nm, or 430 nm ± 5 nm. In some examples, the multi-chromophore is linked to the receptor signal chromophore, and the polymer tandem dye has an emission maximum wavelength selected from the group consisting of: 395nm±5nm, 460nm±5nm, 490nm±5nm, 550nm±5nm, 560nm±5nm, 605nm±5nm, 650nm±5nm, 680nm±5nm, 700nm±5nm, and 805nm±5nm. In some examples, the polymer tandem dye has an emission maximum wavelength in the range of 375 to 900nm (e.g., in the range of 380nm to 900nm, 390nm to 900nm, or 400nm to 900nm).

[0109] In some examples, the multichromophore has 5 x 10 5 cm -1 M -1 or larger, such as 6x 10 5 cm -1 M -1 or larger, 7x10 5 cm -1 M -1 or larger, 8x 10 5 cm -1 M -1 or larger, 9x 10 5 cm -1 M -1 or larger, such as 1x 10 6 cm -1 M -1 or larger, 1.5x 10 6 cm -1 M -1 or larger, 2x 10 6 cm -1 M -1 or larger, 2.5x 10 6 cm -1 M -1 or larger, 3x 10 6 cm-1 M -1 or larger, 4x 10 6 cm -1 M -1 or larger, 5x 10 6 cm -1 M -1 or larger, 6x 10 6 cm -1 M -1 or larger, 7x 10 6 cm -1 M -1 , or larger or 8x10 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, 50 or more, 100 or more, or even more repeating units. In some embodiments, the multichromophore has 5 x 10 5 M -1 cm -1 In certain embodiments, the multichromophore has a molar extinction coefficient of 1 x 10 6 M -1 cm -1 or greater molar extinction coefficient.

[0110] In some examples, the multichromophore has a wavelength of 40,000 cm -1 M -1 / repeat unit or greater extinction coefficient, e.g. 45,000 cm -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 / Repeating unit or larger, 90,000cm -1 M -1 / Repeat unit or larger, 100,000cm -1 M -1 / repeat unit or greater, or even greater extinction coefficient. In some examples, the 40,000 cm-1 M -1 / repeating 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 multichromophore has a wavelength of 40,000 cm -1 M -1 / comonomer or greater extinction coefficient, e.g. 45,000 cm -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 extinction coefficient. In some examples, 40,000 cm -1 M -1 / comonomer or greater is the average extinction coefficient.

[0111] π -Conjugated segment

[0112] As outlined above, the subject polymer dye includes a π-conjugated segment whose effective conjugation length is controlled by incorporating a conjugated modified repeat unit (e.g., as described herein) into the polymer. The terms "π-conjugated segment" and "segment of a π-conjugated comonomer" are used interchangeably herein and refer to the region of a pi-conjugated comonomer in a polymer backbone or on a conjugated branch of a polymer backbone. The terms "π-conjugated (π-conjugated)" and "pi-conjugated (pi-conjugated)" are used interchangeably herein. Any suitable arrangement of π-conjugated segments and conjugated modified repeat units can be used to provide the desired effective conjugation length and the resulting spectral characteristics. It should be understood that in some cases, the subject multi-chromophore can include a random arrangement of co-blocks (e.g., n and m co-blocks) or repeat units or co-monomers. It should be understood that the formula herein depicts a representation of a polymer dye, and the length of the chemical structure of the polymer and / or the specific arrangement of the co-monomer may vary, depending on the synthesis method. The subject multi-chromophore can include any suitable linear arrangement of n and m co-blocks of various lengths within the structure of the whole polymer. The subject multi-chromophore can include any suitable linear arrangement of comonomers within the structure of the whole polymer, depending on the specific synthesis and polymerization method used. In some cases, the linear arrangement of comonomers shown in the structure of this article is random. In addition, the multi-chromophore can include any suitable arrangement of comonomers within such n and / or m co-blocks, including random, alternating or block arrangements of comonomers. Unless otherwise indicated, all possible arrangements of comonomers are intended to be included in the polymer dyes described herein. In the preparation of the subject multi-chromophore, various polymer synthesis methods can be used to prepare comonomers and co-blocks of interest. It should be understood that in some cases, the polymerization method can produce a composition including a conjugated polymer population, and the composition includes some changes in the specific length and / or end group (i.e., end group) present in each conjugated polymer of the population. The formula shown herein can refer to a population or subpopulation of a single compound or a polymerized compound. As used herein, * indicates a site of covalent attachment to the unsaturated backbone of a conjugated polymer. It is understood that the formula shown herein for a subject polymer is one representation of the structure and that other representations, such as those indicating the mol% ratio of a particular comonomer in a conjugated polymer, may also be used.

[0113] The π-conjugated segments of the subject polymer dye can be composed of any suitable comonomers capable of π-conjugation, such as aryl or heteroaryl comonomers, alkynyl comonomers (e.g., ethynyl) or segments and alkenyl comonomers (e.g., vinylidene) or segments. The aryl or heteroaryl comonomers of interest that can be used in the π-conjugated segments include, but are not limited to, phenyl comonomers, biphenyl comonomers, benzoxazole comonomers, benzothiazole comonomers, polyphenylene comonomers and fused tricyclic comonomers such as fluorene comonomers, carbazole comonomers, silole (silole) comonomers and bridged biphenyl comonomers. Aryl or heteroaryl comonomers can be optionally further substituted, for example, as described herein.

[0114] Various comonomers that can be used in the π-conjugated segments of the subject polymeric dyes are now described in more detail.

[0115] In some embodiments, the polymeric dye comprises a plurality of π-conjugated segments, each π-conjugated segment comprising one or more comonomers selected from the following structures (a)-(k):

[0116]

[0117] in:

[0118] X is O or S;

[0119] n is 1-6 (e.g., 1, 2 or 3);

[0120] Y 1 Selected from -C(R 11 )2-、-Si(R 11 )2-、-NR 11 -、-C(R 11 )2C(R 11 )2- and -C(R 11 )2C(R 11 )2-;

[0121] R 11 is one or more optional substituents each independently selected from hydrogen, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro or bromo), sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide, and a water-solubilizing group; and

[0122] Each * is a site for covalent attachment to the polymer backbone.

[0123] In certain cases of structures (a)-(k), each R 11 are independently selected from H, F, methyl, trifluoromethyl, cyano,

[0124]

[0125] in:

[0126] X is O or S;

[0127] m is an integer from 0 to 10; and

[0128] R 5 -(CH2CH2O) p R', wherein R' is H, alkyl or substituted alkyl (eg, methyl), and p is an integer from 0 to 40.

[0129] In some embodiments, the polymeric dye comprises a plurality of π-conjugated segments, each π-conjugated segment comprising one or more comonomers selected from alkynyl comonomers (e.g., ethynylene) and alkenyl comonomers (e.g., vinylene) of structures (j) to (k):

[0130]

[0131] Where R 11 is selected from hydrogen, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro or bromo), sulfonate / ester, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide, and a water-solubilizing group (e.g., as described herein).

[0132] Aromatic or heteroaromatic comonomers

[0133] As outlined above, the subject polymeric dye comprises the conjugated segments of the aryl or heteroaryl comonomers connected via covalent bonds, vinylidene or ethynylene groups. In addition to the comonomers described in the above exemplary formula, a variety of aryl and heteroaryl comonomers can also 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.

[0134] 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 comonomer is an optionally substituted aryl or heteroaryl comonomer selected from one of the following structures:

[0135]

[0136] 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 them are N. In some examples, 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 examples, R 15 and R 16 Each is H. In some cases, the halogen is fluoro.

[0137] In some cases, the aryl or heteroaryl comonomer (eg, structure (a)) is selected from one of the following:

[0138]

[0139] Where R 11 and R 12 independently hydrogen, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro or bromo), sulfonate / ester, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide, and water solubilizing groups. In some cases, the aryl or heteroaryl comonomer is selected from one of the following:

[0140]

[0141] wherein n is 1 to 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 from 12 to 20 or from 12 to 16 or from 16 to 20.

[0142] Also provided are substituted phenylene comonomers substituted with further aryl or heteroaryl groups, which are themselves optionally further substituted. The aryl or heteroaryl substituted phenylene comonomers can provide extended pi-conjugation into the side chain groups of the comonomer. In some cases, the comonomer is a heteroaryl substituted phenylene comonomer. Selecting specific aryl and heteroaryl substituents as extensions of the conjugated side chain groups from 1,4-phenylene comonomers or 1,3-phenylene comonomers can fine-tune the spectral properties of the subject deep UV absorbing polymeric dyes. In some embodiments, the substituted phenylene comonomers are selected from the following structures:

[0143]

[0144] in

[0145] X is O or S;

[0146] Z 1 and Z 2 Independently selected from CR 12 and N; and

[0147] Each R 11 To R 12 independently hydrogen, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro or bromo), sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide, and a water-solubilizing group.

[0148] In some cases, the aryl or heteroaryl comonomer has structure (h):

[0149]

[0150] Where X is O or S; and R 11 is one or more substituents, each independently selected from hydrogen, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro or bromo), sulfonate / ester, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide and water solubilizing groups. In some cases, the aryl or heteroaryl comonomer of structure (h) is selected from the following comonomers:

[0151]

[0152] In one example, the aromatic or heteroaromatic comonomer is a substituted or unsubstituted polyphenylene comonomer such as the above structure (g). In some examples, the comonomer of structure (g) is selected from:

[0153]

[0154] Where R 11 is one or more optional substituents, each independently selected from hydrogen, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro or bromo), sulfonate / ester, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide and water-solubilizing groups; and each * is a site for covalent attachment to the polymer backbone.

[0155] In certain examples of aryl or heteroaryl comonomers described herein, each R 11 To R 14 are independently selected from H, F, methyl, trifluoromethyl, cyano,

[0156]

[0157] in:

[0158] X is O or S;

[0159] m is an integer from 0 to 10; and

[0160] R 5 -(CH2CH2O) p R', wherein R' is H, alkyl or substituted alkyl (eg, methyl), and p is an integer from 0 to 40.

[0161] In some embodiments, the multichromophore includes a substituted aryl or heteroaryl comonomer described by one of the following structures:

[0162]

[0163]

[0164]

[0165]

[0166] In some embodiments, the multichromophore includes a substituted aromatic comonomer described by the following structure:

[0167]

[0168] wherein n is 1 to 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:

[0169]

[0170] 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:

[0171]

[0172] In some embodiments, the multichromophore includes a substituted aromatic comonomer described by the following structure:

[0173]

[0174] In some embodiments of any of the formulae described herein (e.g., formulae (I)-(V)), the multichromophore includes a substituted aromatic comonomer (e.g., M1-M3) having the following structure:

[0175]

[0176] wherein s is 1-50 (e.g., 6-50, 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:

[0177]

[0178] In some embodiments of the formulae described herein (eg, formulae (I)-(IV)), an aryl or heteroaryl comonomer, such as L 1 , L 2 、M 1 、M 2 and M 3 Independently selected from one of formula (XXIII)-(XXVI):

[0179]

[0180]

[0181] in

[0182] Cy 2-7 refers to an aryl or heteroaryl group containing 2 to 7 fused and / or non-fused rings;

[0183] 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;

[0184] Each R 3 is one or more ring substituents 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 ;

[0185] 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;

[0186] Y 5 N or CR 5 , and Y 7 N or CR 7 ;

[0187] 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 ;

[0188] Z 1 is a chemoselective functional group or an attached signaling chromophore; and

[0189] T 1 It's a connector.

[0190] Cy 2-7 is an aryl or heteroaryl group which may include 2, 3, 4, 5, 6, or 7 rings which may be fused together to form a fused ring system or may not be fused (i.e., linked together by covalent single bonds). 2-7Includes 2 or 3 unfused aryl and / or heteroaryl rings. In certain instances, 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 aryl or heteroaryl group, such as naphthalene, anthracene, acridine or quinoline.

[0191] 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):

[0192]

[0193]

[0194]

[0195] in:

[0196] 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;

[0197] 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 ;

[0198] 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 substituted with a 5-membered or 6-membered fused aryl, heteroaryl, cycloalkyl or heterocyclic ring;

[0199] Z1 is a chemoselective functional group or an attached signaling chromophore; and

[0200] T 1 It's a connector.

[0201] In some cases, Y 8 C(R 3 )2. In some cases, Y 8 -C(R 3 )2C(R 3 )2-. In some cases, Y 8 NR 3 In some cases, 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 formula (a)-(x), the comonomer includes a water solubilizing group (e.g., as described herein). In certain embodiments of formula (s), R 3 is H, halogen (e.g., F), or alkoxy (e.g., methoxy). It should be understood that in any of the structures (a)-(x) described herein, N heteroatoms 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 formula (s), wherein the benzo ring is replaced by a pyrido ring:

[0202]

[0203] where Y 8 Independently C(R 4 )2. -C(R 4 )2C(R 4 )2-、NR 4 or Si(R 4 )2.

[0204] In some examples of formulas (XXIII)-(XXVI) and (a)-(x), the comonomer (M 2 、M 3 and M 4 ) is independently selected from one of the following structures (ba) to (cd):

[0205]

[0206]

[0207]

[0208] in:

[0209] Y 9 C(R 4 )2. -C(R 4 )2C(R 4 )2- or Si(R 4 )2;

[0210] X is S or O;

[0211] 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

[0212] 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 an optionally substituted 5-membered or 6-membered fused aryl or heteroaryl ring; in certain embodiments of formula (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).

[0213] Fused tricyclic comonomer

[0214] The aryl and heteroaryl comonomers of interest include fused tricyclic comonomers. A fused tricyclic comonomer is a comonomer comprising a tricyclic aromatic group having three fused rings, the three fused rings being in a configuration in which two aryl or heteroaryl 6-rings are fused to a central 5- or 6-membered carbocyclic or heterocyclic ring. In some cases, a fused tricyclic comonomer comprises two benzo or pyrido rings fused to a central 5- or 6-membered carbocyclic or heterocyclic ring. A fused tricyclic comonomer can be pi-conjugated with an adjacent comonomer of the polymer backbone through any suitable ring atom of the fused ring. The central 5- or 6-membered ring can be an aromatic or partially saturated carbocyclic or heterocyclic ring, and can further include a side chain substituent, such as WSG and / or a linker for 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 through a central 6-membered carbocyclic or heterocyclic ring. In certain examples, the fused tricyclic comonomer is described by the following structure:

[0215]

[0216] in:

[0217] 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;

[0218] Each Z is independently CH, CR or N;

[0219] 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 connector and Z 1 is a chemoselective tag (e.g., a tag comprising a chemoselective functional group) and WSG; and

[0220] 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 / ester, alkoxy, substituted alkoxy, alkyl and substituted alkyl, and any two suitable R groups are optionally cyclically connected. In some examples, at least two of the Z in each ring are CH or CR. In some examples, one of the Z in each ring and only one is N.

[0221] In certain examples, the fused tricyclic comonomer is described by one of the following structures:

[0222]

[0223] in:

[0224] 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;

[0225] 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 connector and Z 1 is a chemoselective tag (e.g., a tag comprising a chemoselective functional group) and WSG; and

[0226] Each R is independently H, R 3Or 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 / ester, alkoxy, substituted alkoxy, alkyl and substituted alkyl, and any two suitable R groups are optionally cyclically connected. The symbol "*" represents the site of covalent connection to the unsaturated backbone of the conjugated polymer, such as π conjugated segments, end groups, joints and connected specific binding members. It should be understood that for any formula including 2,7-pi-conjugated fused tricyclic comonomers described herein, similar formulas including similar 3,6-pi-conjugated comonomers can also be shown, and are intended to be covered by the present disclosure. 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-or-C(R 3 )2Si(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, fluoro, chloro, methoxy, substituted alkoxy, alkyl, and substituted alkyl.

[0227] In certain embodiments of the fused tricyclic comonomer, the comonomer includes two R substituents cyclically linked to provide a carbocyclic or heterocyclic ring A which is optionally further substituted:

[0228]

[0229] 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 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 connector and Z 1 is a chemoselective tag (e.g., a tag comprising a chemoselective functional group) and WSG. In some cases, the fused tricyclic comonomer has the following structure:

[0230]

[0231] 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 connector 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.

[0232] In certain examples, the fused tricyclic comonomer is described by one of the following structures:

[0233]

[0234] in:

[0235] 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 connector and Z 1 is a chemoselective tag (e.g., a tag comprising a chemoselective functional group) and WSG (e.g., as described herein); and

[0236] Each R is independently H, R 3or 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 .

[0237] In certain examples, the fused tricyclic comonomer is described by the following structure:

[0238]

[0239] 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 tricyclic comonomer is described by the following structure:

[0240]

[0241] Where T 5 is an optional linker; 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.

[0242] A fused 6-5-6 tricyclic comonomer is a comonomer comprising a tricyclic aromatic group having three fused rings in a 6-5-6 configuration, i.e., two benzo rings fused to a central 5-membered ring. The 5-membered ring may be a carbocyclic or heterocyclic ring and may further include side chain substituents on the ring atoms not fused to the benzo ring. In certain examples, a fused 6-5-6 tricyclic comonomer is described by the following structure:

[0243]

[0244] 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 1 independently selected from a branched nonionic water-soluble group (WSG), an alkyl group, a substituted alkyl group, an aralkyl group, a substituted aralkyl group, a PEG moiety, and a -L 11 -Z 1 , where L 11 is a connector and Z 1is a chemoselective tag (e.g., a tag comprising a chemoselective functional group) or WSG. As used in any formula described herein, * represents a site of covalent attachment to an unsaturated backbone or end 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 used 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 used in the subject multichromophore. In certain examples of fused 6-5-6 tricyclic comonomers, 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 It should be understood that for any fluorene comonomer described herein, the corresponding comonomer in which the C atom of Z is replaced by Si is also included in the present disclosure.

[0245] 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 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 adjacent polymer backbone groups through positions 2 and 7. In certain embodiments, the fluorene comonomer is described by the following structure:

[0246]

[0247] Among them: Each R 1 independently selected from a branched nonionic water-soluble group (WSG), an alkyl group, a substituted alkyl group, an aralkyl group, a substituted aralkyl group, a PEG moiety, and a -L 11 -Z 1 , where L 11 is a connector and Z 1 is a chemoselective tag (eg, a tag comprising a chemoselective functional group) or WSG. In certain examples of fluorene comonomers, each R 1 is a branched nonionic water-soluble group (WSG). In certain examples of fluorene comonomers, each R 1 is selected from benzyl substituted with one, two or more PEG moieties or alkyl substituted with two or more PEG moieties. 1Functional 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. 1 Includes a carboxylic acid group or a derivative thereof for covalent attachment to an acceptor chromophore. In certain embodiments, L 11 is connected to two or more Z 1 In some examples, the fluorene comonomer is further provided with R at one, two or more positions 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).

[0248] In certain examples, the fluorene comonomer is described by the following structure:

[0249]

[0250] 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).

[0251] In some examples, the fluorene comonomer is described by the following structure:

[0252]

[0253] 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 fluorene comonomers, 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 fluorene comonomers, each R 2 is replaced by a -O(CH2CH2O) n R' groups (e.g., at the 2, 3, or 4 positions) are substituted benzyl, wherein R' is H or alkyl, and n is 1-20, e.g., 3-16, such as n is 8-16. In certain examples of fluorene comonomers, each R 2 It is formed by two -O (CH2CH2O) nR' groups (e.g., at the 2,4-position, 3,4-position, or 3,5-position) 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 It is composed of 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 group substituted with a trivalent branched group each substituted with two PEG moieties (e.g., -CO-NR"2 or -O(CH2R")2 trivalent branched group, 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 fluorene comonomers, each R 2 is a branched nonionic water-soluble group (WSG), for example, as described herein.

[0254] In certain embodiments, the fluorene comonomer is described by the following structure:

[0255]

[0256] 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 YesL 2 -Z 2 , where L 2 is a connector and Z 2 is a chemoselective tag (e.g., for conjugation to an acceptor chromophore). In some examples, the fluorene comonomer is described by the following structure:

[0257]

[0258] Where: R 3 is 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 connector and Z 2 is a chemoselective tag (eg, for conjugation to a receptor chromophore); and R5 and R 6 is independently selected from H, a water solubilizing group, and an aromatic substituent (e.g., an alkyl, substituted alkyl, alkoxy, substituted alkoxy, halogen, or nitro). In certain examples of fluorene comonomers, R 3 is a lower alkyl group, which is substituted with a trivalent branched group (e.g., -CO-NR"2 or -O(CH2R")2, each of which is substituted with two PEG moieties, 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)) substituted. In some examples of fluorene comonomers, R 3 is a branched nonionic water-soluble group (WSG), for example, as described herein.

[0259] 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 comprises one of the following structures as part of the polymer backbone:

[0260]

[0261] 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.

[0262] Any comonomer described herein (e.g., comonomers of formula (I)-(V), (a)-(k), (a)-(x), and (ba)-(cd)) can include a water solubilizing group WSG (e.g., as described herein). In some embodiments, the aromatic and heteroaromatic comonomers include a branched nonionic WSG selected from one of the following structures:

[0263]

[0264] Where: T 5is an optional linker 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.

[0265] 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 ):

[0266]

[0267] 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 instances, each s is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In some instances, each s is 3. In some instances, each s is 4. In some instances, each s is 5. In some instances, each s is 6. In some instances, each s is 7. In some instances, each s is 8. In some instances, each s is 9. In some instances, each s is 10. In some instances, each s is 11. 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 5is a substituted alkoxy group.

[0268] In certain embodiments of the formulae described herein (eg, formulae (I)-(IV)), the comonomer (eg, M 1 、M 2 and / or M 3 ) are independently selected from one of the following structures:

[0269]

[0270]

[0271]

[0272]

[0273] wherein each s is independently 1 to 30, such as 6 to 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.

[0274] In some examples, a multichromophore (eg, a multichromophore of Formula (I)-(IV)) includes a comonomer (eg, L 1 ):

[0275]

[0276] Wherein WSG is an optional water-soluble group; T 2 is a linker; and the dye is a signal chromophore.

[0277] 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(thiophene-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 in the subject multichromophore are selected from a'-m' having the following structures:

[0278]

[0279] in * = a site of covalent attachment to the unsaturated backbone, and each R is independently H, a nonionic side group capable of imparting water solubility (e.g., WSG), or -L 2 -Z 2 , where L 2 is a joint 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 certain 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 instances of a'-m', each R is H or nonionic branched WSG, for example, as described herein.

[0280] In some embodiments, the multichromophore includes an absorption-modified comonomer having the following structure:

[0281]

[0282] 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 42Each is H. In certain embodiments, X is S and R 41 and R 42 Each is H.

[0283] Any of the absorption-modifying comonomers described above may be used in the subject multichromophores, such as those of formulae (I)-(IV).

[0284] Water solubilizing group

[0285] The repeating unit of the subject polymer dye can be substituted by a water-solubilizing group at any suitable position. As used herein, the terms "water-solubilizing group", "water-soluble group" and WSG are used interchangeably, and refer to a group or substituent that can dissolve well in an aqueous environment, for example, under physiological conditions and impart improved water solubility to the molecule 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 can dissolve well in an aqueous environment.

[0286] The water-soluble polymer dyes disclosed herein have solubility under aqueous conditions, making them particularly suitable for application in various 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 dyes is undesirable because it can, for example, cause fluorescence quenching of the dye by aggregation, resulting in a reduction in the fluorescent signal. The subject water-soluble conjugated polymer dyes can be used as fluorescent reporters for a variety of biosensors and provide exceptionally bright signals with a variety of excitation and emission wavelength options for applications such as flow cytometry and imaging.

[0287] In some examples, the solubility of the water-soluble polymer dye in water or buffer is 10mg / mL or higher, for example 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 the water-soluble polymer dye can be configured to form discrete water-solvated nanoparticle in aqueous systems under certain conditions.In some cases, the water-solvated nanoparticle has resistance to gathering and can be used for various bioassays.

[0288] Any suitable water solubilizing group (WSG) may be included in the multichromophore described herein (e.g., the multichromophore of formula (I)-(IV)) to provide increased water solubility. Although the increase in solubility can vary, in some examples, it is increased by 2 times or more, such as 5 times, 10 times, 25 times, 50 times, 100 times or more (compared to compounds without WSG). In some cases, the hydrophilic water solubilizing group is charged, such as 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 examples, WSG is linear. In some embodiments, WSG is a hydrophilic polymer, such as polyethylene glycol, modified PEG, peptide sequence, peptidomimetic, carbohydrate, oxazoline, polyol, dendron, dendritic polyglycerol, cellulose, chitosan or its derivatives. 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, guanidinium, pyridinium, polyamines and sulfoniums, polyols, linear or cyclic sugars, primary, secondary, tertiary or quaternary amines and polyamines, phosphonate groups, phosphinate groups, ascorbate groups, glycols (including polyethers, -COOM', -SO3M', -PO3M', -NR3 + 、Y′、(CH2CH2O) p R and mixtures thereof, wherein Y′ can be any halogen, sulfate, sulfonate or oxyanion; p can be 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 group).

[0289] WSG can include water-soluble polymers. Water-soluble polymers of interest include, but are not limited to, polyalkylene oxide-based polymers, such as polyethylene glycol "PEG", (see, for example, "Poly (ethylene glycol) Chemistry: Biotechnical and Biomedical Applications", JM Harris, ed., Plenum Press, New York, NY (1992) and "Poly (ethylene glycol) Chemistry and Biological Applications", JM Harris and S. Zalipsky, Eds., ACS (1997); and international patent applications: WO 90 / 13540, WO 92 / 00748, WO 92 / 16555, WO 94 / 04193, WO 94 / 14758, WO 94 / 17039, WO 94 / 18247, WO 94 / 28937, WO 95 / 11924, WO 96 / 00080, WO 96 / 23794, WO 98 / 07713, WO 98 / 41562, WO 98 / 48837, WO 99 / 30727, WO 99 / 32134, WO 99 / 33483, WO 99 / 53951, WO 01 / 26692, WO 95 / 13312, WO 96 / 21469, WO 97 / 03106, WO 99 / 45964 and U.S. Patent Nos. 4,179,337; 5,075,046; 5,089,261; 5,100,992; 5,134,192; 5,166,309; 5,171,264; 5,213,891; 5,219,564; 5,275,838; 5,281,698; 5,298,643; 5,312,808; 5,321,095; 5,324,844; 5,349,001; 5,352,756; 5,405,877; 5,455,027; 5,446,090; 5,470,829; 5,478,805; 5,567,422; 5,605,976; 5,612,460; 5,614,549; 5,618,528; 5,672,662; 5,637,749; 5,643,575; 5,650,388; 5,681,567; 5,686,110; 5,730,990; 5,739,208; 5,756,593; 5,808,096; 5,824,778; 5,824,784; 5,840,900; 5,874,500; 5,880,131; 5,900,461; 5,902,588; 5,919,442; 5,919,455; 5,932,462; 5,965,119; 5,965,566; 5,985,263; 5,990,237; 6,011,042; 6,013,283; 6,077,939; 6,113,906; 6,127,355; 6,177,087; 6,180,095; 6,194,580; 6,214,966).

[0290] Examples of water-soluble polymers of interest include, but are not limited to, those containing polyalkylene oxides, polyamide alkylene oxides, or derivatives thereof (including polyalkylene oxides and polyamide alkylene oxides containing ethylene oxide repeating units of the formula -(CH2-CH2-O)-). Further examples of polymers of interest include those 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 which may be the same or different and may be branched or linear, and n is 2-100, for example, a discontinuous integer from 2 to 50, and wherein either or both of X and Y comprise biocompatible, substantially non-antigenic water-soluble repeating units which may be linear or branched. Further examples of water-soluble repeating units include ethylene oxide of the formula -(CH2-CH2-O)- or -(O-CH2-CH2)-. The number of such water soluble repeating units can vary significantly, with the number of such units being 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, 6 to 100, for example 2 to 50 or 6 to 50, such as 6 to 40, 6 to 30, 6 to 20, 8 to 20 or 10 to 20, for example 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. An example of an embodiment is one in which one or both of X and Y are selected from the following: -((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, and wherein n2 is 2 to 50, 2 to 25, 2 to 15, 2 to 10, 2 to 8, or 2 to 5. Further examples of embodiments are those 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)-. In certain instances, the comonomer comprises 2, 3 or 4 WSGs, each independently having the formula -(CH2) n1 -(CH2-CH2-O) n2 R', wherein R' is H, C1-6 alkyl or substituted C1-6 alkyl, n1 is 1 to 6, 1 to 5, 1 to 4 or 1 to 3, and n2 is 2 to 50, for example 6 to 40, 6 to 30, 6 to 20, 8 to 20 or 10 to 20, for example 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0291] As used herein, a modified polymer such as a modified PEG refers to a water-soluble polymer that has been modified or derivatized at one or both ends, e.g., to incorporate a terminal substituent (e.g., a terminal alkyl, substituted alkyl, alkoxy or substituted alkoxy, etc.) and / or a terminal connecting functional group (e.g., an amino or carboxylic acid group suitable for connecting via amide bond formation) suitable for connecting the polymer (e.g., via a branching group) to a multichromophore. The subject water-soluble polymer can be modified to include any suitable connecting group.

[0292] It should be understood that in some cases, the water-soluble polymers may include a certain dispersity related to the polymer length, depending on the preparation and / or purification method of the polymer starting material. In some examples, the subject water-soluble polymers are monodisperse. In some examples, the subject water-soluble polymers are substantially monodisperse, for example, containing 20 wt% or less of non-target substances, such as 15 wt% or less, 10 wt% or less, 5 wt% or less, 2 wt% or less or 1 wt% or less.

[0293] 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., and may contain, for example, up to 18 carbon atoms or even additional polymer chains.

[0294] One or more spacers or joints of the water-soluble polymer part or the polymer part, when present, can include biostable or biodegradable polymer chains or units. For example, polymers with repeated linkages have varying degrees of stability under physiological conditions, depending on the instability of the bond. Polymers with such bonds can be classified according to their relative hydrolysis rates under physiological conditions according to the hydrolysis rates of known low molecular weight analogs, for example, from less stable to more stable, such as polyurethane (-NH-C (O)-O-)> polyorthoester (-OC ((OR) (R '))-O-)> polyamide (-C (O)-NH-). Similarly, the linkage system by which the water-soluble polymer is connected to the target molecule can be biostable or biodegradable, for example, from less stable to more stable: carbonate (-OC (O)-O-)> ester (-C (O)-O-)> carbamate (-NH-C (O)-O-)> orthoester (-OC ((OR) (R '))-O-)> amide (-C (O)-NH-). In general, it may be desirable to avoid the use of sulfated polysaccharides, depending on the instability of the sulfate group. In addition, polycarbonates and polyesters may be less desirable. These bonds are provided by way of example and are not intended to limit the types of bonds that may be employed in the polymer chains or linker systems of the water-soluble polymers that may be used in the modified aldehyde-labeled polypeptides disclosed herein.

[0295] Branched nonionic water-soluble group

[0296] In some cases, WSG is a branched nonionic water-soluble group. The branched nonionic water-soluble group can be attached to the comonomer at any suitable position to provide a side chain group protruding from the conjugated polymer backbone, thereby providing high solvation in an aqueous environment. In some cases, the branched nonionic water-soluble group (WSG) comprises two or more water-soluble polymers (e.g., as described herein), each of which has 6 to 100, such as 6 to 50, 6 to 40 or 6 to 30 monomer units.

[0297] The nonionic water-soluble group (WSG) of tool side chain comprises branching group, and this branching group is connected with the comonomer to which it is connected, and provides further connection key with two, three or more nonionic water-soluble polymers.In some examples, the two or more water-soluble polymers adopted in WSG are polyethylene glycol (PEG) group or modified PEG group.Can adopt and provide 3 or more connection points (for example, 4 or more), such as one connection point with the comonomer of conjugated polymer backbone and two or more (for example, 2,3,4 or more) any suitable branching group with the connection point of nonionic water-soluble polymer.This branching group can be connected with comonomer and nonionic water-soluble polymer by any suitable connection functional group and connection chemical property through optional joint.In some examples, branching group is non-aromatic.In some examples, branching group is cyclic. In some cases, the branching group is a substituted aryl ring or heteroaryl ring, for example a trisubstituted or tetrasubstituted aryl ring, such as a trisubstituted or tetrasubstituted phenyl ring; or a trisubstituted or tetrasubstituted heteroaryl ring, such as a trisubstituted or tetrasubstituted pyridyl ring. In some examples, the branching group is acyclic. In some examples, the branching group is an atom, such as C, Si, N. In some examples, the branching group is a connecting functional group, such as an amide or sulfonamide group. In some examples, the branching group is an amino acid residue or a branched linker, such as glycerol or amino-glycerol.

[0298] 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.

[0299] In some examples, the branched nonionic WSG has one of the following formulas:

[0300]

[0301] in:

[0302] Each B 1 and B 2 are independently branched groups;

[0303] Each W 1 independently a nonionic water-soluble polymer, for example comprising 6 or more monomer units;

[0304] T 3 is an optional linker for the fused 6-5-6 tricyclic comonomer; and

[0305] Each p and q is independently 0 or 1, where each T, if present 1 and each T 2 In some examples, 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 to 30 monomeric units, such as 6 to 24 or 10 to 30, 10 to 24 or 10 to 20, 12 to 24, 12 to 20, 12 to 16 or 16 to 20 monomeric units.

[0306] In some examples, the branched nonionic WSG has the formula:

[0307]

[0308] in:

[0309] Each B 1 is a branching group;

[0310] Each W 1 independently a nonionic water-soluble polymer, for example comprising 6 or more monomer units;

[0311] T 3 is an optional linker for the fused 6-5-6 tricyclic comonomer; and

[0312] Each p is independently 0 or 1, where each T, if present 1 In some examples, each W 1W 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 to 30 monomer units, such as 6 to 24 or 10 to 30, 10 to 24 or 10 to 20, 12 to 24, 12 to 20, 12 to 16 or 16 to 20 monomer units. In some embodiments of the branched nonionic WSG, B 1 is a tetrasubstituted aryl group (eg, 1,3,4,5-phenyl).

[0313] In some embodiments of branched nonionic WSG (e.g., 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 is a C1-C12-alkyl linker, such as a C1-C6-alkyl linker, wherein one or more backbone atoms are optionally substituted by heteroatoms.

[0314] In some embodiments, the branched nonionic WSG is selected from one of the following structures:

[0315]

[0316]

[0317] in:

[0318] T 5 is an optional linker for a comonomer (e.g., a fused 6-5-6 tricyclic comonomer);

[0319] T 6 It is a joint;

[0320] Each s is independently an integer from 6 to 100 (e.g., 6 to 50); and

[0321] Each R 11are independently hydrogen, alkyl or substituted alkyl.

[0322] In some 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 some 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 to 30 monomer units, such as 6 to 24 or 10 to 30, 10 to 24 or 10 to 20, 12 to 24, 12 to 20, 12 to 16 or 16 to 20 monomer units. In some cases, each s is the same. In some embodiments of the 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's methyl.

[0323] In some embodiments, the π-conjugated segment includes 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 (e.g., as described herein) (e.g., disubstituted at the 9 position). In some embodiments, the fluorene comonomer has the following structure:

[0324]

[0325] wherein s is 6 to 60; t is 0 to 10 (e.g., 0 to 6, such as 0, 1, 2 or 3); and R 11 is H, alkyl or substituted alkyl. In certain cases, each R 11 It's methyl.

[0326] 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 optionally substituted by one or more halogen, hydroxyl, C1-C 12 Alkoxy or (OCH2CH2) z OCH3 substituted benzyl, 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 benzyl substituted with at least one WSG group (e.g., one or two WSG groups) selected from: (CH2) x (OCH2CH2) y OCH3, wherein each x is independently an integer from 0 to 20, and each y is independently an integer from 0 to 50.

[0327] Multiple WSG can be included in a single position in the subject multi-chromophore by 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 a 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 by branched linkers to give the required solubility of the multi-chromophore. In some examples, WSG is a nonionic side chain group that can give a solubility in water exceeding 50mg / mL. In some examples, WSG is a nonionic side chain group that can give a solubility in water exceeding 100mg / mL. In some embodiments, the multi-chromophore includes one or more substituents selected from the group consisting of: alkyl, aralkyl and heterocyclic groups, each of which is further replaced by a water-solubilizing group hydrophilic polymer group such as polyethylene glycol (PEG) (e.g., PEG groups of 2 to 20 units).

[0328] 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 modified 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 independently selected from one of the following structures:

[0329]

[0330]

[0331] Among them: Each R 11 is H, alkyl (eg, methyl) or substituted alkyl; T 5 is an optional connector; T 6 is a linker; and each s is independently an integer from 1 to 100 (e.g., 1 to 50, or 6 to 50). In certain examples, 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 certain cases, 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 to C12 or C1 to 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 should be 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.

[0332] 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:

[0333]

[0334]

[0335] Where: T 5 is an optional linker; and each s is an integer from 1 to 50. In certain examples of WSG, T 5does not exist. In some examples, 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 to C12 or C1 to 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 in any of the WSG groups described above, a hydroxyl-terminated PEG chain can be used instead of a methoxy-terminated PEG chain. In certain examples of any of the formulas described herein, one or more of the comonomers is replaced by WSG, which is a dendrimer selected from one of the following structures:

[0336]

[0337] In certain examples of any of the formulas described herein, one or more of the comonomers is replaced by WSG, which is a polyol selected from one of the following structures:

[0338]

[0339] 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:

[0340]

[0341] In certain examples of any of the formulas described herein, one or more of the comonomers is replaced by WSG, which is a peptidomimetic selected from one of the following structures:

[0342]

[0343] Polymer Dyes

[0344] Further details of the subject polymeric dyes are provided in the exemplary formulas below. In some embodiments, the subject polymeric dyes have the structure of formula (I):

[0345]

[0346] in:

[0347] Each M 1 independently selected from substituted or unsubstituted aryl comonomers, substituted or unsubstituted heteroaryl comonomers, alkynyl comonomers, and substituted or unsubstituted alkenyl comonomers (e.g., as described herein);

[0348] Each M 2 independently a conjugated modified repeat unit (e.g., as described herein);

[0349] L 1 and L 2 Each independently is a linker unit;

[0350] Z 1 is a chemically selective tag or a signaling chromophore (-Z 1 );

[0351] Z 2 It is a branched π-conjugated chain segment;

[0352] a to h are each independently 0 or 1, wherein a+b≥1 and b+e+h≥1;

[0353] n is 1 to 10,000;

[0354] m and o are each independently 0 to 10,000;

[0355] p is from 1 to 100,000; and

[0356] G 1 and G 2 In some examples of formula (I), m is 0. In some examples of formula (I), m is 1 to 10,000, such as 1 to 1000, 1 to 100 or 1 to 10. In some examples of formula (I), o is 0. In some examples of formula (I), o is 1 to 10,000, such as 1 to 1000, 1 to 100 or 1 to 10. In some examples of formula (I), d is 1. In some examples of formula (I), g is 1. In some examples of formula (I), a+b=2.

[0357] In some embodiments of Formula (I), M 2is a conjugated modified repeating unit of formula (XI) (e.g., as described herein). Likewise, in some embodiments of formula (I), the subject polymeric dye has a structure of formula (II):

[0358]

[0359] in:

[0360] M 1 and M 3 independently selected from aryl comonomers, heteroaryl comonomers, alkynyl comonomers and alkenyl comonomers, each substituted or unsubstituted;

[0361] Z is selected from a covalent bond, a saturated atom or a saturated group, and a functional group;

[0362] R 11 and R 12 Each is independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide and a water solubilizing group;

[0363] a, b, c and e are each independently 0 or 1, wherein a+b≥1 and c+e≥1.

[0364] In some examples of formula (II), m is 0. In some examples of formula (II), m is 1 to 10,000, such as 1 to 1000, 1 to 100, or 1 to 10. In some examples of formula (II), a is 0. In some examples of formula (II), a is 1. In some examples of formula (II), b is 1. In some examples of formula (II), c is 0. In some examples of formula (II), e is 1. In some examples of formula (II), c is 1. In some examples of formula (II), a+b=2. In some examples of formula (II), b+e=2. In some examples of formula (II), a+c=0.

[0365] In some embodiments of Formula (I), the subject polymeric dye has the structure of Formula (III):

[0366]

[0367] in:

[0368] L 1 is a linker unit comprising an aryl comonomer, a heteroaryl comonomer, an alkynyl comonomer, and an alkene comonomer (e.g., as described herein);

[0369] Z 1 is a linked chemically selective tag or a linked signaling chromophore;

[0370] Z is selected from a covalent bond, a saturated atom or group, and a functional group (e.g., as described herein);

[0371] R 11 and R 12 Each is independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide and a water solubilizing group;

[0372] a, b, d and e are each independently 0 or 1, wherein a+b≥1 and d+e≥1. In certain examples of formula (III), m is 0. In certain examples of formula (III), n is 1 to 10,000, such as 1 to 1000, 1 to 100, or 1 to 10. In certain examples of formula (III), m is 1 to 10,000, such as 1 to 1000, 1 to 100, or 1 to 10. In certain examples of formula (III), a is 1. In certain examples of formula (III), b is 1. In certain examples of formula (III), d is 1. In certain examples of formula (III), e is 1. In certain examples of formula (III), a+b=2.

[0373] In certain embodiments of formula (III), Z 1 is a chemical selective tag for attachment. In certain examples of formula (III), Z 1 is a linked signal chromophore, and the polymer dye can be referred to as a polymer tandem dye.

[0374] Also provided are polymeric dyes comprising branching points in the conjugated polymer backbone to which pi-conjugated segments of comonomers can be attached. Such branched pi-conjugated segments of comonomers can be pi-conjugated with the main backbone of the polymer, or can be separated from the backbone, for example, by conjugated modified repeating units. In some embodiments of formula (I), the subject polymeric dye has a structure of formula (I), wherein a, b, g, and h are each 1; and Z 2 A branched π-conjugated segment of formula (IV):

[0375]

[0376] Among them, M 1 、M 2 , L 1 , Z 1 As defined in formula (I); and G 3 is a terminal group, a π-conjugated segment, a linker, or a linked specific binding member. In certain examples, the polymeric dye has formula (I), wherein a, b, d, e, g, h are each 1; and Z 1is the connected functional signal chromophore.

[0377] In some embodiments of Formula (I), the subject polymeric dye has the structure of Formula (V):

[0378]

[0379] in

[0380] Z is selected from a covalent bond, a saturated atom or group, and a functional group (e.g., as described herein);

[0381] R 1 To R 3 , R 11 and R 12 each independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide, a water solubilizing group, an attached chemoselective tag, and an attached signaling chromophore;

[0382] a, b, c and e are each independently 0 or 1, wherein a+b≥1 and c+d≥1;

[0383] n is 1 to 10,000;

[0384] m is 0 to 10,000;

[0385] p is from 1 to 10,000; and

[0386] G 1 and G 2 is independently selected from terminal groups, π-conjugated segments, linkers, and linked specific binding members. In certain examples of Formula (V), R 1 -R 3 are each independently a water-solubilizing group; and R 2 and R 3 are the same.

[0387] In certain embodiments of Formula (V), R 1 and R 2 are each independently a water-solubilizing group; and R 3is a linked chemoselective tag or a linked signal chromophore. In some examples of Formula (V), m is 0. In some examples of Formula (V), n is 1 to 10,000, such as 1 to 1000, 1 to 100, or 1 to 10. In some examples of Formula (V), n is 2 to 10,000, such as 2 to 1000, 2 to 100, or 2 to 10. In some examples of Formula (V), m is 1 to 10,000, such as 1 to 1000, 1 to 100, or 1 to 10. In some examples of Formula (V), m is 2 to 10,000, such as 2 to 1000, 2 to 100, or 2 to 10. In some examples of Formula (V), a is 1. In some examples of Formula (V), b is 1. In some examples of Formula (V), c is 1. In some examples of Formula (V), e is 1. In some examples of Formula (V), a+b=2. Any suitable embodiment of the Z group of the conjugated modified repeat unit described herein can be used in the polymeric dye of formula (V). Any suitable embodiment of WSG described herein can be used as R in the polymeric dye of formula (V). 1 -R 3 , R 11 and R 12 .

[0388] In certain examples of Formula (V), the subject polymeric dye has Formula (VI):

[0389]

[0390] in:

[0391] The dye is the attached signal chromophore and L is the linker.

[0392] WSG1 and WSG2 are each independently a water solubilizing group (WSG) (e.g., as described herein);

[0393] Z is selected from -S-, -S(O)-, -SO2-, -SO2NR'-, -S(O)O-, -O-, -C(O)-, -C(O)NR'-, -C(O)O-, -OC(O)NR'-, -OC(O)O-, -NR'C(O)NR'-, -C(R 5 )(R 6 )-、-Si(R 5 )(R 6 )-, -N(R')-, alkyl linkers (e.g. C2-C 12 , such as C2-C6 substituted alkyl linkers or unsubstituted alkyl linkers) and polyethylene glycol linkers;

[0394] Each R', R 5 and R 6are independently selected from H, alkyl, substituted alkyl, and wherein R 5 and / or R 6 Optionally with R 12 or R 12 The rings are linked to provide a benzo-fused carbocyclic or heterocyclic ring; and

[0395] x and y represent mol% values. In some cases, y is 1 mol% to 25 mol% and x is 99 mol% or less.

[0396] In some instances of Formula (VI), Z is selected from -S-, -S(O)-, -SO2-, -O-, -C(O)-, -C(O)NR'-, -OC(O)NR'-, -OC(O)O-, and -NR'C(O)NR'-. In some instances of Formula (VI), Z is -SO2-. In some instances of (VI), Z is C(R 5 )(R 6 )-or-Si(R 5 )(R 6 )-. In some examples of formula (VI), each WSG 1 and WSG 2 Each is independently a water solubilizing group selected from the following:

[0397] wherein m is 0 to 10 (e.g., 1 to 6, such as 1, 2, 3, 4, 5 or 6), and each R 5 independently -(CH2CH2O) p R', wherein p is an integer from 0 to 50 (e.g., 6 to 50, 6 to 40, 6 to 30, 8 to 30, 10 to 30, or 10 to 20, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and R' is H, alkyl, or substituted alkyl; and X is O. Figure 3 and Figure 6C Exemplary polymeric dyes of formula (V) and (VI) are shown.

[0398] In certain examples of Formula (V), the subject polymeric dye has Formula (VII):

[0399]

[0400] in:

[0401] R 1 -R 4each independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide, a water solubilizing group (WSG) (e.g., as described herein), an attached chemoselective tag, and an attached signaling chromophore;

[0402] a, b, c and e are each independently 0 or 1, wherein a+b≥1 and c+d≥1;

[0403] x and y represent mol% values;

[0404] p is from 1 to 10,000; and

[0405] G 1 and G 2 is independently selected from terminal groups, π-conjugated segments, linkers, and linked specific binding members. In certain examples of Formula (VII), R 1 -R 3 are each independently a water-solubilizing group; and R 2 and R 3 In certain examples of formula (VII), R 3 or R 4 is an attached chemoselective tag or an attached signaling chromophore. In some cases, y is 1 mol % to 25 mol % and x is 99 mol % or less.

[0406] In certain examples of Formula (V), the subject polymeric dye has Formula (VIII):

[0407]

[0408] in:

[0409] The dye is the attached signal chromophore and L is the linker.

[0410] Each WSG1, each WSG2, and WSG3 is independently a water solubilizing group (WSG) (e.g., as described herein); x and y represent mol% values. In some cases, y is 1 mol% to 25 mol%, and x is 99 mol% or less.

[0411] In certain examples of Formula (VIII), WSG1, WSG2 and WSG3 are independently water solubilizing groups selected from the following:

[0412] wherein m is 0 to 10 (e.g., 1 to 6, such as 1, 2, 3, 4, 5 or 6), and each R 5 independently -(CH2CH2O) pR', wherein p is an integer from 0 to 50 (e.g., 6 to 50, 6 to 40, 6 to 30, 8 to 30, 10 to 30, or 10 to 20, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and R' is H, alkyl, or substituted alkyl; and X is O. Figure 4 and Fig. 6A -B shows exemplary polymeric dyes of formula (V), (VII) and (VIII).

[0413] Polymer tandem dyes

[0414] The water-soluble light-gathering multichromophore itself can be fluorescent and capable of transferring energy to an attached acceptor signal chromophore. Therefore, the subject polymer tandem dye further comprises a covalently linked acceptor signal chromophore that is energy-receiving proximity to the donor water-solvated light-gathering multichromophore. Therefore, excitation of the multichromophore donor results in energy transfer to the covalently linked acceptor signal chromophore and emission from the chromophore. The number of signal chromophore acceptor units attached to the donor water-solvated light-gathering multichromophore can vary, wherein in some examples, the number ranges from 1 mol% to 50 mol%, such as 5 mol% to 25 mol% or 10 mol% to 25 mol%.

[0415] Energy transfer mechanisms from a fluorescent water-solvated light-collecting multichromophore donor to an attached acceptor signaling chromophore include, for example, resonance energy transfer (e.g., (or fluorescence) resonance energy transfer, FRET), quantum charge exchange (Dexter energy transfer), etc. In some examples, these energy transfer mechanisms have a relatively short range; that is, the close proximity of the light-collecting multi-chromophore system to the acceptor provides efficient energy transfer. In some examples, under conditions of efficient energy transfer, if the emission from the luminescent signal chromophore is more intense when the incident light ("pump light") is at a wavelength absorbed by the light-collecting multi-chromophore than when the pump light is used to directly excite the luminescent signal chromophore, then the emission from the acceptor is amplified.

[0416] "Effective" energy transfer means that 10% or more of the energy harvested by the donor is transferred to the acceptor, for example, 20% or more or 30% or more. "Amplification" means that the signal from the signal chromophore is 1.5 times or more larger when excited by energy transfer from the donor light-gathering multichromophore compared to direct excitation with incident light of equal intensity. The signal can be measured using any suitable method. In some cases, 1.5 times or more of the signal refers to the intensity of the emitted light. In some cases, 1.5 times or more of the signal refers to the increased signal-to-noise ratio. In certain embodiments of polymer tandem dyes, compared to directly exciting the signal chromophore with incident light, the signal chromophore emits 1.5 times or more when excited by the multichromophore, for example, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 8 times or more, 10 times or more, 20 times or more, 50 times or more, 100 times or more, or even more times, compared to directly exciting the signal chromophore with incident light.

[0417] The linked luminescent signal chromophore emission of the polymer tandem dye can 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 a larger extinction coefficient, such as 6x 10 5 cm -1 M -1 or larger, 7x 10 5 cm -1 M -1 or larger, 8x 10 5 cm -1 M -1 or larger, 9x 10 5 cm -1 M -1 or larger, such as 1x 10 6 cm -1 M -1 or larger, 1.5x 10 6 cm -1 M -1 or larger, 2x 10 6 cm -1 M -1 or larger, 2.5x 10 6 cm -1 M -1 or larger, 3x 10 6cm -1 M -1 or larger, 4x10 6 cm -1 M -1 or larger, 5x 10 6 cm -1 M -1 or larger, 6x 10 6 cm -1 M -1 or larger, 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 an extinction coefficient of 5 x 10 5 M -1 cm -1 In 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.

[0418] The subject polymer tandem dye provides fluorescent emission from a luminescent signal chromophore dye that is brighter than the emission from such a luminescent dye in isolation. The linked luminescent signal chromophore emission of the polymer tandem dye may have a fluorescence emission of 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 -1In some examples, the brightness of the emission of the linked signal chromophore of the polymer tandem dye is at least 5 times higher than the brightness of the directly excited luminescent dye, for example, at least 10 times higher, at least 20 times higher, at least 30 times higher, at least 50 times higher, at least 100 times higher, at least 300 times higher, or even greater than the brightness of the directly excited luminescent dye.

[0419] In some embodiments, a polymer tandem dye comprises: a water-solvable polymer dye having a deep ultraviolet excitation spectrum and comprising a π-conjugated comonomer segment and a conjugated modifying repeat unit; and a signal chromophore covalently linked to the multichromophore in energy-sucking proximity to the multichromophore.

[0420] In certain embodiments or formulae described herein, such as formulae (I) and (III), a linked signal chromophore (e.g., as described herein) is incorporated as a substituent of a comonomer. Any suitable signal chromophore may be linked to any suitable polymeric dye described herein by coupling with a compatible chemoselective functional group. The signal chromophore may be selected to provide a desired emission spectrum and emission maximum wavelength.

[0421] As used herein, the terms "chemoselective functional group" and "chemoselective tag" are used interchangeably and refer to a functional group that is capable of reacting selectively with another compatible functional group to form a covalent bond (in some cases, after optionally activating one of the functional groups). Chemoselective functional groups of interest include, but are not limited to, thiols and maleimides or iodoacetamides, amines and carboxylic acids or their active esters, and groups that can react with each other via click chemistry, such as azides and alkyne groups (e.g., cyclooctyne groups), as well as hydroxyls, hydrazides, hydrazines, aldehydes, ketones, azides, alkynes, phosphines, epoxides, and the like.

[0422] 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 formulas 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.

[0423] 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.

[0424] Any suitable chemoselective functional group may be included in the subject polychromophore (e.g., in -Z 1 , -Z 2 and / or in G 1 or G 2 terminal groups), including but not limited to carboxylic acid, active ester (e.g., NHS or sulfo-NHS ester), amino, hydroxyl, sulfhydryl, 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 structures, these groups are used interchangeably in the structures to refer to either an attached signal chromophore or a chemoselective functional group that can subsequently be conjugated to a suitable chromophore precursor to produce an attached signal chromophore.

[0425] 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 an acceptor chromophore in a 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, dipyrromethene boron difluoride, 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, DY485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY731, 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, Cascade Blue, 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, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY FL, BODIPY FL-Br2, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY R6G, BODIPY TMR, BODIPY TR, 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, DY 530, DY610, DY 633, DY 640, DY 651, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY732, DY 734, DY 752, DY 754, DY 778, DY 782, DY 800, or DY 831), Biotium CF 555, Cy 3.5, and diethylaminocoumarin.

[0426] In some embodiments, the maximum emission 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.

[0427] Terminal Group

[0428] 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 groups include, but are not limited to, terminal capping groups, π-conjugated segments, linkers, and linked specific binding members. In some embodiments, the terminal capping group is a monovalent group that is conjugated to the backbone of the multichromophore after polymerization. In some examples, the terminal capping group is an aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, or substituted alkyl. In some cases, the terminal comonomer is directly connected to a chemoselective tag or linker. 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 examples, G 1 and / or G 2 As used herein, a π-conjugated segment refers to any suitable segment of a conjugated polymer that may be conjugated to a multichromophore, i.e., allows pi electrons to be delocalized across adjacent units. In certain embodiments, G 1 and / or G 2 is a linker, for example, a linker comprising a functional group suitable for conjugation with a specific binding moiety. It should be understood that G can be selected to be located at the polychromophore. 1 and / or G 2 The linkers are positioned so that they can be attached to the side chains that may be present in the polychromophore (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, a carboxylic acid functional group or a derivative thereof is included in G 1 and / or G 2 and an amino functional group or a derivative thereof is included in Z 2 Place.

[0429] 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 modified for use in the subject multichromophore. Chemoselective tags of interest include, but are not limited to, amines, active esters, maleimides, sulfhydryls, 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 group, 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:

[0430] *-Ar-LZ

[0431] wherein Ar is a π-conjugated aromatic group, L is a linker, and Z is a chemoselective tag or a specific binding member. In some cases, the LZ group can be directly attached to a terminal comonomer. In certain embodiments of the formulas described herein, G 1 and G 2 At least one of is described by the following structure:

[0432]

[0433] in:

[0434] q is 0 or an integer from 1 to 12;

[0435] L is an optional linker; and

[0436] Z is a chemoselective tag or a specific binding member.

[0437] 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 examples, Z is an antibody. In some examples, 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.

[0438] It should be understood that for any structure and formula shown herein, in some cases of the subject multichromophores, the terminal group shown may be located at the opposite end to the terminal group shown, for example, terminal group G 1In some embodiments of the multichromophores described herein (eg, Formulas (II) to (V)), G 1 and G 2 At least one of the following structures 1 to 33 is selected:

[0439]

[0440]

[0441]

[0442] * = site of covalent attachment to the unsaturated backbone;

[0443] 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;

[0444] Where k is 2, 4, 8, 12 or 24;

[0445] Where R 15 Selected from the group consisting of lu having the following structure:

[0446] * = site of covalent attachment to the backbone.

[0447] In some embodiments of the multichromophores described herein (eg, Formulas (I)-(V)), 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:

[0448]

[0449] 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.

[0450] 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:

[0451]

[0452]

[0453] Labeled SBM

[0454] Aspects of the present disclosure include the specific binding members of labeling.The specific binding members of labeling are the conjugates of theme polymer dye (for example, as described herein) and specific binding members.Any polymer dye or polymer tandem dye as described herein can be conjugated with specific binding members.Specific binding members and polymer dye can be conjugated to each other (for example, covalently linked) at any suitable position of two molecules by optional joints.In some embodiments, the specific binding members of labeling have aggregation resistance.As used herein, "aggregation resistance" means the specific binding members of such labeling, and the specific binding members of the labeling can form the specific binding members concentration of labeling in the aqueous buffer of interest for 1mg / ml or higher, for example 2mg / ml or higher, 3mg / ml or higher, 4mg / ml or higher, 5mg / ml or higher, 6mg / ml or higher, 7mg / ml or higher, 8mg / ml or higher, 9mg / ml or higher, 10mg / mL or higher or even higher uniform aqueous compositions without aggregation precipitation.

[0455] In certain embodiments, the labeled specific binding member comprises: a water-solvable polymeric dye having a deep ultraviolet excitation spectrum and comprising a π-conjugated comonomer segment and a conjugated modifying repeat unit; and a specific binding member covalently linked to a multichromophore.

[0456] 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 of molecules may have an area on its surface, or a cavity, that specifically binds to an area on the surface of the other member of the pair of molecules or a cavity in the other member of the pair of molecules. Thus, the members of the pair of molecules 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, e.g. 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 -13 M 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 -9M or smaller, e.g. 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.

[0457] Specific binding members can be proteinaceous. As used herein, the term "proteinaceous" refers to a portion consisting of amino acid residues. The portion of proteinaceous can be a polypeptide. In some cases, the specific binding member of proteinaceous is an antibody. In certain embodiments, the specific binding member of proteinaceous 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 essentially of one or more polypeptides encoded by immunoglobulin genes that are recognized in whole or in part. For example, the recognized immunoglobulin genes in humans include kappa (k), lambda (I) and heavy chain genetic loci that together constitute countless variable region genes, and constant region genes μ (u), δ (d), γ (g), σ (e) and α (a) that encode 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., U.S. Department of Health and Human Services (1991)). The numbering of all antibody amino acid sequences discussed herein conforms to the Kabat system. The sequences of the framework regions of different light or heavy chains are relatively conserved within 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-chain length antibodies and may refer to natural antibodies, engineered antibodies, or recombinantly produced antibodies from any organism used for experiments, treatments, or other purposes as further defined below.

[0458] Antibody fragments of interest include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, or other antigen-binding subsequences of antibodies, which are produced by modifying whole antibodies or antibodies 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.

[0459] 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 mouse antibody or a binding fragment thereof. In some examples, the specific binding member is a recombinant antibody or a binding fragment thereof.

[0460] In some embodiments, the labeled specific binding member comprises: a water-solvated light-gathering multichromophore (e.g., as described herein); and a signal chromophore covalently linked to the multichromophore in proximity to the multichromophore energy receiving region (e.g., as described herein); and a specific binding member covalently linked to the multichromophore. In some examples of a labeled specific binding member, a multichromophore of any of the formulae described herein (e.g., formula (I)-(V)), 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.

[0461] method

[0462] As outlined above, aspects of the present invention include methods for evaluating the presence of a target analyte in a sample. Aspects of the method include contacting a sample with a polymer dye conjugate that specifically binds to a target analyte to produce a sample contacted with a labeling 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 examples, the polymer dye conjugate further includes a signal chromophore covalently attached to the multichromophore energy receiving proximity to the polymer dye.

[0463] Any suitable method can be utilized to contact the sample with a polymer dye conjugate, and the polymer dye conjugate specifically binds the target analyte to produce a sample contacted by the labeling composition. In some examples, the sample is contacted with a polymer dye conjugate under certain conditions, wherein 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 can be used, and the solution maintains the biological activity of the sample components and the specific binding members. The solution can be combined with an acceptable low concentration such as a 5-25mM buffer, and is suitably supplemented with a balanced salt solution of fetal calf serum, human platelet lysate or other factors, such as normal saline, PBS, Hank's balanced salt solution, etc. Suitable buffer includes HEPES, phosphate buffer, lactate buffer, etc. Various culture media can be obtained from the market, and can be used according to the properties of the target analyte, and these culture media include dMEM, HBSS, dPBS, RPMI, Iscove culture media, etc., wherein fetal calf serum or human platelet lysate are supplemented in some cases. The final component of the solution can be selected according to the sample components included.

[0464] The temperature at which the specific binding member of the conjugate specifically binds to the target analyte can vary, and in some examples can be in the range of 5°C to 50°C, such as 10°C to 40°C, 15°C to 40°C, 20°C to 40°C, for example 20°C, 25°C, 30°C, 35°C or 37°C (e.g., as described herein). In some examples, 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 examples, 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 examples, 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.

[0465] Any suitable specific binding member can be used in the conjugate. Interested specific binding members include but are not limited to those factors that specifically bind to the cell surface proteins of various cell types, including but 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, which 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. Any suitable cell surface protein or cell marker can be used as a target for specific binding to the polymer dye conjugate in the subject method. In some embodiments, target cells include cell surface markers selected from cell receptors and cell surface antigens. In some cases, the target 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.

[0466] 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 purchased from the market. In some embodiments, the polymer dye conjugate includes an antibody or an antibody fragment. Any suitable target analyte that specifically binds to an antibody or antibody fragment of interest can be used as a target in the subject method.

[0467] In some embodiments, the target analyte is associated with a cell. In some examples, 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 examples, the target analyte is an intracellular target, and the method further comprises lysing the cell.

[0468] In some embodiments, the sample may include a heterogeneous cell population for separating target cells. In some examples, the sample includes peripheral whole blood, peripheral whole blood in which red blood cells have been lysed before cell separation, cord blood, bone marrow, density gradient purified peripheral blood mononuclear cells or homogenized tissue. In some cases, the sample includes hematopoietic progenitor cells (e.g., CD34+ cells) in whole blood, bone marrow or cord blood. In certain embodiments, the sample includes tumor cells in peripheral blood. In some examples, the sample is a sample including (or suspected to include) viral cells (e.g., HIV).

[0469] The specific binding members of labeling can be used in the 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 cells to be processed (for example, detection, analysis and / or sorting) in labeling flow cytometer.The specific binding members of labeling can include antibodies that specifically bind to cell surface proteins (for example, as described herein) 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 including (or possibly including) antibody-mediated cells, particles or analyte labels.

[0470] In some examples of the methods, the labeled specific binding member comprises a multichromophore as described herein (e.g., 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.

[0471] Aspects of the method include measuring the presence of polymer dye conjugate-target analyte binding complex in the sample contacted by the marker composition to evaluate 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 presence of polymer dye conjugate-target analyte binding complex in the sample contacted by the marker composition produced. The polymer dye conjugate-target analyte binding complex is a binding complex produced after the specific binding member of the conjugate specifically binds to the target analyte (if present). The sample contacted by the marker 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, such as 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 specific binding members can be used (such as avidin-biotin or hapten-antihapten antibody) and the specific binding members need to be present. Methods and assay formats of interest that may be modified for use with the subject compositions include, but are not limited to, flow cytometry, in situ hybridization methods, enzyme-linked immunosorbent assays (ELISAs), Western blot analysis, magnetic cell separation assays, and fluorescent dye purification chromatography.

[0472] In certain embodiments, the method further includes contacting the sample with a second specific binding member that specifically binds to the target analyte. In some examples, the second specific binding member is bound by a carrier. Any suitable carrier can be used to fix the component (e.g., the second specific binding member) of the subject method. In some examples, the carrier is a particle, such as a magnetic particle. In some examples, the second specific binding member and the polymer dye conjugate produce a sandwich complex, and the sandwich complex (if present) can be separated and detected using any suitable method. In some embodiments, the method further includes analyzing polymer dye conjugate-target analyte binding complex by flow cytometry, i.e., fluorescently labeled target analyte. Determining the presence of polymer dye conjugate-target analyte binding complex can provide a measurement result (e.g., qualitative or quantitative measurement data) that can be used to evaluate whether there is a target analyte in the sample.

[0473] 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 substrate, wherein the substrate 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 examples, the carrier is selected from the group consisting of particles, planar solid substrates, fiber meshes, hydrogels, porous matrices, needles, microarray surfaces, and chromatographic carriers. The carrier can be incorporated into a system that provides cell separation assisted by any suitable method, such as a manually operated syringe, a centrifuge, or an automated liquid handling system. In some cases, the carrier can be used in an automated liquid handling system for high-throughput cell separation, such as a flow cytometer.

[0474] 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 magnetic field gradient source, i.e., near the surface of the magnet.

[0475] The separation may further include one or more optional washing steps to remove unbound material from the carrier. Any suitable washing method may be used, such as washing the immobilized carrier with a biocompatible buffer that retains the specific binding interactions of the polymeric dye and the specific binding member. Separating and optionally washing away unbound material from the carrier provides an enriched target cell population in which unwanted cells and material can be removed.

[0476] In certain embodiments, the method further includes detecting a labeled target. Detecting a labeled target can include exciting a multichromophore with one or more lasers, followed by detecting the fluorescence emission from a polymer tandem dye using one or more optical detectors. The labeled target can be detected using any suitable instrument and method, including but not limited to flow cytometry, FACS systems, fluorescence microscopy; fluorescence, luminescence, ultraviolet and / or visible light detection using a microplate 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 the following literature, for example, Lakowicz, JR, Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983); Herman, B., Resonance energy transfer microscopy, see Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, Vol. 30, Taylor, DL & Wang, Y.-L. Eds., San Diego: Academic Press (1989), pp. 219-243; Turro, NJ, Modern Molecular Photochemistry, Menlo Park: Benjamin / Cummings Publishing Col, Inc. (1978), pp. 296-361.

[0477] 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, a 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 for maintaining the sample at a specific temperature while the sample is scanned. In some examples, 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 function, and electronic devices associated with imaging and data collection can be managed with a properly programmed digital computer. The computer can also convert the data collected during the assay into another format for presentation.

[0478] In some embodiments, the method for evaluating the presence of a target analyte in a sample further comprises detecting fluorescence in a flow cytometer. In some embodiments, the method for evaluating the presence of a target analyte in a sample further comprises imaging the sample contacted with the labeling composition using fluorescence microscopy. Fluorescence microscopy imaging can be used to identify polymer dye conjugate-target analyte binding complexes in the contacted sample to evaluate whether the target analyte exists. Microscopic methods of interest that can be used in the subject methods include laser scanning confocal microscopy.

[0479] 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 includes: contacting the target molecule with a polymer dye (e.g., as described herein) to produce a labeled target molecule, wherein the polymer dye includes a conjugated tag covalently linked to the target molecule. In some cases, the polymer dye further includes a signal chromophore covalently linked to the multichromophore energy receiving proximity to the polymer dye. 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, and G 1 and G 2 The other one in is a conjugated label.

[0480] 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 used 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 sulfhydryl group, a hydroxyl group, a hydrazine, a hydrazide, an azide, an alkyne, and a protein reactive group (e.g., amino-reactive, sulfhydryl-reactive, hydroxyl-reactive, imidazole-reactive, or guanidino-reactive).

[0481] Any suitable method and reagent can be modified for the subject labeling method so that the conjugated tag is covalently linked to the target molecule. Interested methods for labeling targets include, but are not limited to, those methods and reagents described in the following literature: Hermanson, Bioconjugate Techniques, Third Edition, Academic Press, 2013. The contacting step can be performed in an aqueous solution. In some examples, the conjugated tag 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 examples, the conjugated tag includes a maleimide functional group, and the target molecule includes a thiol functional group, or vice versa. In some examples, the conjugated tag 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.

[0482] 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 purchased from the market. In some embodiments, the target molecule is a specific binding member (e.g., as described herein). In some examples, the specific binding member is an antibody. In some examples, 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.

[0483] In some cases, the method includes a separation step of separating the labeled target molecule 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.

[0484] In some examples, the method further includes detecting and / or analyzing the target molecule of the label. In some examples, the method further includes fluorescent detection of the target molecule of the label. Any suitable method can be combined with the subject method and composition for detecting and / or analyzing the target molecule of the label. The method of analyzing the target of interest that can be used in 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.

[0485] 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 field 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 they are used simultaneously or sequentially to enhance target detection, identification and / or analysis.

[0486] system

[0487] Aspects of the present invention further include systems for practicing subject methods and compositions. The sample analysis system may include a sample field of view or a flow channel loaded with a sample and a labeled specific binding member. In some embodiments, the system is a flow cell system, comprising: a flow cytometer comprising a flow path; a composition in the flow path, wherein the composition comprises: a sample; and a labeled specific binding member (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 placed in the sample field of view, wherein the composition comprises a sample; and a labeled specific binding member (e.g., as described herein).

[0488] In some examples of this system, the labeled specific binding member includes: a water-solvated light-gathering 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 energy receiving proximity to the polymer dye. 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.

[0489] In certain embodiments of the system, the composition further comprises a second specific binding member that is bound by a carrier and specifically binds to the target analyte. In some cases, the carrier comprises magnetic particles. Thus, in certain examples, the system may further comprise a controllable external paramagnetic field configured to be applied to the assay region of the flow channel.

[0490] The sample may include cells. In some cases, the sample is a biological sample containing cells. In some cases, the sample comprises a specific binding member of a label that specifically binds to a target cell. In some cases, the target analyte specifically bound by the specific binding member is a cell surface marker of the cell. In some cases, the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen.

[0491] In certain aspects, the system may further include a light source configured to direct light to an assay region or sample field of view of a flow channel. The system may include a detector configured to receive a signal from an assay region or sample field of view of a flow channel, wherein the signal is provided by a fluorescent composition. Optionally further, the sample analysis system may include one or more additional detectors and / or light sources for detecting one or more additional signals.

[0492] In some aspects, the system may further include a computer-based system configured to detect the presence of a fluorescent signal. "Computer-based system" refers to 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 of the currently available computer-based systems is suitable for the subject system. The data storage device can include any product, including a record of current information as described above, or a memory access device that can access such a product.

[0493] To "record" data, programming or other information on a computer readable medium refers to the process of storing 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.

[0494] "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 is available in the form of an electronic controller, mainframe, server, or personal computer (desktop or portable). If the processor is programmable, appropriate 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 magnetic, optical, 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 corresponding station.

[0495] In addition to the sensor devices and signal processing modules (e.g., as described above), the system of the present invention may include many 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.

[0496] 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 which are incorporated herein by reference.

[0497] Other systems can be used to practice the subject method. In some aspects, the system can be a fluorometer or a microscope loaded with a sample, and the sample has the fluorescent composition of any embodiment discussed herein. The fluorometer or microscope can include a light source, and the light source is configured to guide light to the assay area or sample field of flow channel. The fluorometer or microscope can also include a detector, and the detector is configured to receive a signal from the assay area or sample field of flow channel, wherein the signal is provided by the fluorescent composition.

[0498] Reagent test kit

[0499] Aspects of the invention further include kits for practicing the subject methods and compositions. The compositions of the invention can be incorporated into kits as reagents, as starting materials, or provided for use in methods such as those described above.

[0500] The kit may include: a polymer dye comprising a water-solvated light-gathering multichromophore (e.g., as described herein); and a container. 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 further 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 specific binding member bound by a carrier, 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 examples, the specific binding member is an antibody. In some examples, 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-chain antibody, and a three-chain antibody.

[0501] In certain embodiments, the kit can be used to evaluate the presence of a target analyte in a sample, such as an intracellular target. Thus, in some examples, the kit includes one or more components suitable for cell lysis. One or more additional components of the kit can be provided in a separate container (e.g., a separate tube, bottle, or well in a porous strip or plate).

[0502] In some aspects, the kit further includes a reagent for performing flow cytometry. Interested reagents include but are not limited to the buffer for redissolving and dilution, the buffer for contacting the cell sample with multiple chromophores, the wash buffer, the control cells, the control beads, the fluorescent beads for flow cytometer calibration and their combinations. The kit can also include one or more cell fixatives, such as paraformaldehyde, glutaraldehyde, methanol, acetone, formalin or any combination or buffer thereof. Further, the kit can include a cell permeabilizing agent, such as methanol, acetone or detergent (for example, triton, NP-40, saponin, tween 20, digitonin, leucoperm), or any combination or buffer thereof. Other protein transport inhibitors, cell fixatives and cell permeabilizing agents familiar to the technician are all within the scope of the subject kit.

[0503] The composition of the kit can be provided in a liquid composition, such as any suitable buffer. Alternatively, the composition of the kit can be provided as a dry composition (e.g., can be lyophilized), and the kit can optionally include one or more buffers for reconstituting 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 wells in a porous strip or plate).

[0504] In addition, one or more components can be combined in a single container, such as a glass or plastic vial, tube or bottle. In some examples, the kit can further include a container (e.g., such as a box, bag, insulating container, bottle, tube, etc.) in which all components (and their individual containers) are provided. The kit can further include packaging that is separate from or attached to the kit container and printed with information about the kit, the components of the kit, and / or the instructions for use of the kit.

[0505] In addition to the above-mentioned components, the subject test kit can further include the instructions for practicing the subject method. These instructions can be present in the subject test kit in various forms, and one or more of them can be present in the test kit. A form in which these instructions can 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. Another mode will be a computer-readable medium having information recorded thereon, such as a floppy disk, CD, DVD, portable flash drive etc. Another mode that can exist is to be used for accessing the website address of information by the Internet at a remote site. Any suitable device can be present in the test kit.

[0506] Practicality

[0507] As described herein, polymer dyes, compositions, methods and systems can be used for various applications including diagnostic and research applications that require labeling, detection and / or analysis of targets of interest. Such applications include methodologies such as cytometry, microscopy, immunoassays (e.g., competitive or non-competitive), evaluation of free analytes, evaluation of receptor binding ligands, etc. The compositions, systems and methods described herein can be used to analyze any of a variety of samples, including but not limited to biological fluids, cell culture samples and tissue samples. In some aspects, the compositions, systems and methods described herein can be used for methods for detecting analytes (if present) in samples using fluorescent labels (e.g., in fluorescence-activated cell sorting or analysis, immunoassays, immunostaining, etc.). In some examples, the compositions and methods can be used for various applications where the presence of target analytes in samples needs to be evaluated.

[0508] In some cases, the methods and compositions can be used in any assay format requiring detection and / or analysis of a target from a sample, including but not limited to flow cytometry, fluorescence microscopy, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assays, and fluorescent dye purification chromatography. In some instances, the methods and compositions can be used in any application requiring fluorescent labeling of a target molecule. The subject compositions can be modified for use in any suitable application in which specific binding member pairs (e.g., biotin-streptavidin and hapten-anti-hapten antibodies) can be used.

[0509] Additional Implementation

[0510] Notwithstanding the appended claims, the disclosure set forth herein is also defined by the following terms:

[0511] Item 1. A water-solvated polymer dye, comprising:

[0512] a segment of a π-conjugated comonomer; and

[0513] Conjugated modified repeating unit;

[0514] The polymer dye has a deep ultraviolet excitation spectrum.

[0515] Item 2. The polymeric dye according to Item 1, wherein the polymeric dye comprises a plurality of π-conjugated segments separated by conjugated modifying repeating units.

[0516] Item 3. The polymer dye according to Item 2, wherein each of the plurality of π-conjugated segments has a conjugation length of 20 or less.

[0517] Item 4. The polymer dye according to any one of Items 1 to 3, wherein the excitation spectrum of the polymer dye has a λ of 350 nm or less. max .

[0518] Item 5. The polymer dye according to any one of Items 1 to 4, wherein the conjugated modified repeating unit comprises the following structure:

[0519]

[0520] in:

[0521] Z is selected from the group consisting of a covalent bond, a saturated atom, a saturated group, and a functional group, provided that when Z is a covalent bond, R 22 , R 23 , R 25 and R 28 At least two of them are not H (for example, R 22 and R 23 At least one of R 25 and R 28 At least one of is not H);

[0522] R 21 To R 28 are independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide, and a water solubilizing group, and wherein R 22 , R 23 , R 25 and R 28 One or more of are optionally cyclically linked to Z to provide a benzo-fused carbocyclic or heterocyclic ring; and

[0523] Each * It is the site for covalent attachment to the polymer dye backbone.

[0524] Item 6. The polymeric dye according to Item 5, wherein Z is selected from the group consisting of a saturated carbon atom, a saturated heteroatom, a functional group, and a saturated linker.

[0525] Clause 7. The polymeric dye according to any one of clauses 5 to 6, wherein:

[0526] Z is selected from -S-, -S(O)-, -SO2-, -SO2NR'-, -S(O)O-, -O-, -C(O)-, -C(O)NR'-, -C(O)O-, -OC(O)NR'-, -OC(O)O-, -NR'C(O)NR'-, -C(R 5 )(R 6 )-、-Si(R5 )(R 6 )-, -N(R')-, alkyl linkers, and polyethylene glycol linkers; and

[0527] Each R', R 5 and R 6 are independently selected from H, alkyl, substituted alkyl, or wherein R 5 and / or R 6 Optionally with R 22 , R 23 , R 25 or R 28 The rings are linked to provide benzo-fused carbocyclic or heterocyclic rings.

[0528] Item 8. The polymer dye according to Item 7, wherein Z is selected from -S-, -S(O)-, -SO2-, -O-, -C(O)-, -C(O)NR'-, -OC(O)NR'-, -OC(O)O- and -NR'C(O)NR'-.

[0529] Item 9. The polymer dye according to Item 7, wherein Z is C(R 5 )(R 6 )-or-Si(R 5 )(R 6 )-.

[0530] Item 10. The polymer dye according to Item 9, wherein the conjugated modified repeating unit comprises the following structure:

[0531]

[0532] in:

[0533] n 1 、n 2 、m 1 and m 2 independently 0 to 3, wherein n 1 +m 1 and n 2 +m 2 each independently 2 to 3;

[0534] R 16 and R 17 are each independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide, and a water-solubilizing group; and

[0535] Each * It is the site for covalent attachment to the polymer dye backbone.

[0536] Item 11. The polymer dye according to Item 5, wherein Z is a covalent bond, and the conjugated modified repeating unit comprises the following structure:

[0537]

[0538] in:

[0539] R 1 To R 4 are independently selected from hydrogen, alkyl, substituted alkyl, halogen (eg, fluorine), cyano,

[0540] The condition is R 1 and R 2 Not hydrogen;

[0541] Each R 5 are independently -(CH2CH2O) p R', wherein each p is independently an integer from 0 to 50 (e.g., from 6 to 50, from 6 to 40, from 6 to 30, from 8 to 30, from 10 to 30, or from 10 to 20, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and R' is H, alkyl, or substituted alkyl;

[0542] X is O or S; and

[0543] m is an integer from 0 to 10.

[0544] Item 12. The polymer dye according to any one of items 1 to 11, wherein the polymer dye comprises a plurality of π-conjugated segments, each π-conjugated segment comprising one or more comonomers selected from the group consisting of substituted or unsubstituted aryl or heteroaryl comonomers, alkynyl comonomers and substituted or unsubstituted olefinic comonomers.

[0545] Item 13. A polymer dye according to Item 12, wherein the polymer dye comprises a plurality of π-conjugated segments, each π-conjugated segment comprising one or more comonomers selected from the group consisting of substituted or unsubstituted polyparaphenylene, substituted or unsubstituted biphenylene, substituted or unsubstituted phenylene, substituted or unsubstituted fluorene, substituted or unsubstituted carbazole, substituted or unsubstituted silole, substituted or unsubstituted benzoxazole, substituted or unsubstituted benzothiazole, alkynyl, and substituted or unsubstituted alkenyl.

[0546] Item 14. The polymeric dye according to any one of Items 1 to 13, wherein the polymeric dye comprises a plurality of π-conjugated segments, each π-conjugated segment comprising one or more comonomers selected from the following structures (a) to (k):

[0547]

[0548] in:

[0549] X is O or S;

[0550] n is 1 to 6 (e.g., 1, 2 or 3);

[0551] R 11 is one or more optional substituents each independently selected from hydrogen, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro or bromo), sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide, and a water-solubilizing group; and

[0552] Each * It is the site of covalent attachment to the polymer backbone.

[0553] Item 15. The polymer dye according to Item 14, wherein:

[0554] Structure (a) is selected from:

[0555]

[0556] Structure (g) is selected from:

[0557]

[0558] The structure (h) is selected from:

[0559]

[0560] Structure (i) is selected from:

[0561]

[0562] in:

[0563] X is O or S;

[0564] Z 1 and Z 2 Independently selected from CR 12 and N;

[0565] Y 3 is C or Si; and

[0566] Each R 11 To R 14are independently hydrogen, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide, and a water-solubilizing group, and wherein two R 14 The groups are optionally cyclically linked to form a fused carbocyclic or heterocyclic ring (eg, a partially saturated seven-membered heterocyclic ring).

[0567] Item 16. The polymer dye according to Item 15, wherein each R 11 To R 14 are independently selected from H, F, methyl, trifluoromethyl, cyano,

[0568]

[0569] in:

[0570] X is O or S;

[0571] m is an integer from 0 to 10; and

[0572] R 5 -(CH2CH2O) p R′, wherein R′ is H, alkyl or substituted alkyl (e.g., methyl), and each p is independently an integer from 0 to 40 (e.g., 6 to 40, 6 to 30, 8 to 30, 10 to 30, or 10 to 20, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

[0573] Item 17. The polymeric dye according to any one of Items 1 to 16, wherein the polymeric dye has formula (I):

[0574]

[0575] in:

[0576] Each M 1 independently selected from substituted or unsubstituted aryl comonomers, substituted or unsubstituted heteroaryl comonomers, alkynyl comonomers, and substituted or unsubstituted alkenyl comonomers;

[0577] Each M 2 are independently conjugated modified repeating units;

[0578] L 1 and L 2 Each independently is a linker unit;

[0579] Z 1 is a chemically selective tag or a signaling chromophore (-Z 1 );

[0580] Z 2 It is a branched π-conjugated chain segment;

[0581] a to h are each independently 0 or 1, wherein a+b≥1 and b+e+h≥1;

[0582] n is 1 to 10,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 250, e.g., 2 to 250, 5 to 250, or 10 to 250);

[0583] m and o are each independently 0 to 10,000 (e.g., 0 to 1,000, 0 to 500, or 0 to 250, such as 1 to 150, 2 to 250, 5 to 250, or 10 to 250);

[0584] p is 1 to 100,000 (eg, 1 to 10,000, 1 to 1,000, 1 to 500, or 1 to 250, such as 2 to 250, 5 to 250, or 10 to 250); and

[0585] G 1 and G 2 Independently selected from terminal groups, π-conjugated segments, linkers, and linked specific binding members.

[0586] Item 18. The polymer dye according to Item 17, wherein the polymer dye has formula (II):

[0587]

[0588] in:

[0589] M 1 and M 3 independently selected from aromatic comonomers, heteroaromatic comonomers, alkynyl comonomers, and alkenyl comonomers;

[0590] Z is selected from a covalent bond, a saturated atom or a saturated group, and a functional group;

[0591] R 11 and R 12 Each is independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide and a water solubilizing group;

[0592] a, b, c and e are each independently 0 or 1, wherein a+b≥1 and c+d≥1;

[0593] n is 1 to 10,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 250, e.g., 2 to 250, 5 to 250, or 10 to 250);

[0594] m is 0 to 10,000 (e.g., 0 to 1,000, 0 to 500, or 0 to 250, e.g., 1 to 150, 2 to 250, 5 to 250, or 10 to 250);

[0595] p is 1 to 100,000 (eg, 1 to 10,000, 1 to 1,000, 1 to 500, or 1 to 250, such as 2 to 250, 5 to 250, or 10 to 250); and

[0596] G 1 and G 2 Independently selected from terminal groups, π-conjugated segments, linkers, and linked specific binding members.

[0597] Item 19. The polymer dye according to Item 17, wherein the polymer dye has formula (III):

[0598]

[0599] in:

[0600] M 1 selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, alkynyl comonomers and alkenyl comonomers;

[0601] L 1 is a linker unit comprising an aromatic comonomer, a heteroaromatic comonomer, an alkynyl comonomer, and an alkenyl comonomer;

[0602] Z 1 is a chemically selective tag or a signaling chromophore (-Z 1 );

[0603] Z is selected from a covalent bond, a saturated atom or a saturated group, and a functional group;

[0604] R 11 and R 12 Each is independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide and a water solubilizing group;

[0605] a, b, d and e are each independently 0 or 1, wherein a+b≥1 and d+e≥1;

[0606] n is 1 to 10,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 250, e.g., 2 to 250, 5 to 250, or 10 to 250);

[0607] m is 0 to 10,000 (e.g., 0 to 1,000, 0 to 500, or 0 to 250, e.g., 1 to 150, 2 to 250, 5 to 250, or 10 to 250);

[0608] p is 1 to 10,000 (eg, 1 to 1,000, 1 to 500, or 1 to 250, such as 2 to 250, 5 to 250, or 10 to 250); and

[0609] G 1 and G 2 Independently selected from terminal groups, π-conjugated segments, linkers, and linked specific binding members.

[0610] Clause 20. The polymer dye according to Clause 17, wherein:

[0611] The polymeric dye has formula (I), wherein a, b, g and h are each 1;

[0612] Z 2 is a branched π-conjugated segment of formula (IV):

[0613]

[0614] Among them, M 1 、M 2 , L 1 , Z 1 As defined in clause 17; and

[0615] G 3 It is a terminal group, a π-conjugated segment, a linker or a connected specific binding member.

[0616] Item 21. The polymer dye according to Item 20, wherein:

[0617] The polymeric dye has formula (I), wherein a, b, d, e, g, h are each 1; and

[0618] Z 1 A functional signaling chromophore is attached.

[0619] Clause 22. The polymeric dye according to any one of clauses 17 to 19, wherein the polymeric dye has formula (V):

[0620]

[0621] in

[0622] Z is selected from a covalent bond, a saturated atom or a saturated group, and a functional group;

[0623] R 1 To R 3 , R11 and R 12 each independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide, a water solubilizing group, an attached chemoselective tag, and an attached signaling chromophore;

[0624] a, b, c and e are each independently 0 or 1, wherein a+b≥1 and c+d≥1;

[0625] n is 1 to 10,000 (e.g., 1 to 1,000, 1 to 500, or 1 to 250, e.g., 2 to 250, 5 to 250, or 10 to 250);

[0626] m is 0 to 10,000 (e.g., 0 to 1,000, 0 to 500, or 0 to 250, e.g., 1 to 150, 2 to 250, 5 to 250, or 10 to 250);

[0627] p is 1 to 10,000 (eg, 1 to 1,000, 1 to 500, or 1 to 250, such as 2 to 250, 5 to 250, or 10 to 250); and

[0628] G 1 and G 2 Independently selected from terminal groups, π-conjugated segments, linkers, and linked specific binding members.

[0629] Item 23. The polymer dye according to Item 22, wherein:

[0630] R 1 To R 3 are each independently a water-solubilizing group; and

[0631] R 2 and R 3 are the same.

[0632] Item 24. The polymer dye according to Item 22, wherein:

[0633] Each R 1 and R 2 are each independently a water-solubilizing group; and

[0634] R 3 is an attached chemically selective tag or an attached signaling chromophore.

[0635] Item 25. A polymer dye according to any one of items 18 to 24, wherein Z is selected from saturated carbon atoms, saturated heteroatoms, functional groups (e.g., oxygen and / or nitrogen containing functional groups such as sulfonyl, keto, amide, ester, carbonate, carbamate or urea) and saturated linkers.

[0636] Clause 26. The polymer dye according to Clause 25, wherein:

[0637] Z is selected from -S-, -S(O)-, -SO2-, -SO2NR'-, -S(O)O-, -O-, -C(O)-, -C(O)NR'-, -C(O)O-, -OC(O)NR'-, -OC(O)O-, -NR'C(O)NR'-, -C(R 5 )(R 6 )-、-Si(R 5 )(R 6 )-, -N(R')-, alkyl linkers (e.g. C2-C 12 , such as a C2 to C6 substituted or unsubstituted alkyl linker) and a polyethylene glycol linker; and

[0638] Each R', R 5 and R 6 are independently selected from H, alkyl, substituted alkyl, and wherein R 5 and / or R 6 Optionally with R 12 or R 12 The rings are linked to provide benzo-fused carbocyclic or heterocyclic rings.

[0639] Item 27. The polymer dye according to Item 26, wherein Z is selected from -S-, -S(O)-, -SO2-, -O-, -C(O)-, -C(O)NR'-, -OC(O)NR'-, -OC(O)O- and -NR'C(O)NR'-.

[0640] Item 28. The polymer dye according to Item 27, wherein each Z is -SO2-.

[0641] Item 29. The polymer dye according to Item 26, wherein Z is C(R 5 )(R 6 )-or-Si(R 5 )(R 6 )-.

[0642] Item 30. The polymer dye according to any one of Items 22 to 29, wherein each R 1 and R 2 Each is independently a water-solubilizing group, and the water-solubilizing group is selected from:

[0643] wherein m is 0 to 10 (e.g., 1 to 6, such as 1, 2, 3, 4, 5 or 6), and each R 5 independently -(CH2CH2O) pR', wherein p is an integer from 0 to 50 (e.g., 6 to 50, 6 to 40, 6 to 30, 8 to 30, 10 to 30, or 10 to 20, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and R' is H, alkyl, or substituted alkyl; and X is O.

[0644] Item 31. The polymeric dye according to Item 22, wherein the polymeric dye has formula (VI):

[0645]

[0646] in:

[0647] The dye is the attached signal chromophore and L is the linker.

[0648] WSG1 and WSG2 are each independently a water solubilizing group (WSG) (e.g., as described herein);

[0649] Z is selected from -S-, -S(O)-, -SO2-, -SO2NR'-, -S(O)O-, -O-, -C(O)-, -C(O)NR'-, -C(O)O-, -OC(O)NR'-, -OC(O)O-, -NR'C(O)NR'-, -C(R 5 )(R 6 )-、-Si(R 5 )(R 6 )-, -N(R')-, alkyl linkers (e.g. C2-C 12 , such as C2-C6 substituted or unsubstituted alkyl linkers) and polyethylene glycol linkers;

[0650] Each R', R 5 and R 6 are independently selected from H, alkyl, substituted alkyl, and wherein R 5 and / or R 6 Optionally with R 12 or R 12 cyclically linked to provide a benzo-fused carbocyclic or heterocyclic ring; and

[0651] X and y represent mol % values ​​(eg, y is 1 mol % to 25 mol %, and x is 99 mol % or less).

[0652] Item 33. The polymer dye according to Item 32, wherein Z is selected from -S-, -S(O)-, -SO2-, -O-, -C(O)-, -C(O)NR'-, -OC(O)NR'-, -OC(O)O- and -NR'C(O)NR'-.

[0653] Item 34. The polymer dye according to Item 32, wherein Z is -SO2-.

[0654] Item 35. The polymer dye according to Item 32, wherein Z is C(R 5 )(R 6 )-or-Si(R 5 )(R 6 )-.

[0655] Item 36. The polymer dye according to any one of Items 32 to 35, wherein each WSG 1 and WSG 2 Each is independently a water-solubilizing group, and the water-solubilizing group is selected from:

[0656] wherein m is 0 to 10 (e.g., 1 to 6, such as 1, 2, 3, 4, 5 or 6), and each R 5 independently -(CH2CH2O) p R', wherein p is an integer from 0 to 50 (e.g., 6 to 50, 6 to 40, 6 to 30, 8 to 30, 10 to 30, or 10 to 20, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and R' is H, alkyl, or substituted alkyl; and X is O.

[0657] Clause 37. The polymeric dye according to Clause 22, wherein the polymeric dye has formula (VII):

[0658]

[0659] in:

[0660] R 1 -R 4 each independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide, a water solubilizing group (WSG) (e.g., as described herein), an attached chemoselective tag, and an attached signaling chromophore;

[0661] a, b, c and e are each independently 0 or 1, wherein a+b≥1 and c+d≥1;

[0662] X and y represent mol % values ​​(eg, y is 1 mol % to 25 mol %, and x is 99 mol % or less).

[0663] p is 1 to 10,000 (eg, 1 to 1,000, 1 to 500, or 1 to 250, such as 2 to 250, 5 to 250, or 10 to 250); and

[0664] G 1 and G 2 Independently selected from terminal groups, π-conjugated segments, linkers, and linked specific binding members.

[0665] Item 38. The polymer dye according to Item 37, wherein R 1 To R 3 are each independently a water-solubilizing group; and R 2 and R 3 are the same.

[0666] Clause 39. The polymer dye according to Clause 37, wherein the subject polymer dye has formula (VIII):

[0667]

[0668] in:

[0669] Dye is the attached signal chromophore, and L is the linker;

[0670] Each WSG1, each WSG2, and WSG3 is independently a water solubilizing group (WSG) (e.g., as described herein); and x and y represent mol% values. In some cases, y is 1 mol% to 25 mol%, and x is 99 mol% or less.

[0671] Clause 40. The polymer dye according to clause 39, wherein WSG1, each WSG2 and WSG3 are independently water-solubilizing groups selected from:

[0672] wherein m is 0 to 10 (e.g., 1 to 6, such as 1, 2, 3, 4, 5 or 6), and each R 5 independently -(CH2CH2O) p R', wherein p is an integer from 0 to 50 (e.g., 6 to 50, 6 to 40, 6 to 30, 8 to 30, 10 to 30, or 10 to 20, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and R' is H, alkyl, or substituted alkyl; and X is O.

[0673] Item 41. A polymeric dye according to any one of Items 17 to 21, wherein each M 1 and each M 3 are independently selected from the following structures (a) to (k):

[0674]

[0675] in:

[0676] X is O or S;

[0677] n is 1-6 (e.g., 1, 2 or 3);

[0678] Y 1 Selected from -C(R 11 )2-、-Si(R 11 )2-、-NR 11 -、-C(R 11 )2C(R 11 )2- and -C(R 11 )2C(R 11 )2-;

[0679] R 11 R is one or more optional substituents, each independently selected from hydrogen, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro or bromo), sulfonate / ester, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide and water solubilizing group, or any two adjacent R 11 The groups are optionally linked cyclically (e.g., to provide a carbocyclic or heterocyclic ring); and

[0680] Each * It is the site of covalent attachment to the polymer backbone.

[0681] Item 42. The polymer dye according to Item 41, wherein:

[0682] Structure (a) is selected from:

[0683]

[0684] Structure (g) is selected from:

[0685]

[0686] The structure (h) is selected from:

[0687]

[0688] Structure (i) is selected from:

[0689]

[0690] in:

[0691] X is O or S;

[0692] Z 1 and Z 2 Independently selected from CR 12 and N;

[0693] Y 3 is C or Si; and

[0694] Each R 11 To R 14 are independently hydrogen, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro or bromo), sulfonate / ester, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide and water-solubilizing group, and wherein two R 14 The groups are optionally cyclically linked to form a fused carbocyclic or heterocyclic ring (eg, a partially saturated seven-membered heterocyclic ring).

[0695] Item 43. A polymer tandem dye comprising:

[0696] A water-solvated polymer dye having a deep ultraviolet excitation spectrum and comprising segments of π-conjugated comonomers and conjugated modified repeating units; and

[0697] A signal chromophore is covalently attached to the water-solvated polymeric dye in energy-accepting proximity to the water-solvated polymeric dye.

[0698] Item 44. The polymer tandem dye according to Item 43, wherein the conjugated modified repeating unit comprises the following structure:

[0699]

[0700] in:

[0701] Z is selected from the group consisting of a covalent bond, a saturated atom, a saturated group, and a functional group, provided that when Z is a covalent bond, R 22 , R 23 , R 25 and R 28 At least two of them are not H (for example, R 22 and R 23 At least one of R 25 and R 28 At least one of is not H);

[0702] R 21 -R 28 are independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro or bromo), sulfonate / ester, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide and water solubilizing groups, and wherein R 22 , R 23 , R 25 and R28 One or more of are optionally cyclically linked to Z to provide a benzo-fused carbocyclic or heterocyclic ring; and

[0703] Each * It is the site for covalent attachment to the polymer dye backbone.

[0704] Item 45. The polymer tandem dye of Item 43, wherein the signal chromophore emission has a quantum yield of 0.1 or greater.

[0705] Item 46. The polymer tandem dye according to Item 43, wherein the extinction coefficient of the polymer dye is 1x10 6 M -1 cm -1 Or higher.

[0706] Item 47. The polymer tandem dye according to any one of Items 43 to 46, wherein the multichromophore is substituted with non-ionic side groups capable of conferring a solubility in water exceeding 10 mg / mL.

[0707] Item 48. The polymer tandem dye according to any one of Items 43 to 47, wherein the signal chromophore is a fluorophore.

[0708] Item 49. The polymer tandem dye according to any one of Items 43 to 47, wherein the signal chromophore is a quencher.

[0709] Item 50. A polymer tandem dye according to any one of Items 43 to 49, wherein the signal chromophore is selected from rhodamine, coumarin, xanthene, cyanine, polymethine, pyrene, dipyrromethene boron difluoride, naphthylimide, phycobiliprotein, peridinin chlorophyll protein, conjugates thereof, and combinations thereof.

[0710] Item 51. A polymer tandem dye according to any one of items 43 to 48 and 50, wherein the signal chromophore is selected from fluorescein, 6-FAM, rhodamine, Texas Red, California Red, iFluor594, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodamine, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy-Chrome, DyLight 350, 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, OregonGreen 500, 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.

[0711] Clause 52. The polymer tandem dye according to any one of clauses 43 to 51, wherein:

[0712] The signal chromophore is linked to a comonomer that constitutes 1% to 20% by mole of the multichromophore; and

[0713] The polymer dye is a conjugated polymer comprising 5 or more repeating units.

[0714] Item 53. The polymer tandem dye of any one of items 43 to 48 and 50 to 52, wherein the signal chromophore emits 1.5 or more times greater when excited by the polymer dye compared to direct excitation of the acceptor chromophore with incident light.

[0715] Clause 54. A polymeric tandem dye according to any one of clauses 43 to 53, wherein the polymeric dye comprises a terminal group -L 3 -Z, where L 3 is a linker, and Z is a specific binding member.

[0716] Clause 55. The polymer tandem dye according to Clause 54, wherein the linker is selected from the group consisting of: alkyl, substituted alkyl, alkyl-amido, alkyl-amido-alkyl, and PEG moieties.

[0717] Item 56. The polymer tandem dye according to Item 55, wherein -L 3 -Z is described by the following structure:

[0718]

[0719] wherein: q is 0 to 12; and Z is a specific binding member.

[0720] Item 57. The polymer tandem dye according to any one of items 54 to 56, wherein Z is a biomolecule.

[0721] Item 58. The polymer tandem dye according to Item 57, wherein Z is an antibody.

[0722] Item 59. The polymer tandem dye according to Item 57, wherein Z is an antibody fragment or a binding derivative thereof.

[0723] Clause 60. The polymer tandem dye according to Clause 59, 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.

[0724] Item 61. The polymer tandem dye according to any one of items 53 to 60, wherein the water-solvated polymeric dye is a polymeric dye according to any one of items 2 to 42.

[0725] Clause 62. A labeled specific binding member comprising:

[0726] A water-solvated polymer dye having a deep ultraviolet excitation spectrum and comprising segments of π-conjugated comonomers and conjugated modified repeating units; and

[0727] A specific binding member is covalently linked to the polymeric dye.

[0728] Clause 63. A labeled specific binding member according to Clause 62, wherein the conjugated modified repeat unit comprises the following structure:

[0729]

[0730] in:

[0731] Z is selected from the group consisting of a covalent bond, a saturated atom, a saturated group, and a functional group, provided that when Z is a covalent bond, R 22 , R 23 , R 25 and R 28 At least two of them are not H (for example, R 22 and R 23 At least one of R 25 and R 28 At least one of is not H);

[0732] R 21 -R 28 are independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro or bromo), sulfonate / ester, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide and water solubilizing groups, and wherein R 22 , R 23 , R 25 and R 28 One or more of are optionally cyclically linked to Z to provide a benzo-fused carbocyclic or heterocyclic ring; and

[0733] Each * is the site of covalent attachment to the polymeric dye backbone.

[0734] Clause 64. A labeled specific binding member according to any one of clauses 62 to 63, wherein the polymeric dye comprises a signal chromophore covalently attached to the polymeric dye in energy-accepting proximity to the polymeric dye.

[0735] Clause 65. A labeled specific binding member according to any one of clauses 62 to 64, wherein the specific binding member is an antibody.

[0736] Clause 66. A labeled specific binding member according to any one of clauses 62 to 64, wherein the specific binding member is an antibody fragment or a binding derivative thereof.

[0737] Clause 67. A labeled specific binding member according to Clause 66, 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.

[0738] Item 68. A labeled specific binding member according to Item 64, wherein the signal chromophore is selected from rhodamine, coumarin, xanthene, cyanine, polymethine, pyrene, dipyrromethene boron difluoride, naphthylimide, phycobiliprotein, peridinin chlorophyll protein, conjugates thereof and combinations thereof.

[0739] Item 69. A labeled specific binding member according to any one of items 62 to 68, wherein the water-solvable polymeric dye is a polymeric dye according to any one of items 2 to 42.

[0740] Clause 70. A method of evaluating a sample for the presence of a target analyte, the method comprising:

[0741] (a) contacting the sample with a polymer dye conjugate that specifically binds to the target analyte to produce a labeled composition-contacted sample, wherein the polymer dye conjugate comprises:

[0742] (i) a polymer dye having a deep ultraviolet excitation spectrum according to any one of clauses 1 to 42; and

[0743] (ii) a specific binding member covalently linked to the polymeric dye; and

[0744] (b) determining the presence of the polymer dye conjugate-target analyte binding complex in the sample contacted with the labeling composition to assess the presence or absence of the target analyte in the sample.

[0745] Clause 71. The method of Clause 70, wherein the polymeric dye comprises a signaling chromophore covalently linked to the multichromophore in proximity to the multichromophore energy receiving group.

[0746] Clause 72. The method according to any one of clauses 70 to 71, further comprising contacting the sample with a second specific binding member that is bound to a support and specifically binds to the target analyte.

[0747] Clause 73. The method according to Clause 72, wherein the carrier comprises magnetic particles.

[0748] Clause 74. The method according to any one of clauses 70 to 73, wherein the target analyte is associated with a cell.

[0749] Clause 75. The method according to Clause 75, wherein the target analyte is a cell surface marker of the cell.

[0750] Clause 76. The method according to Clause 75, wherein the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen.

[0751] Clause 77. The method according to Clause 74, wherein the target analyte is an intracellular target, and the method further comprises lysing the cell.

[0752] Clause 78. The method according to any one of clauses 70 to 77, wherein the method further comprises analyzing the fluorescently labeled target analyte by flow cytometry.

[0753] Clause 79. A method of labeling a target molecule, the method comprising:

[0754] contacting the target molecule with a water-solvated polymeric dye to produce a labeled target molecule, wherein the water-solvated polymeric dye has a deep ultraviolet excitation spectrum and comprises:

[0755] a segment of a π-conjugated comonomer;

[0756] a conjugated modified repeating unit; and

[0757] A conjugated label is covalently linked to the target molecule.

[0758] Clause 80. The method according to Clause 79, wherein the conjugated modified repeating unit comprises the following structure:

[0759]

[0760] in:

[0761] Z is selected from the group consisting of a covalent bond, a saturated atom, a saturated group, and a functional group, provided that when Z is a covalent bond, R 22 , R 23 , R 25 and R 28 At least two of them are not H (for example, R 22 and R 23 At least one of R 25 and R 28 At least one of is not H);

[0762] R 21 -R 28are independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro or bromo), sulfonate / ester, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide and water solubilizing groups, and wherein R 22 , R 23 , R 25 and R 28 One or more of are optionally cyclically linked to Z to provide a benzo-fused carbocyclic or heterocyclic ring; and

[0763] Each * is the site of covalent attachment to the polymeric dye backbone.

[0764] Clause 81. The method according to any one of Clauses 79 to 80, wherein the polymeric dye comprises a signal chromophore covalently attached to the polymeric dye in energy-accepting proximity to the polymeric dye.

[0765] Clause 82. The method according to any one of clauses 79 to 81, further comprising fluorescent detection of the labeled target molecule.

[0766] Clause 83. A method according to any one of clauses 79 to 82, 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.

[0767] Clause 84. The method according to any one of clauses 79 to 83, wherein the target molecule is a specific binding member.

[0768] Clause 85. The method according to Clause 84, wherein the specific binding member is an antibody.

[0769] Clause 86. The method according to Clause 85, wherein the specific binding member is an antibody fragment or a binding derivative thereof.

[0770] Clause 87. The method according to Clause 86, 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.

[0771] Clause 88. A flow cytometric analysis system, the system comprising:

[0772] a flow cytometer including a flow path;

[0773] A composition in the flow path, wherein the composition comprises:

[0774] samples; and

[0775] A labeled specific binding member comprising:

[0776] A water-solvated polymer dye having a deep ultraviolet excitation spectrum according to any one of clauses 1 to 30; and

[0777] A specific binding member that specifically binds the target analyte and is covalently linked to the water-solvated polymeric dye.

[0778] Clause 89. The system of Clause 88, wherein the labeled specific binding member further comprises a signaling multichromophore covalently attached to the polymeric dye in energy-receiving proximity to the polymeric dye.

[0779] Clause 90. The system according to any one of clauses 88 to 89, wherein the composition further comprises a second specific binding member bound by a carrier and specifically binding to the target analyte.

[0780] Clause 91. The system of clause 90, wherein the carrier comprises magnetic particles.

[0781] Clause 92. The system of any one of clauses 88 to 91, wherein the sample comprises cells.

[0782] Clause 93. The system of Clause 92, wherein the target analyte is a cell surface marker of the cell.

[0783] Clause 94. The system according to Clause 93, wherein the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen.

[0784] Clause 95. A kit comprising: a polymer dye having a deep ultraviolet excitation spectrum according to any one of Clauses 1 to 42; and a container.

[0785] Item 96. The kit according to Item 95, 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.

[0786] Clause 97. The kit according to any one of clauses 95 to 96, wherein the polymeric dye is covalently linked to a specific binding member.

[0787] Clause 98. The kit according to Clause 97, wherein the specific binding member is an antibody.

[0788] Clause 99. The kit according to Clause 97, wherein the specific binding member is an antibody fragment or a binding derivative thereof.

[0789] Clause 100. The kit according to Clause 99, 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.

[0790] Clause 101. The kit according to any one of Clauses 95 to 100, wherein the polymeric dye further comprises an acceptor signal chromophore covalently attached to the polymeric dye in energy-accepting proximity to the polymeric dye.

[0791] Example

[0792] Example 1: Synthesis of conjugated polymers via CH bond arylation

[0793] The synthesis of the subject polymer dye can be achieved by a technique known as CH bond arylation (Scheme 1). Other methods can also be utilized, such as the Suzuki coupling method (Scheme 2) or the Stille coupling method (Scheme 3). The following exemplary synthesis scheme can be modified for the synthesis of the subject polymer dye with a variety of comonomers, as described herein.

[0794] Scheme 1: CH bond arylation method

[0795]

[0796] Scheme 2: Suzuki coupling method

[0797]

[0798] Scheme 3: Stille coupling method

[0799]

[0800] 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.

[0801] Therefore, the foregoing merely illustrates the principles 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 principles of the present invention and are included in its spirit and scope.In addition, all examples and conditional language described herein are mainly intended to help readers understand the principles of the present invention and the conception provided by the inventor to promote the development of the art, and should be interpreted as not being limited to such specific examples and conditions.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, regardless of the structure, any element developed to perform the same function.In addition, the contents disclosed herein are not intended to contribute to the public, regardless of whether the disclosure is explicitly described in the claims.

[0802] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Instead, the scope and spirit of the present invention are embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined to be invoked only when the exact phrase "meaning of..." or the exact phrase "step of..." is recited at the beginning of the limitation in the claim; if such exact phrase is not used in the limitation of the claim, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) will not be invoked.

Claims

1. A water-solvated polymer dye having a deep ultraviolet excitation spectrum ranging from 200 nm to 350 nm, the polymer dye comprising: a segment of a π-conjugated comonomer; and Conjugated modified repeating units comprising the following structures: in: Z is selected from the group consisting of a covalent bond, a saturated atom, a saturated group, and a functional group, provided that when Z is a covalent bond, R 22 , R 23 , R 25 and R 28 At least two of them are not H; R 21 To R 28 are independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide, and a water solubilizing group, and wherein R 22 , R 23 , R 25 and R 28 One or more of are optionally cyclically linked to Z to provide a benzo-fused carbocyclic or heterocyclic ring; and Each * is the site of covalent attachment to the polymeric dye backbone.

2. The polymer dye according to claim 1, wherein: a) Z is selected from the group consisting of a saturated carbon atom, a saturated heteroatom, a functional group and a saturated linker; b)Z is selected from -S-, -S(O)-, -SO2-, -SO2NR'-, -S(O)O-, -O-, -C(O)-, -C(O)NR'-, -C(O)O-, -OC(O)NR'-, -OC(O)O-, -NR'C(O)NR'-, -C(R 5 )(R 6 )-、-Si(R 5 )(R 6 )-, -N(R')-, an alkyl linker, and a polyethylene glycol linker; and each R', R 5 and R 6 are independently selected from H, alkyl, substituted alkyl, or wherein R 5 and / or R 6 Optionally with R 22 , R 23 , R 25 or R 28 cyclically linked to provide a benzo-fused carbocyclic or heterocyclic ring; c) Z is selected from -S-, -S(O)-, -SO2-, -O-, -C(O)-, -C(O)NR'-, -OC(O)NR'-, -OC(O)O- and -NR'C(O)NR'-; or d) Z is C(R 5 )(R 6 )- or -Si(R 5 )(R 6 )-.

3. The polymer dye according to any one of claims 1 to 2, wherein the conjugated modified repeating unit comprises the following structure: in: n 1 、n 2 、m 1 and m 2 independently 0 to 3, wherein n 1 +m 1 and n 2 +m 2 each independently 2 to 3; R 16 and R 17 are each independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide, and a water-solubilizing group; and Each * is the site of covalent attachment to the polymeric dye backbone.

4. The polymer dye according to claim 1, wherein Z is a covalent bond, and the conjugated modified repeating unit comprises the following structure: in: R 1 To R 4 are independently selected from hydrogen, alkyl, substituted alkyl, halogen, cyano, The condition is R 1 and R 2 Not hydrogen; Each R 5 independently -(CH2CH2O) p R′, wherein p is an integer from 0 to 50, and R′ is H, alkyl, or substituted alkyl; X is O or S; and m is an integer from 0 to 10.

5. The polymer dye according to any one of claims 1 to 4, wherein the polymer dye comprises a plurality of π-conjugated segments, each of which comprises one or more comonomers selected from: a) substituted or unsubstituted aryl or heteroaryl comonomers, alkynyl comonomers and substituted or unsubstituted alkenyl comonomers; b) substituted or unsubstituted poly(p-phenylene), substituted or unsubstituted biphenyl, substituted or unsubstituted phenylene, substituted or unsubstituted fluorene, substituted or unsubstituted carbazole, substituted or unsubstituted silole, substituted or unsubstituted benzoxazole, substituted or unsubstituted benzothiazole, alkynyl, and substituted or unsubstituted alkenyl; c) The following structures (a) to (k): in: X is O or S; n is 1 to 6 (e.g., 1, 2 or 3); R 11 is one or more optional substituents each independently selected from hydrogen, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro or bromo), sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide, and a water-solubilizing group; and Each * is the site of covalent attachment to the polymer backbone.

6. The polymeric dye according to any one of claims 1 to 5, wherein the polymeric dye has formula (I): in: Each M 1 independently selected from substituted or unsubstituted aryl comonomers, substituted or unsubstituted heteroaryl comonomers, alkynyl comonomers, and substituted or unsubstituted alkenyl comonomers; Each M 2 are independently conjugated modified repeating units; L 1 and L 2 Each independently is a linker unit; Z 1 is a chemically selective tag or a signaling chromophore (-Z 1 ); Z 2 It is a branched π-conjugated chain segment; a to h are each independently 0 or 1, wherein a+b≥1 and b+e+h≥1; n is 1 to 250; m and o are each independently 0 to 250; p is from 1 to 100,000; and G 1 and G 2 Independently selected from terminal groups, π-conjugated segments, linkers, and linked specific binding members.

7. The polymer dye according to claim 6, wherein: a) The polymeric dye has formula (II): in: M 1 and M 3 independently selected from aromatic comonomers, heteroaromatic comonomers, alkynyl comonomers, and alkenyl comonomers; Z is selected from a covalent bond, a saturated atom or a saturated group, and a functional group; R 11 and R 12 Each is independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide and a water solubilizing group; a, b, c and e are each independently 0 or 1, wherein a+b≥1 and c+d≥1; n is 1 to 250; m is 0 to 250; p is from 1 to 100,000; and G 1 and G 2 independently selected from terminal groups, π-conjugated segments, linkers, and linked specific binding members; or b) The polymeric dye has the formula (III): in: M 1 selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, alkynyl comonomers and alkenyl comonomers; L1 is a linker unit comprising an aromatic comonomer, a heteroaromatic comonomer, an alkynyl comonomer and an alkenyl comonomer; Z 1 is a chemically selective tag or a signaling chromophore (-Z 1 ); Z is selected from a covalent bond, a saturated atom or a saturated group, and a functional group; R 11 and R 12 Each is independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide and a water solubilizing group; a, b, d and e are each independently 0 or 1, wherein a+b≥1 and d+e≥1; n is 1 to 250; m is 0 to 250; p is from 1 to 10,000; and G 1 and G 2 Independently selected from terminal groups, π-conjugated segments, linkers, and linked specific binding members.

8. The polymer dye according to claim 6, wherein: The polymeric dye has formula (I), wherein a, b, g and h are each 1; Z 2 is a branched π-conjugated segment of formula (IV): Among them, M 1 、M 2 , L 1 , Z 1 As defined in claim 7; and G 3 It is a terminal group, a π-conjugated segment, a linker or a connected specific binding member.

9. The polymeric dye according to any one of claims 6 to 8, wherein the polymeric dye has formula (V): in Z is selected from a covalent bond, a saturated atom or a saturated group, and a functional group; R 1 To R 3 , R 11 and R 12 each independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen, sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide, a water solubilizing group, an attached chemoselective tag, and an attached signaling chromophore; a, b, c and e are each independently 0 or 1, wherein a+b≥1 and c+d≥1; n is 1 to 250; m is 0 to 250; p is from 1 to 10,000; and G 1 and G 2 Independently selected from terminal groups, π-conjugated segments, linkers, and linked specific binding members.

10. The polymer dye according to claim 9, wherein: a) R 1 To R 3 are each independently a water-solubilizing group; and R 2 and R 3 are the same; or b) R 1 and R 2 are each independently a water-solubilizing group; and R 3 is an attached chemically selective tag or an attached signaling chromophore.

11. The polymer dye according to any one of claims 6 to 10, wherein: a) Z is selected from saturated carbon atoms, saturated heteroatoms, functional groups (e.g., oxygen- and / or nitrogen-containing functional groups such as sulfonyl, keto, amide, ester, carbonate, carbamate or urea) and saturated linkers; b)Z is selected from –S–, –S(O)–, –SO2–, –SO2NR’–, –S(O)O–, –O–, –C(O)–, –C(O)NR’–, –C(O)O–, –OC(O)NR’–, –OC(O)O–, –NR’C(O)NR’–, –C(R 5 )(R 6 )–、–Si(R 5 )(R 6 )–, –N(R')–, alkyl linkers (e.g., C2 to C 12 , such as C2 to C6 substituted or unsubstituted alkyl linker) and polyethylene glycol linker; and each R', R 5 and R 6 are independently selected from H, alkyl, substituted alkyl, and wherein R 5 and / or R 6 Optionally with R 12 or R 12 cyclically linked to provide a benzo-fused carbocyclic or heterocyclic ring; c) Z is selected from -S-, -S(O)-, -SO2-, -O-, -C(O)-, -C(O)NR'-, -OC(O)NR'-, -OC(O)O- and -NR'C(O)NR'-; or d) Z is C(R 5 )(R 6 )- or -Si(R 5 )(R 6 )-.

12. A polymer tandem dye comprising: The water-solvated polymer dye according to any one of claims 1 to 11; and A signal chromophore is covalently attached to the water-solvated polymeric dye in energy-accepting proximity to the water-solvated polymeric dye.

13. A labeled specific binding member comprising: The water-solvated polymer dye according to any one of claims 1 to 11; and A specific binding member is covalently linked to the polymeric dye.

14. A kit comprising: A water-solvated polymer dye according to any one of claims 1 to 11, a polymer tandem dye according to claim 12, or a labeled specific binding member according to claim 13.

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