Water-solvated polymer dye capable of exciting blue color

Abstract

Hydrosolvated polymeric dyes and polymeric tandem dyes having a blue excitation spectrum are provided. The polymeric dye is a conjugated polymer that may include a thiophene-containing comonomer. The polymer tandem dye further comprises a signal chromophore covalently attached to the conjugated polymer and close to the conjugated polymer and capable of receiving energy. Also provided are labeled specific binding members comprising the subject polymeric dyes. Also provided are methods of assessing the presence of a target analyte in a sample and methods of labeling a target molecule in which the subject polymeric dyes can be used. Systems and kits for practicing the subject methods are also provided.

Description

[0001] This application is a divisional application of the Chinese patent application with application number "201780055238.6", application date "July 5, 2017", and invention name "Water-solvable polymer dye that can excite blue".

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority under 35 U.S.C. § 119(e) to the filing date of U.S. Provisional Patent Application Serial No. 62 / 360,882, filed on July 11, 2016, the disclosure of which is incorporated herein by reference. Technical Field

[0004] The present application relates to the technical field of polymer dyes, in particular to water-solvated polymer dyes and polymer tandem dyes with blue excitation spectra. Background Art

[0005] Fluorescent dyes are compounds that emit (usually) different wavelengths of light when irradiated with the wavelength light absorbed. Fluorescent dyes can be used for a variety of applications in biochemistry, biology and medicine, such as diagnostic kits, microscopy or drug screening. Fluorescent dyes are characterized by many parameters that allow users to select suitable dyes for desired purposes. 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, such as to allow excitation radiation to penetrate into biological samples, minimize background fluorescence and / or achieve high signal-to-noise ratios.

[0006] 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

[0007] A water-solvated polymer dye and a polymer tandem dye with a blue excitation spectrum are provided. The polymer dye is a conjugated polymer that can include a comonomer containing thiophene. The polymer tandem dye also includes a signal chromophore covalently attached to the conjugated polymer that is close to it and can receive energy. A specific binding member comprising a label of the subject polymer dye is also provided. A method for assessing the presence of a target analyte in a sample and a method for labeling a target molecule are also provided, and the subject polymer dye can be used in the method. A system and a kit for practicing the subject method are also provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] It should be understood that the drawings described below are for illustration purposes only and are not intended to limit the scope of the teachings of the present invention in any way.

[0009] Figure 1 The structures of exemplary polymeric dyes 1-7 of the present disclosure are shown.

[0010] Figure 2 Normalized absorption spectra of exemplary dyes 1-7 are shown. DETAILED DESCRIPTION

[0011] definition

[0012] 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 description.

[0013] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. However, for clarity and ease of reference, certain terms are defined below.

[0014] It must be noted that, as used herein and in the appended claims, the singular forms "a / an" and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "primer" refers to one or more primers, i.e., a single primer and multiple primers. It is further noted that the claims can be drafted to exclude any optional elements. Thus, this statement is intended to serve as a prior basis for using exclusive terms such as "solely," "only," etc., with the recitation of claim elements, or for adopting a "negative" limitation.

[0015] As used herein, the term "sample" refers to a substance or mixture of substances containing one or more analytes of interest, in some cases in liquid form. In some embodiments, the term, as used in the broadest sense, refers to any plant, animal or bacterial substance containing cells or producing cell metabolites, such as tissues or fluids separated from individuals (including but not limited to plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections) or tissues or fluids separated from in vitro cell culture components, 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 whole organism or a subset of its tissues, cells or constituent parts (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" can also refer to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or constituent parts, or a portion or part thereof, including but not limited to plasma, serum, cerebrospinal fluid, lymph, skin, respiratory tract, intestinal tract and urogenital tract external sections, tears, saliva, milk, blood cells, tumors and organs. In certain embodiments, the sample has been removed 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 of a eukaryote and a prokaryote, which has at least a nucleus and a cell membrane. In certain embodiments, the cell includes, for example, a prokaryotic cell from a bacterium. In other embodiments, the cell includes a eukaryotic cell, such as a cell obtained from a biological sample from an animal, a plant or a fungus.

[0016] As used herein, the terms "binding carrier" and "attached to a carrier" are used interchangeably and refer to a portion (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 portions of interest (e.g., a carrier and a specific binding member). In some cases, non-covalent attachment can involve specific binding between the two portions 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 into a substrate.

[0017] As used herein, the term "polypeptide" refers to a polymer form of amino acids of any length, including peptides with a length in the range of 2-50 amino acids and polypeptides with a length of more 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 with non-natural or synthetic backbones. The polypeptide can have any suitable length, such as 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.

[0018] As used herein, the term "isolated" means that the fraction of interest is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free, and even at least 99% free from other components with which the fraction was associated prior to purification.

[0019] A "plurality" contains at least 2 members. In some cases, a plurality can have 10 or more, such as 100 or more, 1000 or more, 10,000 or more, 10 ... 6 or more, 10 7 or more, 10 8 or more or 10 9 or more members.

[0020] Numerical ranges are inclusive of the numbers defining the range.

[0021] 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 particular analyte (or a second member of a specific binding pair) present, for example, in a homogeneous mixture of different analytes. In some cases, the specific binding interaction will discriminate between desirable and undesirable analytes in a sample with a specificity of 10-fold or more, such as 100-fold or more or 1000-fold or more for desirable analytes over undesirable analytes. In some cases, the affinity between the capture agent and the analyte when they are specifically bound in the capture agent / analyte complex is at least 10 -8 M, at least 10 -9 M, e.g. up to 10 -10M.

[0022] 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.

[0023] Method as described herein comprises a plurality of steps.Each step can be carried out after the predetermined amount of time between steps as needed.Thus, the time for carrying out each step can be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more and include 5 hours or more.In certain embodiments, each subsequent step is carried out immediately after the preceding step is completed.In other embodiments, step can be carried out after incubation or waiting time after the preceding step is completed, for example, a few minutes to overnight waiting time.

[0024] As used herein, the terms "assessing," "determining," "measuring," and "assessing" and "assaying" are used interchangeably and include both quantitative and qualitative assays.

[0025] As used herein, the term "separation" refers to the physical separation of two elements (eg, by size or affinity, etc.), as well as the degradation of one element, leaving the other element intact.

[0026] As used herein, the term "joint" or "connection" refers to a connecting portion connecting two groups and having a main chain length of 100 atoms or less. A joint or connection can be a covalent bond connecting two groups or a chain of length between 1 atom 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 a straight chain, a branched chain, a cyclic or a single atom. In some cases, the joint is a branched joint, which refers to a connecting portion connecting three or more groups. In some cases, one, two, three, four or five or more carbon atoms of the joint main chain can be optionally substituted with sulfur, nitrogen or oxygen heteroatoms. In some cases, the joint main chain includes a connecting functional group, such as ether, thioether, amino, amide, sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester or imine. The key between the backbone atoms can be saturated or unsaturated, and in some cases there are at most one, two or three unsaturated bonds in the joint backbone. The joint can include one or more substituents, such as alkyl, aryl or alkenyl. The joint can include but is not limited to polyethylene glycol; ether, thioether, tertiary amine, alkyl, alkyl can be straight or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl) and the like. The joint backbone can include cyclic groups, such as aryl, heterocyclic or cycloalkyl, wherein 2 or more atoms of the cyclic group, such as 2, 3 or 4 atoms are included in the backbone. The joint can be cleavable or non-cleavable.

[0027] 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 polymer group of the chain 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 a variety of end groups and / or other substitution groups, including but not limited to alkyl, aryl, hydroxyl, amino, acyl, acyloxy and amido end groups and / or substitution groups. Suitable PEG groups for use in the subject multichromophores include those described in S. Zalipsky, “Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates”, Bioconjugate Chemistry 1995, 6(2), 150-165; and Zhu et al., “Water-Soluble Conjugated Polymers for Imaging, Diagnosis, and Therapy”, Chem. Rev., 2012, 112(8), pp. 4687-4735.

[0028] As used herein, the term "alkyl" alone or as part of another substituent refers to a saturated branched or straight-chain monovalent hydrocarbon radical derived by removing one hydrogen atom from a single carbon atom of a parent alkane. Alkyl groups of interest include, but are not limited to, methyl; ethyl; propyl, such as prop-1-yl or prop-2-yl; and butyl, such as but-1-yl, but-2-yl, 2-methyl-prop-1-yl or 2-methyl-prop-2-yl. In some embodiments, the alkyl group includes 1 to 20 carbon atoms. In some embodiments, the alkyl group includes 1 to 10 carbon atoms. In certain embodiments, the alkyl group includes 1 to 6 carbon atoms, such as 1 to 4 carbon atoms. For example, the term includes straight and branched chain hydrocarbon groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), tert-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0029] The term "substituted alkyl" refers to an alkyl group as defined herein in which one or more carbon atoms in the alkyl chain have been optionally substituted with, for example, -O-, -N-, -S-, -S(O)- n-(wherein n is 0 to 2), -NR-(wherein R is hydrogen or alkyl), and has 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NR- a R b , where R a and R b and 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.

[0030] "Alkoxy" refers to the group -O-alkyl, where alkyl is as defined herein. For example, alkoxy includes methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, and the like. The term "alkoxy" also refers to the groups alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0031] 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.

[0032] "Amino" refers to the group -NH2.

[0033] The term "substituted amino" refers to the group -NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl, with the proviso that at least one R is not hydrogen.

[0034] "Aryl" by itself or as part of another substituent refers to a monovalent aromatic hydrocarbon derived by removing one hydrogen atom from a single carbon atom of an aromatic ring system. Aryl groups of interest include, but are not limited to, those derived from aceanthrene, 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.

[0035] "Heteroaryl" by itself or as part of another substituent refers to a monovalent heteroaryl radical derived by the removal of one hydrogen atom from a single atom of a heteroaromatic ring system. Heteroaryl groups of interest include, but are not limited to, groups derived from acridine, arsindene, carbazole, β-carboline, chroman, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perylene, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, triazole, benzotriazole, thiophene, triazole, xanthene, benzodioxole, and the like. In certain embodiments, heteroaryl is a 5-20 membered heteroaryl. In certain embodiments, heteroaryl is a 5-10 membered heteroaryl. In certain embodiments, the heteroaryl group is a heteroaryl group derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole, and pyrazine.

[0036] "Heterocycle", "heterocyclic", "heterocycloalkyl" and "heterocyclyl" refer to saturated or unsaturated groups having a single ring or multiple condensed rings, including fused bridged and spiro 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, with the proviso that the point of attachment is through a non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atoms of the heterocyclyl are optionally oxidized to provide N-oxides, -S(O)- or -SO2- moieties.

[0037] 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, oxazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also known as thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, etc.

[0038] Unless otherwise limited by the definition of heterocyclic substituents, such heterocyclic groups may be optionally substituted with 1 to 5 or 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, 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, -SO2-heteroaryl, and fused heterocycle.

[0039] The term "alkaryl" or "aralkyl" refers to the groups -alkylene-aryl and substituted alkylene-aryl, wherein alkylene, substituted alkylene and aryl are defined herein.

[0040] "Alkylene" refers to a group preferably having 1 to 6 and more preferably 1 to 3 carbon atoms and optionally interrupted by one or more selected from -O-, -NR 10 -、-NR 10 C(O)-、-C(O)NR 10-, etc. This 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-), and the like. "Substituted alkylene" refers to an alkylene group in which 1 to 3 hydrogens are replaced with substituents as described for the carbon in "Substitution" below.

[0041] "Substituted" refers to a group in which one or more hydrogen atoms are independently replaced with the same or different substituents. 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 )NR60 R 61 , where M is halogen; R 60 , R 61 , R 62 and R 63 R is independently hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, or optionally R 60 and R 61 Together with the nitrogen atom to which it is bonded, it forms a cycloheteroalkyl ring or a substituted cycloheteroalkyl ring; and R 64 and R 65 R is independently hydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, or optionally R 64 and R 65 Together with the nitrogen atom to which it is bonded, it forms a cycloheteroalkyl ring or a 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, -SC N, -NO, -NO2, =N2, -N3, -S(O)2R 60 、-OS(O)2O - 、-OS(O)2R 60 、-P(O)(O - )2、-P(O)(OR 60 )(O - )、-OP(O)(OR 60 )(OR 61 )、-C(O)R 60 、-C(S)R 60 、-C(O)OR 60 、-C(O)NR 60 R 61 、-C(O)O - 、-NR 62 C(O)NR 60 R 61 In certain embodiments, the substituents include -M, -R 60 ,=O,-OR 60 、-SR 60 、-NR 60 R 61, -CF3, -CN, -NO2, -S(O)2R 60 、-P(O)(OR 60 )(O - )、-OP(O)(OR 60 )(OR 61 )、-C(O)R 60 、-C(O)OR 60 、-C(O)NR 60 R 61 、-C(O)O - In certain embodiments, the substituents include -M, -R 60 ,=O,-OR 60 、-SR 60 、-NR 60 R 61 , -CF3, -CN, -NO2, -S(O)2R 60 、-OP(O)(OR 60 )(OR 61 )、-C(O)R 60 、-C(O)OR 60 、-C(O)O-, where R 60 , R 61 and R 62 As defined above. For example, the 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 aryl or heteroaryl, the substituent (e.g. as described herein) may be referred to as an "aryl substituent".

[0042] Additional definitions of terms may appear throughout the specification.

[0044] As outlined above, water-solvated polymer dyes and polymer tandem dyes with blue excitation spectra are provided. The polymer dye can be a conjugated polymer comprising a comonomer containing thiophene. The polymer tandem dye also includes a signal chromophore covalently attached to a multichromophore close to it and capable of receiving energy. A specific binding member comprising a labeling of the subject polymer dye is also provided. A method for the presence of a target analyte in an assessment sample and a method for labeling a target molecule are also provided, and the subject polymer dye can be used in the method. A system and a kit for practicing the subject method are also provided.

[0045] Before describing various embodiments in more detail, it should be understood that the teachings of the present disclosure are not limited to the specific embodiments described, and thus can certainly 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 limiting, as the scope of the teachings of the present invention is limited only by the appended claims.

[0046] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described in any way. Although the teachings of the present invention are described in conjunction with a variety of embodiments, it is not intended that the teachings of the present invention be limited to such embodiments. On the contrary, as will be appreciated by those skilled in the art, the teachings of the present invention encompass a variety of alternatives, modifications, and equivalents.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used to practice or test the teachings of the present invention, some exemplary methods and materials are now described.

[0048] The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present claims are not entitled to antedate such publication by virtue of prior invention. Further, publication dates may be different from actual publication dates which may be independently verified.

[0049] As will be apparent to those skilled in the art after reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that are readily separated or combined with the features of any other several embodiments without departing from the scope or spirit of the teachings of the present invention. Any recited method may be performed in the order of events recited or in any other order that is logically possible.

[0050] All patents and publications mentioned herein, including all sequences disclosed within such patents and publications, are expressly incorporated by reference.

[0051] In further describing the present invention, polymeric dyes comprising light-harvesting multichromophores and related polymeric tandem dyes also comprising signal chromophores are first described in more detail. Then, labeled specific binding members comprising the subject polymeric dyes are described. Next, methods of interest for which the subject polymeric dyes can be used are reviewed. Systems and kits that can be used to practice the methods of the present disclosure are also described.

[0052] Polymer Dyes

[0053] As outlined above, the present disclosure provides water-solvated polymer dyes with blue excitation spectra. The subject polymer dye can be a π-conjugated polymer comprising a comonomer based on thiophene. Polymer can be an alternating copolymer or a random copolymer of a comonomer. In some cases, the subject polymer dye provides a narrow endothermic peak width relative to other dyes of interest. In polychromatic applications, it is essential to be able to use multiple excitation light sources for achieving diversity and a large number of parameters. When the peak width is narrow, the amount excited by the source other than the expected wavelength is reduced. Except for the narrow peak width, it is essential to minimize the absorption at other common excitation light sources for higher parameter experiments. The cross-light source excitation degree can be adjusted for the subject polymer dye by selecting a mixture of specific structure, comonomer and copolymerization block.

[0054] The subject polymer dye is characterized by the end on the conjugated polymer chain, which can include a free chemical selective functional group providing a bioconjugation. In some cases, such functional groups are called terminal joints. Under these terminal joints, a covalent bond can be formed to connect biomolecules, such as antibodies, polynucleotides or aptamers. For example, antibodies labeled with polymer dyes can be used as reagents showing large brightness for flow cytometry. In addition, orthogonal chemical selective functional groups can be placed along the conjugated polymer chain, which can be used for the connection of receptor signal chromophores in bioconjugation or donor-acceptor polymer tandem dyes.

[0055] As used herein, the terms "light-harvesting multichromophore," "polymer dye," and "conjugated polymer" are used interchangeably and refer to a conjugated polymer having a structure capable of capturing light having a specific maximum absorption wavelength and converting it into emitted light having a longer emission maximum wavelength. In some cases, the light-harvesting multichromophore itself fluoresces. Conjugated polymers (CPs) are characterized by a delocalized electronic structure and can have an effective conjugation length that is substantially shorter than the length of the polymer chain because the backbone can contain many conjugated segments in close proximity. In some cases, conjugated polymers effectively harvest light and provide light amplification via Forster energy transfer to an acceptor.

[0056] As used herein, the term "unit" refers to a structural subunit of a polymer. The term unit is intended to include monomers, comonomers, copolymerization blocks, conjugated segments, repeating units, etc. "Repeat unit" is a subunit of a polymer, which is defined by the different structural features of the minimum number of units required to be considered as a monomer, so 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 a polymer is a random arrangement of a multi-block polymer or unit, each block may define different repeating units, such as n blocks and m blocks. It should be understood that a variety of arrangements of n and / or m repeating units or blocks are possible, and in the depicted formula of the subject multi-chromophore described herein, any suitable linear arrangement of n and m copolymerization blocks of various lengths is included in the structure of the total polymer. In some cases, the repeating unit of a polymer includes a single monomer group. In some cases, the repeating unit of a polymer includes two or more monomer groups, i.e., comonomer groups, such as two, three, four or more comonomer groups. As used herein, the term "comonomer" or "comonomer group" refers to a structural unit of a polymer that can itself be part of a repeating unit of a polymer. In some embodiments, the conjugated polymer includes a block copolymer consisting of blocks of polymerized monomers. In such cases, the block copolymer can be described as having different repeating units each corresponding to a different common block of the polymer. In some cases, the polymer is a diblock copolymer containing two different copolymer blocks. In such cases, the polymer can be described as including copolymer blocks, wherein each copolymer block can be composed of comonomers, such as one, two, three or more comonomers.

[0057] The multichromophore can have any suitable length. In some cases, the specific number of monomer repeat units or segments of the multichromophore can be in the range of 2 to 500,000, e.g., 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 e.g., 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 cases, the specific number of monomer repeat units or segments of the multichromophore can range from 2 to 1,000, e.g., 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.

[0058] 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 cases, 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.

[0059] The subject polymer dye can include a certain minimum content of thiophene-containing comonomers that can provide desirable spectral properties of interest. The content (e.g., mole % value) of the comonomer containing thiophene in the polymer dye (e.g., as described herein) can be measured and mentioned according to the specific comonomer containing thiophene or according to the cumulative total of all comonomers containing thiophene in the polymer dye. In some embodiments, the multichromophore includes 5% (e.g., 5 mole %) or more, such as 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more or even more comonomers containing thiophene based on the molar concentration of the multichromophore. In such cases, the multichromophore can include 5 or more repeating units, such as 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more, 500 or more, 1000 or more, 10,000 or more, or even more repeating units. In such cases, the multichromophore 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 more comonomer units. In certain embodiments, the thiophene-containing comonomer of interest comprises 25% by molar concentration of the polychromophore, e.g., 30% or more, 40% or more, 45% or more, 50% or more, or even more by molar concentration of the polychromophore, comprising 5 or more repeating units, e.g., 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.

[0060] In some embodiments, the multichromophore includes a plurality of first optically active units forming a conjugated system having an absorption wavelength (e.g., as described herein) at which the first optically active units absorb light, thereby forming an excited state. In some cases, the multichromophore includes a conjugated polymer segment or an oligomeric structure including n conjugated repeating units that reduce the band gap.

[0061] The subject multichromophores may possess one or more desirable spectral properties, such as a specific absorption maximum wavelength, a specific emission maximum wavelength, an extinction coefficient, a quantum yield, etc. The subject polymeric dyes and polymeric tandem dyes provide a variety of absorption and emission characteristics, which depend on a variety of factors, such as the selected comonomers, linking groups, substituents, and optionally attached signal chromophores constituting the polymer.

[0062] The subject water-solvated polymer dye has a blue excitation spectrum. As used herein, the blue excitation spectrum is an excitation spectrum with a full width at half maximum (FWHM) within the range of 375nm to 575nm. The full width at half maximum (FWHM) is a statement of the degree of the function given by the difference between the two extreme values ​​of the independent variable when the dependent variable is equal to half of its maximum value. In other words, it is the width of the excitation spectrum curve measured between those points of half the maximum amplitude on the y-axis (the difference or range of two specific wavelengths). In some cases, FWHM can be referred to as the difference between wavelengths. In some cases, FWHM can be referred to as a specific region of the spectrum where the wavelength defining FWHM is located and measured.

[0063] In certain embodiments, polymer dye has a blue excitation spectrum with a full width at half maximum (FWHM) located in the range of 375nm to 550nm. In certain embodiments, polymer dye has a blue excitation spectrum with a full width at half maximum (FWHM) located in the range of 375nm to 525nm. In certain embodiments, polymer dye has a blue excitation spectrum with a full width at half maximum (FWHM) located in the range of 375nm to 500nm. In certain embodiments, polymer dye has a blue excitation spectrum with a full width at half maximum (FWHM) located in the range of 375nm to 475nm. In certain embodiments, polymer dye has a blue excitation spectrum with a full width at half maximum (FWHM) located in the range of 375nm to 450nm. In certain embodiments, polymer dye has a blue excitation spectrum with a full width at half maximum (FWHM) located in the range of 400nm to 575nm. In certain embodiments, polymer dye has a blue excitation spectrum with a full width at half maximum (FWHM) located in the range of 410nm to 575nm. In certain embodiments, polymeric dye has a blue excitation spectrum with a full width at half maximum (FWHM) positioned at 420nm to 575nm. In certain embodiments, polymeric dye has a blue excitation spectrum with a full width at half maximum (FWHM) positioned at 430nm to 575nm. In certain embodiments, polymeric dye has a blue excitation spectrum with a full width at half maximum (FWHM) positioned at 440nm to 575nm. In certain embodiments, polymeric dye has a blue excitation spectrum with a full width at half maximum (FWHM) positioned at 450nm to 575nm. In certain embodiments, polymeric dye has a blue excitation spectrum with a full width at half maximum (FWHM) positioned at 460nm to 575nm. In some cases, the water-solvated polymeric dye has a blue excitation spectrum with a FWHM of 200 nm or less, e.g., 180 nm or less, 175 nm or less, 170 nm or less, 160 nm or less, 150 nm or less, 140 nm or less, 130 nm or less, 120 nm or less, 110 nm or less, 100 nm or less, or even less.

[0064] In some cases, the polymer dye absorbs a maximum wavelength of 375 to 575nm, for example, 400nm to 550nm, 410nm to 550nm, 420nm to 540nm, 430nm to 540nm or 440nm to 530nm. In certain embodiments, the multichromophore has a maximum absorption wavelength of 550nm or less, for example, 525nm or less, 500nm or less, 490nm or less, 480nm or less, 470nm or less, 460nm or less, 450nm or less, 425nm or less or even less. In some cases, the multichromophore has a maximum absorption wavelength in the range of 375nm to 440nm. In some cases, the multichromophore has a maximum absorption wavelength in the range of 400nm to 450nm. In some cases, the multichromophore has a maximum absorption wavelength in the range of 450nm to 500nm. In some cases, the multichromophore has a maximum absorption wavelength in the range of 500nm to 550nm. In some cases, polymeric dyes disclosed herein can provide a maximum absorption wavelength within the range of 440nm to 530nm. In some cases, polymeric dyes provide high extinction coefficients and high quantum yields, which produce bright fluorescent reagents. In some cases, polymeric dyes have no yellow-green absorption. In some cases, polymeric dyes have no absorption at 562nm or greater, such as 570nm or greater, 580nm or greater, 590nm or greater, 600nm or greater, 610nm or greater, or 620nm or greater.

[0065] In some cases, the multichromophore has a maximum absorption wavelength of 380nm±5nm. In some cases, the multichromophore has a maximum absorption wavelength of 390nm±5nm. In some cases, the multichromophore has a maximum absorption wavelength of 400nm±5nm. In some cases, the multichromophore has a maximum absorption wavelength of 410nm±5nm. In some cases, the multichromophore has a maximum absorption wavelength of 420nm±5nm. In some cases, the multichromophore has a maximum absorption wavelength of 430nm±5nm. In some cases, the multichromophore has a maximum absorption wavelength of 440nm±5nm. In some cases, the multichromophore has a maximum absorption wavelength of 450nm±5nm. In some cases, the multichromophore has a maximum absorption wavelength of 460nm±5nm. In some cases, the multichromophore has a maximum absorption wavelength of 470nm±5nm. In some cases, the multichromophore has a maximum absorption wavelength of 480nm±20nm. In some cases, the multichromophore has a maximum absorption wavelength of 480nm±10nm. In some cases, the multichromophore has a maximum absorption wavelength of 480nm ± 5nm. In some cases, the multichromophore has a maximum absorption wavelength of 490nm ± 5nm. In some cases, the multichromophore has a maximum absorption wavelength of 500nm ± 5nm. In some cases, the multichromophore has a maximum absorption wavelength of 510nm ± 5nm. In some cases, the multichromophore has a maximum absorption wavelength of 520nm ± 5nm. In some cases, the multichromophore has a maximum absorption wavelength of 530nm ± 5nm. In some cases, the multichromophore has a maximum absorption wavelength of 540nm ± 5nm. In some cases, the multichromophore has a maximum absorption wavelength of 550nm ± 5nm. In some cases, the multichromophore has a maximum absorption wavelength of 560nm ± 5nm. In some cases, the multichromophore has a maximum absorption wavelength of 570nm ± 5nm.

[0066] In some embodiments, the multichromophore has an emission maximum wavelength in the range of 300 to 900 nm, e.g., 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, where specific examples of emission maxima of interest include, but are not limited to, 395 nm ± 5 nm, 460 nm ± 5 nm, 490 nm ± 5 nm, 550 nm ± 5 nm, 560 nm ± 5 nm, 605 nm ± 5 nm, 650 nm ± 5 nm, 680 nm ± 5 nm, 700 nm ± 5 nm, 805 nm ± 5 nm. In some cases, the multichromophore has an emission maximum wavelength selected from the group consisting of 395 nm, 460 nm, 490 nm, 550 nm, 560 nm, 605 nm, 650 nm, 680 nm, 700 nm, and 805 nm. In some cases, the multichromophore has an emission maximum wavelength of 395 nm ± 5 nm. In some cases, the multichromophore itself has an emission maximum wavelength in the range of 375 to 900 nm (e.g., 380 nm to 900 nm, 390 nm to 900 nm, or 400 nm to 900 nm).

[0067] In some cases, the multichromophore has 5×10 5 cm -1 M -1 or more, e.g. 6×10 5 cm -1 M -1 or more, 7×10 5 cm -1 M -1 or more, 8×10 5 cm -1 M -1 or more, 9×10 5 cm -1 M -1 or more, for example 1×10 6 cm -1 M -1 or more, 1.5×10 6 cm -1 M -1 or more, 2×10 6 cm -1 M -1 or more, 2.5×10 6 cm -1 M -1 or more, 3×10 6 cm -1 M -1 or more, 4×10 6 cm-1 M -1 or more, 5×10 6 cm -1 M -1 or more, 6×10 6 cm -1 M -1 or more, 7×10 6 cm -1 M -1 or more or 8×10 6 cm -1 M -1 In such cases, the multichromophore may have 5 or more repeating units, such as 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or even more repeating units. In some embodiments, the multichromophore has 5×10 5 M -1 cm -1 In some embodiments, the multichromophore has a molar extinction coefficient of 1×10 6 M -1 cm -1 or more molar extinction coefficient.

[0068] In some cases, the multichromophore has a wavelength of 40,000 cm per repeating unit. -1 M -1 or more, e.g. 45,000 cm per repeating unit -1 M -1 or more, 50,000cm per repeating unit -1 M -1 or more, 55,000cm per repeating unit -1 M -1 or more, 60,000cm per repeating unit -1 M -1 or more, 70,000cm per repeating unit -1 M -1 or more, 80,000cm per repeating unit -1 M -1 or more, 90,000cm per repeating unit -1 M -1 or more, 100,000cm per repeating unit -1 M -1 In some cases, each repeating unit described herein has an extinction coefficient of 40,000 cm -1 M -1or more is an average extinction coefficient. In some cases, the repeating unit of the multichromophore can include a single monomer, two comonomers, or three or more comonomers. In some cases, the multichromophore has a comonomer of 40,000 cm -1 M -1 or more, e.g. 45,000 cm per comonomer -1 M -1 or more, 50,000cm per comonomer -1 M -1 or more, 55,000cm per comonomer -1 M -1 or more, 60,000cm per comonomer -1 M -1 or more, 70,000cm per comonomer - 1 M -1 or more, 80,000cm per comonomer -1 M -1 or more, 90,000cm per comonomer -1 M -1 or more, 100,000cm per comonomer -1 M -1 In some cases, each comonomer has an extinction coefficient of 40,000 cm -1 M -1 or more is the average extinction coefficient.

[0069] It should be understood that in some cases the subject multichromophore may include random arrangements of copolymer blocks (e.g., n and m copolymer blocks) or n and m repeating units. The subject multichromophore may include any suitable linear arrangement of n and m copolymer blocks of various lengths within the structure of the total polymer. In addition, the multichromophore may include any suitable arrangement of comonomers within such n and / or m copolymer blocks. Unless otherwise indicated, all possible arrangements of comonomers are intended to be included in the polymer dyes described herein. A variety of polymer synthesis methods can be used to prepare comonomers and copolymer blocks of interest in the preparation of the subject multichromophore. It should be understood that in some cases, the polymerization method can produce a composition including a group of conjugated polymers, which includes some variations in the specific length and / or the end groups (i.e., terminal groups) present in each conjugated polymer of the group. The formulas depicted herein may refer to a single compound or a group of polymeric compounds or a subgroup of polymeric compounds. As used herein, * Represents the point of covalent attachment to the unsaturated backbone of a conjugated polymer.

[0070] In some embodiments, the subject polymer dye is a π conjugated polymer including a comonomer based on thiophene and an aryl or heteroaryl comonomer. The aryl or heteroaryl comonomer of interest includes but is not limited to a fused tricyclic comonomer, such as a fluorene comonomer, a carbazole comonomer, a thiole comonomer or a bridged biphenyl comonomer. The fused tricyclic comonomer is a comonomer including a tricyclic aromatic group, which has three fused rings in the following configuration, wherein two aryl or heteroaryl 6-rings are fused with a central 5 or 6-membered carbocyclic ring or a heterocyclic ring. In some cases, the fused tricyclic comonomer includes two benzo or pyridine rings fused with a central 5 or 6-membered carbocyclic ring or a heterocyclic ring. The fused tricyclic comonomer can be π conjugated via any suitable ring atom of the fused ring and the comonomer adjacent to the polymer backbone. The central 5 or 6 membered ring may be carbocyclic or heterocyclic, aromatic or partially saturated, and may further include side chain substituents, such as WSG and / or linkers to chemoselective tags. A bridged biphenyl comonomer is a fused tricyclic comonomer having a biphenyl group in which the two phenyl rings are further connected to each other via a central 6 membered ring. In some cases, the fused tricyclic comonomer is described by the following structure:

[0071]

[0072] in:

[0073] Y is C(R 3 )2、-C(R 3 )2C(R 3 )2-、NR 3 、Si(R 3 )2 or Se;

[0074] Z is CH, CR or N;

[0075] Every R 3 independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido, aralkyl, substituted aralkyl, PEG moiety, -L 1 -Z 1 , where L 1 is a joint and Z 1 is a chemoselective tag (e.g., a tag comprising a chemoselective functional group) and WSG; and

[0076] Each R is independently H or one or more aryl or heteroaryl substituents. In some cases, each R refers to one or two ring substituents independently selected from halogen, sulfonate, alkoxy, substituted alkoxy, alkyl and substituted alkyl. In some cases, at least two Z in each ring are CH or CR. In some cases, one and only one Z in each ring is N.

[0077] In some cases, the fused three-ring comonomer is described by one of the following structures:

[0078]

[0079] in:

[0080] Y is C(R 3 )2、-C(R 3 )2C(R 3 )2-、NR 3 、Si(R 3 )2 or Se;

[0081] Every R 3 independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido, aralkyl, substituted aralkyl, PEG moiety, -L 1 -Z 1 , where L 1 is a joint and Z 1 is a chemoselective tag (e.g., a tag comprising a chemoselective functional group) and WSG; and

[0082] Each R is independently H, R 3 Or one or more aryl or heteroaryl substituents. In some cases, each R refers to one or two ring substituents independently selected from halogen, sulfonate, alkoxy, substituted alkoxy, alkyl and substituted alkyl. The symbol "*" represents a covalent attachment site to the unsaturated backbone of the conjugated polymer, such as a π-conjugated segment, an end group, a linker, and a connected specific binding member. It should be understood that for any formula of a fused tricyclic comonomer comprising 2,7-π conjugation described herein, a similar formula comprising a similar 3,6-π conjugated comonomer can also be depicted. 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 In some cases, the fused tricyclic comonomer is a bridged biphenyl comonomer, wherein Y is -CHR 3 CHR 3-. In certain instances of any fused tricyclic comonomer described herein, each R is independently selected from H, halogen, alkoxy, substituted alkoxy, alkyl, and substituted alkyl. In certain instances, each R is independently selected from H, fluorine, chlorine, methoxy, substituted alkoxy, alkyl, and substituted alkyl.

[0083] In some cases, the fused three-ring comonomer is described by one of the following structures:

[0084]

[0085] in:

[0086] Every R 3 independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido, aralkyl, substituted aralkyl, PEG moiety, -L 1 -Z 1 , where L 1 is a joint and Z 1 is a chemoselective tag (e.g., a tag comprising a chemoselective functional group) and WSG; and

[0087] Each R is independently H, R 3 or one or more aryl or heteroaryl substituents. In some cases, each R refers to one or two ring substituents independently selected from halogen, sulfonate, alkoxy, substituted alkoxy, alkyl, and substituted alkyl. In some cases, each R is fluoro or methoxy. In some cases, at least one R 3 is WSG. In some cases, at least one R 3 For -L 1 -Z 1 .

[0088] In some cases, the multichromophore includes a conjugated segment having a structure of Formula (I):

[0089]

[0090] in:

[0091] M 1 、M 2 and M 3 are independently aromatic or heterocyclic comonomers, wherein M 1 、M 2 and M 3 At least one of the monomers is a thiophene-containing comonomer;

[0092] Z 1 is a chemoselective functional group or a linked signaling chromophore;

[0093] Z 5 and Z 6 are independently CR or N, wherein R is selected from H, halogen, alkoxy, substituted alkoxy, alkyl, and substituted alkyl;

[0094] G 1 and G 2 each independently selected from a terminal group, a π-conjugated segment, a linker, and a linked specific binding member;

[0095] Y 1 , Y 2 and Y 3 Independently C(R 3 )2、-C(R 3 )2C(R 3 )2-、NR 3 、Si(R 3 )2 or Se;

[0096] Every R 3 independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido, and WSG;

[0097] ai is independently 0 or 1;

[0098] n, m and o are each independently 0 or an integer from 1 to 10,000; and

[0099] p is an integer from 1 to 100,000. In some cases of formula (I), the comonomer M 2 and M 3 One of the molecules comprises an attached chemoselective functional group or an attached signaling chromophore.

[0100] In certain embodiments of Formula (I), Y 1 C(R 3 ) 2. In some embodiments of formula (I), Y 1 NR 3 In certain cases of Formula (I), Y 1 Si(R 3 ) 2. In certain cases of formula (I), Y 1 -C(R 3 )2C(R 3 )2-. In some cases of Formula (I), Y 1 -CHR 3 CHR 3 -. In certain cases of formula (I), Y 1In certain embodiments of formula (I), Y 2 C(R 3 ) 2. In some cases of Formula (I), Y 2 NR 3 In certain embodiments of Formula (I), Y 2 Si(R 3 ) 2. In certain cases of formula (I), Y 2 -C(R 3 )2C(R 3 )2-. In some cases of Formula (I), Y 2 -CHR 3 CHR 3 -. In some embodiments of Formula (I), Y 2 In certain cases of formula (I), Y 3 C(R 3 )2, one of the R 3 Contains Z 1 In some cases of Formula (I), Y 3 NR 3 , where R 3 Contains Z 1 In certain cases of Formula (I), Y 3 Si(R 3 )2, one of the R 3 Contains Z 1 In certain cases of Formula (I), Y 3 -C(R 3 )2C(R 3 )2-, one of the R 3 Contains Z 1 In some cases of Formula (I), Y 3 -CHR 3 CHR 3 -, one of the R 3 Contains Z 1 In certain embodiments of Formula (I), Z 1 is a chemoselective functional group. In certain cases of formula (I), Z 1 In certain embodiments of formula (I), G 1 and G 2 In certain embodiments of Formula (I), G 1 and G 2 At least one of the is a linked specific binding member.

[0101] In some cases of formula (I), each fused tricyclic comonomer has the same core structure, such as fluorene, carbazole, silole, or bridged biphenyl comonomer. In some cases of formula (I), each fused tricyclic comonomer is independently selected from fluorene, carbazole, silole, and bridged biphenyl comonomer. In certain cases of the fused tricyclic comonomers of formula (I), Z 5 and Z 6 In some cases of the fused tricyclic comonomer of formula (I), Z 5 and Z 6 Both are independently CR. In some embodiments of the fused three-ring comonomer of formula (I), Z 5 CR and Z 6 is N. In certain cases of the fused tricyclic comonomer of formula (I), Z 5 N and Z 6 In certain cases of the fused tricyclic comonomer of formula (I), Z 5 and Z 6 In certain cases of the fused tricyclic comonomer of formula (I), Z 5 and Z 6 Both are CH. In some embodiments of the fused tricyclic comonomer of formula (I), Z 5 CR and Z 6 is N. In certain cases of the fused tricyclic comonomer of formula (I), Z 5 N and Z 6 For CR.

[0102] In certain cases of Formula (I), M 1 、M 2 and M 3 are independently heterocyclic comonomers, wherein M 1 、M 2 and M 3 At least one of the monomers is a thiophene-containing comonomer.

[0103] In some cases of Formula (I), the multichromophore includes a conjugated segment having a structure of Formula (Ia):

[0104]

[0105] In certain embodiments of Formula (I), M 1 、M 2 and M 3Each is independently a thiophene-containing comonomer. In certain cases of formula (I), a+c≤1. In certain cases of formula (I), d+f≤1. In certain cases of formula (I), g+i≤1. In certain cases of formula (I), b+e+h≥1. In certain cases of formula (I), n+m+o≥1. In some cases, a+c≤1; d+f≤1; g+i≤1; b+e+h≥1; and n+m+o≥1. In certain cases, n≥1. In certain cases, m=0. In certain cases, o=0. In certain cases, a+c=1. In certain cases, b=1 and e=1. In certain cases, a+c=1. In certain cases, b+e+h=2. In certain cases, d+f=1. In certain cases, b=1, e=1 and h=0. In certain cases, g+i=1. In certain cases, b=1, e=0 and h=1.

[0106] In some embodiments of Formula (I), the polymeric dye has the structure of Formula (Ib):

[0107]

[0108] In some cases of formula (Ib), each fused tricyclic comonomer has the same core structure, such as fluorene, carbazole, silole, or bridged biphenyl comonomer. In some cases of formula (Ib), each fused tricyclic comonomer is independently selected from fluorene, carbazole, silole, and bridged biphenyl comonomer. In certain cases of fused tricyclic comonomers of formula (Ib), Z 5 and Z 6 In some cases of the fused tricyclic comonomer of formula (Ib), Z 5 and Z 6 Both are independently CR. In some embodiments of the fused tricyclic comonomer of formula (Ib), Z 5 CR and Z 6 is N. In certain cases of the fused tricyclic comonomer of formula (Ib), Z 5 N and Z 6 In certain cases of the fused tricyclic comonomer of formula (Ib), Z 5 and Z 6 In certain cases of the fused tricyclic comonomer of formula (Ib), Z 5 and Z 6 Both are CH. In some embodiments of the fused tricyclic comonomer of formula (Ib), Z 5 CR and Z 6 is N. In certain cases of the fused tricyclic comonomer of formula (Ib), Z 5 N and Z 6 For CR.

[0109] In some embodiments of Formulas (I) and (Ib), the polymeric dye has the structure of Formula (Ic):

[0110]

[0111] In certain instances of Formulae (Ib)-(Ic), h, i, and o are each 0. In certain instances of Formulae (Ib)-(Ic), b and c are each 1. In certain instances of Formulae (Ib)-(Ic), e and f are each 1. In certain instances of Formulae (Ib)-(Ic), e, f, and m are each 0. In certain instances of Formulae (Ib)-(Ic), h and i are each 1. In certain instances of Formulae (Ib)-(Ic), h, i, and o are each 0; b and c are each 1; and e and f are each 1. In certain instances, M 2 is attached to a signal chromophore or a chemoselective functional group. In certain instances of Formulas (Ib)-(Ic), e, f, and m are each 0; b and c are each 1; and h and i are each 1. In certain instances, Z 1 is a linked signal chromophore. In some cases, Z 1 A chemically selective functional group for attachment.

[0112] In some embodiments of Formula (I), the polymeric dye has the structure of Formula (II):

[0113]

[0114] wherein u, v, w, x, y and z represent the mole % values ​​of each comonomer in the multichromophore. In some cases of formula (IIa), each fused tricyclic comonomer has the same core structure, such as fluorene, carbazole, silole or bridged biphenyl comonomer. In some cases of formula (IIa), each fused tricyclic comonomer is independently selected from fluorene, carbazole, silole and bridged biphenyl comonomer. In certain cases of fused tricyclic comonomers of formula (IIa), Z 5 and Z 6 In some cases of the fused tricyclic comonomer of formula (IIa), Z 5 and Z 6 Both are independently CR. In some embodiments of the fused tricyclic comonomer of formula (IIa), Z 5 CR and Z 6 is N. In certain cases of the fused tricyclic comonomer of formula (IIa), Z 5 N and Z 6 In certain cases of the fused tricyclic comonomer of formula (IIa), Z 5 and Z6 In certain cases of the fused tricyclic comonomer of formula (IIa), Z 5 and Z 6 Both are CH. In some embodiments of the fused tricyclic comonomer of formula (IIa), Z 5 CR and Z 6 is N. In certain cases of the fused tricyclic comonomer of formula (IIa), Z 5 N and Z 6 In some cases, R is H, halogen, alkoxy, substituted alkoxy, alkyl, and substituted alkyl. In some cases, R is fluoro, methoxy, lower alkyl, or substituted lower alkyl.

[0115] In some embodiments, the polymeric dye has the structure of Formula (IIb):

[0116]

[0117] Wherein u, v, w, x, y and z represent the mole % value of each comonomer in the multichromophore. It should be understood that any polymer dye described herein can also be described by a formula including a mole % value for each comonomer present, such as a formula corresponding to formula (IIa)-(IIb). It should be understood that any polymer dye of formula (Ia) and (Ic) can be represented by formula (II). In some cases of formula (IIa)-(IIb), u+w+y accounts for 5% or more (e.g., 5 mole %) of the molar concentration of the multichromophore, such as 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more or even more. In some cases, z is 0. In some cases of formula (IIa)-(IIb), M 1 、M 2 and M 3 Each is a thiophene-containing comonomer. In some cases of formula (II), M 1 is a thiophene-containing comonomer. In some cases of Formulas (IIa)-(IIb), M 2 is a thiophene-containing comonomer. In some cases of Formulas (IIa)-(IIb), M 3is a thiophene-containing comonomer. In some cases of Formulas (IIa)-(IIb), v+x+z accounts for 80% or less (e.g., 80 mol%), such as 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or even less, based on the molar concentration of the multichromophore.

[0118] In some cases of Formulas (IIa)-(IIb), u is 5% or more, e.g., 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or even more. In some cases of Formulas (IIa)-(IIb), w is 5% or more, e.g., 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or even more. In some cases of Formula (IIa)-(IIb), y is 1% or more, e.g., 2% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or even more. In some cases of Formula (IIa)-(IIb), v is 80% or less, e.g., 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or even less. In some cases of Formula (IIa)-(IIb), x is 80% or less, e.g., 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or even less. In some cases of Formula (IIa)-(IIb), z is 80% or less, e.g., 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or even less.

[0119] In some embodiments of Formulas (I)-(IIb), the polymeric dye has the structure of Formula (IIIa):

[0120]

[0121] In some cases of formula (IIIa), the fused tricyclic comonomer is selected from fluorene, carbazole, silole, and bridged biphenyl comonomers. In some cases of formula (IIIa), Z 5 and Z 6 In some cases of Formula (IIIa), Z 5 and Z 6 Both are independently CR. In some embodiments of Formula (IIIa), Z 5 CR and Z 6 is N. In certain cases of Formula (IIIa), Z 5 N and Z 6 In certain cases of Formula (IIIa), Z 5 and Z 6 In certain cases of Formula (IIIa), Z 5 and Z 6 Both are CH. In some embodiments of Formula (IIIa), Z 5 CR and Z 6 is N. In certain cases of Formula (IIIa), Z 5 N and Z 6 In some cases, R is H, halogen, alkoxy, substituted alkoxy, alkyl, and substituted alkyl. In some cases, R is fluoro, methoxy lower alkyl, or substituted lower alkyl.

[0122] In some embodiments of Formulas (I)-(IIIa), the polymeric dye has the structure of Formula (IIIb):

[0123]

[0124] Where n is an integer from 1 to 100,000 and M 1 is a comonomer containing thiophene. In certain embodiments of formula (III)-(IIIb), Y 1 C(R 3 )2. In certain embodiments of formula (III)-(IIIb), Y 1 -C(R 3 )2C(R 3 )2-. In some cases of Formula (IIIa)-(IIIb), Y 2 -CHR 3 CHR 3 -. In certain cases of Formula (IIIa)-(IIIb), Y 1 NR 3 In certain embodiments of Formula (III)-(IIIb), Y 1 Si(R3 )2. In some cases, each R 3 R is independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido, substituted sulfonamido, and WSG. In certain cases, at least one R 3 In some cases, two R 3 The groups are independently WSG.

[0125] In some embodiments of Formulas (I)-(IIa), the polymeric dye has the structure of Formula (IVa):

[0126]

[0127] Where: M 1 and M 2 Each is independently a thiophene-containing comonomer; each n and m is independently an integer from 1 to 10,000; and p is an integer from 1 to 100,000. In some cases of formula (IVa), each fused tricyclic comonomer has the same core structure, such as a fluorene, carbazole, silole, or bridged biphenyl comonomer. In some cases of formula (IVa), each fused tricyclic comonomer is independently selected from fluorene, carbazole, silole, and bridged biphenyl comonomers. In some cases of formula (IVa), the fused tricyclic comonomer is selected from fluorene, carbazole, silole, and bridged biphenyl comonomers. In certain cases of formula (IVa), Z 5 and Z 6 In some cases of Formula (IVa), Z 5 and Z 6 Both are independently CR. In some embodiments of Formula (IVa), Z 5 CR and Z 6 is N. In certain cases of Formula (IVa), Z 5 N and Z 6 In certain cases of Formula (IVa), Z 5 and Z 6 In certain cases of Formula (IVa), Z 5 and Z 6 Both are CH. In some embodiments of Formula (IVa), Z 5 CR and Z 6 is N. In certain cases of Formula (IVa), Z 5 N and Z 6In some cases, R is H, halogen, alkoxy, substituted alkoxy, alkyl, and substituted alkyl. In some cases, R is fluoro, methoxy lower alkyl, or substituted lower alkyl.

[0128] In some embodiments of Formulas (I)-(IVa), the polymeric dye has the structure of Formula (IVb):

[0129]

[0130] Where: M 1 and M 2 each is independently a thiophene-containing comonomer; each n and m is independently an integer from 1 to 10,000; and p is an integer from 1 to 100,000. In certain instances of Formulas (IVa)-(IVb), Y 1 NR 3 ; Y 2 C(R 3 )2; and M 1 and M 2 are the same thiophene-containing comonomer. In certain cases of formula (IVa)-(IVb), Y 1 C(R 3 )2;Y 2 NR 3 ; and M 1 and M 2 are the same thiophene-containing comonomer. In certain cases of formula (IVa)-(IVb), Y 1 and Y 2 Each is independently C(R 3 )2; and M 1 and M 2 are the same thiophene-containing comonomer. In certain cases of formula (IVa)-(IVb), Y 1 NR 3 ; Y 2 C(R 3 )2; and M 1 and M 2 are different thiophene-containing comonomers. In certain cases of formula (IVa)-(IVb), Y 1 C(R 3 )2;Y 2 NR 3 ; and M 1 and M 2 are different thiophene-containing comonomers. In certain cases of formula (IVa)-(IVb), Y 1 and Y 2 Each is independently C(R 3 )2; and M1 and M 2 are different thiophene-containing comonomers. In certain cases of formula (IVa)-(IVb), Y 1 and Y 2 Each is independently Si(R 3 )2. In certain cases of Formula (IVa)-(IVb), Y 1 Si(R 3 )2. In certain cases of Formula (IVa)-(IVb), Y 2 Si(R 3 )2. In some cases of Formulas (IVa)-(IVb), Y 1 -C(R 3 )2C(R 3 )2-. In some cases of Formula (IVa)-(IVb), Y 1 -CHR 3 CHR 3 In some cases of Formulas (IVa)-(IVb), Y 2 -C(R 3 )2C(R 3 )2-. In some cases of Formula (IVa)-(IVb), Y 2 -CHR 3 CHR 3 In some cases, each R 3 R is independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido, and WSG. In certain cases, at least one R 3 In some cases, two R 3 The groups are independently WSG.

[0131] In certain embodiments of Formulas (I)-(IIb), the polymeric dye has the structure of Formula (Va):

[0132]

[0133] Where: M 1 and M 3Each is independently a thiophene-containing comonomer; each n and each o is independently an integer from 1 to 10,000; and p is an integer from 1 to 100,000. In some cases of Formula (Va), each fused tricyclic comonomer has the same core structure, such as a fluorene, carbazole, silole, or bridged biphenyl comonomer. In some cases of Formula (Va), each fused tricyclic comonomer is independently selected from fluorene, carbazole, silole, and bridged biphenyl comonomers. In some cases of Formula (Va), the fused tricyclic comonomer is selected from fluorene, carbazole, silole, and bridged biphenyl comonomers. In certain cases of Formula (Va), Z 5 and Z 6 In some cases of Formula (Va), Z 5 and Z 6 Both are independently CR. In some embodiments of Formula (Va), Z 5 CR and Z 6 is N. In certain cases of Formula (Va), Z 5 N and Z 6 In certain cases of formula (Va), Z 5 and Z 6 In certain cases of Formula (Va), Z 5 and Z 6 Both are CH. In some embodiments of Formula (Va), Z 5 CR and Z 6 is N. In certain cases of Formula (Va), Z 5 N and Z 6 In some cases, R is H, halogen, alkoxy, substituted alkoxy, alkyl, and substituted alkyl. In some cases, R is fluoro, methoxy lower alkyl, or substituted lower alkyl.

[0134] In certain embodiments of Formulas (I)-(IIb), the polymeric dye has the structure of Formula (Vb):

[0135]

[0136] Where: M 1 and M 3 each is independently a thiophene-containing comonomer; each n and each o is independently an integer from 1 to 10,000; and p is an integer from 1 to 100,000. In some cases of Formulas (Va)-(Vb), Y 3 -Z 1 C(R 3 )(T 1 -Z 1 ), where Z 1is a chemoselective functional group or a linked signal chromophore and T 1 In some cases of Formulas (Va)-(Vb), Y 3 -Z 1 NT 1 -Z 1 , where Z 1 is a chemoselective functional group or a linked signal chromophore and T 1 In some cases of Formulas (Va)-(Vb), Y 3 -Z 1 Si(R 3 )(T 1 -Z 1 ), where Z 1 is a chemoselective functional group or a linked signal chromophore and T 1 In some cases, Z 1 is a chemically selective functional group. 1 is a linked signal chromophore. In some embodiments of Formulas (Va)-(Vb), Y 1 NR 3 ; and M 1 and M 3 are the same thiophene-containing comonomer. In some embodiments of Formulas (Va)-(Vb), Y 1 C(R 3 )2; and M 1 and M 3 are the same thiophene-containing comonomer. In some cases of Formulas (Va)-(Vb), Y 1 NR 3 ; and M 1 and M 3 are different thiophene-containing comonomers. In some embodiments of Formulas (Va)-(Vb), Y 1 C(R 3 )2; and M 1 and M 3 are different thiophene-containing comonomers. In some cases of formula (Va)-(Vb), Y 1 -C(R 3 )2C(R 3 )2-. In some cases of Formulas (Va)-(Vb), Y 1 -CHR 3 CHR 3 -. In some embodiments of Formula (Va)-(Vb), Y 1 Si(R 3 )2. In some embodiments of Formula (Va)-(Vb), M 1 and M3 are the same thiophene-containing comonomer. In some embodiments of Formulas (Va)-(Vb), M 1 and M 3 are different comonomers containing thiophene.

[0137] In certain embodiments of Formulas (I)-(II), the polymeric dye has the structure of Formula (VIa):

[0138]

[0139] Where: M 1 and M 2 At least one of the monomers is a thiophene-containing comonomer; each n and each o are independently an integer from 1 to 10,000; Z 1 is a linked chemoselective functional group or a linked signal chromophore; and p is an integer from 1 to 100,000. In some cases of Formula (VIa), each fused tricyclic comonomer has the same core structure, such as fluorene, carbazole, silole, or a bridged biphenyl comonomer. In some cases of Formula (VIa), each fused tricyclic comonomer is independently selected from fluorene, carbazole, silole, and a bridged biphenyl comonomer. In certain cases of the fused tricyclic comonomer of Formula (VIa), Z 5 and Z 6 In some cases of the fused tricyclic comonomer of formula (VIa), Z 5 and Z 6 Both are independently CR. In some embodiments of the fused tricyclic comonomer of formula (VIa), Z 5 CR and Z 6 is N. In certain cases of the fused tricyclic comonomer of formula (VIa), Z 5 N and Z 6 In certain cases of the fused tricyclic comonomer of formula (VIa), Z 5 and Z 6 In certain cases of the fused tricyclic comonomer of formula (VIa), Z 5 and Z 6 Both are CH. In some embodiments of the fused tricyclic comonomer of formula (VIa), Z 5 CR and Z 6 is N. In certain cases of the fused tricyclic comonomer of formula (VIa), Z 5 N and Z 6 In some cases, R is H, halogen, alkoxy, substituted alkoxy, alkyl, and substituted alkyl. In some cases, R is fluoro, methoxy lower alkyl, or substituted lower alkyl.

[0140] In certain embodiments of Formulas (I)-(VIa), the polymeric dye has the structure of Formula (VIb):

[0141]

[0142] Where: M 1 and M 2 At least one of the monomers is a thiophene-containing comonomer; each n and each o are independently an integer from 1 to 10,000; Z 1 is an attached chemoselective functional group or an attached signal chromophore; and p is an integer from 1 to 100,000. In some cases of Formulas (VIa)-(VIb), M 1 and M 2 Each is independently a thiophene-containing comonomer. In some cases of Formulas (VIa)-(VIb), Z 1 is a chemoselective functional group attached. In some cases of Formulas (VIa)-(VIb), Z 1 is a linked signal chromophore. In some embodiments of Formulas (VIa)-(VIb), Y 1 NR 3 In some embodiments of Formula (VIa)-(VIb), Y 1 C(R 3 )2. In some embodiments of Formula (VIa)-(VIb), Y 2 NR 3 In some embodiments of Formula (VIa)-(VIb), Y 2 C(R 3 )2. In certain cases of Formulas (VIa)-(VIb), Y 1 and Y 2 Each is independently Si(R 3 )2. In certain cases of Formulas (VIa)-(VIb), Y 1 Si(R 3 )2. In certain cases of Formulas (VIa)-(VIb), Y 2 Si(R 3 )2. In some cases of Formulas (VIa)-(VIb), Y 1 -C(R 3 )2C(R 3 )2-. In some cases of Formulas (VIa)-(VIb), Y 1 -CHR 3 CHR 3 In some cases of Formulas (VIa)-(VIb), Y 2 -C(R 3 )2C(R3 )2-. In some cases of Formulas (VIa)-(VIb), Y 2 -CHR 3 CHR 3 In some cases of Formulas (VIa)-(VIb), M 1 and M 2 having the same essential unsubstituted thiophene-containing comonomer, except that M 2 Includes connected Z 1 , and M 1 Optionally, R 3 In some cases of Formulas (VIa)-(VIb), M 1 and M 2 are different thiophene-containing comonomers. In some cases, each R 3 R is independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido, and WSG. In certain cases, at least one R 3 In some cases, two R 3 The groups are independently WSG.

[0143] Thiophene-containing comonomers

[0144] Aspects of the present disclosure include water-solvated polymeric dyes (e.g., as described herein, e.g., polymeric dyes of one of formulas (I)-(VIb)), which include a comonomer containing thiophene. The term "comonomer containing thiophene" refers to a heterocyclic group or substituted heterocyclic group that includes a thiophene ring and is capable of conjugating to a π-conjugated polymer backbone via an adjacent comonomer. Thiophene is a 5-membered aromatic heterocyclic ring having the formula C4H4S, which may be substituted with any suitable substituent (e.g., at the 2, 3, 4 and / or 5 positions and / or connected or fused to any suitable aryl or heteroaryl ring system (e.g., fused to an additional ring at the 2,3-position, 3,4-position and / or 4,5-position). The comonomer is divalent and is thus substituted at two sites on the ring system to covalently attach to the unsaturated backbone of the conjugated polymer. In some cases, the thiophene ring of the comonomer is attached to the conjugated polymer backbone via the 2 and / or 5 positions of the thiophene ring.

[0145]

[0146] Any suitable thiophene-containing heterocyclic group is suitable for use as a thiophene-containing comonomer in the subject conjugated polymer. Thiophene-containing heterocyclic groups of interest include, but are not limited to, thiophene, 1-benzothiophene, 2-benzothiophene, dibenzothiophene, and polythiophenes, such as fused dithiophenes or fused terthiophenes, which groups may be attached to the conjugated polymer via any two suitable positions of the group and may be optionally further substituted with, for example, WSG. In some cases, the thiophene-containing comonomer is a single ring (e.g., a substituted thiophene). In some cases, the thiophene-containing comonomer comprises a fused thiophene ring, such as 1-benzothiophene, 2-benzothiophene, and substituted versions thereof. In some cases, the thiophene-containing comonomer comprises an aryl or heteroaryl ring system as a substituent not directly attached to the conjugated polymer backbone.

[0147] In some embodiments of Formulas (I)-(VIb), the thiophene-containing comonomer (M 1 -M 3 ) is selected from the group consisting of formula (VII), (VIII) and (IX):

[0148]

[0149] in:

[0150] A1 and A2 are each a fused monocyclic or bicyclic group;

[0151] Every R 3 independently selected from H, amino, substituted amino, halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido, substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy, WSG, and -T 2 -Z 2 , where Z 2 is a chemoselective functional group or a linked acceptor chromophore, and T 2 For the joint;

[0152] n is 0, 1, 2, 3 or 4; and

[0153] Each * is a covalent attachment site to the unsaturated backbone of the conjugated polymer. In some cases of Formulas (I)-(VIb), the thiophene-containing comonomer (e.g., M 1 、M 2 and / or M 3 ) is described by formula (VII). In some cases of formulas (I)-(VIb), the thiophene-containing comonomer (e.g., M 1 、M 2 and / or M 3) is described by formula (VIII). In some cases of formulas (I)-(VIb), the thiophene-containing comonomer (e.g., M 1 、M 2 and / or M 3 ) is described by formula (IX).

[0154] Condensed monocyclic or bicyclic groups of interest include fused carbocyclic and heterocyclic groups having one or two 5, 6 and / or 7-membered rings, which when fused to a thiophene ring form a fused bicyclic or tricyclic ring system. The fused carbocyclic and heterocyclic rings may be saturated or unsaturated. In some cases of formula (VIII)-(IX), A1 and A2 are fused monocyclic groups, such as fused benzo rings, fused pyridine rings, fused benzothiophene ring systems or fused 7-membered heterocycles. In some cases of formula (VIII)-(IX), A1 and A2 are selected from fused monocyclic heterocycles, fused monocyclic heteroaryls and fused bicyclic heteroaryls.

[0155] In some embodiments of Formulas (VII)-(IX), the thiophene-containing comonomer is selected from Formulas (X)-(XVII):

[0156]

[0157] in:

[0158] X 1 CR 3 NR 3 , O or S;

[0159] Every X 2 are independently N, O or S, wherein when X 2 When N, the adjacent is a double bond and when X 2 When it is O or S, the adjacent is a single bond;

[0160] Every X 3 are independently O, S or CH;

[0161] R 1 and R 2 together to form optionally one or more R 3 A 5- or 6-membered fused aromatic or heteroaromatic ring substituted with a group; and

[0162] Y 4 NR 3 , C(R 3 )2 or Si(R 3 )2.

[0163] In certain embodiments of Formula (XV), X 1 CR 3In certain embodiments of Formula (XV), X 1 In certain cases of Formula (XV), X 1 NR 3 In certain cases of Formula (XV), X 1 is O. In some embodiments of Formula (XV), X 1 is S. In certain cases of Formula (XV), R 3 is WSG and X 1 It is O or S.

[0164] In certain embodiments of Formulas (XII)-(XIII), each X 2 is N. In certain embodiments of Formulas (XIII)-(XIV), each X 2 is O. In certain embodiments of Formulas (XIII)-(XIV), each X 2 is S. In certain embodiments of Formula (XII), each X 3 is O. In certain embodiments of Formula (XII), each X 3 is S. In certain embodiments of Formula (XII), each X 3 For CH.

[0165] In some embodiments of the polymeric dye, the thiophene-containing comonomer (e.g., comonomer M 1 -M 3 ) are independently selected from formula (XVIII) and (XIX):

[0166]

[0167] in:

[0168] R 1 and R 2 together to form optionally one or more R 3 A 5- or 6-membered fused aromatic or heteroaromatic ring substituted with a group; and

[0169] Every R 3 independently selected from H, amino, substituted amino, halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido, substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy, and -T 2 -Z 2 , where Z 2 is a chemoselective functional group or a linked acceptor chromophore, and T 2 For the connector.

[0170] In some embodiments of Formulas (I)-(XVII), the thiophene-containing comonomer (eg, M 1 、M 2 and / M 3 ) is described by one of the following structures (a) to (q):

[0171]

[0172]

[0173] in:

[0174] Every R 4 independently selected from H, amino, substituted amino, halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido, substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy, WSG, and -T 2 -Z 2 , where Z 2 is a chemoselective functional group or a linked signal chromophore, and T 2 is a connector; and

[0175] Each s is 0 or an integer from 1 to 50. In some cases, each s 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 is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some cases, each s is 3. In some cases, each s is 4. In some cases, each s is 5. In some cases, each s is 6. In some cases, each s is 7. In some cases, each s is 8. In some cases, each s is 9. In some cases, each s is 10. In some cases, each s is 11. In some cases, at least one R 4 -T 2 -Z 2 In some embodiments, T 2 is an alkyl linker, such as a C1-C6 alkyl linker. 2 is a substituted alkyl linker. In some embodiments, T 2 is an alkoxy linker (eg, -O-alkyl-). In some embodiments, T 2 is a substituted alkoxy group. 2 is a chemically selective functional group. 2 is a linked signal chromophore. In some cases, each R 4 is WSG. In some cases, each R 4is a substituted alkyl group. In some cases, each R 4 is an alkoxy group or a substituted alkoxy group.

[0176] Thiophene-containing comonomers (e.g., comonomer M 1 -M 3 ) can be any suitable fused three-ring comonomer. Three-ring comonomers of interest include, but are not limited to, those comprising two thiophene rings connected via a fused central 5- or 6-membered aryl or heteroaryl ring. The fused central ring can be a pyrrole, a benzo ring, or a cyclopentadiene. In some embodiments of the polymeric dye, a thiophene-containing comonomer (e.g., comonomer M 1 -M 3 ) are independently selected from formula (e)-(i):

[0177]

[0178] Among them: each R 4 independently selected from H, amino, substituted amino, halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido, substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy, and -T 2 -Z 2 , where Z 2 is a chemoselective functional group or a linked acceptor chromophore, and T 2 is a linker; and each s is 0 or an integer from 1 to 50 (eg, s is 1 to 20, etc., for example, s is 11).

[0179] In some embodiments of formula (I)-(XVI), at least one thiophene-containing comonomer is described by structure (a). In some embodiments of formula (I)-(XVI), at least one thiophene-containing comonomer is described by structure (b). In some embodiments of formula (I)-(XVI), at least one thiophene-containing comonomer is described by structure (c). In some embodiments of formula (I)-(XVI), at least one thiophene-containing comonomer is described by structure (d). In some embodiments of formula (I)-(XVI), at least one thiophene-containing comonomer is described by structure (e). In some embodiments of formula (I)-(XVI), at least one thiophene-containing comonomer is described by structure (f). In some embodiments of formula (I)-(XVI), at least one thiophene-containing comonomer is described by structure (g). In some embodiments of formula (I)-(XVI), at least one thiophene-containing comonomer is described by structure (h). In some embodiments of formula (I)-(XVI), at least one thiophene-containing comonomer is described by structure (i). In some embodiments of formula (I)-(XVI), at least one thiophene-containing comonomer is described by structure (j). In some embodiments of formula (I)-(XVI), at least one thiophene-containing comonomer is described by structure (k). In some embodiments of formula (I)-(XVI), at least one thiophene-containing comonomer is described by structure (l). In some embodiments of formula (I)-(XVI), at least one thiophene-containing comonomer is described by structure (m). In some embodiments of formula (I)-(XVI), at least one thiophene-containing comonomer is described by structure (n). In some embodiments of formula (I)-(XVI), at least one thiophene-containing comonomer is described by structure (o). In some embodiments of formula (I)-(XVI), at least one thiophene-containing comonomer is described by structure (p). In some embodiments of Formulae (I)-(XVI), at least one thiophene-containing comonomer is described by structure (q).

[0180] In some embodiments of Formulas (I)-(XVII) and (a)-(q), the thiophene-containing comonomer (e.g., M 1 、M 2 and / M 3 ) is described by one of the following structures (ba) to (bu):

[0181]

[0182]

[0183]

[0184] In some cases, at least one thiophene-containing comonomer (e.g., M 1 、M 2 and / M 3 ) has structure (ba). In some cases, at least one thiophene-containing comonomer (e.g., M 1 、M 2 and / M 3 ) has structure (bb). In some cases, at least one thiophene-containing comonomer (e.g., M 1 、M 2 and / M 3 ) has structure (bc). In some cases, at least one thiophene-containing comonomer (e.g., M 1 、M 2 and / M 3 ) has structure (bd). In some cases, at least one thiophene-containing comonomer (e.g., M 1 、M 2 and / M 3 ) has structure (be). In some cases, at least one thiophene-containing comonomer (e.g., M 1 、M 2 and / M 3 ) has structure (bf). In some cases, at least one thiophene-containing comonomer (e.g., M 1 、M 2 and / M 3 ) has structure (bg). In some cases, at least one thiophene-containing comonomer (e.g., M 1 、M 2 and / M 3 ) has structure (bh). In some cases, at least one thiophene-containing comonomer (e.g., M 1 、M 2 and / M 3 ) has structure (bi). In some cases, at least one thiophene-containing comonomer (e.g., M 1 、M 2 and / M 3 ) has structure (bj). In some cases, at least one thiophene-containing comonomer (e.g., M 1 、M 2 and / M 3 ) has structure (bk). In some cases, at least one thiophene-containing comonomer (e.g., M 1 、M 2 and / M 3 ) has structure (bl). In some cases, at least one thiophene-containing comonomer (e.g., M 1 、M 2 and / M3 ) has the structure (bm). In some cases, at least one thiophene-containing comonomer (e.g., M 1 、M 2 and / M 3 ) has structure (bn). In some cases, at least one thiophene-containing comonomer (e.g., M 1 、M 2 and / M 3 ) has structure (bo). In some cases, at least one thiophene-containing comonomer (e.g., M 1 、M 2 and / M 3 ) has the structure (bp). In some cases, at least one thiophene-containing comonomer (e.g., M 1 、M 2 and / M 3 ) has structure (bq). In some cases, at least one thiophene-containing comonomer (e.g., M 1 、M 2 and / M 3 ) has the structure (br). In some cases, at least one thiophene-containing comonomer (e.g., M 1 、M 2 and / M 3 ) has structure (bs). In some cases, at least one thiophene-containing comonomer (e.g., M 1 、M 2 and / M 3 ) has structure (bt). In some cases, at least one thiophene-containing comonomer (e.g., M 1 、M 2 and / M 3 ) has structure (bu).

[0185] In some embodiments of Formulas (I)-(VI), the polymeric dye includes a conjugated segment having one of the following structures:

[0186]

[0187]

[0188]

[0189] Where n is 1 to 100,000 and each R 11 , R 12 and R 13 are independently substituted alkyl, substituted aralkyl or WSG.

[0190] In some embodiments of Formulas (I), (Ib), (II), and (V), the polymeric dye includes a conjugated segment having one of the following structures:

[0191]

[0192]

[0193] wherein n and o are independently 1 to 10,000 and each R 11 , R 12 and R 13 is independently a substituted alkyl, a substituted aralkyl or WSG. In some embodiments of formula (I), (Ib), (II) and (VI), the polymeric dye comprises a conjugated segment having the following structure:

[0194]

[0195] wherein n and m are independently 1 to 10,000 and each R 11 is independently substituted alkyl, substituted aralkyl or WSG. In some embodiments of Formulas (I), (Ib), (II) and (VI), the polymeric dye includes a conjugated segment having one of the following structures:

[0196]

[0197] wherein n and m are independently 1 to 10,000 and each R 11 , R 12 and R 13 are independently substituted alkyl, substituted aralkyl or WSG.

[0198] In some cases, each R of the conjugated segment 11 , R 12 and R 13 Independently selected from the following substituents:

[0199]

[0200] wherein each s is an integer from 1 to 50. In some cases, each s 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 is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some cases, each s is 3. In some cases, each s is 4. In some cases, each s is 5. In some cases, each s is 6. In some cases, each s is 7. In some cases, each s is 8. In some cases, each s is 9. In some cases, each s is 10. In some cases, each s is 11. In some cases, each R 11 is a substituent (yy), wherein s is 3 to 20 (e.g., 11). In certain cases, each R 12 is a substituent (zz), wherein s is 3 to 20 (e.g., 11). In certain cases, each R 13 is a substituent (xx), wherein s is 3 to 20 (eg, 11). In certain embodiments of any of the conjugated segment structures, the terminal end of the polymer dye includes the following linker.

[0201]

[0202] Water-solubilizing substituents

[0203] The subject polymer dye can be water solvated.Any suitable water solubilizing group (WSG) can be included in the multichromophore described herein (for example the multichromophore of formula (I)-(VIb), formula (VII)-(XVII) and the comonomer of (a)-(q)), to provide the water solubility of increase.Although the increase of solubility may change, in some cases, increase (compared with the compound without WSG) 2 times or more, for example 5 times, 10 times, 25 times, 50 times, 100 times or more.As used herein, the terms "water solubilizing group", "water-soluble group" and WSG are used interchangeably and refer to in aqueous environment, for example, fully solvated under physiological conditions and give the molecule connected thereto with the water-soluble group or substituent group of raising.In some embodiments, compared with the contrast multichromophore lacking WSG, WSG increases the solubility of multichromophore in main aqueous solution.In some cases, the WSG of multichromophore is a water-soluble nonionic side group that can give more than 10mg / mL. The water solubilizing group can be any suitable hydrophilic group that is fully solvated in an aqueous environment. 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 a hydrophilic polymer, such as polyethylene glycol, modified PEG, peptide sequence, peptoid, carbohydrate, oxazoline, polyol, dendritic structure, 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 glycol (PEG) and modified PEGs, hydroxyls, amines, amino acids, ammonium, guanidinium, pyridinium, polyamines and sulfoniums, polyols, linear or cyclic sugars, primary, secondary, tertiary or quaternary ammonium and polyamines, phosphonate groups, phosphinate groups, ascorbic acid groups, glycols including polyethers, -COOM', -SO3M', -PO3M', -NR3 + 、Y′、(CH2CH2O) p R and mixtures thereof, wherein Y' may be any halogen, sulfate, sulfonate or oxyanion, p may be from 1 to 500, each R may independently be H or an alkyl group (e.g., methyl) and M' may be a cationic counterion or hydrogen, --(CH2CH2O) yy CH2CH2XR yy 、--(CH2CH2O) yy CH2CH2X--、--X(CH2CH2O) yy CH2CH2--, glycol and polyethylene glycol, wherein yy is selected from 1 to 1000, and X is selected from O, S and NR ZZ , and R ZZ and R YY Independently selected from H and C 1-3In 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).

[0204] 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 with 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 cases, the substituent is (CH2)3(OCH2CH2) 11 In some embodiments, one or more of the substituents is selected from (CH2) x (OCH2CH2) y A benzyl group substituted with a WSG group (e.g., one or two WSG groups) of OCH3, wherein each x is independently an integer from 0 to 20, and each y is independently an integer from 0 to 50.

[0205] Multiple WSG can be included in a single position in the subject multichromophore via a branching linker. In certain embodiments, the branching linker is an aralkyl substituent, further substituted with a water-solubilizing group. Thus, in some cases, the branching linker group is a substituent of a multichromophore that connects the multichromophore to two or more water-solubilizing groups. In certain embodiments, the branching linker is an amino acid, such as a lysine amino acid that is connected to three groups via an amino group and a carboxylic acid group. In some cases, the incorporation of multiple WSGs into the multichromophore via branching linkers gives the multichromophore a desirable solubility. In some cases, WSG is a nonionic side chain group that can impart a water solubility exceeding 10 mg / mL.

[0206] In some embodiments, the multichromophore includes substituents selected from the group consisting of alkyl, aralkyl, and heterocyclic groups, each of which is further substituted with a water solubilizing group, a hydrophilic polymer group, such as polyethylene glycol (PEG), such as 2-20 units of a PEG group.

[0207] In certain cases of any of Formulae (I)-(XVII) and (a)-(q), one or more of the comonomers may be substituted independently selected from H, substituted aryl, alkyl, and a group of one of the following structures (e.g., R of the depicted formula: 3 and R 4 Substituent) substituted:

[0208]

[0209] Among them: Each T 5 is independently an optional linker; and each s is an integer from 1 to 50. In some cases, each s 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 is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some cases, each s is 3. In some cases, each s is 4. In some cases, each s is 5. In some cases, each s is 6. In some cases, each s is 7. In some cases, each s is 8. In some cases, each s is 9. In some cases, each s is 10. In some cases, each s is 11. In some 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-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 a hydroxyl-terminated PEG chain can be used in place of a methoxy-terminated PEG chain in any of the WSG groups described herein.

[0210] In some cases, the WSG is a dendritic structure selected from one of the following structures:

[0211]

[0212] In some cases, WSG is a polyol selected from one of the following structures:

[0213]

[0214] In some cases, WSG is an oxazoline of the following structure:

[0215]

[0216] In some cases, WSG is a peptoid selected from one of the following structures:

[0217]

[0218] Polymer tandem dyes

[0219] The water-soluble light-harvesting multichromophore itself can fluoresce and can transfer energy to the connected acceptor signal chromophore. Thus, the subject polymer tandem dye also includes a covalently linked acceptor signal chromophore that is close to the donor water-solvated light-harvesting multichromophore in energy reception. Thus, excitation of the multichromophore donor causes energy transfer to the covalently linked acceptor signal chromophore and emission from the covalently linked acceptor signal chromophore. The number of signal chromophore acceptor units connected to the donor water-solvated light-harvesting multichromophore can vary, with values ​​in some cases ranging from 1 mol % to 50 mol %, such as 5 mol % to 25 mol % or 10 mol % to 25 mol %.

[0220] Mechanisms for energy transfer from a fluorescent water-solvated light-harvesting multichromophore donor to an attached acceptor signal chromophore include, for example, resonance energy transfer (e.g., Forster (or fluorescence) resonance energy transfer, FRET), quantum charge exchange (Dexter energy transfer), and the like. In some cases, these energy transfer mechanisms are relatively short-range; that is, the close proximity of the light-harvesting multichromophore system to the acceptor provides efficient energy transfer. In some cases, under conditions of efficient energy transfer, emission from the acceptor is enhanced, wherein emission from the luminescent signal chromophore is more intense when the incident light ("pump light") is at a wavelength absorbed by the light-harvesting multichromophore than when the luminescent signal chromophore is directly excited by the pump light.

[0221] "Effective" energy transfer means that 10% or more, such as 20% or more or 30% or more of the energy captured by the donor is transferred to the acceptor. "Enhancement" means that the signal from the signal chromophore is 1.5 times or more when excited by energy transfer from the donor light-harvesting 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 direct excitation of the signal chromophore with incident light, the signal chromophore emits 1.5 times or more, such as 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 greater than direct excitation of the signal chromophore with incident light.

[0222] The linked luminescent signal chromophore emission of the polymer tandem dye can have a quantum yield of 0.03 or more, such as 0.04 or more, 0.05 or more, 0.06 or more, 0.07 or more, 0.08 or more, 0.09 or more, 0.1 or more, 0.15 or more, 0.2 or more, 0.3 or more, or even more. In some cases, the polymer tandem dye has a quantum yield of 5×10 5 cm -1 M -1 or more, e.g. 6×10 5 cm -1 M -1 or more, 7×10 5 cm -1 M -1 or more, 8×10 5 cm - 1 M -1 or more, 9×10 5 cm -1 M -1 or more, e.g. 1×10 6 cm -1 M -1 or more, 1.5×10 6 cm -1 M -1 or more, 2×10 6 cm -1 M -1 or more, 2.5×10 6 cm -1 M -1 or more, 3×10 6 cm -1 M -1 or more, 4×10 6 cm -1 M -1 or more, 5×10 6 cm -1 M -1 or more, 6×10 6 cm -1 M -1 or more, 7×10 6 cm -1 M -1 or more or 8×10 6 cm -1 M -1 In some embodiments, the polymer tandem dye has an extinction coefficient of 5×10 5 M -1 cm -1In some embodiments, the polymer tandem dye has a molar extinction coefficient of 1×10 6 M -1 cm -1 or more molar extinction coefficient.

[0223] The subject polymer tandem dye provides fluorescent emission from the luminescent signal chromophore dye that is brighter than the emission from such fluorescent dye alone. The linked luminescent signal chromophore emission of the polymer tandem dye may have a 50 mM -1 cm -1 or more, e.g. 60 mM -1 cm -1 or more, 70mM -1 cm -1 or more, 80mM -1 cm -1 or more, 90mM -1 cm -1 or more, 100mM -1 cm -1 or more, 150mM -1 cm -1 or more, 200mM -1 cm -1 or more, 250mM -1 cm -1 or more, 300mM -1 cm -1 In some cases, the brightness of the emission of the linked signal chromophore of the polymer tandem dye is at least 5 times greater than the brightness of the directly excited luminescent dye, such as at least 10 times greater, at least 20 times greater, at least 30 times greater, at least 50 times greater, at least 100 times greater, at least 300 times greater, or even greater than the brightness of the directly excited luminescent dye.

[0224] In some embodiments, the polymer tandem dye has one of formula (I)-(VI), wherein the attached signal chromophore (e.g., as described herein) is included as a substituent of a comonomer. In some cases of formula (I)-(VI), the polymer tandem dye includes one or more comonomers as described by (VII)-(XVII), (a)-(q), and (ba)-(bu). Any suitable signal chromophore can be attached to any suitable polymer dye described herein via coupling of compatible chemoselective functional groups. The signal chromophore can be selected to provide a desirable emission spectrum and emission maximum wavelength.

[0225] The subject polymeric dye can include any suitable moiety of interest that can be conjugated to a side chain chemoselective functional group. Comonomers comprising such side chain groups may be referred to herein as linking comonomers. Polymeric dyes may be conjugated to signal chromophores to produce polymer tandem dyes having desirable spectral properties. In some cases, multichromophores (e.g., formula (I), (II), or (VI)) include thiophene-containing comonomers (e.g., M-1, M-2, M-3, M-4, M-5, M-6, M-7, M-8, M-9, M-10, M-11, M-12, M-13, M-14, M-15, M-16, M-17, M-18, M-19, M-21, M-22, M-23, M-24, M-25, M-26, M-27, M-28, M-29, M-30, M-31, M-32, M-33, M-34, M-35, M-36, M-37, M-38, M-39, M-40, M-41, M-42, M-43, M-44, M-45, M-46, M-47, M-48, M-49, M-50, M-51, M-52, M-53, M-54, M-55, M-56, M-57, M-58, M-59, M-60, M-61, M-62, M-63, M-74, M-75 2 or M 3 In some cases, the multichromophore (eg, Formula (I), (II), or (V)) includes a comonomer having a structure of one of Formulas (XVIII)-(XIX):

[0226]

[0227] Where: W 1 is alkyl, substituted alkyl, or WSG (e.g., as described herein); and the dye is a signal chromophore. In certain embodiments, the comonomer is attached to the signal chromophore and has one of the following structures:

[0228]

[0229] As used herein, the terms "chemoselective functional group" and "chemoselective tag" are used interchangeably and refer to a functional group that can selectively react with another compatible functional group to form a covalent bond, in some cases after optionally activating one of the functional groups. Any suitable chemoselective tag and conjugation chemistry is suitable for use in the subject multichromophores. Chemoselective functional groups of interest include, but are not limited to, thiols and thiol-reactive groups such as maleimide, iodoacetamide, or vinylsulfone, amines and carboxylic acids or their active esters, and groups that can react with each other via click chemistry, such as azides and alkynyl groups (e.g., cyclooctynyl), sulfur (VI) fluoride exchange chemistry (SuFEX), sulfonyl fluorides, tetrazines, alkynes, and aldehydes, hydroxyls, hydrazides, hydrazines, aldehydes, ketones, alkoxyamines, phosphine, epoxides, and the like.

[0230] 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 comonomers described in the formulae herein (e.g., Formulae (XVIII)-(XIX)), fluorene comonomers, phenylene vinylene comonomers, phenylene ethynylene comonomers, carbazole comonomers, C2-C 12In some cases, the linking comonomer is a thiophene-containing comonomer (e.g., as described herein). In some cases, the linking comonomer is a substituted aryl or heteroaryl comonomer, such as a fluorene comonomer (e.g., as described herein).

[0231] In some cases of any of the formulae described herein, the signal chromophore is linked to a comonomer that constitutes 1% to 50% by molar concentration of the multichromophore, e.g., 1% to 20%, 1% to 10%, or 11 to 20% by molar concentration. In some cases, the multichromophore is a conjugated polymer comprising 5 or more repeating units.

[0232] Any suitable chemoselective functional group may be included in the subject polychromophore (e.g., in -Z 1 , -Z 2 and / or G 1 or G 2 Terminal groups include, but are not limited to, carboxylic acids, active esters (e.g., NHS or sulfo-NHS esters), amino groups, hydroxyl groups, thiols, maleimides, iodoacetyl groups, hydrazides, hydrazines, aldehydes, ketones, azides, alkynes, phosphine groups, and epoxides. In some cases, the chemoselective functional groups or linkers can be directly attached to the terminal comonomers. It should be understood that in the polymer tandem dye structures described herein, in some cases, the group Z 1 and Z 2 Appearing at equivalent positions in the structure these groups may be used interchangeably to refer to either an attached signal chromophore or a chemoselective functional group that can subsequently be conjugated with a suitable chromophore precursor to produce an attached signal chromophore.

[0233] 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, naphthalimide, 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 cases, the signal chromophore (Z 1) is selected from DY 431, DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY732, 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, or an acridine dye. Fluorescent dyes of interest include, but are not limited to, fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodamine, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, Coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP (peridin 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, Sea Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY FL, BODIPY FL-Br.sub.2, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY R6G, BODIPY TMR, BODIPY TR, 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 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 a Dyomics dye (e.g., DY 431, DY 485XL, DY 500XL, DY530, DY 610, DY 633, DY 640, DY 651, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY731, DY 732, DY 734, DY 752, DY 754, DY 778, DY 782, DY 800, or DY 831), Biotium CF 555, Cy 3.5, and diethylaminocoumarin.

[0234] In some embodiments, the selected signal chromophore has an emission maximum wavelength in the range of 300 to 900 nm, e.g., 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 signal chromophores 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 ± 5 nm, 620 nm ± 5 nm, 630 nm ± 5 nm, 640 nm ± 5 nm, 650 nm ± 5 nm, m, 460nm±5nm, 470nm±5nm, 480nm±5nm, 490nm±5nm, 500nm±5nm, 510nm±5nm, 520nm±5nm, 530nm±5nm, 540nm±5nm, 550n m±5nm, 560nm±5nm, 570nm±5nm, 580nm±5nm, 590nm±5nm, 605nm±5nm, 650nm±5nm, 680nm±5nm, 700nm±5nm, 805nm±5nm.

[0235] Terminal Group

[0236] Any suitable terminal groups (e.g., G 1 and G 2 As used herein, the terms "terminal group" and "end group" are used interchangeably to refer to a group located at the end of a polymer structure, such as a multichromophore as described herein. 1 and G 2The groups include, but are not limited to, end-capping groups, π-conjugated segments, linkers, and linked specific binding members. In some embodiments, the end-capping group is a monovalent group that is conjugated to the backbone of the multichromophore after polymerization. In some cases, the end-capping group is an aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, or substituted alkyl. In some cases, the end-capping group is derived from the monomer used in the polymerization process, such as an end group that is capable of further conjugation, such as a halogen (e.g., Br), boric acid, or borate. In some cases, G 1 and / or G 2 As used herein, a π-conjugated segment refers to any suitable segment of a conjugated polymer that allows the multi-chromophore to be conjugated, i.e., allows π 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. 1 and / or G 2 The linker at the position can be selected to include side chains that may be present in multiple chromophores (e.g., Z 2 ) is orthogonal to any other linker of the chemoselective tag at G. In certain 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 .

[0237] 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 3 is a chemoselective tag (e.g., as described herein). 3 is selected from carboxylic acid, active ester (e.g., N-hydroxysuccinimidyl ester (NHS) or sulfo-NHS), amino, maleimide, iodoacetyl, and thiol.3 is a chemoselective functional group useful in bioorthogonal conjugation chemistry, such as an azide or an alkyne (e.g., cyclooctyne), sulfur(VI) fluoride exchange chemistry (SuFEX), sulfonyl fluoride, or tetrazine. In certain embodiments of the formulae described herein, G 1 and G 2 At least one of is described by the following structure:

[0238] *-Ar-LZ

[0239] wherein Ar is a π-conjugated aromatic group, L is a linker and Z is a chemoselective tag or specific binding member. In some cases, the LZ group can be directly attached to a terminal comonomer. In certain embodiments of the formula described herein, G 1 and G 2 At least one of is described by the following structure:

[0240]

[0241] in:

[0242] q is 0 or an integer from 1 to 12;

[0243] L is an optional linker; and

[0244] Z is a chemoselective tag or a specific binding member.

[0245] In certain embodiments, Z is a biomolecule. Biomolecules of interest include, but are not limited to, polypeptides, polynucleotides, carbohydrates, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs thereof, and combinations thereof. In some cases, Z is an antibody. In some cases, 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 double antibody, and a three-chain antibody. Z can be any suitable chemical selective tag. In some cases, Z is a chemical selective functional group that can be used for bioorthogonal click chemistry, such as an azide or an alkyne (e.g., a cyclooctyne), a tetrazine or an alkyne, or a sulfonyl fluoride. In some cases, Z is selected from carboxylic acids, active esters (e.g., N-hydroxysuccinimidyl esters (NHS) or sulfo-NHS), amino groups, hydroxyl groups, hydrazide groups, hydrazine groups, aldehydes, ketones, alkoxyamines, azides, alkynes, phosphine, epoxide maleimide, iodoacetyl groups, and thiols.

[0246] It will be appreciated that for any of the structures and formulae depicted herein, in some instances of the subject multichromophores, the depicted terminal groups may be located at the opposite end to the terminal group shown, e.g., terminal group G 1In some embodiments of the multichromophores described herein (eg, Formula (I)-(VIb), G 1 and G 2 At least one of the following structures 1-33 is selected:

[0247]

[0248]

[0249]

[0250] * = site of covalent attachment to the unsaturated backbone;

[0251] Wherein R' is independently H, halogen, C1-C 12 Alkyl, (C1-C 12 Alkyl)NH2, C2-C 12 Olefins, C2-C 12 Alkyne, C3-C 12 Cycloalkyl, C1-C 12 Haloalkyl, 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 with 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;

[0252] Where k is 2, 4, 8, 12 or 24;

[0253] Where R 15 Select from the group consisting of lu having structure:

[0254] * = site of covalent attachment to the backbone.

[0255] In some embodiments of the multichromophores described herein (eg, Formula (I)-(VIb)), at least one terminal group (eg, T 2 -Z 2 , G 1 , G 2 , -LZ, -L 3 -Z) or a side chain group is selected from one of the following structures:

[0256]

[0257] wherein r is 0 or an integer of 1-50; k is 0 or an integer of 1-50 (e.g., 1-20); 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 cases, 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. In some embodiments of the multichromophores described herein (e.g., Formula (I)-(VIb)), at least one terminal group (e.g., T 2 -Z 2 , G 1 , G 2 , -LZ, -L 3 -Z)

[0258] Labeled SBM

[0259] Aspects of the present disclosure include labeled specific binding members. Labeled specific binding members are conjugates of a subject polymer dye (e.g., as described herein) and a specific binding member. Any of the polymer dyes and polymer tandem dyes described herein can be bound to a specific binding member. The specific binding member and the polymer dye can be conjugated (e.g., covalently linked) to each other via an optional linker at any suitable position of the two molecules. In some cases, the end groups (e.g., G 1 or G 2) comprises at least one of the linked specific binding members.

[0260] As used herein, the term "specific binding member" refers to one member of a pair of molecules that have binding specificity for each other. One member of the pair may have an area or cavity on its surface that specifically binds to an area or cavity on the surface of the other member of the pair. Thus, the members of the pair have the property of specifically binding to each other, thereby producing a binding complex. In some embodiments, the affinity between the specific binding members in the binding complex is characterized by 10 -6 M or less, e.g. 10 -7 M or less, including 10 -8 M or less, e.g. 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -13 M or less, 10 -14 M or less, including 10 -15 M or less K d (dissociation constant). In some embodiments, the specific binding member specifically binds with high affinity. High affinity means that the binding member binds with a specific affinity determined by K d Characterization of 10×10 -9 M or less, e.g. 1x 10 -9 M or less, 3×10 -10 M or less, 1×10 -10 M or less, 3×10 -11 M or less, 1×10 -11 M or less, 3×10 -12 M or less or 1×10 -12 Specific binding with an apparent affinity of M or less.

[0261] Specific binding members can be proteins. As used herein, the term "protein" refers to a portion consisting of amino acid residues. The protein portion can be a polypeptide. In some cases, the protein-specific binding member is an antibody. In certain embodiments, the protein-specific binding member is an antibody fragment, such as a binding fragment of an antibody that specifically binds to a polymer dye. As used herein, the terms "antibody" and "antibody molecule" are used interchangeably and refer to a protein consisting of one or more polypeptides encoded by all or a portion of the immunoglobulin genes identified. For example, the immunoglobulin genes identified in humans include κ (k), λ (l) and heavy chain 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) interspersed with three hypervariable regions (also known as "complementarity determining regions" or "CDRs"). The extent of the framework regions and CDRs has been precisely defined (see "Sequences of Proteins of "Immunological Interest", E. Kabat et al., US Department of Health and Human Services, (1991)). The numbering of all antibody amino acid sequences discussed in this article follows the Kabat system. The sequences of the framework regions of different light chains or heavy chains are relatively conserved within a species. The antibody framework region, which is the combined framework region of the component light chain and heavy chain, is used to position and align the CDR. The CDR is primarily responsible for binding to the antigenic epitope of the antigen. The term antibody is intended to include full-length antibodies and may refer to natural antibodies from any organism, engineered antibodies, or antibodies recombinantly produced for experimental, therapeutic or other purposes as further defined below.

[0262] Antibody fragments of interest include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv or other antigen-binding subsequences of antibodies, antibodies produced by modification of whole antibodies or synthesized de novo using recombinant DNA technology. Antibodies may be monoclonal or polyclonal, and may have other specific activities on cells (e.g., antagonists, agonists, neutralizing, inhibitory or stimulating antibodies). It should be understood that antibodies may have additional conservative amino acid substitutions that have substantially no effect on antigen binding or other antibody functions.

[0263] In certain embodiments, the specific binding member is a Fab fragment, a F(ab')2 fragment, a scFv, a diabody or a triabody. In certain embodiments, the specific binding member is an antibody. In some cases, the specific binding member is a murine antibody or a binding fragment thereof. In some cases, the specific binding member is a recombinant antibody or a binding fragment thereof.

[0264] In some embodiments, the labeled specific binding member comprises: a water-solvated light-harvesting multichromophore (e.g., as described herein); and a signal chromophore covalently linked to the multichromophore in energy-accepting proximity (e.g., as described herein); and a specific binding member covalently linked to the multichromophore. In some cases of the labeled specific binding member, a multichromophore of any formula described herein (e.g., formula (V) and (VII)-(VIII)), wherein: G 1 and G 2 Each is independently selected from 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.

[0265] method

[0266] As outlined above, aspects of the present invention include methods for assessing the presence of a sample for a target analyte. Aspects of the method include contacting a sample with a polymer dye conjugate that specifically binds to a target analyte, producing a sample contacted with a labeled composition. As used herein, the terms "polymer dye conjugate" and "labeled specific binding member" are used interchangeably. Thus, 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 cases, the polymer dye conjugate also includes a signal chromophore that is covalently attached to a multichromophore that is close in energy reception.

[0267] Any suitable method can be used to contact the sample with the polymer dye conjugate that specifically binds to the target analyte, and produce the sample that the labeling composition contacts. In some cases, the sample is contacted with the polymer dye conjugate under the condition that the specific binding member is specifically bound to the target analyte (if present). In order to make the specific binding member of the conjugate specifically bind to the target analyte, an appropriate solution that maintains the biological activity of the sample components and the specific binding member can be used. The solution can be a balanced salt solution, such as normal saline, PBS, Hank's balanced salt solution, etc., preferably supplemented with fetal bovine serum, human platelet lysate or other factors, combined with low concentrations, such as 5-25mM acceptable buffer. Suitable buffers include HEPES, phosphate buffer, lactate buffer, etc. A variety of culture media are available and can be used according to the properties of the target analyte, including dMEM, HBSS, dPBS, RPMI, Iscove culture media, etc., supplemented with fetal bovine serum or human platelet lysate in some cases. The final component of the solution can be selected depending on the sample components included.

[0268] The temperature at which specific binding of the specific binding member of the conjugate to the target analyte occurs can vary, and in some cases, 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, such as 20°C, 25°C, 30°C, 35°C or 37°C (e.g., as described above). In some cases, the temperature at which specific binding occurs is selected to be compatible with the biological activity of the specific binding member and / or the target analyte. In some cases, the temperature is 25°C, 30°C, 35°C or 37°C. In some cases, the specific binding member is an antibody or 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 the desired amount of binding complex, and in some cases, can be 1 minute (min) or more, such as 2min or more, 10min or more, 30min or more, 1 hour or more, 2 hours or more or even 6 hours or more.

[0269] Any suitable specific binding member can be used in the conjugate. The specific binding members of interest include, but are not limited to, those agents that specifically bind to cell surface proteins of various cell types, including, but not limited to, stem cells, such as 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 erythroid cells. The target cells of interest include cells with suitable cell surface markers or antigens that can be captured by suitable specific binding member conjugates. In some embodiments, the target cells are selected from cells containing HIV, Treg cells, antigen-specific T cell colonies, tumor cells, or hematopoietic progenitor cells (CD34+) from whole blood, bone marrow, or umbilical cord blood. For polymer dye conjugates specifically bound to the subject method, any suitable cell surface protein or cell marker can be targeted. In some embodiments, the target cells include cell surface markers selected from cell receptors and cell surface antigens. In some cases, the target cells can include cell surface antigens, such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor α / β, T cell receptor γ / δ, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminal), SSEA-3, TRA-1-60 antigen, disialylganglioside GD2, and CD71.

[0270] Any suitable target can be selected for evaluation using the subject method. The target of interest includes, but is 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, glycosylated, ubiquitinated, ubiquitinated 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 its fragments. 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 and synthetic molecules, which can be prepared or commercially purchased using any suitable recombinant expression method or using any suitable synthesis method. In some embodiments, the polymer dye conjugate includes an antibody or antibody fragment. In the subject method, any suitable target analyte that can be targeted to specifically bind to an antibody or antibody fragment of interest.

[0271] In some embodiments, the target analyte is associated with a cell. In some cases, the target analyte is a cell surface marker of a cell. In some cases, the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen. In some cases, the target analyte is an intracellular target, and the method further comprises lysing the cell.

[0272] In some embodiments, sample can include the heterogeneous cell colony that isolates target cell.In some cases, sample includes peripheral whole blood, peripheral whole blood, cord blood, bone marrow, peripheral blood mononuclear cell or homogenized tissue of density gradient purification wherein red blood cell has dissolved before cell separation.In some cases, sample includes hematopoietic progenitor cell (such as CD34+ cell), bone marrow or cord blood in whole blood.In certain embodiments, sample includes tumor cell in peripheral blood.In some cases, sample is for including (or suspecting to include) virus cell (such as HIV) sample.

[0273] The specific binding members of the labeling can be used in the subject method, for example, target cells, particles, targets or analytes are labeled with polymer dyes or polymer tandem dyes. For example, the specific binding members of the labeling can be used to label cells to be processed (e.g., detected, analyzed and / or sorted) in a flow cytometer. The specific binding members of the labeling can include antibodies that specifically bind to cell surface proteins (e.g., as described herein) of, for example, various cell types. The specific binding members of the labeling can be used to study various biological (e.g., cell) properties or processes, such as cell cycle, cell proliferation, cell differentiation, DNA repair, T cell signaling, apoptosis, cell surface protein expression and / or presentation, etc. The specific binding members of the labeling can be used to include (or can include) antibody-mediated cells, any application of the labeling of particles or analytes.

[0274] In some cases of the methods, the labeled specific binding member comprises a multichromophore according to any one of formulas (I)-(VI) (e.g., as described herein). 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.

[0275] Aspects of the method include measuring the sample contacted by the labeling composition for the presence of the polymer dye conjugate-target analyte binding complex to assess whether the target analyte is present in the sample. Once the sample has been contacted with the polymer dye conjugate, any suitable method can be used to measure the sample contacted by the labeling composition for the presence of the polymer dye conjugate-target analyte binding complex. The polymer dye conjugate-target analyte binding complex is a binding complex produced after the specific binding member of the conjugate specifically binds to the target analyte (if present). Measuring the sample contacted by the labeling composition may include detecting a 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, such as via fixation on a carrier, to separate the target analyte from the sample. The determination method of interest includes, but is not limited to, any suitable method and determination format for using, paying attention to, for example, avidin-biotin or hapten-anti-hapten antibodies in pairs of specific binding members. Methods and assay formats of interest suitable for use with the subject compositions include, but are not limited to, flow cytometry, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assays, and fluorescent dye purification chromatography.

[0276] In certain embodiments, the method also includes contacting the sample with a second specific binding member that specifically binds to the target analyte. In some cases, the second specific binding member is a carrier of combination. The component (e.g., second specific binding member) of the subject method can be fixed using any suitable carrier. In some cases, the carrier is a particle, such as a magnetic particle. In some cases, the second specific binding member and the polymer dye conjugate produce a sandwich complex that can be separated and detected (if present) using any suitable method. In some embodiments, the method also includes analyzing polymer dye conjugate-target analyte binding complex, i.e., fluorescently labeled target analyte, by flow cytometry. The determination of the presence of polymer dye conjugate-target analyte binding complex can provide a determination result (e.g., qualitative or quantitative determination data), which can be used to assess whether the target analyte is present in the sample.

[0277] Any suitable component of the method can be fixed by any suitable carrier 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 paper, beads or other structures, such as a flat plate with holes; beads, polymers, particles, fiber meshes, hydrogels, porous matrices, nails, microarray surfaces, chromatography carriers, etc. In some cases, the carrier is selected from the group consisting of particles, planar solid substrates, fiber meshes, hydrogels, porous matrices, nails, microarray surfaces, and chromatography carriers. The carrier can be incorporated into a system that provides cell separation assisted by any suitable method, such as a manually operated syringe, centrifuge, or automated liquid handling system. In some cases, the carrier can be used for an automated liquid handling system for high throughput separation of cells, such as a flow cytometer.

[0278] 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 are close to the surface accumulation closest to the magnetic field gradient source, i.e., the magnet.

[0279] Separation can also include one or more optional washing steps to remove the unbound material of the sample from the carrier. Any suitable washing method can be used, such as washing the fixed carrier with a biocompatible buffer solution that keeps the specific binding interaction of the polymeric dye and the specific binding member. Separating and optionally washing the unbound material of the sample from the carrier provides an enriched target cell population in which undesirable cells and materials can be removed.

[0280] In certain embodiments, the method further comprises detecting a labeled target. Detecting a labeled target may include exciting a multichromophore with one or more lasers and subsequently detecting fluorescence emission from a polymer tandem dye using one or more photodetectors. The labeled target may 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 plate reader; high performance liquid chromatography (HPLC); and mass spectrometry. When using fluorescently labeled components in the methods and compositions of the present disclosure, it is recognized that different types of fluorescence detection systems may be used to practice the subject method. In some cases, high throughput screening may be performed, such as a system using a 96-well or larger microtiter plate. A variety of methods for performing measurements on fluorescent materials may be used, such as those described in: Lakowicz, JR, Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983); Herman, B., Resonance energy transfer microscopy, Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, Vol. 30, ed. Taylor, DL & Wang, Y.-L., San Diego: Academic Press (1989), pp. 219-243; Turro, NJ, Modern Molecular Photochemistry, Menlo Park: Benjamin / Cummings Publishing Co. (1978), pp. 296-361.

[0281] Fluorescence in a sample can be measured using a fluorometer. In some cases, excitation radiation from an excitation light source having a first wavelength passes through an excitation optical device. The excitation optical device causes the excitation radiation to excite the sample. In response, the target emission wavelength of the fluorescent marker in the sample is different from the excitation wavelength. Then the converging optical device converges the emission from the sample. The device can include a temperature controller to maintain the sample at a specific temperature during scanning. In some cases, a multi-axis translation stage moves a microtiter plate storing multiple samples to place different holes to be exposed. The multi-axis translation stage, temperature controller, self-focusing features and electronic equipment related to imaging and data collection can be managed by a properly programmed digital computer. The computer can also convert the data collected during the assay into another presentation format.

[0282] 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 conjugates-target analyte binding complexes in the contacted sample to assess the presence or absence of the target analyte. Microscopy methods of interest that can be used for the subject method include laser scanning confocal microscopy.

[0283] Also provided is a method for labeling a target molecule. The subject polymer dye can be used in a variety of methods for labeling, separation, detection and / or analysis. In some embodiments, the method comprises: contacting the target molecule with a polymer dye (e.g., as described herein) to produce a labeled target molecule, wherein the polymer dye comprises a conjugated label covalently attached to the target molecule. In some cases, the polymer dye also includes a signal chromophore that is close to the energy receiving multichromophore of the polymer dye covalently attached. In some cases of the method, the polymer dye member includes a multichromophore according to any one of formulas (I)-(VI) (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.

[0284] 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 attached to a compatible functional group of a target molecule after optional activation and / or removal of the protecting group. 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 following: amino, carboxylic acid or derivatives thereof, thiol, hydroxyl, hydrazine, hydrazide, azide, alkyne, and protein reactive groups (e.g., amino reactive, thiol reactive, hydroxyl reactive, imidazole reactive, or guanidyl reactive).

[0285] Any suitable method and reagent are applicable to the subject labeling method to covalently link the conjugated label to the target molecule. The method for labeling the target of interest includes but is not limited to those methods and reagents described in Hermanson, Bioconjugate Techniques, 3rd edition, Academic Press, 2013. The contacting step can be carried out in an aqueous solution. In some cases, the conjugated label includes an amino functional group and the target molecule includes an activated ester functional group, such as an NHS ester or a sulfo-NHS ester, or vice versa. In some cases, the conjugated label includes a maleimide functional group and the target molecule includes a thiol functional group, or vice versa. In some cases, the conjugated label includes an alkyne (e.g., cyclooctyne group) functional group and the target molecule includes an azide functional group, or vice versa, the functional group can be conjugated via click chemistry.

[0286] Any suitable target molecule can be selected for labeling using the subject method. The 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, glycosylated, ubiquitinated, ubiquitinated 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 their fragments. 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 and synthetic molecules, which can be prepared or commercially purchased using any suitable recombinant expression method or using any suitable synthesis method. In some embodiments, the target molecule is a specific binding member (e.g., as described herein). In some cases, the specific binding member is an antibody. In some cases, 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.

[0287] In some cases, the method includes a separation step, wherein the labeled target molecule is separated from the reaction mixture, such as excess reagents or unlabeled target. The target can be separated from the sample using a variety of methods, such as via immobilization on a support, precipitation, chromatography, and the like.

[0288] In some cases, the method also includes detecting and / or analyzing the target molecule of the label. In some cases, the method also includes the target molecule of the fluorescent detection label. Any suitable method can be used to detect and / or analyze the target molecule of the label in combination with the subject method and composition. The method of analyzing the target of interest that can be used for 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. The detection method of interest includes, but is not limited to, fluorescence spectroscopy, fluorescence microscopy, nucleic acid sequencing, fluorescence in situ hybridization (FISH), protein mass spectrometry, flow cytometry, etc.

[0289] Detection can be achieved directly via polymer dyes or polymer tandem dyes, or indirectly through a second 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 specific and / or non-specific antibody can be labeled and used simultaneously or continuously to enhance target detection, identification and / or analysis.

[0290] system

[0291] Aspects of the present invention also include systems for practicing subject methods and compositions. The sample analysis system may include a sample field of view or a flow cell for loading sample and labeled specific binding members. In some embodiments, the system is a flow cytometry system, comprising: a flow cytometer, comprising a flow path; a composition in the flow path, wherein the composition comprises: a sample; and a labeled specific binding member (e.g., as described herein).

[0292] 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 disposed in the sample field of view, wherein the composition comprises the sample; and a labeled specific binding member (e.g., as described herein).

[0293] In some cases of the system, the labeled specific binding member includes: a water-solvated light-harvesting multichromophore (e.g., as described herein) and a specific binding member that specifically binds to a target analyte covalently linked to the multichromophore. In some cases, the labeled specific binding member also comprises a signal chromophore that is covalently linked to a multichromophore of a polymeric dye in close energy receiving proximity. In some cases of the subject system, the labeled specific binding member, the multichromophore is described by any one of formulas (I)-(VI) (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.

[0294] In certain embodiments of the system, the composition further comprises a second specific binding member that is bound to the carrier and specifically binds to the target analyte. In some cases, the carrier comprises magnetic particles. Thus, in some cases, the system may further comprise a controllable external paramagnetic field configured to be applied to the assay zone of the flow cell.

[0295] The sample may include cells. In some cases, the sample is a biological sample containing cells. In some cases, the sample includes a specific binding member specifically bound to a label of a target cell. In some cases, the target analyte specifically bound by the specific binding member is a cell surface marker of a cell. In some cases, the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen.

[0296] In some aspects, the system may further include a light source configured to direct light to the assay area or sample field of view of the flow cell. The system may include a detector configured to receive a signal from the assay area or sample field of view of the flow cell, wherein the signal is provided by a fluorescent composition. Optionally, the sample analysis system may further include one or more additional detectors and / or light sources for detecting one or more additional signals.

[0297] In some aspects, the system may also include a computer-based system configured to detect the presence of a fluorescent signal. "Computer-based system" refers to a hardware device, software device, and data storage device for analyzing the information of the present invention. The minimum hardware of the computer-based system of the present invention includes a central processing unit (CPU), an input device, an output device, and a data storage device. A skilled technician will readily appreciate that any existing computer-based system is suitable for the subject system. The data storage device may include any product, including a memory access device that records the information of the present invention as described above, or can access such a product.

[0298] To "record" data, programming or other information about a computer readable medium refers to a method of storing information using any such method as is 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 processing programs and formats may be used for storage, such as word processing text files, database formats, and the like.

[0299] "Processor" refers to any hardware and / or software combination that will perform its required functions. For example, any processor herein may be a programmable digital microprocessor, such as may be available in the form of an electronic controller host, server, or personal computer (desktop or portable). Where the processor is programmable, suitable programming may be communicated to the processor from a remote location, or may be pre-stored in a computer program product (e.g., a portable or fixed computer readable storage medium, based on magnetic, optical, or solid-state devices). For example, a magnetic medium or optical disk may carry the programming, and may be read by a suitable reader that communicates with each processor at its corresponding station.

[0300] In addition to sensor devices and signal processing modules such as those described above, the system of the present invention may include many additional components, such as data output devices, such as monitors and / or speakers; data input devices, such as interface ports, keyboards, etc.; fluid handling components, power supplies, etc.

[0301] In certain aspects, the system includes a flow cytometer. Flow cytometers of interest include, but are not limited to, those devices described in U.S. Patent 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.

[0302] Other systems can be used to practice the subject method. In some aspects, the system can be a fluorometer or a microscope for loading a sample of the fluorescent composition of any embodiment discussed herein. The fluorometer or microscope can include a light source configured to direct light to the assay area or sample field of view of the flow cell. The fluorometer or microscope can also include a detector configured to receive a signal from the assay area or sample field of view of the flow cell, wherein the signal is provided by the fluorescent composition.

[0303] Reagent test kit

[0304] Aspects of the invention also include kits for practicing the subject methods and compositions. The compositions of the invention can be included as reagents in a kit, as starting materials or for use in, for example, the methods described above.

[0305] The kit can include a polymer dye and a container containing a water-solvated light-harvesting multichromophore (e.g., as described herein). Any suitable container can 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 can also include one or more components selected from the following: polymer tandem dyes, fluorophores, specific binding members, specific binding member conjugates, specific binding members of a carrier, cells, carriers, biocompatible aqueous elution buffers, and instructions for use. In some embodiments of the kit, the multichromophore is covalently linked to the specific binding member. In some cases, the specific binding member is an antibody. In some cases, the specific binding member is an antibody fragment or its binding derivatives. In some cases, the antibody fragment or its binding derivatives are selected from the group consisting of: Fab fragments, F(ab')2 fragments, scFv, double antibodies, and three chain antibodies.

[0306] In certain embodiments, the kit can be used to assess a sample for the presence of a target analyte, such as an intracellular target. Thus, in some cases, the kit includes one or more components suitable for lysing cells. One or more additional components of the kit can be provided in separate containers (e.g., separate tubes, bottles, or wells in a multi-porous strip or plate).

[0307] In certain aspects, the kit also includes reagents for performing flow cytometry assays. Reagents of interest include, but are not limited to, buffers for reconstitution and dilution, buffers for contacting cell sample multichromophores, wash buffers, control cells, control beads, fluorescent beads for flow cytometer calibration, and combinations thereof. The kit may also include one or more cell fixation reagents, such as paraformaldehyde, glutaraldehyde, methanol, acetone, formalin, or any combination thereof or buffers. In addition, the kit may include cell permeabilization reagents, such as methanol, acetone, or detergents (e.g., triton, NP-40, saponin, Tween 20, digitonin, leucoperm, or any combination thereof or buffers. Other protein transport inhibitors, cell fixation reagents, and cell permeabilization reagents familiar to skilled artisans are within the scope of the subject kits.

[0308] The composition of the kit can be provided as a liquid composition, such as any suitable buffer. Alternatively, the composition of the kit can be provided as a dry composition (e.g., it can be lyophilized), and the kit can optionally include one or more buffers for 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 holes in porous strips or plates).

[0309] In addition, one or more components can be combined into a single container, such as a glass or plastic vial, tube or bottle. In some cases, the kit can also include a container (e.g., a box, bag, insulated container, bottle, tube, etc.) in which all components (and their separate containers) are present. The kit can also include packaging, which is separate from or connected to the kit container and on which information about the kit, kit components and / or instructions for use are printed.

[0310] In addition to the above components, the subject test kit can also include 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 such as printing information on suitable media or substrate (a piece or several sheets of paper such as printed information), in the packaging of the test kit, in the package instructions, etc. Another device will be a computer-readable medium for recording information above, such as a disk, CD, DVD, portable flash drive, etc. Another device that can exist is to use the website address of accessing information in a far place via the Internet. Any suitable device can be present in the test kit.

[0311] utility

[0312] Polymer dyes, compositions, methods and systems as described herein can be used for multiple applications of targets that need to be marked, detected and / or analyzed, including diagnosis and research applications. The subject polymer dye can be used as a fluorescent marker in multiple applications to utilize under an excitation light source of 430nm to 530nm. The key laser source used in multiple biological detection techniques is an argon (Ar) ion laser, which is used as a continuous wave source of 488nm light. Due to the early use of such laser sources, in fact all flow cytometers have a 488nm source. Therefore, attention is paid to the subject polymer dye excited at 488nm and can be used for flow cytometry instruments and experimental equipment. In addition, the Ar ion laser has several lines with significant intensity close to 488nm. Along with the use of the wide range wavelength of modern diode lasers and the central position of this range in the UV-visible spectrum, attention is paid to the region of 440nm to 530nm for exciting the subject polymer dye.

[0313] Applications of interest include, but are not limited to, methods such as cytometry, microscopy, immunoassays (e.g., competitive or non-competitive), assessment of free analytes, assessment of receptor-bound ligands, and the like. The compositions, systems, and methods described herein can be used to analyze any of a number of samples including, but not limited to, biological fluids, cell culture samples, and tissue samples. In certain aspects, the compositions, systems, and methods described herein can be used in methods where a fluorescent marker is used to detect an analyte in a sample (if present), such as fluorescence-activated cell sorting or analysis, immunoassays, immunostaining, and the like. In some cases, the compositions and methods can be used in applications where it is of interest to assess the presence of a target analyte in a sample.

[0314] In some cases, the methods and compositions can be used in any assay format where detection and / or analysis of a target from a sample is of interest, including but not limited to flow cytometry, fluorescence microscopy, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assays, and fluorescent dye purification chromatography. In some cases, the methods and compositions can be used in any application where fluorescent labeling of a target molecule is of interest. The subject compositions are suitable for use in any suitable application where paired specific binding members, such as biotin-streptavidin and hapten-anti-hapten antibodies, are used.

[0315] Example

[0316] Example 1: Synthesis of conjugated polymers via CH bond arylation

[0317] The synthesis of the subject dye can be achieved by a technique known as C-H bond arylation (Scheme 1). Other methods such as the Suzuki coupling method (Scheme 2) or the Stille coupling method (Scheme 3) can also be utilized. Unlike the Suzuki coupling involving the synthesis of boronate / boronic acid monomers, or the Stille coupling involving toxic tin reagents, this method uses simpler monomers and achieves high molecular weights relative to other coupling reactions that can be employed. Figure 2 Several exemplary dyes prepared according to the methods described herein are shown in Figure 1 ) absorption characteristics.

[0318] Scheme 1: CH bond arylation method

[0319]

[0320] Exemplary Procedure for C-H Bond Arylation

[0321]

[0322] Polymer synthesis procedure:

[0323] Monomer A (446.2 mg, 0.1775 mmol), monomer B (212.9 mg, 0.1775 mmol), potassium carbonate (61.3 mg, 0.444 mmol), Pd(OAc)2 (2.0 mg), pivalic acid (5.4 mg) and dimethylacetamide solvent (1.25 mL) were added to a single-necked round-bottom flask (with side arms) equipped with a Teflon-coated stirring rod. The flask was equipped with a condenser and connected to a Schlenk line. The solution was degassed by nitrogen blowing and three freeze-pump-thaw cycles. The mixture was then stirred at 80 ° C overnight under nitrogen protection.

[0324] The resulting polymer solution was cooled to room temperature, diluted with 20% EtOH / H2O, and stirred with EDTA (60 mg) at 23°C for 1 h. The mixture was spin filtered and rinsed with 20% EtOH / H2O. The polymer (550 mg) was collected after vacuum drying. Mn = 105 kDa; Mw = 191 kDa; Absorption λ max =471nm; fluorescence λ max =516nm.

[0325] Solution 2: Suzuki Puppetry

[0326]

[0327] Scheme 3: Stiller coupling method

[0328]

[0329] Notwithstanding the appended claims, the disclosure set forth herein is also defined by the following terms:

[0330] Item 1. A water-solvated polymer dye having a blue excitation spectrum.

[0331] Item 2. The polymeric dye according to Item 1, wherein the polymeric dye comprises a thiophene-containing comonomer.

[0332] Item 3. The polymeric dye according to any one of items 1-2, wherein the polymeric dye has no yellow-green absorption at 562 nm.

[0333] Item 4. The polymer dye according to any one of Items 1 to 3, wherein the excitation spectrum of the polymer dye has a full width at half maximum (FWHM) with a width of 100 nm or less.

[0334] Item 5. The polymer dye according to any one of Items 1 to 4, wherein the polymer dye has an absorption maximum wavelength of 488 nm ± 20 nm.

[0335] Item 6. The polymer dye according to any one of Items 1 to 5, wherein the polymer dye has 1×10 6 M - 1 cm -1 or more extinction coefficient.

[0336] Item 7. The polymeric dye according to any one of items 1 to 6, wherein the polymeric dye is substituted with non-ionic side groups capable of imparting a water solubility exceeding 10 mg / mL.

[0337] Clause 8. The polymeric dye according to any one of clauses 1 to 7, wherein the polymeric dye comprises a conjugated segment having a structure of formula (I):

[0338]

[0339] in:

[0340] Every M 1 、M 2 and M 3 are independently aromatic or heterocyclic comonomers, wherein M 1 、M 2 and M 3 At least one of the monomers is a thiophene-containing comonomer;

[0341] Z 1 is a chemoselective functional group or a linked acceptor chromophore;

[0342] Z 5 and Z 6 are independently CR or N, wherein R is H, halogen, alkoxy, substituted alkoxy, alkyl, and substituted alkyl;

[0343] G 1 and G 2 each independently selected from a terminal group, a π-conjugated segment, a linker, and a linked specific binding member;

[0344] Y 1 , Y 2 and Y 3 Independently C(R 3 )2、-C(R 3 )2C(R 3 )2-、NR 3 、Si(R 3 )2 or Se;

[0345] Every R 3independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido, and WSG (e.g., a water-soluble group);

[0346] ai is independently 0 or 1;

[0347] n, m and o are each independently 0 or an integer from 1 to 10,000; and

[0348] p is an integer from 1 to 100,000.

[0349] Item 9. The polymer dye according to Item 8, wherein M 1 、M 2 and M 3 Each is independently a thiophene-containing comonomer.

[0350] Clause 10. A polymeric dye according to any one of clauses 8-9, wherein:

[0351] a+c≤1;

[0352] d+f≤1;

[0353] g+i≤1;

[0354] b+e+h≥1; and

[0355] n+m+o≥1.

[0356] Item 11. The polymeric dye according to any one of Items 8 to 10, wherein the polymeric dye has a structure of one of Formulas (Ib) to (Ic):

[0357]

[0358]

[0359] Item 12. The polymeric dye according to any one of Items 8 to 11, wherein the polymeric dye has a structure of one of Formulas (IIa) to (IIb):

[0360]

[0361] wherein u, v, w, x, y and z represent the mole % values ​​of each comonomer in the multichromophore.

[0362] Item 13. The polymeric dye according to any one of Items 8 to 12, wherein the polymeric dye has a structure of one of Formulas (IIIa) to (IIIb):

[0363]

[0364]

[0365] wherein n is an integer from 1 to 100,000.

[0366] Item 14. The polymer dye according to Item 13, wherein Y 1 C(R 3 )2.

[0367] Item 15. The polymer dye according to Item 13, wherein Y 1 NR 3 .

[0368] Item 16. The polymeric dye according to any one of Items 8 to 12, wherein the polymeric dye has a structure of one of Formulas (IVa) to (IVb):

[0369]

[0370] in:

[0371] M 1 and M 2 each independently a thiophene-containing comonomer;

[0372] Each n and m is independently an integer from 1 to 10,000;

[0373] p is an integer from 1 to 100,000.

[0374] Item 17. A polymeric dye according to Item 16, wherein:

[0375] Y 1 NR 3 ; Y 2 C(R 3 )2; and M 1 and M 2 are the same thiophene-containing comonomer; or

[0376] Y 1 C(R 3 )2;Y 2 NR 3 ; and M 1 and M 2 The same thiophene-containing comonomer.

[0377] Item 18. A polymer dye according to Item 16, wherein: Y 1 and Y 2 Each is independently C(R 3 )2; and M 1 and M 2The same thiophene-containing comonomer.

[0378] Item 19. The polymeric dye according to Item 8 or 11, wherein the polymeric dye has a structure of one of Formulas (Va)-(Vb):

[0379]

[0380] in:

[0381] M 1 and M 3 each independently a thiophene-containing comonomer;

[0382] Each n and each o is independently an integer from 1 to 10,000; and

[0383] p is an integer from 1 to 100,000.

[0384] Item 20. The polymer dye according to Item 19, wherein Y 3 -Z 1 C(R 3 )(T 1 -Z 1 ), where Z 1 is the connected signal chromophore and T 1 For the connector.

[0385] Item 21. The polymer dye according to Item 19, wherein Y 3 -Z 1 NT 1 -Z 1 , where Z 1 is the connected signal chromophore and T 1 For the connector.

[0386] Item 22. A polymer dye according to any one of Items 19 to 21, wherein: Y 1 NR 3 ; and M 1 and M 3 The same thiophene-containing comonomer.

[0387] Item 23. A polymer dye according to any one of Items 19 to 21, wherein: Y 1 C(R 3 )2; and M 1 and M 3 The same thiophene-containing comonomer.

[0388] Item 24. The polymeric dye according to Item 8 or 11, wherein the polymeric dye has a structure of one of Formulas (VIa)-(VIb):

[0389]

[0390] in:

[0391] M 1 and M 2 each independently a thiophene-containing comonomer;

[0392] Each n and each m is independently an integer from 1 to 10,000;

[0393] Z 1 is a linked chemoselective functional group or a linked signaling chromophore; and

[0394] p is an integer from 1 to 100,000.

[0395] Item 25. The polymer dye according to Item 24, wherein Y 1 and Y 2 NR 3 .

[0396] Item 26. The polymer dye according to Item 24, wherein Y 1 and Y 2 C(R 3 )2.

[0397] Item 27. A polymer dye according to Item 24, wherein Y 1 NR 3 And Y 2 C(R 3 )2, or Y 1 C(R 3 )2 and Y 2 NR 3 .

[0398] Item 28. The polymeric dye according to any one of Items 2 to 27, wherein the thiophene-containing comonomer is described by formula (VII), (VIII) or (IX):

[0399]

[0400] in:

[0401] A1 and A2 are each a fused monocyclic or bicyclic group;

[0402] Every R 3 independently selected from H, amino, substituted amino, halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido, substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy, WSG, and -T 2 -Z2 , where Z 2 is a chemoselective functional group or a linked signal chromophore, and T 2 For the joint;

[0403] n is 0, 1, 2, 3 or 4; and

[0404] Every * It is a covalent attachment site to the unsaturated backbone of the conjugated polymer.

[0405] Item 29. The polymeric dye according to Item 28, wherein A1 and A2 are selected from the group consisting of a fused monocyclic heterocycle, a fused monocyclic heteroaryl, and a fused bicyclic heteroaryl.

[0406] Clause 30. The polymeric dye according to any one of clauses 28-29, wherein the thiophene-containing comonomer is described by one of formulas (X)-(XVII):

[0407]

[0408] in:

[0409] X 1 CR 3 NR 3 , O or S;

[0410] X 2 is N, O or S, when X 2 When N, each is a double bond and when X 2 When it is O or S, each is a single bond;

[0411] R 1 and R 2 together to form optionally one or more R 3 A 5- or 6-membered fused aromatic or heteroaromatic ring substituted with a group; and

[0412] Y 4 NR 3 , C(R 3 )2 or Si(R 3 )2.

[0413] Item 31. The polymeric dye according to Item 30, wherein the thiophene-containing comonomer is described by one of the following structures (a) to (q):

[0414]

[0415] in:

[0416] Every R 4independently selected from H, amino, substituted amino, halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido, substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy, and -T 2 -Z 2 , where Z 2 is a chemoselective functional group or a linked acceptor chromophore, and T 2 is a connector; and

[0417] Each s is 0 or an integer from 1 to 50.

[0418] Item 32. A polymer dye according to any one of Items 8 to 31, wherein each R 3 And every R 4 Independently selected from H, substituted aryl, alkyl and the following structures:

[0419]

[0420] Among them: Each T 5 is independently an optional linker; and each s is 0 or an integer from 1 to 50.

[0421] Clause 33. The polymeric dye according to any one of clauses 28 to 32, wherein the thiophene-containing comonomer is described by one of the following structures (ba) to (bu):

[0422]

[0423]

[0424] Item 34. The polymeric dye according to any one of Items 8 to 33, wherein the polymeric dye comprises a comonomer attached to a signal chromophore having a structure of formula (XVI):

[0425]

[0426] wherein: W is an alkyl group, a substituted alkyl group or WSG; and the dye is a signal chromophore.

[0427] Item 35. The polymeric dye according to Item 34, wherein the polymeric dye comprises a comonomer linked to a signal chromophore having one of the following structures:

[0428]

[0429] Item 36. A polymer dye according to any one of Items 8 to 30, wherein the comonomer M 1 -M 3 Independently selected from formula (XVIII) and (XIX):

[0430]

[0431] in:

[0432] R 1 and R 2 together to form optionally one or more R 3 A 5- or 6-membered fused aromatic or heteroaromatic ring substituted with a group; and

[0433] Every R 3 independently selected from H, amino, substituted amino, halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido, substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy, and -T 2 -Z 2 , where Z 2 is a chemoselective functional group or a linked acceptor chromophore, and T 2 For the connector.

[0434] Item 37. A polymer dye according to any one of Items 8 to 30, wherein the comonomer M 1 -M 3 Independently selected from formula (e)-(i):

[0435]

[0436] in:

[0437] Every R 4 independently selected from H, amino, substituted amino, halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido, substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy, and -T 2 -Z 2 , where Z 2 is a chemoselective functional group or a linked acceptor chromophore, and T 2 is a connector; and

[0438] Each s is 0 or an integer from 1 to 50.

[0439] Item 38. A polymer dye according to any one of Items 36-37, wherein each R 3 And every R 4 Independently selected from H, substituted aryl, alkyl and the following structures:

[0440]

[0441] in:

[0442] Every T 5 are independently an optional linker; and

[0443] Each s is 0 or an integer from 1 to 50.

[0444] Item 39. A polymer tandem dye comprising:

[0445] A polymeric dye having a blue excitation spectrum according to any one of clauses 1 to 38; and

[0446] A signal chromophore covalently attached to the polymer dye is in close proximity thereto and capable of receiving energy.

[0447] Item 40. The polymeric tandem dye according to Item 39, wherein the polymeric dye comprises a thiophene-containing comonomer.

[0448] Item 41. The polymer tandem dye according to Item 39, wherein the polymer dye has a maximum absorption wavelength of 440 nm to 530 nm.

[0449] Item 42. The polymer tandem dye according to Item 39, wherein the signal chromophore emission has a quantum yield of 0.1 or more.

[0450] Item 43. A polymer tandem dye according to Item 39, wherein the polymer dye has 1×10 6 M -1 cm -1 or more extinction coefficient.

[0451] Item 44. The polymeric tandem dye according to any one of items 39-43, wherein the multichromophore is substituted with non-ionic side groups capable of conferring a water solubility of more than 10 mg / mL.

[0452] Item 45. The polymer tandem dye according to any one of items 39-44, wherein the signal chromophore is a fluorophore.

[0453] Item 46. The polymer tandem dye according to any one of items 39-44, wherein the signal chromophore is a quencher.

[0454] Item 47. A polymer tandem dye according to any one of Items 39-46, wherein the signal chromophore is selected from rhodamine, coumarin, xanthene, cyanine, polymethine, pyrene, dipyrromethene boron difluoride, napthalimide, phycobiliprotein, peridinin chlorophyll protein, conjugates thereof, and combinations thereof.

[0455] Item 48. A polymer tandem dye according to any one of items 39-45 and 47, wherein the signal chromophore is selected from fluorescein, 6-FAM, rhodamine, Texas Red, California Red, iFluor594, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxy-p-aminophenol, 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, Sea Blue, Oregon Green 488, Oregon Green 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, its conjugates, and combinations thereof.

[0456] Clause 49. A polymer tandem dye according to any one of clauses 39 to 49, wherein:

[0457] The signal chromophore is linked to a comonomer that constitutes 1% to 20% of the molar concentration of the multichromophore; and

[0458] The polymer dye is a conjugated polymer comprising 5 or more repeating units.

[0459] Item 50. A polymer tandem dye according to any one of items 39 to 45 and 47-49, wherein the signal chromophore emits 1.5 times greater or more when excited by the polymer dye than when the acceptor chromophore is excited directly with incident light.

[0460] Clause 51. A polymeric tandem dye according to any one of clauses 39 to 50, wherein the polymeric dye comprises a terminal group -L 3 -Z, where L 3 is a linker and Z is a specific binding member.

[0461] Item 52. A polymer tandem dye according to Item 51, wherein the linker is selected from the group consisting of alkyl, substituted alkyl, alkyl-amido, alkyl-amido-alkyl and PEG moieties.

[0462] Item 53. A polymer tandem dye according to Item 51, wherein -L 3 -Z is described by the following structure:

[0463]

[0464] wherein: q is 0 or an integer from 1 to 12; and Z is the specific binding member.

[0465] Item 54. The polymer tandem dye according to any one of items 51-53, wherein Z is a biomolecule.

[0466] Item 55. The polymer tandem dye according to any one of items 51-54, wherein Z is an antibody.

[0467] Item 56. A polymer tandem dye according to any one of items 51-54, wherein Z is an antibody fragment or a binding derivative thereof.

[0468] Item 57. A polymer tandem dye according to Item 56, 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.

[0469] Clause 58. A labeled specific binding member comprising:

[0470] A polymeric dye having a blue excitation spectrum according to any one of clauses 1 to 38; and

[0471] A specific binding member is covalently attached to the polymeric dye.

[0472] Item 59. A labeled specific binding member according to Item 58, wherein the polymeric dye comprises a thiophene-containing comonomer.

[0473] Item 60. A labeled specific binding member according to any one of Items 58-59, wherein the polymeric dye comprises a signaling chromophore covalently linked to the polymeric dye in proximity thereto capable of receiving energy.

[0474] Item 61. A labelled specific binding member according to any one of items 59-60, wherein the specific binding member is an antibody.

[0475] Clause 62. A labelled specific binding member according to any one of clauses 59-60, wherein the specific binding member is an antibody fragment or a binding derivative thereof.

[0476] Clause 63. A labelled specific binding member according to Clause 62, wherein said 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.

[0477] Item 64. A labeled specific binding member according to Item 60, wherein the signal chromophore is selected from rhodamine, coumarin, xanthene, cyanine, polymethine, pyrene, dipyrromethene boron difluoride, naphthalimide, phycobiliprotein, peridinin chlorophyll protein, conjugates thereof and combinations thereof.

[0478] Clause 65. A method of assessing the presence of a target analyte in a sample, the method comprising:

[0479] (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:

[0480] (i) a polymeric dye having a blue excitation spectrum according to any one of clauses 1 to 38; and

[0481] (ii) a specific binding member covalently attached to the polymeric dye; and

[0482] (b) assaying the sample contacted with the labeling composition for the presence of the polymer dye conjugate-target analyte binding complex to assess whether the target analyte is present in the sample.

[0483] Clause 66. The method according to Clause 65, wherein the polymeric dye comprises a thiophene-containing comonomer.

[0484] Item 67. The method according to any one of Items 65-66, wherein the polymeric dye comprises a signal chromophore covalently linked to the multichromophore in proximity thereto capable of receiving energy.

[0485] Clause 68. The method according to any one of clauses 65-67, further comprising contacting the sample with a second specific binding member that is bound to a support and specifically binds to the target analyte.

[0486] Clause 69. The method according to Clause 68, wherein the carrier comprises magnetic particles.

[0487] Clause 70. The method according to any one of clauses 65-69, wherein the target analyte is associated with a cell.

[0488] Clause 71. The method according to Clause 70, wherein the target analyte is a cell surface marker of the cell.

[0489] Clause 72. The method according to Clause 71, wherein the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen.

[0490] Clause 73. The method according to Clause 70, wherein the target analyte is an intracellular target, and the method further comprises lysing the cell.

[0491] Clause 74. The method according to any one of clauses 65-73, wherein the method further comprises analyzing the fluorescently labeled target analyte by flow cytometry.

[0492] Clause 75. A method of labeling a target molecule, the method comprising:

[0493] The target molecule is contacted with a polymer dye to produce a labeled target molecule, wherein the polymer dye has a blue excitation spectrum and comprises a conjugated tag covalently attached to the target molecule.

[0494] Clause 76. The method according to Clause 75, wherein the polymeric dye comprises a thiophene-containing comonomer.

[0495] Item 77. The method according to any one of Items 75-76, wherein the polymeric dye comprises a signal chromophore covalently attached to the polymeric dye in proximity thereto capable of receiving energy.

[0496] Clause 78. The method according to any one of clauses 75-77, further comprising fluorescently detecting the labeled target molecule.

[0497] Clause 79. A method according to any one of clauses 75 to 78, 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.

[0498] Clause 80. The method according to any one of clauses 75 to 79, wherein the target molecule is a specific binding member.

[0499] Clause 81. The method according to Clause 80, wherein the specific binding member is an antibody.

[0500] Clause 82. The method according to Clause 80, wherein the specific binding member is an antibody fragment or a binding derivative thereof.

[0501] Clause 83. The method according to Clause 82, 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.

[0502] Clause 84. A flow cytometric system comprising:

[0503] Flow cytometers containing flowpaths;

[0504] A composition in the flow path, wherein the composition comprises:

[0505] samples; and

[0506] A labeled specific binding member comprising:

[0507] A polymeric dye having a blue excitation spectrum according to any one of clauses 1 to 38; and

[0508] A specific binding member that specifically binds to the target analyte and is covalently attached to the polymeric dye.

[0509] Clause 85. The system of Clause 84, wherein the polymeric dye comprises a thiophene-containing comonomer.

[0510] Clause 86. The system according to any one of clauses 84-85, wherein the labeled specific binding member further comprises a signal chromophore covalently linked to the polymer dye in proximity thereto capable of receiving energy.

[0511] Clause 87. The system according to any one of clauses 84-86, wherein the composition further comprises a second specific binding member which is bound to a carrier and which specifically binds to the target analyte.

[0512] Clause 88. The system according to Clause 85, wherein the carrier comprises magnetic particles.

[0513] Clause 89. The system according to any one of clauses 84-88, wherein the sample comprises cells.

[0514] Clause 90. The system according to Clause 89, wherein the target analyte is a cell surface marker of the cell.

[0515] Clause 91. The system according to Clause 90, wherein said cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen.

[0516] Clause 92. A kit comprising: a polymer dye having a blue excitation spectrum; and a container.

[0517] Item 93. The kit according to Item 92, wherein the polymeric dye comprises a thiophene-containing comonomer.

[0518] Item 94. A kit according to any one of items 92-93, 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.

[0519] Item 95. The kit according to any one of items 92-94, wherein the polymeric dye is described by any one of items 2-53.

[0520] Item 96. The kit according to any one of items 92-95, wherein the polymeric dye is covalently linked to the specific binding member.

[0521] Clause 97. The kit according to Clause 96, wherein the specific binding member is an antibody.

[0522] Clause 98. The kit according to Clause 96, wherein the specific binding member is an antibody fragment or a binding derivative thereof.

[0523] Clause 99. A kit according to clause 98, 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.

[0524] Item 100. The kit according to any one of Items 92-99, wherein the polymeric dye further comprises an acceptor signal chromophore covalently linked to the polymeric dye and in proximity thereto capable of receiving energy.

[0525] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to one skilled in the art in light of the teachings of this invention that certain changes and modifications may be made without departing from the spirit or scope of the appended claims.

[0526] 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 a variety of arrangements, although these arrangements are not explicitly described or shown herein, but they embody the principles of the present invention and are included in its spirit and scope. In addition, all embodiments and conditional language described herein are mainly intended to help readers understand the principles of the present invention and the inventors for promoting the concept of this field, and will be regarded as not limited to such specific narrated embodiments and conditions. In addition, all statements describing the principles, aspects and embodiments of the present invention herein and their specific embodiments are intended to cover their structural 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 research and development elements that perform the same function. Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. More specifically, the scope and spirit of the present invention are embodied as follows.

Claims

1. A water-soluble polymer dye having a blue excitation spectrum in the range of 375 nm to 500 nm, wherein the polymer dye is described by the structure of formula (I): in: Every M 1 、M 2 and M 3 are independently aromatic or heteroaromatic comonomers, wherein M 1 、M 2 and M 3 At least one of the monomers is a thiophene-containing comonomer; Z 1 is a chemoselective functional group or a linked acceptor chromophore; Every Z 5 and Z 6 are independently CR or N, wherein R is selected from H, halogen, alkoxy, substituted alkoxy, alkyl, and substituted alkyl; G 1 and G 2 each independently selected from a terminal group, a π-conjugated segment, a linker, and a linked specific binding member; Y 1 , Y 2 and Y 3 Independently C(R 3 )2、-C(R 3 )2C(R 3 )2-、NR 3 、Si(R 3 )2 or Se; Every R 3 independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido, and WSG; ai is independently 0 or 1; Each of n, m and o is independently 0 or an integer from 1 to 10000; and p is an integer from 1 to 100,000. 2 . The polymer dye according to claim 1 , wherein an excitation spectrum of the polymer dye has a full width at half maximum (FWHM) of 100 nm or less.

3. The polymer dye according to claim 1 or 2, wherein: a+c≤1; d+f≤1; g+i≤1; b+e+h≥1; and n+m+o≥1.

4. The polymeric dye according to any one of the preceding claims, wherein the polymeric dye has the structure of formula (Ic):

5. The polymer dye according to any one of claims 1 to 2, wherein the polymer dye has a structure of formula (IIIb): Wherein n is an integer from 1 to 100,000.

6. The polymer dye according to any one of claims 1 to 2, wherein the polymer dye has a structure of formula (IVb): in: M 1 and M 2 each independently a thiophene-containing comonomer; Each n and m is independently an integer from 1 to 10000; p is an integer from 1 to 100,000.

7. The polymer dye according to claim 4, wherein the polymer dye has: a) Structure of formula (Vb): in: M 1 and M 3 each independently a thiophene-containing comonomer; Each n and each o is independently an integer from 1 to 10000; and p is an integer from 1 to 100000; or b) Structure of formula (VIb): in: M 1 and M 2 each independently a thiophene-containing comonomer; Each n and each m is independently an integer from 1 to 10000; Z 1 is a linked chemoselective functional group or a linked signaling chromophore; and p is an integer from 1 to 100,000.

8. The polymer dye according to claim 7, wherein Y 3 -Z 1 C(R 3 )(T 1 -Z 1 ), where Z 1 is the connected signal chromophore and T 1 For the connector.

9. The polymer dye according to claim 7, wherein: Y 1 and Y 2 Each is independently C(R 3 )2; and M 1 、M 2 and M 2 The same thiophene-containing comonomer.

10. The polymeric dye according to any one of the preceding claims, wherein the thiophene-containing comonomer is described by formula (VII), (VIII) or (IX): in: A1 and A2 are each a fused monocyclic or bicyclic group; Every R 3 independently selected from H, amino, substituted amino, halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido, substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy, WSG, and -T 2 -Z 2 , where Z 2 is a chemoselective functional group or a linked signal chromophore, and T 2 For the joint; n is 0, 1, 2, 3 or 4; and Every * It is a covalent attachment site to the unsaturated backbone of the conjugated polymer.

11. The polymer dye according to claim 10, wherein the thiophene-containing comonomer is described by one of formulas (X)-(XVII): in, X 1 CR 3 NR 3 , O or S; X 2 is N, O or S, when X 2 When N, each is a double bond and when X 2 When it is O or S, each is a single bond; R 1 and R 2 together to form a 5- or 6-membered fused aromatic or heteroaromatic ring, which is optionally fused via one or more than one R 3 group substitution; and Y 4 NR 3 , C(R 3 )2 or Si(R 3 )2.

12. The polymer dye of claim 11, wherein the thiophene-containing comonomer is described by one of the following structures (a) to (q): in: Every R 4 independently selected from H, amino, substituted amino, halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido, substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy, and -T 2 -Z 2 , where Z 2 is a chemoselective functional group or a linked acceptor chromophore, and T 2 is a connector; and Each s is 0 or an integer from 1 to 50.

13. The polymeric dye according to any one of claims 10 to 12, wherein the thiophene-containing comonomer is described by one of the following structures (ba) to (bu):

14. The polymer dye according to any one of claims 1 to 9, wherein the comonomer M 1 -M 3 Comonomers independently selected from formula (XVIII) and (XIX): in: R 1 and R 2 together to form a 5- or 6-membered fused aromatic or heteroaromatic ring, which is optionally fused via one or more than one R 3 group substitution; and Every R 3 independently selected from H, amino, substituted amino, halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido, substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy, and -T 2 -Z 2 , where Z 2 is a chemoselective functional group or a linked acceptor chromophore, and T 2 For the connector.

15. The polymer dye according to any one of claims 1 to 9, wherein the comonomer M 1 -M 3 Comonomers independently selected from formula (e)-(i): in: Every R 4 independently selected from H, amino, substituted amino, halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, amido, substituted amido, sulfonic acid, cyano, alkoxy, substituted alkoxy, and -T 2 -Z 2 , where Z 2 is a chemoselective functional group or a linked acceptor chromophore, and T 2 is a connector; and Each s is 0 or an integer from 1 to 50.

16. The polymer dye according to any one of claims 10 to 15, wherein each R 3 And every R 4 Independently selected from H, substituted aryl, alkyl and the following structures: in: Every T 5 are independently an optional linker; and Each s is 0 or an integer from 1 to 50.

17. A polymer tandem dye comprising: The polymer dye having a blue excitation spectrum according to any one of claims 1 to 16; and A signal chromophore covalently attached to the polymer dye is in close proximity thereto and capable of receiving energy.

18. A labeled specific binding member comprising: The polymer dye having a blue excitation spectrum according to any one of claims 1 to 16; and A specific binding member is covalently attached to the polymeric dye.

19. A kit comprising the polymer dye according to any one of claims 1 to 17 or the labeled specific binding member according to claim 18.

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