UV-absorbing polymer dyes and methods for using them

Abstract

To provide water soluble light harvesting multichromophores that have an ultraviolet absorption maximum, methods of labelling a target molecule using compositions including the light harvesting multichromophores, and methods of and kits and systems for evaluating a sample for the presence of a target analyte.SOLUTION: Water soluble light harvesting multichromophores that have an ultraviolet absorption maximum are provided. In some embodiments, the multichromophores include a conjugated segment including a fused 6-5-6 tricyclic co-monomer and a UV absorbance-modifying co-monomer. The multichromophores may include an acceptor chromophore covalently linked to the multichromophore in energy-receiving proximity therewith. In some embodiments, a specific binding member is covalently linked to the multichromophore. Also provided are methods of evaluating a sample for the presence of a target analyte and methods of labelling a target molecule using compositions including the light harvesting multichromophores. Kits and systems for practicing the subject methods are also provided.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Cross-reference of related applications In accordance with 35 U.S. SC § 119(e), this application claims priority to the filing date of U.S. Provisional Patent Application No. 62 / 132,449, filed March 12, 2015, the disclosure of which is incorporated herein by reference. [Background technology]

[0002] Fluorescent dyes are compounds that, when irradiated with light of a wavelength they absorb, (usually) emit light of a different wavelength. Fluorescent dyes have been found in a variety of applications in biochemistry, biology, and medicine, for example, in diagnostic kits, microscopy, or drug screening. Fluorescent dyes are characterized by many parameters that allow the user to select the appropriate dye according to the desired purpose. These parameters include the maximum excitation wavelength, maximum emission wavelength, Stokes shift, extinction coefficient, fluorescence quantum yield, and fluorescence lifetime. Dyes may be selected according to the intended application, for example, to allow the transmission of excitation radiation into a biological sample, to minimize background fluorescence, and / or to achieve a high signal-to-noise ratio.

[0003] Molecular recognition requires the specific binding of two molecules. Molecules with binding specificity to target biomolecules have been found for use in various research and diagnostic applications, such as sample labeling and separation, flow cytometry, in situ hybridization, enzyme immunosorbent assay (ELISA), Western blotting, magnetic cell separation, and chromatography. Target biomolecules can be detected by labeling with fluorescent dyes. [Overview of the project] [Means for solving the problem]

[0004] There is provided a water-soluble light-harvesting multichromophore having a maximum ultraviolet absorption. In some embodiments, the multichromophore includes a conjugated segment containing a 6-5-6 fused tricyclic comonomer and an ultraviolet absorbance-modifying comonomer. The multichromophore can include an acceptor chromophore covalently linked to the multichromophore proximal to its energy acceptance. In some embodiments, a specific binding member is covalently linked to the multichromophore. There are also provided a method of evaluating a sample for the presence of a target analyte, and a method of labeling a target molecule using a composition comprising a light-harvesting multichromophore. Kits and systems for carrying out the subject methods are also provided.

[0005] It should be understood that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the teachings of the present invention in any way.

Brief Description of the Drawings

[0006] [Figure 1] Exemplary fluorescence emission profiles of various polymer tandem dyes based on an exemplary multichromophore core structure of the disclosure of the present invention linked to various different acceptor chromophores are illustrated. [Figure 2] Exemplary ultraviolet absorption spectra of various multichromophores of the control, MC-1 to MC-5, are illustrated.

Modes for Carrying Out the Invention

[0007] Definitions Before describing more particularly exemplary embodiments, the meanings and ranges of terms used herein are set forth and defined in the following definitions.

[0008] Unless otherwise specified, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Many of the general meanings of the terms used in this specification are provided to one of ordinary skill in the art from Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale&Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991). Additionally, specific terms are defined below for clarity and to facilitate reference.

[0009] It should be noted that as used in this specification and the appended claims, the singular forms "a", "an", and "the" include the plural unless the context clearly dictates otherwise. For example, the term "primer" means one or more primers, i.e., a single primer and multiple primers. It is further noted that the claims may be drafted to exclude any element. In and of itself, this recitation serves as a basis for using such exclusive terms as "solely", "only", etc., in relation to the detailed description of the elements of the claim, or for using "negative" limitations.

[0010] As used in this specification, the term "sample" means a material or mixture of materials, sometimes liquid, containing one or more specimens of a subject. In some embodiments, the term used in its broad sense means plant, animal or bacterial material that contains cells or gives rise to intracellular metabolites, such as tissue or body fluid isolated from an individual (including, but not limited to, plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections), or components of an in vitro cell culture, and samples from its environment. The term "sample" may also mean a "biological sample". As used herein, the term “biological sample” means a whole organism or a subset of its tissues, cells, or components (including, but not limited to, bodily fluids such as blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic umbilical cord blood, urine, vaginal fluid, and semen). “Biological sample” may also mean, but not limited to, a whole organism or a subset of its tissues, cells, or components, or a fraction or part thereof, such as plasma, serum, cerebrospinal fluid, lymph, skin, respiratory tract, intestinal tract, and the external sections of the urogenital tract, tears, saliva, milk, blood cells, tumors, and organs, or homogenates, lysates, or extracts prepared from such fraction or part thereof. In certain embodiments, the sample is taken from an animal or a plant. The biological sample may include cells. The term “cell” is used in its conventional sense to mean the basic structural unit of both eukaryotic and prokaryotic organisms having at least a nucleus and a cell membrane. In certain embodiments, cells include prokaryotic cells, such as cells derived from bacteria. In other embodiments, cells include eukaryotic cells, such as cells obtained from biological samples derived from animals, plants, or fungi.

[0011] As used herein, the terms “affinity” and “binding activity” have the same meaning and are used interchangeably. “Affinity” refers to the strength of the bond, and higher binding affinity correlates with lower Kd.

[0012] As used herein, the terms “determination,” “measurement,” and “evaluation,” as well as “assay,” are used without distinction and include both quantitative and qualitative determinations.

[0013] As used herein, the terms “carrier-bound” and “carrier-bound” are used without distinction. This refers to a site (e.g., a specific binding site) that is used and covalently or non-covalently linked to the target support. Covalent linkage may involve a chemical reaction between two compatible functional groups (e.g., two chemoselective functional groups, an electrophile and a nucleophile) to form a covalent bond between two sites of the target (e.g., a support and a specific binding member). In some cases, non-covalent linkage may involve specific binding between two sites of the target (e.g., two affinity sites, e.g., a hapten and an antibody, or a biotin site and streptavidin). In certain cases, non-covalent linkage may involve absorption into a substrate.

[0014] As used herein, the term “biomolecule” means organic molecules or macromolecules of the natural class of molecules, or derivatives thereof. Biomolecules encompass polypeptides (e.g., peptides, antibodies, or antibody fragments), polynucleotides, carbohydrates (e.g., sugars), and lipids. In some cases, biomolecules are specific binding members (e.g., members described herein).

[0015] As used herein, the term “polypeptide” refers to a macromolecular form of an amino acid of any length, including peptides ranging in length from 2 to 50 amino acids and polypeptides longer than 50 amino acids. “Polypeptide” and “protein” are used interchangeably herein. The term “polypeptide” includes polymers of coding and non-coding amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a modified peptide backbone in which the conventional backbone is replaced by a non-natural or synthetic backbone. Polypeptides can be of a convenient length, for example, two or more amino acids, such as four or more amino acids, ten or more amino acids, twenty or more amino acids, fifty or more amino acids, one hundred or more amino acids, three hundred or more amino acids, up to five hundred, or up to one thousand or more amino acids. "Peptides" can be two or more amino acids, such as four or more amino acids, ten or more amino acids, twenty or more amino acids, up to fifty amino acids. In some embodiments, peptides are 5 to 30 amino acids long.

[0016] As used herein, the term “isolated” means a site of interest that has been separated from other components with which it is associated by at least 60%, at least 75%, at least 90%, at least 95%, at least 98%, and further at least 99% prior to purification.

[0017] "Multiple" means including at least two types of members. In certain cases, multiple means 100 or more, 1000 or more, 10,000 or more, 100,000 or more, 10 6 The above 10 7 The above 10 8 or 10 9 The company may have more than 10 members, including those listed above.

[0018] A range of numbers encompasses the numbers that define that range.

[0019] As used herein, the term “separation” means physically separating two elements (for example, by size or affinity, etc.), as well as decomposing one element while leaving the other elements intact.

[0020] As used herein, the term “specific binding” refers to the ability of a capture agent (or the first member of a specific binding pair) to preferentially bind to a particular sample (or the second member of a specific binding pair) present in a homogeneous mixture of different samples. In some cases, specific binding interactions distinguish desirable and undesirable samples in a sample where the desirable sample has more than 10 times the specificity of the undesirable sample, such as more than 100 times or more, or more than 1000 times. In some cases, they may be present in the capture agent / sample complex. When specifically bound, the affinity between the capture agent and the sample is at least 10 -8 M, at least 10 -9 M, for example 10 -10 It goes up to M.

[0021] The methods described herein include multiple steps. Each step may be performed after a predetermined length of time has elapsed between steps, as necessary. For example, the time between each step may be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more, and 5 hours or more. In certain embodiments, each subsequent step is performed immediately after the completion of the previous step. In other embodiments, a step may be performed after an incubation or waiting period following the completion of the previous step, for example, after a waiting period of several minutes to overnight.

[0022] As used herein, the terms “linker” or “linking” mean a linking site that connects two groups and has a back chain of no more than 100 atoms in length. A linker or linking can be a covalent bond connecting two groups, or a chain of 1 to 100 atoms in length, e.g., a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, or 20 or more carbon atoms, and the linker can be linear, branched, cyclic, or single-atom. In some cases, a linker is a branched linker, meaning a linking site that connects three or more groups. In certain cases, 1, 2, 3, 4, or 5 or more carbon atoms in the linker back chain may be optionally substituted with heteroatoms of sulfur, nitrogen, or oxygen. The bonds between the main chain atoms are saturated or unsaturated, and in some cases, one, two, or three or fewer unsaturated bonds are present in the linker main chain. The linker may contain one or more substituents, such as alkyl, aryl, or alkenyl groups. Examples of linkers, but not limited to, are polyethylene glycol; ethers, thioethers, tertiary amines, alkyls (which may be linear or branched), such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), etc. The linker main chain may contain cyclic groups, such as aryl, heterocyclic, or cycloalkyl groups, and two or more atoms of the cyclic group, for example, two, three, or four atoms, are included in the main chain. The linker may be cleavable or incleavable.

[0023] As used herein, the terms “polyethylene oxide,” “PEO,” “polyethylene glycol,” “PEG site,” and “PEG” are used interchangeably and refer to the formula --(CH2--CH2--O--) n - refers to a polymer group or derivative thereof containing a chain indicated by -. In some embodiments, "n" is 5000 or less, e.g., 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, e.g., 3 to 15, or 10 to 15. It should be understood that the PEG polymer group can be of a convenient length and may include various terminal groups and / or further substituents, but are not limited to alkyl, aryl, hydroxyl, amino, acyl, acyloxy, and amide terminal groups and / or substituents. The PEG group that can be adapted for use with the target multichromophore is "Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates" by S. Zalipsky, Bioconjugate Chemistry. Examples include PEG as described in 1995, 6(2), 150 - 165; and "Water - Soluble Conjugated Polymers for Imaging, Diagnosis, and Therapy" by Zhu et al., Chem. Rev., 2012, 112(8), pp4687 - 4735.

[0024] As used herein, the term "alkyl", either by itself or as part of another substituent, means a saturated branched or straight - chain monovalent hydrocarbon radical derived by removing one hydrogen atom from a single carbon atom of the parent alkane. Examples of the alkyl groups include, but are not limited to, methyl; propyl such as ethyl, propane - 1 - yl or propane - 2 - yl; and butyl such as butane - 1 - yl, butane - 2 - yl, 2 - methylpropane - 1 - yl or 2 - methylpropane - 2 - yl. In some embodiments, the alkyl group contains 1 to 20 carbon atoms. In some embodiments, the alkyl group contains 1 to 10 carbon atoms. In certain embodiments, the alkyl group contains 1 to 6 carbon atoms, such as 1 to 4 carbon atoms. This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH3-), ethyl (CH3CH2-), n - propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n - butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), s - butyl ((CH3 -)(CH3CH2)CH-), t - butyl ((CH3)3C-), n - pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0025] The term "substituted alkyl" means that one or more carbon atoms in its alkyl chain are optionally - O -, - N -, - S -, - S(O) n-(n is 0 to 2), -NR- (R is hydrogen or alkyl), etc., are substituted with heteroatoms, and are alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thioketone, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, SO2-heteroaryl, and -NR a R b (R’ and R” may be the same or different, and are selected from the group consisting of hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic), and means an alkyl group defined herein having 1 to 5 substituents selected therefrom.

[0026] "Alkoxy" means an -O-alkyl group where alkyl is as defined herein. As an example of alkoxy, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, s-butoxy, n-pentoxy, etc. are included. The term "alkoxy" also means alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O- groups, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0027] The term "substituted alkoxy" means substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- groups, where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0028] The terms "alkynyl" and "alkyne" refer to linear or branched monovalent hydrocarbyl groups having 2 to 6 carbon atoms, preferably 2 to 3 carbon atoms, and at least one, preferably 1 to 2, unsaturated triple bond sites. Examples of such alkynyl groups include acetylenyl (-C≡CH) and propagyl (-CH2C≡CH). Unless otherwise specified, these terms include both substituted and unsubstituted forms.

[0029] The term "substituted alkynyl" includes alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, This refers to an alkynyl group as defined herein, having 1 to 5 substituents or 1 to 3 substituents, selected from acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, 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, and -SO2-heteroaryl.

[0030] The term "aryl," either by itself or as part of another substituent, refers to a monovalent aromatic hydrocarbon radical derived by removing a single hydrogen atom from a single carbon atom in an aromatic ring structure. Examples of aryl groups include, but are not limited to, groups derived from acetantrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluorantene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indan, indene, naphthalene, octacene, octafen, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentafen, perylene, phenalene, phenanthrene, picene, pleiaden, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene, etc. In certain embodiments, the aryl group contains 6 to 20 carbon atoms. In certain embodiments, the aryl group contains 6 to 12 carbon atoms. Examples of aryl groups are phenyl and naphthyl.

[0031] "Heteroaryl," either by itself or as part of another substituent, refers to a monovalent heteroaromatic radical induced by removing a single hydrogen atom from a single atom of an aromatic heterocyclic system. The target heteroaryl groups are not limited to those derived from acridine, arsindol, carbazole, β-carbolin, chroman, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indidine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthoridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, quinazoline, quinoline, quinoridine, quinoxaline, tetrazoline, thiadiazole, thiazoline, triazole, benzotriazole, thiophene, triazole, xanthene, benzodioxole, etc. In certain embodiments, the heteroaryl group is a 5- to 20-membered heteroaryl. In certain embodiments, the heteroaryl group is a 5- to 10-membered heteroaryl. In certain embodiments, the heteroaryl group is a group derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole, and pyrazine.

[0032] The terms "alkalyl" or "aralkyl" refer to alkylene-aryl groups and substituted alkylene-aryl groups, where alkylene, substituted alkylene, and aryl are as defined herein.

[0033] "Alkylene" refers to a linear or branched chain that is -O- or -NR. 10 -, NR 10 C(O)-, -C(O)NR 10 - This term refers to a divalent aliphatic hydrocarbyl group having 1 to 6 carbon atoms, more preferably 1 to 3 carbon atoms, in which one or more groups selected from the above are optionally interposed. Examples of this term include methylene (-CH2-), ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2) This includes (-), isopropylene (-CH2CH(CH3)-), (-C(CH3)2CH2CH2-), (-C(CH3)2CH2C(O)-), (-C(CH3)2CH2C(O)NH-), (-CH(CH3)CH2-), etc. "Substituted alkylene" refers to an alkylene group in which 1 to 3 hydrogen atoms are substituted by substituents, as described for carbon in the definition of "substitution" below.

[0034] "Substituted" means a group in which one or more hydrogen atoms are independently substituted with the same or different substituents. Substituents include, but are not limited to, alkylenedioxy (such as methylenedioxy), -M, and -R. 60 , -O - ,=O,-OR 60 , -SR 60 , -S - ,=S,-NR60 R 61 ,=NR 60 , -CF3, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -S(O)2O - -S(O)2OH, -S(O)2R 60 -OS(O)2O - -OS(O)2R 60 ,-P(O)(O - )2, -P(O)(OR 60 )(O - ), -OP(O)(OR 60 )(OR 61 ), -C(O)R 60 ,-C(S)R 60 , -C(O)OR 60 -C(O)NR 60 R 61 ,-C(O)O - , -C(S)OR 60 , -NR 62 C(O)NR 60 R 61 , -NR 62 C(S)NR 60 R 61 , -NR 62 C(NR 63 )NR 60 R 61 and -C(NR 62 )NR 60 R 61 The following are listed, where M is a halogen and R 60 , R 61 , R 62 and R 63 independently of R, is 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 However, together with the nitrogen atom to which they are bonded, they form a cycloheteroalkyl or substituted cycloheteroalkyl ring; R 64 and R 65is independently hydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, or optionally R 64 and R 65 together with the nitrogen atom to which they are attached form a cycloheteroalkyl or substituted cycloheteroalkyl ring. In certain embodiments, substituents include -M, -R 60 , =O, -OR 60 , -SR 60 , -S - , =S, -NR 60 R 61 , =NR 60 , -CF3, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -S(O)2R 60 , -OS(O)2O - , -OS(O)2R 60 , -P(O)(O - ), -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 are included. In certain embodiments, 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)NR60 R 61 ,-C(O)O - These are some examples. In certain embodiments, the substituents are -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 - These are listed, and in the formula, R 60 , R 61 and R 62 A substituent is defined as follows: For example, a substituent may have a methylenedioxy group or one, two, or three substituents selected from halogen atoms, (1-4C)alkyl groups, and (1-4C)alkoxy groups. If the substituted group is an aryl or heteroaryl group, the substituent (as described herein, for example) may be called an "aryl substituent."

[0035] Other definitions of terms may appear throughout this specification.

[0036] A water-soluble, light-gathering, multi-chromophore is provided, as summarized below. In some embodiments, The multichromophore comprises a conjugated segment such as a 6-5-6 condensed tricyclic comonomer and an ultraviolet absorbance-modified comonomer, and the multichromophore has an ultraviolet absorption maximum. The multichromophore may include an acceptor chromophore covalently linked to the multichromophore near its energy acceptor. In some embodiments, a specific binding member is covalently linked to the multichromophore. Methods for evaluating a sample for the presence of a target sample and methods for labeling a target molecule using a composition containing a light-harvesting multichromophore are also provided. Kits and systems for carrying out the methods of the subject are also provided.

[0037] Before describing the various embodiments in more detail, it should be understood that the teachings in this disclosure are not limited to the specific embodiments described and, therefore, may naturally differ. It should also be understood that the terms used herein are provided solely for the purpose of describing specific embodiments, and are not intended to limit them, as the scope of the teachings of the invention is limited only by the appended claims.

[0038] The section titles used in this invention are provided for structuring purposes only and should not be construed as limiting the subject matter described. Although the teachings of this invention are described in conjunction with various embodiments, the teachings of this invention are not intended to be limited to such embodiments. On the contrary, the teachings of this invention encompass various alternative forms, modifications, and equivalents, as will be understood by those skilled in the art.

[0039] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure pertains. Similar or equivalent methods and materials may also be used in carrying out and testing the teachings of the present invention, but several exemplary methods and materials are described.

[0040] Citation of a published patent application should not be interpreted as an acknowledgment that the claims of the present invention are not granted prior rights to such disclosure by prior inventions, as the disclosures in the application date are shown prior to the filing date. Furthermore, the date of the published patent application may differ from the actual publication date, which may be separately confirmed.

[0041] As will be apparent to those skilled in the art upon reading this disclosure, the individual embodiments described and illustrated herein have individual components and features that can be readily separated from or combined with any of the features of several other embodiments without departing from the scope or spirit of the teachings of the invention. The methods described may be carried out in the order of the events described or in any other logically possible order.

[0042] All patents and published patents, including all sequences, that are referenced herein and disclosed within such patents and published patents are explicitly incorporated by reference.

[0043] In further description of the present invention, light-gathering multichromophores are first described in more detail. Next, tandem dyes containing the multichromophores of the subject and their conjugates are described. Then, methods in which compositions containing the multichromophores of the subject are used are outlined. Systems and kits that may be used in carrying out the methods of the present invention are also described.

[0044] Light-gathering, multi-color chromatophores As summarized above, the disclosure of the present invention provides a light-gathering multichromophore having an ultraviolet absorption maximum. In some embodiments, the multichromophore comprises a conjugated segment having a 6-5-6 condensed tricyclic comonomer and an ultraviolet absorbance-modified comonomer, and the multichromophore has an ultraviolet absorption maximum. As used herein, the term “ultraviolet absorption maximum” refers to an absorption maximum wavelength in the ultraviolet region of the electromagnetic spectrum, for example, an absorption maximum in the range of 10 to 400 nm. This refers to wavelengths below 400 nm, such as long wavelengths.

[0045] As used herein, the terms “light-harvesting multichromophore,” “polymer dye,” and “conjugated polymer” are used without distinction and refer to conjugated polymers having a structure capable of collecting light with a specific absorption maximum wavelength and converting it into synchrotron radiation at a longer emission maximum wavelength. In some cases, light-harvesting multichromophore is fluorescent itself. Conjugated polymers (CPs) are characterized by a delocalized electronic structure, and their main chain may contain a large number of conjugated segments in the vicinity, thus having an effective conjugation length substantially shorter than the polymer chain length. In some cases, conjugated polymers are effective in light-harvesting and confer light amplification to acceptors via Forster energy transfer.

[0046] As used herein, the term “unit” means a structural subunit of a polymer. The term “unit” includes monomers, comonomers, coblocks, conjugated segments, repeating units, etc. A “repeating unit” is a subunit of a polymer defined by the minimum number of distinct structural features required for the unit to be considered a monomer, such that the polymer or block is represented by the structure obtained when the unit is repeated n times. In some cases, a polymer may contain two or more distinct repeating units; for example, if the polymer is a multiblock polymer, each block may define a different repeating unit. In some cases, a repeating unit of a polymer may contain a single monomer group. In certain cases, the repeating units of a polymer include two or more monomer groups, i.e., comonomer groups such as two, three, or four or more comonomer groups. As used herein, the terms “comonomer” or “comonomer group” mean a structural unit of a polymer that may itself be part of the repeating units of the polymer. In some embodiments, the conjugated polymer includes a block copolymer composed of blocks of polymer monomers. In such cases, the block copolymer may be described as having different repeating units, each corresponding to a separate coblock of the polymer. In some cases, the polymer is a diblock copolymer containing two different coblocks. In such cases, the polymer may be described as containing coblocks, each coblock may be composed of comonomers such as one, two, or three or more comonomers.

[0047] As used herein, the term “ultraviolet absorbance modified comonomer” means a comonomer that imparts a maximum absorbance to a multichromophore that shifts the maximum absorbance to shorter wavelengths in the ultraviolet region (e.g., up to wavelengths below 400 nm) compared to the maximum absorbance of a control multichromophore, for example, the maximum absorbance of a conjugated polymer in which the ultraviolet absorbance modified comonomer is not present in the repeating units. In some cases, the control multichromophore is a polyfluorene multichromophore. In some cases, the control multichromophore is a polycarbazole multichromophore.

[0048] To provide a multi-chromophore having an ultraviolet absorption maximum (for example, an absorption maximum at wavelengths below 400 nm), any simple light-gathering multi-chromophore can be adapted to include an absorbance-modified comonomer. Light-gathering multi-chromophores of subjects that can be modified to contain absorbance-modified como-Mmer include, but are not limited to, U.S. Patent Publication Nos. 20040142344, 20080293164, 20080064042, 20100136702, 20110256549, 20120028828, 20120252986, and 20130190193, as well as U.S. Patent Nos. 8,575,303 and 8,802,450 by Gaylord et al., whose entire disclosures are incorporated herein by reference; al., J.Am.Chem.Soc.,2001,123(26),pp6417-6418;Feng et al. Examples include the multichromophores described in Chem. Soc. Rev., 2010, 39, 2411-2419; and Traina et al., J. Am. Chem. Soc., 2011, 133(32), pp12600-12607.

[0049] In some embodiments, the multichromophore comprises a plurality of first optically active units that form a conjugated system, each having an absorption wavelength (e.g., as described herein) that absorbs light to form an excited state. In certain cases, the multichromophore comprises an oligomeric structure such as a conjugated polymer segment or a bandgap-reduced n-conjugated repeat unit.

[0050] The subject polychromophore may be water-soluble. Water solubility can be enhanced by incorporating a simple water-soluble group into the polychromophore. The increase in water solubility can vary, but in some cases, the increase is more than twofold (compared to compounds without WSGs), for example, fivefold, tenfold, 25fold, 50fold, or 100fold or more. The term "water-soluble group" (WSG) refers to a group that is well solvable in an aqueous environment, for example under physiological conditions, and that confers improved water solubility to the molecule to which it is bound. In some embodiments, the WSG enhances the water solubility of the polychromophore, primarily in aqueous solutions, compared to a control polychromophore without a WSG. The water-soluble group may be any simple hydrophilic group that is well solvable in an aqueous environment. In some cases, hydrophilic water-soluble groups are charged, for example, having a positive or negative charge. In certain cases, the hydrophilic water-soluble group is a neutral hydrophilic group. In some embodiments, the WSG is a hydrophilic polymer, e.g., polyethylene glycol, cellulose, chitosan, or derivatives thereof. The water-soluble groups in question include, but are not limited to, carboxylates, phosphonates, phosphates, sulfonates, sulfates, sulfinates, sulfonium, esters, polyethylene glycol (PEG) and modified PEG, hydroxyls, amines, ammonium, guanidinium, pyridinium, polyamines and sulfonium, polyhydric alcohols, linear or cyclic saccharides, primary, secondary, tertiary, or quaternary amines and polyamines, phosphonate groups, phosphinate groups, ascorbic acid groups, glycols, e.g., polyethers, -COOM', -SO3M', -PO3M', -NR3 + ,Y',(CH2CH2O) p R and mixtures thereof (where Y' can be a halogen, sulfate, sulfonate, or oxygen-containing anion, p can be 1-500, R can independently be H or alkyl (such as methyl), and M' can be a cationic counterion or hydrogen, --(CH2CH2O) yy CH2CH2XR yy ,--(CH2CH2O) yy CH2CH2X--, --X(CH2CH2O) yyIt can be CH2CH2--, glycol, and polyethylene glycol, where yy is selected from 1 to 1000 and X is O, S, and NR. ZZ Selected from, R ZZ and R YY H and C are independent of each other. 1~3 Examples include (selected from alkyl groups).

[0051] Multiple WSGs may be incorporated into the subject's multichromophore at a single position by a branched linker. In certain embodiments, the branched linker is further an aralkyl substituent disubstituted with a water-soluble group. Thus, the branched linker group is optionally a substituent of the multichromophore that links the multichromophore to two or more water-soluble groups. Optionally, the incorporation of multiple WSGs by the branched linker imparts desirable water solubility to the multichromophore.

[0052] In some embodiments, the chromophore comprises substituents selected from alkyl, aralkyl, and heterocyclic groups, each of which is further substituted with a water-soluble group, a hydrophilic polymer group, such as polyethyl glycol (PEG) (e.g., 2 to 20 units of PEG).

[0053] In some embodiments, the polychromophore is a water-soluble, light-harvesting polychromophore comprising a conjugated segment containing a fluorene comonomer; an ultraviolet absorbance-modified comonomer, and having an ultraviolet absorption maximum. In some cases, the polychromophore is a conjugated segment containing a carbazole comonomer; an ultraviolet absorbance-modified comonomer. A water-soluble, light-gathering, multi-chromophore containing a monomer-containing conjugated segment and having an ultraviolet absorption maximum. In certain embodiments, the multi-chromophore has an absorption maximum wavelength of 400 nm or less, such as wavelengths in the range of 10-400 nm, 100-400 nm, 200-400 nm, 300-400 nm, 300-390 nm, 300-380 nm, 300-370 nm, 300-360 nm, 300-350 nm, 300-340 nm, 300-330 nm, or 300-325 nm. In certain embodiments, the multi-chromophore absorbs only ultraviolet light, i.e., only light of 400 nm or less, and does not absorb light of wavelengths greater than 400 nm. In certain cases, multichromophores have an absorption maximum wavelength in the 300-400 nm range. In some cases, multichromophores have an absorption maximum wavelength in the 300-400 nm range (such as 300-390 nm, 300-380 nm, 300-370 nm, 300-360 nm, or 300-325 nm range) and an emission maximum wavelength in the 375-900 nm range (such as 380-900 nm, 390-900 nm, or 400-900 nm range). In certain cases, multichromophores do not absorb visible light and do not absorb wavelengths above 400 nm, such as wavelengths above 405 nm. In certain cases, a multichromophore has an absorption spectrum in which 80% or more of its integrated absorption intensity (area below the absorption line) is in the ultraviolet region with a wavelength of 400 nm or less, such as 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more.

[0054] A multichromophore may have any convenient length. Depending on the case, the specific number of monomer repeat units or segments of a multichromophore may fall within the range of 2 to 500,000, such as 2 to 100,000, 2 to 30,000, 2 to 10,000, 2 to 3,000, or 2 to 1,000 units or segments, or 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 repeating units or segments of a multichromophore can fall within the range of 2 to 1,000, such as 2 to 500, 2 to 100, 3 to 100, 4 to 100, 5 to 100, 6 to 100, 7 to 100, 8 to 100, 9 to 100, or 10 to 100 units or segments.

[0055] Multichromophores may have a convenient molecular weight (MW). In some cases, the MW of a multichromophore is expressed as an average molecular weight. In some cases, polymer dyes have an average molecular weight of 500 to 500,000, such as 1,000 to 100,000, 2,000 to 100,000, or 10,000 to 100,000, or an average molecular weight of 50,000 to 100,000.

[0056] In some embodiments, the UV absorbance-modified comonomer accounts for 25% or more (by molar concentration) of the polychromophore, such as 30% or more, 40% or more, 45% or more, 50% or more, 60% or more, or 70% or more. In such cases, the polychromophore may contain five 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, or 10,000 or more. In such cases, the multichromophore may contain five 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, or 10,000 or more. In certain embodiments, the UV absorbance-modified comonomer accounts for 25% or more (by molar concentration) or more of the multichromophore, such as 30% or more, 40% or more, 45% or more, or 50% or more, and contains five 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, or 100 or more repeating units.

[0057] The subject chromophore may have one or more desirable spectroscopic properties, such as a specific absorption maximum wavelength, a specific emission maximum wavelength, an extinction coefficient, a quantum yield, etc. In some embodiments, the chromophore may have wavelengths such as 350-850 nm, 350-600 nm, 360-500 nm, 370-500 nm, 380-500 nm, 390-500 nm, or 400-500 nm. The chromophore has an emission maximum wavelength in the range of 300 to 900 nm, and specific examples of the emission maximum, though not limited to, include 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, and 805 nm ± 5 nm. In specific cases, the chromophore has an emission maximum wavelength selected from 395 nm, 460 nm, 490 nm, 550 nm, 560 nm, 605 nm, 650 nm, 680 nm, 700 nm, and 805 nm. In specific cases, the chromophore has an emission maximum wavelength of 395 nm ± 5 nm.

[0058] In some cases, the chromophore has an absorption coefficient of 5 × 10⁻⁶. 5 cm -1 M -1 For example, 6 x 10 5 cm -1 M -1 The above 7 x 10 5 cm -1 M -1 The above 8 x 10 5 cm -1 M -1 The above is 9 x 10 5 cm -1 M -1 For example, 1 × 10 6 cm -1 M -1 The above is 1.5 × 10 6 cm -1 M -1 The above is 2 x 10 6 cm -1 M -1 The above is 2.5 × 10 6 cm -1 M -1 The above is 3 x 10 6 cm -1 M -1 The above is 4 x 10 6 cm -1 M -1 The above 5 x 10 6 cm -1 M -1 The above 6 x 10 6 cm -1 M -1 The above 7 x 106 cm -1 M -1 or more, or 8 x 10 6 cm -1 M -1 The above is achieved. In such cases, the multichromophore may have five or more repeating units, such as 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or more repeating units. In some embodiments, the multichromophore has a molar extinction coefficient of 5 × 10 5 M -1 cm -1 The above is achieved. In a particular embodiment, the multichromophore has a molar extinction coefficient of 1 × 10⁻⁶. 6 M -1 cm -1 That concludes my statement.

[0059] In some cases, the multi-chromophore has an absorption coefficient of 40,000 cm⁻¹. -1 M -1 Above / repeating units, e.g., 45,000 cm -1 M -1 Above / repetition unit, 50,000 cm -1 M -1 Above / repetition unit, 55,000 cm -1 M -1 Over / repetition units, 60,000 cm -1 M -1 Over / repetition units, 70,000 cm -1 M -1 Above / repetition unit, 80,000 cm -1 M -1 Over / repetition units, 90,000 cm -1 M -1 Above / repetition unit, 100,000 cm -1 M -1 Having an absorption coefficient of 40,000 cm² or more per repeat unit. In some cases, 40,000 cm² as specified herein. -1 M -1 The above / repeating unit is the average absorption coefficient. In certain cases, the repeating units of a polychromophore may consist of one monomer, two comonomers, or three or more comonomers. In some cases, the polychromophore may have a density of 40,000 cm². -1 M -1 More than / comonomer, for example 45,000 cm³-1 M -1 Over / commonomer, 50,000 cm -1 M -1 Above / commonomer, 55,000 cm -1 M -1 Over / commonomer, 60,000 cm -1 M -1 Over / commonomer, 70,000 cm -1 M -1 Over / commonomer, 80,000 cm³ -1 M -1 Over / commonomer, 90,000 cm³ -1 M -1 Over / commonomer, 100,000 cm -1 M -1 Having an absorption coefficient of 40,000 cm² or more. In some cases, 40,000 cm². -1 M -1 The above / comonomer is the average extinction coefficient.

[0060] In certain cases, multichromophores do not absorb visible light, such as light above 400 nm, including light above 405 nm. In certain cases, multichromophores do not show significant absorption at wavelengths above 400 nm, for example, the absorption coefficient at wavelengths above that (such as 405 nm, e.g., 405 nm) is 1 × 10⁻¹⁰. 5 cm -1 M -1 For example, 9 x 10 4 cm -1 M -1 Below, 8 x 10 4 cm -1 M -1 Below, 7 x 10 4 cm -1 M -1 Below, 6 x 10 4 cm -1 M -1 Below, 5 x 10 4 cm -1 M -1 Below, 4 x 10 4 cm -1 M -1 Below, 3 x 10 4 cm -1 M -1 Below, 2 x 10 4cm -1 M -1 Below, 1 x 10 4 cm -1 M -1 Below, 5 x 10 3 cm -1 M -1 Below, 1 x 10 3 cm -1 M -1 It has an absorption coefficient of less than or equal to the following.

[0061] In a particular embodiment, the chromophore is 0.35 or greater, 0.4 or greater, 0.45 or greater, 0 These polymer dyes have a quantum yield of 0.3 or higher, such as 0.5 or higher, 0.55 or higher, 0.6 or higher, 0.65 or higher, 0.7 or higher, or a quantum yield exceeding that. In certain cases, the multi-chromophore has a quantum yield of 0.4 or higher. In certain cases, the polymer dye has a quantum yield of 0.5 or higher.

[0062] It is understood that, depending on the case, the subject polychromophore may contain coblocks (e.g., n and m coblocks). The subject polychromophore may contain any convenient linear sequence of n and m coblocks of various lengths within the overall polymer structure. Furthermore, the polychromophore may contain any convenient sequence of comonomers within such n and / or m coblocks. In the production of the subject polychromophore, various polymer synthesis methods can be used to produce the comonomers and coblocks of interest. It is understood that, depending on the case, compositions containing a collection of conjugated polymers, including some variant forms, with respect to terminal groups (i.e., end groups) present in each CP of a particular length and / or set, may be produced by this polymerization method. Formulas shown herein may mean a single compound, or a collection or subset of polymer compounds.

[0063] In some cases, the multiple chromophore is given by formula (I):

[0064] [ka]

[0065] This is shown by, in the formula, F 1 It is a 6-5-6 condensed tricyclic comonomer; M 1 and M 2 These are, independently, UV absorbance-modified comonomers; b is either 1 or 2; a, c, d, e, and f are each independently either 0 or 1, where a + c + d + f ≥ 1; L 1 This is a chemoselective tag-Z 1 It is a binding comonomer containing; n is an integer between 1 and 100,000; m is an integer between 0 and 1,000; p is an integer between 1 and 100,000; and G 1 and G 2 Each of these is independently selected from terminal groups, π-conjugated segments, linkers, and linked specific-binding members.

[0066] In some cases of equation (I), F 1 is a fluorene comonomer. In some cases of formula (I), F 1 It is a carbazole comonomer. In the case of formula (I), L 1 L is a fluorene comonomer. In a particular embodiment of formula (I), L 1 It is a carbazole comonomer.

[0067] In some embodiments of formula (I), b is 1. In certain embodiments of formula (I), b is 2. In some cases of formula (I), a is 0. In all cases, c is 0. In certain embodiments of equation (I), d is 0. In certain cases of equation (I), e is 0. In certain cases of equation (I), f is 0. In some embodiments of equation (I), a+c+d+f=1 (i.e., a is 1, or c is 1, or d is 1, or f is 1). In some embodiments of equation (I), a+c+d+f=2. In some embodiments of equation (I), a+c+d+f=3. In some embodiments of equation (I), a+c+d+f=4. In some embodiments of equation (I), f is 1. In certain embodiments of equation (I), e is 1 and d or f is 1 such that d+e+f=2. In certain cases of equation (I), e is 1 and d and f are 0, respectively.

[0068] In certain embodiments of formula (I), e is 0, and d, f, and m are each 0. In certain cases, e is 1, d+f≦1, and m≧1. In certain cases, e is 1, d and f are each 0, and m≧1. In certain cases, e is 1; d+f=1, and m≧1. In some cases, d is 1 and f is 0. In some cases, d is 0 and f is 1. In some embodiments of formula (I), n, m, and p are selected such that the multichromophore contains a total of 2 to 100,000 repeating units (i.e., monomer repeating units), and the multichromophore may contain a variety of different monomer repeating units. In some cases, when m is 0, p is 1, and n is 2 to 100,000. In some embodiments of formula (I), L 1 It is a fluorene comonomer.

[0069] 6-5-6 fused tricyclic comonomers are comonomers containing a tricyclic aromatic group having three fused rings in a 6-5-6 configuration, namely two benzo rings fused to a central five-membered ring. The five-membered ring can be a carbocyclic or heterocyclic ring, and may further contain side-chain substituents on the ring atoms not fused to the benzo ring. In certain cases, 6-5-6 fused tricyclic comonomers have the following structure:

[0070] [ka]

[0071] This is shown by, in the formula, Z is -C(R 1 )2- or -N(R 1 )-and; Each R is independently H or one or more aryl substituents; R 1 These are, independently, alkyl, substituted alkyl, aralkyl, substituted aralkyl, PEG moiety, and -L. 1 -Z 1 (L 1 It is a linker, and Z 1 is selected from chemoselective tags (e.g., tags containing chemoselective functional groups) or WSGs used in any of the formulas described herein. * This refers to the site or terminal group of covalent bonding to the unsaturated main chain of a conjugated polymer. In some embodiments, Z is -N(R 1 If )-, the 6-5-6 condensed tricyclic comonomer is a carbazole comonomer. A simple carbazole comonomer can be used in the subject's polychromophore. In some embodiments, Z is -C(R 1 )2- If the case is 6-5-6 condensed tricyclic comonomer is a fluorene comonomer. A simple fluorene comonomer can be used in the subject's polychromophore. In the specific case of the 6-5-6 condensed tricyclic comonomer, R 1 Each is selected from a benzyl group substituted with one or more PEG moieties or an alkyl group substituted with two or more PEG moieties.

[0072] Fluorene comonomers are comonomers containing an aromatic group having a 9H-fluorene core structure substituted at position 9 with a convenient side-chain substituent. In some cases, the fluorene comonomer is a 9,9-disubstituted fluorene. The fluorene comonomer is bonded to adjacent polymer backbone groups via convenient positions of the fluorene core structure, such as any two positions from 1 to 8 (see the numbered scheme below). In some embodiments, the fluorene core structure is bonded to adjacent groups of the polymer backbone via positions 2 and 7.

[0073] In certain embodiments, the fluorene comonomer has the following structure:

[0074] [ka]

[0075] This is shown by, in the formula, R 1 These are, independently, alkyl, substituted alkyl, aralkyl, substituted aralkyl, PEG moiety, and -L. 1 -Z 1 (L 1 It is a linker, and Z 1 R is selected from chemoselective tags (e.g., tags containing chemoselective functional groups) or WSGs. In the particular case of fluorene comonomers, R 1 Each is selected from a benzyl group substituted with one or more PEG moieties or an alkyl group substituted with two or more PEG moieties. 1 Functional groups are found to be used in the covalent linkage of multiple chromophores to acceptor chromophores (e.g., as described herein). In certain cases, Z 1 It contains an amino group for covalent bonding to the acceptor chromophore. In certain cases, Z 1 It comprises a carboxylic acid group or a derivative thereof for covalent linkage to the acceptor chromophore. In certain embodiments, L 1 is two or more Z 1It is a branched linker that connects to a base (e.g., WSG). In a particular case, the fluorene comonomer is located at one, two or more positions selected from positions 1, 3, 4, 5, 6 and 8. 5 and / or R 6 It is further substituted with substituents, R 5 and R 6 These are independently selected from water-soluble groups (WSGs) and aryl substituents (e.g., as described herein).

[0076] In certain cases, fluorene comonomers have the following structure:

[0077] [ka]

[0078] This is shown by, in the formula, R 1 These are defined above; R 5 and R 6 This is independently selected from H, a water-soluble group, or an aryl substituent (for example, as described herein).

[0079] In some cases, fluorene comonomers have the following structure:

[0080] [ka]

[0081] This is shown by, in the formula, R 2 Each of these is an alkyl group substituted with a water-soluble group or a branched linker linked to two or more water-soluble groups (e.g., PEG disubstituted benzyl or PEG substituted alkyl). In certain cases of fluorene comonomers, R 2 Each has one, two, or three PEG sites (e.g., -O(CH2CH2O)) n It is a benzyl group substituted with R' (where R' is H or alkyl, and n is 1 to 20, for example 3 to 16, for example n is 8 to 16). In certain cases of fluorene comonomers, R 2 Each of these is a single -O(CH2CH2O) n A benzyl group substituted with an R' group (for example, at the 2, 3, or 4 position), where R' is H or alkyl, and n is 1 to 20, for example 3 to 16, for example n is 8 to 16. In certain cases of fluorene comonomers, R 2 Each of these consists of two -O(CH2CH2O) n A benzyl group substituted with an R' group (for example, at the 2,4-, 3,4-, or 3,5- positions), where R' is independently H or alkyl, and n is independently 1 to 20, for example 3 to 16, for example n is 8 to 16. In certain cases of fluorene comonomers, R 2 Each of these consists of three -O(CH2CH2O) molecules. n A benzyl group substituted with an R' group (for example, at the 2,2,4,6-, 2,4,5-, or 3,4,5- positions), where R' is H or alkyl, and n is 1 to 20, for example 3 to 16, for example n is 8 to 16. In certain cases of fluorene comonomers, R 2 Each of these is a lower alkyl group substituted with a trivalent branched group, each substituted with two PEG moieties (e.g., -CO-NR''2 or -O(CH2R'')2 trivalent branched group), and each R'' is independently a PEG moiety (e.g., -O(CH2CH2O) n R' (where R' is H or alkyl, and n is 1-20, for example 3-16, for example n is 8-16).

[0082] In certain embodiments, the fluorene comonomer has the following structure:

[0083] [ka]

[0084] This is shown by, in the formula, R 3R is an alkyl group substituted with a water-soluble group (e.g., PEG-substituted alkyl group), 4 is L 2 -Z 2 And L 2 It is a linker, and Z 2 This is a chemoselective tag (for example, for binding to an acceptor chromophore). In some cases, fluorene comonomers have the following structure:

[0085] [ka]

[0086] This is shown by, in the formula, R 3 is a substituent comprising a water-soluble group (for example, as described herein); R 4 L 2 -Z 2 And here, L 2 It is a linker, and Z 2 is a chemoselective tag (for example, for binding to an acceptor chromophore); and R 5 and R 6 R is independently selected from H, a water-soluble group, and an aryl substituent (e.g., alkyl, substituted alkyl, alkoxy, substituted alkoxy, halogen, or nitro). In the particular case of fluorene comonomers, R 3 R'' is a lower alkyl group substituted with a trivalent branched group, each substituted with two PEG moieties (e.g., -CO-NR''2 or -O(CH2R'')2 trivalent branched group), where R'' is a PEG moiety (e.g., -O(CH2CH2O) n R' (where R' is H or alkyl, and n is 1-20, for example 3-16, for example n is 8-16).

[0087] Any of the fluorene comonomers described above can be used in the subject's multichromophore, for example, the multichromophore of formulas (I) to (IV). In some cases, the multichromophore may be part of the polymer backbone, with the following structure:

[0088] [ka]

[0089] It includes one of the following, in the formula, R 3 Each is independently a water-soluble group linked via an optional linker, or an optionally substituted alkyl, aralkyl, or aryl group; Ar is an optionally substituted aryl or heteroaryl group; and n is an integer from 1 to 100,000. In a particular embodiment, R 3 Each of these is independently a substituted alkyl group. In a particular embodiment, R 3 These are each independently substituted aralkyl groups. In some cases, R 3 And Ar are independently substituted with a water-soluble group, an acceptor chromophore, a chemoselective functional group, or a specific binding site (via an optional linker).

[0090] As used herein, the terms “chemoselective functional group” and “chemoselective tag” are used interchangeably and refer to functional groups that, if any, can be selectively activated and then selectively react with other compatible functional groups to form a covalent bond. Examples of chemoselective functional groups include, but are not limited to, thiols and maleimides or iodoacetamides, amines and carboxylic acids or their activated esters, as well as groups that can react with each other via Click chemistry, such as azides and alkynes (e.g., cyclooctin groups), and hydroxyls, hydrazides, hydrazinos, aldehydes, and k Examples include tonates, azides, alkynes, phosphines, and epoxides.

[0091] Any simple binding comonomer (L 1By incorporating the ) into the subject's multichromophore, a binding group can be provided that can be attached to a convenient site on the target. The binding comonomer of the target is not limited to, but includes fluorene comonomer, phenylene vinylene comonomer, phenylene ethynylene comonomer, carbazole comonomer, C2-C 12 Examples include alkyne comonomers, arylene-ethynylene comonomers, heteroarylene-ethynylene comonomers, arylene comonomers, and heteroarylene comonomers.

[0092] The polychromophores of the subject may include, but are not limited to, simple chemoselective functional groups such as carboxylic acids, active esters (e.g., NHS or sulfo-NHS esters), amino, hydroxyl, thiol, maleimide, iodoacetyl, hydrazide, hydrazino, aldehyde, ketone, azide, alkyne, phosphine, and epoxide (e.g., -Z 1 (In which case).

[0093] In certain cases, the linkable comonomers are substituted or unsubstituted 1,4-phenyl, substituted or unsubstituted 1,3-phenyl, substituted or unsubstituted 4,4'-biphenyl, substituted or unsubstituted 2,5-pyridyl, and substituted or unsubstituted 2,6-pyridyl. In some cases, the linkable monomers are fluorene comonomers. In certain cases, the linkable comonomers are ultraviolet absorbance modified comonomers (e.g., as described herein).

[0094] The subject's multi-chromophore has one of the following simple terminal groups (e.g., G 1 and G 2 ) can be used. 1 and G 2The groups include, but are not limited to, terminal cap-forming groups, π-conjugated segments, linkers, and linked specific bonding members. In some embodiments, the terminal cap-forming group is a monovalent group that is bonded to the main chain of the polychromophore after polymerization. In certain cases, the terminal cap-forming group is aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, or substituted alkyl. In certain cases, the terminal cap-forming group is derived from a monomer used in the polymerization method, for example, a terminal group such as a halogen (e.g., Br), a boronic acid, or a boronic acid ester that can receive further bonding. In some cases, G 1 and / or G 2 This is a π-conjugated segment. As used herein, a π-conjugated segment means a simple segment of a conjugated polymer in which multiple chromophores can be conjugated, i.e., π electrons can be delocalized across adjacent units. In certain embodiments, G 1 and / or G 2 These include linkers containing functional groups suitable for conjugation to specific binding sites. (The G of the multichromophore) 1 and / or G 2 Linkers located at position Z are attached to the side chains of the multichromophore (for example, Z 1 It is understood that the linker may be selected orthogonal to other linkers containing chemoselective tags that may be present in the form of G 1 and / or G 2 It is contained in and the carboxylic acid functional group or its derivative is Z 1 It is contained in. In certain embodiments, the carboxylic acid functional group or its derivative is G 1 and / or G 2 It is contained in and the amino functional group or its derivative is Z 1 It is contained in.

[0095] A simple UV absorbance-modified comonomer can be incorporated into the subject's multi-chromophore to impart an UV absorption maximum to the multi-chromophore. In some embodiments, the UV absorbance-modified comonomer has an absorption maximum of 350 nm or less, such as 340 nm or less, 330 nm or less, 320 nm or less, 310 nm or less, 300 nm or less, 290 nm or less, 280 nm or less, 270 nm or less, 260 nm or less, 250 nm or less, 240 nm or less, 230 nm or less, 220 nm or less, 210 nm or less, or 200 nm or less.

[0096] In some embodiments, the UV absorbance-modified comonomer is an optionally substituted aryl or heteroaryl comonomer. In some cases, UV absorbance The modified comonomers are substituted or unsubstituted phenyl, biphenyl, or pyridyl comonomers. In certain embodiments, the UV absorbance modified comonomers are optionally substituted aryl or heteroaryl comonomers selected from substituted or unsubstituted 1,4-phenyl, substituted or unsubstituted 1,3-phenyl, substituted or unsubstituted 4,4'-biphenyl, substituted or unsubstituted 2,5-pyridyl, and substituted or unsubstituted 2,6-pyridyl. In certain cases, the UV absorbance modified comonomer has the following structure:

[0097] [ka]

[0098] (In the formula, Z 2 ~Z 5 Each is independently either CR or N, where Z 2 ~Z 5 At least one of them is N; R and R 11 ~R 16 Each is independently an optionally substituted aryl or heteroaryl comonomer selected from one of the following: hydrogen, a water-soluble group, a halogen, cyano, alkoxy, substituted alkoxy, alkyl, and substituted alkyl. In certain embodiments, Z 2 ~Z5 One of these and only one of them is N. In a particular embodiment, Z 2 ~Z 5 Of these, two and only two are N. In a particular case, R 11 , R 12 and R 14 These are H, respectively. In some cases, R 12 and R 14 These are H, respectively. In some cases, R 11 and R 13 These are H, respectively. Depending on the case, R 15 and R 16 These are H, respectively. In some cases, halogens are fluoro. In certain cases, R 11 ~R 14 One and only one of them are alkyl or substituted alkyl, R 11 ~R 14 The other three are each H. In a particular case, R 11 and R 13 Each of these is an alkyl or substituted alkyl, and R 12 and R 14 These are H, respectively.

[0099] Depending on the circumstances, UV absorption Degree-modified comonomers are as follows:

[0100] [ka]

[0101] (In the formula, n is between 1 and 20, and R' is H or a lower alkyl group.) It is an optionally substituted aryl or heteroaryl comonomer, selected from one of the following. In some embodiments of the substituted aryl or heteroaryl comonomer structure, n is an integer from 3 to 20. In some embodiments, the polychromophore has the following structure:

[0102] [ka]

[0103] (In the formula, n is between 1 and 20, and R' is H or a lower alkyl group.) It contains substituted aryl comonomers represented by . In certain cases, n is 3 to 12. In some embodiments, the polychromophore has the following structure:

[0104] [ka]

[0105] The compounds include substituted aryl comonomers represented by (wherein n is independently 1 to 20, and R' is independently H or a lower alkyl group). In certain embodiments of the substituted aryl or heteroaryl comonomer structures shown above, n is 3. In certain cases, R' is methyl. In certain cases, R' is hydrogen. In some embodiments, the polychromophore has the following structure:

[0106] [ka]

[0107] (In the formula, R 11 and R 13 Each comprises a substituted aryl comonomer, which is independently represented by an alkyl or substituted alkyl, e.g., a lower alkyl or substituted lower alkyl (e.g., a substituted methyl such as trifluoromethyl, difluoromethyl, or monofluoromethyl). In some embodiments, the polychromophore has the following structure:

[0108] [ka]

[0109] (In the formula, R 11 and R 12Each comprises a substituted aryl comonomer, which is independently represented by an alkyl or substituted alkyl, e.g., a lower alkyl or substituted lower alkyl (e.g., a substituted methyl such as trifluoromethyl, difluoromethyl, or monofluoromethyl). In some embodiments, the polychromophore has the following structure:

[0110] [ka]

[0111] Contains substituted aryl comonomers as shown by

[0112] In some embodiments, the multiple chromophore has the following structure:

[0113] [ka]

[0114] (In the formula, R 11 The material comprises a substituted aryl comonomer represented by an alkyl or substituted alkyl, for example, a lower alkyl or substituted lower alkyl (for example, a substituted methyl such as trifluoromethyl, difluoromethyl, or monofluoromethyl).

[0115] In some embodiments, the multiple chromophore has the following structure:

[0116] [ka]

[0117] Contains substituted aryl comonomers as shown by

[0118] Any of the above-mentioned ultraviolet absorbance-modified comonomers can be used in the subject's multichromophore, for example, the multichromophore of formulas (I) to (IV).

[0119] In some embodiments, the multiple chromophore is given by formula (II):

[0120] [ka]

[0121] It is expressed by, in the formula, F 1 M 1 M 2 a, b, c, e, L 1 , Z 1 , p, G 1 and G 2 This is as shown in equation (I). In some cases of equation (II), F 1 is full It is an orencomonomer. In the specific case of formula (II), F 1 L is a carbazole comonomer. In some embodiments of formula (II), L 1 L is a fluorene comonomer. In a particular embodiment of formula (II), L 1 is a carbazole comonomer. In certain embodiments, the chromophore is of formula (III):

[0122] [ka]

[0123] It is expressed by, in the formula, F 1 M 1 M 2 a, b, c, n, G 1 and G 2 This is as shown in equation (I). In some cases of equation (III), F 1 is a fluorene comonomer. In the specific case of formula (III), F 1 It is a carbazole comonomer.

[0124] In some cases of equations (II) and (III), b is 1 and a+c≧1. In certain cases of equations (II) and (III), a+c=1 (for example, a is 1 and c is 0, or a is 0 and c is 1). In certain embodiments of equations (II) and (III), a+c=2. In some cases of equations (II) and (III), G 1 G is a terminal group; 2 G is a terminal group, linker, or linked specific binding member. In certain cases, G 2 It is a linked specific binding member. In some cases, G 2 This is a linker, and that linker may contain chemoselective tags.

[0125] In some cases, the multiple chromophore is given by formula (IV):

[0126] [ka]

[0127] It is expressed by, in the formula, F 1 M 2 , b, c, e, f, L 1 , Z 1 n, m, p, G 1 and G 2 This is as shown in equation (I). In some cases of equation (IV), F 1 is a fluorene comonomer. In the specific case of formula (IV), F 1 It is a carbazole comonomer.

[0128] In some embodiments of equation (IV), b is 1; c is 0 or 1; e and f are 0 or 1, respectively, where e + f ≥ 1; G 1 G is a terminal group; 2 c is a terminal group, a linker, or a linked specific binding member. In the specific case of formula (IV), c is 1. In the specific case of formula (IV), c is 0. In certain cases of equation (IV), e is 1. In certain cases of equation (IV), e is 0. In certain cases of equation (IV), f is 1. In certain cases of equation (IV), f is 0. In certain cases, G 2 It is a linked specific binding member. In some cases, G 2 is a linker, and the linker may contain a chemoselective tag. In some embodiments of formula (IV), L 1 is a fluorene comonomer. In some embodiments of formula (IV), L 1 It is a carbazole comonomer.

[0129] In some embodiments, the multiple chromophore has the following structure:

[0130] [ka]

[0131] It is expressed by, in the formula, R 1 Each is independently an alkyl group substituted with WSG or a branched group further substituted with two or more WSGs; L is a linker and Z is a chemoselective tag or linked specific binding member; G 1 is a terminal group; n is an integer from 1 to 100,000. In some cases, G 1 It is an aryl-terminated group. In some cases, G 1 is phenyl. In certain embodiments, Z is selected from carboxylic acids, aminos, or maleimides. In certain cases, Z is a linked specific bonding member. In some cases, L is an alkyl-carboxylic acid such as -(CH2)3COOH. In certain embodiments, R 1 Each group is a branched group that is further substituted with two or more WSGs. In certain cases, the branched group is a substituted aralkyl group, such as a substituted benzyl group, which is substituted with two PEG groups.

[0132] In some embodiments, the multiple chromophore has the following structure:

[0133] [ka]

[0134] It is expressed by, in the formula, R 1 Each is independently an alkyl group substituted with WSG or a branched group further substituted with two or more WSGs; L is a linker and Z is a chemoselective tag or linked specific binding member; G 1 is a terminal group; n is an integer from 1 to 100,000. In some cases, G 1 It is an aryl-terminated group. In some cases, G 1 is phenyl. In certain embodiments, Z is selected from carboxylic acids, aminos, or maleimides. In certain cases, Z is a linked specific bonding member. In some cases, L is an alkyl-carboxylic acid such as -(CH2)3COOH. In certain embodiments, R 1 Each group is a branched group that is further substituted with two or more WSGs. In certain cases, the branched group is a substituted aralkyl group, such as a substituted benzyl group, which is substituted with two PEG groups.

[0135] In some embodiments, the multiple chromophore has the following structure:

[0136] [ka]

[0137] It is expressed by, in the formula, R 1 Each is independently an alkyl group substituted with WSG or a branched group further substituted with two or more WSGs; L is a linker and Z is a chemoselective tag or linked specific binding member; G 1 is a terminal group; n is an integer from 1 to 100,000. In some cases, G 1 It is an aryl-terminated group. In some cases, G 1is phenyl. In certain cases, Z is selected from carboxylic acids, aminos, or maleimides. In certain cases, Z is a linked specific bonding member. In some cases, L is an alkyl-carboxylic acid such as -(CH2)3COOH. In certain embodiments, R 1 Each group is a branched group further substituted with two or more WSGs. In certain cases, the branched group is a substituted aralkyl group, such as a substituted benzyl group, which is substituted with two PEG groups.

[0138] In some embodiments, the multiple chromophore has the following structure:

[0139] [ka]

[0140] Represented by the formula, where Ak is independently an alkyl group; mPEG is a methyl-capped PEG group, n1 and n2 are independently 3-20; L is a linker, and Z is a chemoselective tag or linked specific binding member; G 1 is a terminal group; n is an integer from 1 to 100,000. In some cases, G 1 is phenyl. In certain embodiments, Z is selected from carboxylic acids, aminos, or maleimides. In certain cases, Z is a linked specific binding member. In some cases, L is an alkyl-carboxylic acid such as -(CH2)3COOH. In some embodiments, Ak is C1-6 alkyl. In certain cases, n1 is 5-15 each. In certain cases, n2 is 3-12, for example, 3.

[0141] In some embodiments, the multiple chromophore has the following structure:

[0142] [ka]

[0143] Represented by the formula, where mPEG is a methyl-capped PEG group; L is a linker, and Z is a chemoselective tag or linked specific binding member; G 1 is a terminal group; n is an integer from 1 to 100,000. In some cases, G 1 is phenyl. In certain embodiments, Z is selected from carboxylic acids, aminos, or maleimides. In certain cases, Z is a linked specific binding member. In some cases, L is an alkyl-carboxylic acid such as -(CH2)3COOH.

[0144] In some embodiments, the multiple chromophore has the following structure:

[0145] [ka]

[0146] As shown by the formula, where Ak is independently an alkyl group; mPEG is a methyl-capped PEG group, n1 and n2 are independently 3 to 20; L is a linker, and Z is a chemoselective tag or linked specific binding member; G 1 is a terminal group; n is an integer from 1 to 100,000. In some cases, G 1 is phenyl. In certain embodiments, Z is selected from carboxylic acids, aminos, or maleimides. In certain cases, Z is a linked specific binding member. In some cases, L is an alkyl-carboxylic acid such as -(CH2)3COOH. In some embodiments, Ak is C1-6 alkyl. In certain cases, n1 is 5-15 each. In certain cases, n2 is 3-12, for example, 3.

[0147] In some embodiments, the multiple chromophore has the following structure:

[0148] [ka]

[0149] As shown by the formula, where mPEG is a methyl-capped PEG group; L is a linker and Z is a chemoselective tag or linked specific binding member; G 1 is a terminal group; n is an integer from 1 to 100,000. In some cases, G 1 is phenyl. In certain embodiments, Z is selected from carboxylic acids, aminos, or maleimides. In certain cases, Z is a linked specific binding member. In some cases, L is an alkyl-carboxylic acid such as -(CH2)3COOH. Optionally, in any of the multichromophore structures described herein, the fluorene comonomer shown is a single side-chain group R linked to the N atom of the corresponding carbazole comonomer in that structure, for example, the carbazole comonomer. 1 It is understood that this can be replaced with carbazole comonomers containing the same compound.

[0150] Polymer tandem dyes In some embodiments, the light-harvesting multichromophore is a polymer tandem dye. The polymer tandem dye comprises two covalent sites: a donor light-harvesting multichromophore (e.g., as described herein) and an acceptor chromophore. In certain embodiments, the polymer tandem dye is a water-soluble light-harvesting multichromophore comprising a conjugated segment such as a 6-5-6 condensed tricyclic comonomer (e.g., as described herein); and an ultraviolet absorbance-modified comonomer (e.g., as described herein), which may comprise a multichromophore having an ultraviolet absorption maximum (e.g., as described herein) and an acceptor chromophore covalently linked to the multichromophore near its energy acceptor. In some cases, acceptor chromophores are inactivators. In certain cases, acceptor chromophores are fluorescent dyes. As used herein, the term “acceptor chromophore” means a photoabsorbing molecule that can receive or absorb energy transferred from a multichromophore. Depending on the case, an acceptor chromophore may emit the energy it receives from a multichromophore as light or dissipate the energy as heat. Unless otherwise specified, in the structures and formulas shown herein, the label “dye” means “acceptor chromophore.” As used herein, the term “inactivator” means an acceptor chromophore that absorbs energy from a multichromophore and does not emit light but can dissipate the energy as heat.

[0151] In some embodiments, a polymer tandem dye may be excited in the UV region at the absorption maximum wavelength of the donor multichromophore and emit light at the emission wavelength of the acceptor chromophore. In some cases, the light-gathering multichromophore may transfer energy to the acceptor chromophore species near the energy acceptor. Mechanisms of energy transfer include, for example, resonance energy transfer (e.g., Förster (or fluorescence) resonance energy transfer, FRET) and quantum charge exchange (Dexter energy transfer). In some cases, these energy transfer mechanisms are over a relatively short range; In other words, efficient energy transfer occurs when a light-gathering multichromophore system is located near an acceptor chromophore. In some cases, under conditions for efficient energy transfer, if there are many individual chromophores in the light-gathering multichromophore system, amplification of emission from the acceptor chromophore occurs; that is, when the incident light ("pump light") is at a wavelength absorbed by the light-gathering multichromophore, the emission from the signaling chromophore is stronger than when the signaling chromophore is directly excited by the pump light.

[0152] "Efficient" energy transfer means that 30% or more of the collected energy is transferred to the acceptor. If the acceptor chromophore is a fluorescent dye, the term efficient energy transfer means a fluorescence quantum yield of 0.3 or greater, e.g., 0.4 or greater, 0.5 or greater, or greater. "Amplified" means that the signal from the acceptor chromophore is 1.5 times or greater when excited by a light-gathering chromophore compared to direct excitation with incident light of equal intensity. This signal can be measured using any convenient method. In some cases, a signal of 1.5 times or greater means the intensity of emitted light. In certain cases, a signal of 1.5 times or greater means an increase in the signal-to-noise ratio. In certain embodiments of polymer tandem dyes, the emission of the acceptor chromophore is 1.5 times or greater when excited by a multichromophore compared to direct excitation of the acceptor chromophore with incident light.

[0153] In some cases, polymer tandem dyes are 5 × 10 5 cm -1 M -1 For example, 6 x 10 5 cm -1 M -1 The above 7 x 10 5 cm -1 M -1 The above 8 x 10 5 cm -1 M -1 The above is 9 x 10 5 cm -1 M -1 For example, 1 × 10 6 cm -1 M -1 The above is 1.5 × 10 6 cm -1 M -1 The above is 2 x 10 6 cm -1 M -1 The above is 2.5 × 10 6 cm -1 M -1 The above is 3 x 10 6 cm -1 M -1 The above is 4 x 10 6 cm-1 M -1 The above 5 x 10 6 cm -1 M -1 The above 6 x 10 6 cm -1 M -1 The above 7 x 10 6 cm -1 M -1 or more, or 8 x 10 6 cm -1 M -1 It has the above absorption coefficient. In some embodiments, the polymer tandem dye is 5 × 10 5 M -1 cm -1 It has the above molar extinction coefficient. In a particular embodiment, the polymer tandem dye is 1 × 10 6 M -1 cm -1 It has the above molar extinction coefficient.

[0154] In certain embodiments, the polymer tandem dye has a quantum yield of 0.3 or more, for example, 0.35 or more, 0.4 or more, 0.45 or more, 0.5 or more, 0.55 or more, 0.6 or more, 0.65 or more, 0.7 or more, or greater. In certain cases, the polymer tandem dye has a quantum yield of 0.4 or more. In certain cases, the polymer tandem dye has a quantum yield of 0.5 or more.

[0155] As acceptor chromophores, simple fluorescent dyes can be used in polymer tandem dyes. The terms "fluorescent dye" and "fluorophore" are used interchangeably herein. In some embodiments, the acceptor chromophores are cyanine dyes, xanthene dyes, coumarin dyes, thiadin dyes, or acridine dyes. The target fluorescent dyes are not limited to, but include fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxylodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-chromium, phycoerythrin, PerCP (peridinin chlorophyll-α protein), PerCP-Cy5.5, JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350, and Alex Examples include Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY FL, BODIPY FL-Br.sub.2, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY R6G, BODIPY TMR, BODIPY TR, their conjugates, and combinations thereof. Examples of lanthanide chelates, though not limited to them, include europium chelate, terbium chelate, and samarium chelate. In some embodiments, the polymer tandem dye means a polymer dye linked to an acceptor fluorophore selected from Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Alexa 488, Alexa 647, and Alexa 700. In certain embodiments, the polymer tandem dye means a Dyomics dye (e.g., DY 431, DY 485XL, DY 500XL, DY 530, DY 610, DY 633, DY 640, DY 651, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY Examples include polymer dyes linked to acceptor fluorophores selected from 734, DY 752, DY 754, DY 778, DY 782, DY 800 or DY 831), Biotium CF 555, Cy 3.5, and diethylaminocoumarin.

[0156] In some embodiments, the polymer tandem dye has an absorption maximum wavelength in the range of 300-400 nm and an emission maximum wavelength in the range of 375-900 nm. In certain embodiments, the polymer tandem dye comprises a water-soluble light-harvesting multichromophore having a conjugated segment containing a 6-5-6 condensed tricyclic comonomer and an ultraviolet absorbance-modified comonomer. In some cases, the ultraviolet absorbance-modified comonomer is present in the polymer tandem dye at a concentration of 25% or more (by molar concentration), and the multichromophore is a conjugated polymer containing 5 or more monomer repeating units.

[0157] In some cases, polymer tandem dyes are of formula (V):

[0158] [ka]

[0159] This is shown by, in the formula, F 1 is a 6-5-6 condensed tricyclic comonomer (e.g., as described herein); M 1 and M2 Each of these is independently an ultraviolet absorbance-modified comonomer (e.g., as described herein); b is either 1 or 2; a, c, d, and f are each independently either 0 or 1, where a + c + d + f ≥ 1; e is either 1 or 2; L 1 This is the Acceptor Chromogroup-C 1 It is a connectable comonomer linked to; n is an integer between 1 and 10,000; m is an integer between 1 and 10,000; p is an integer between 1 and 100,000; and G 1 and G 2 Each is independently selected from terminal groups, π-conjugated segments, linkers, and linked specific-binding members. In certain embodiments of formula (V), e is 1. In some cases of formula (V), d+f≦1 (i.e., d is 1 and f is 0, or d is 0 and f is 1) and m≧1. In some cases of formula (V), F 1 is a fluorene comonomer. In the specific case of formula (V), F 1 It is a carbazole comonomer.

[0160] In some embodiments, the polymer tandem dye is of formula (VI):

[0161] [ka]

[0162] This is shown by, in the formula, F 1 M 1 M 2 a, b, c, e, L 1 , C 1 , p, G 1 and G 2 This is as described for equation (V). In some cases of equation (VI), F 1is a fluorene comonomer. In the specific case of formula (VI), F 1 It is a carbazole comonomer. In some embodiments, the polymer tandem dye is of formula (VII):

[0163] [ka]

[0164] This is shown by, in the formula, F 1 M 2 , b, c, e, f, n, m, L 1 , C 1 , p, G 1 and G 2 This is as described for equation (V). In some cases of equation (VII), F 1 is a fluorene comonomer. In the specific case of formula (VII), F 1 b is a carbazole comonomer. In the specific case of formula (VII), b is 1 and c is 0. c is 1; e is 1; f is 0 or 1. In some cases of formula (VII), c is 1. In some embodiments of formula (VII), c is 0. In some cases of formula (VII), f is 1. In certain cases of formula (VII), f is 0. In certain cases of formula (VII), G 1 G is a terminal group. 2 is a terminal group, linker, or linked specific binding member. In some cases, G 2 This is a linker containing a chemoselective tag. In some cases, G 2 These are linked specific binding members.

[0165] In the polymer tandem dyes of formulas (V) to (VII), any simple fluorene comonomer (e.g., those described herein) may be used. In the polymer tandem dyes of formulas (V) to (VII), any simple carbazole comonomer may be used. In the polymer tandem dyes of formulas (V) to (VII), any simple UV absorbance modified comonomer (e.g., those described herein) may be used. In the polymer tandem dyes of formulas (V) to (VII), any simple linkable comonomer (e.g., those described herein) may be used. In certain embodiments of formulas (V) to (VII), L 1 L is a fluorene comonomer. In certain embodiments of formulas (V) to (VII), L 1 L is a carbazole comonomer. In some cases of formulas (V) to (VII), 1 Structure:

[0166] [ka]

[0167] This is shown by, in the formula, R 1 This is a substituent containing a water-soluble group (e.g., a PEG-substituted alkyl group); R 2 L 2 -Z 2 (L 2 It is a linker, and Z 2 (is an acceptor chromophore); and R 3 and R 4 These are independently H, a water-soluble group, alkyl, substituted alkyl, alkoxy, substituted alkoxy, halogen, and nitro. In certain cases, R 3 and R 4 Each of them is independently hydrogen.

[0168] In some embodiments of formulas (V) to (VII), G 1 and G 2 At least one of them is -L 3-Z, where L 3 is a linker (e.g., as described herein), and Z is a specific bonding member (e.g., as described herein). In some embodiments of formulas (I) to (VII), G 1 and G 2 At least one of them is -L 3 -Z, where L 3 A is a linker (e.g., as described herein), and Z is a chemoselective tag (e.g., as described herein). In some cases, Z is selected from carboxylic acids, active esters (e.g., N-hydroxysuccinimidyl esters (NHS) or sulfo-NHS), aminos, maleimides, iodoacetyls, and thiols. In certain embodiments of formulas (I) to (VII), G 1 and G 2 At least one of them has the following structure: *-Ar-LZ As shown by the formula, where Ar is a π-conjugated aryl group, L is a linker, and Z is a chemoselective tag or specific bonding member. In the embodiment, G 1 and G 2 At least one of them has the following structure:

[0169] [ka]

[0170] This is shown by, in the formula, q is an integer between 0 and 12; L is an optional linker; and Z is a chemoselective tag or specific binding member. In certain embodiments of formulas (I) to (VII), at least one L 2 The base, structure:

[0171] [ka]

[0172] The formula is represented by , where q is an integer from 0 to 12; L is an optional linker; and Z is a chemoselective tag or a specific binding member. In certain cases, -NH-LZ includes an amide bond to a chemoselective tag or a specific binding member. In certain embodiments, Z is a biomolecule. In certain cases, Z is an antibody. In some cases, Z is an antibody fragment or a binding derivative thereof. The antibody fragment or its binding derivative may be selected from Fab fragments, F(ab')2 fragments, scFv, diabody, and triabody.

[0173] In some embodiments of formulas (V) to (VII), C 1 The acceptor chromophore C is selected from cyanine dyes, xanthene dyes, coumarin dyes, thiadin dyes, and acridine dyes linked via an optional linker. In certain cases, the linker is selected from alkyl, substituted alkyl, alkylamide, alkylamide-alkyl, and PEG moieties. In certain embodiments of (V) to (VII), the acceptor chromophore C 1 These are selected from DY 431, DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 778, DY 782, DY 800, DY 831, Biotium CF 555, Cy3.5, and diethylaminocoumarin.

[0174] In some embodiments, the polymer tandem dye has the following structure:

[0175] [ka]

[0176] This is shown by, in the formula, R 1Each is independently a WSG-substituted alkyl group, or a branched group further substituted with two or more WSGs; R 2 is L 2 -Z 2 (L 2 It is a linker, and Z 2 ( is an acceptor chromophore); L is a linker and Z is a chemoselective tag or linked specific binding member; G 1 is a terminal group; n, m, and p are each an integer between 1 and 100,000, independently of each other. In some cases, G 1 It is an aryl-terminated group. In some cases, G 1 is phenyl. In certain embodiments, Z is selected from carboxylic acids, aminos, or maleimides. In certain cases, Z is a linked specific bonding member. In some cases, L is an alkyl-carboxylic acid such as -(CH2)3COOH. In certain embodiments, R 1 One or more of the groups are branched groups further substituted with two or more WSGs. In certain cases, the branched group is a substituted aralkyl group, such as a substituted benzyl group, which is disubstituted with two PEG groups.

[0177] In some embodiments, the multiple chromophore has the following structure:

[0178] [ka]

[0179] This is shown by, in the formula, R 1 Each is independently an alkyl group substituted with WSG or a branched group further substituted with two or more WSGs; R 2 L 2 -Z 2 (L 2 It is a linker, and Z 2 ( is an acceptor chromophore); L is a linker and Z is a chemoselective tag or linked specific binding member; G 1is a terminal group; n, m, and p are each an integer between 1 and 100,000, independently of each other. In some cases, G 1 It is an aryl-terminated group. In some cases, G 1 is phenyl. In certain embodiments, Z is selected from carboxylic acids, aminos, or maleimides. In certain cases, Z is a linked specific bonding member. In some cases, L is an alkyl-carboxylic acid such as -(CH2)3COOH. In certain embodiments, R 1 One or more of the groups are branched groups further substituted with two or more WSGs. In certain cases, the branched group is a substituted aralkyl group, such as a substituted benzyl group, which is disubstituted with two PEG groups.

[0180] In some embodiments, the multiple chromophore has the following structure:

[0181] [ka]

[0182] This is shown by, in the formula, R 1 Each is independently an alkyl group substituted with WSG or a branched group further substituted with two or more WSGs; R 2 L 2 -Z 2 (L 2 It is a linker, and Z 2 ( is an acceptor chromophore); L is a linker and Z is a chemoselective tag or linked specific binding member; G 1 is a terminal group; n is an integer from 1 to 100,000. In some cases, G 1 It is an aryl-terminated group. In some cases, G 1 It is phenyl. In certain embodiments, Z is selected from carboxylic acids, amino acids, or maleimides. In certain cases, Z is a linked specific bonding member. In some cases, L is an alkyl-carboxylic acid such as -(CH2)3COOH. In certain embodiments, R 1 One or more of the groups are branched groups further substituted with two or more WSGs. In certain cases, the branched group is a substituted aralkyl group, such as a substituted benzyl group, which is substituted with two PEG groups.

[0183] In some embodiments, the multiple chromophore has the following structure:

[0184] [ka]

[0185] This is shown by, in the formula, R 1 Each is independently a WSG-substituted alkyl group or a branched group further substituted with more than Q WSGs, -Ak-O-mPEG n1 Ak is an alkyl group independently; mPEG is a methyl-capped PEG group, with n1 and n2 independently being 3-20; R 2 L 2 -Z 2 (L 2 It is a linker, and Z 2 ( is an acceptor chromophore); L is a linker and Z is a chemoselective tag or linked specific binding member; G 1 is a terminal group; n is an integer from 1 to 100,000. In some cases, G 1 is phenyl. In certain embodiments, Z is selected from carboxylic acids, amino acids, or maleimides. In certain cases, Z is a linked specific bonding member. In some cases, L is an alkyl-carboxylic acid such as -(CH2)3COOH. In some embodiments, Ak is C 1~6It is alkyl. In some cases, Ak is -CH2-. In some cases, R 1 These are, respectively, propyl-O-mPEG 11 That is In certain cases, n1 is between 5 and 15. In certain cases, n2 is between 3 and 12, for example, 3.

[0186] In some embodiments, the multiple chromophore has the following structure:

[0187] [ka]

[0188] As shown by the formula, where Ak is independently alkyl; mPEG is a methyl-capped PEG group, and n1 and n2 are independently 3 to 20; L 2 It is a linker, and Z 2 L is an acceptor chromophore; L is a linker, and Z is a chemoselective tag or linked specific binding member; G 1 is a terminal group; n is an integer from 1 to 100,000. In some cases, G 1 is phenyl. In certain embodiments, Z is selected from carboxylic acids, amino acids, or maleimides. In certain cases, Z is a linked specific bonding member. In some cases, L is an alkyl-carboxylic acid such as -(CH2)3COOH. In some embodiments, Ak is C1-6 alkyl. In certain cases, n1 is 5-15 each. In certain cases, n2 is 3-12 each, for example, 3. In some cases, the Ak of ​​UV-modified comonomers is -CH2-. In some cases, the Ak of ​​fluorene comonomers is -(CH2)3- each.

[0189] In any of the structures and formulas shown herein, in the case of a part of the subject's polychromophore, the indicated terminal group may be located at the opposite end from the indicated end, for example, terminal group G1 It is understood that -Ph-LZ can be interchanged.

[0190] Labeled specific binding members Aspects of the disclosure of the present invention include a labeled specific binding member. The labeled specific binding member is a conjugate of a subjective multichromophore (e.g., as described herein) and a specific binding member. The multichromophore may be a polymer dye. The multichromophore may be a polymer tandem dye. Any of the multichromophores described herein can be conjugated with a specific binding member. The specific binding member and the multichromophore may be conjugated (covalently bonded) to each other via an optional linker through a convenient position on the multichromophore.

[0191] As used herein, the term “specifically binding member” means one member of a pair of molecules that have binding specificity to each other. One member of the pair of molecules has a region or cavity on its surface that specifically binds to a region or cavity on the surface of the other member of the pair of molecules. Thus, the members of the pair have the property of specifically binding to each other to form a binding complex. In some embodiments, the affinity between the specifically binding members in the binding complex is 10 -7 10 or less -6 M or less, for example, 10 -8 More than M For example, 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, for example, 10 -15 K below M d It is characterized by (dissociation constant). In some embodiments, the specific binding member binds specifically with high binding activity. High binding activity means that the binding member binds specifically with 10 × 10 -9 M or less, for example, 1 × 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 K that appears to be M or lower d This means that it specifically binds with apparent affinity, which is characterized by [the specified characteristic].

[0192] As used herein, the term "proteinic" refers to a site composed of amino acid residues (e.g., a specific binding member). The proteinic site may be a polypeptide. In some embodiments, the specific binding member is proteinic. In certain cases, the proteinic specific binding member is an antibody. In certain embodiments, the proteinic specific binding member refers to an antibody fragment, for example, 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 substantially encoded by all or part of the recognized immunoglobulin genes. For example in humans, the recognized immunoglobulin genes include the kappa (k), lambda (l), and heavy chain loci (both containing numerous variable region genes), and the constant region genes mu (u), delta (d), gamma (g), sigma (e), and alpha (a) (encoding the IgM, IgD, IgG, IgE, and IgA isotypes, respectively). The immunoglobulin light or heavy chain variable region consists of a “framework” region (FR) interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDR.” The extent of the framework region and CDR is precisely defined (see “Sequences of Proteins of Immunological Interest,” E. Kabat et al., USD Department of Health and Human Services, (1991)). The numbering of all antibody amino acid sequences described herein conforms to the Kabat system. The sequences of different light or heavy chain framework regions are relatively conserved within the species. The antibody framework region, i.e., the combined framework region of the light and heavy chain components, plays a role in positioning and aligning the CDR. The CDR is primarily responsible for binding to the antigen epitope.

[0193] The term antibody encompasses full-length antibodies and may refer to naturally occurring antibodies of any organism, genetically engineered antibodies, or antibodies recombinantly produced for experimental, therapeutic, or other purposes, as further defined below. Antibody fragments of interest include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, or other antigen-binding sequences of antibodies produced by modifications of the whole antibody or de novo-synthesized using recombinant DNA technology. Antibodies can be monoclonal or polyclonal and may possess other specific activities against cells (e.g., antagonist, agonist, neutralizing, blocking, or stimulating antibodies). It is understood that the antibody may have further conserved amino acid substitutions that do not substantially affect its antigen-binding or other antibody functions.

[0194] In certain embodiments, the specific binding member is an antibody. In certain embodiments, the specific binding member is a Fab fragment, an F(ab')2 fragment, an scFv, a diabody, or a triabody. In some cases, the specific binding member is a mouse antibody or a fragment thereof. In certain cases, the specific binding member is a recombinant antibody or a fragment thereof.

[0195] In some embodiments, the labeled specific binding member includes a water-soluble light-harvesting polychromophore comprising a conjugated segment containing a fluorene comonomer (e.g., as described herein) and an ultraviolet absorbance-modified comonomer (e.g., as described herein), wherein the polychromophore comprises an ultraviolet absorption maximum and a specific binding member covalently bonded to the polychromophore. In certain embodiments of the labeled specific binding member, the polychromophore has an absorption maximum wavelength in the range of 300-400 nm and an emission maximum wavelength in the range of 375-900 nm. In some cases of the labeled specific binding member, the ultraviolet absorbance-modified comonomer constitutes 25% or more (by molar concentration) of the polychromophore; the polychromophore is a conjugated polymer comprising five or more repeating units.

[0196] In the specific case of a labeled, specifically binding member, the multichromophore has a molar extinction coefficient of 5 × 10⁻⁶. 5 M -1 cm -1 The member has the above characteristics (for example, as described herein). In certain cases of the labeled specific binding member, the multichromophore has a quantum yield of 0.3 or more (for example, as described herein). In some embodiments, the labeled specific binding member further comprises an acceptor chromophore covalently bonded to the multichromophore near its energy acceptor, for example, the multichromophore being a polymer tandem dye. In certain cases, the acceptor chromophore is a fluorophore. In some embodiments of the labeled specific binding member, the acceptor chromophore emission is 1.5 times or more when excited by the multichromophore compared to the direct excitation of the acceptor chromophore with incident light.

[0197] In some cases of labeled specific binding members, the multichromophore is given by formula (VIII):

[0198] [ka]

[0199] This is shown by, in the formula, F 1It is a 6-5-6 condensed tricyclic comonomer; M 1 and M 2 These are, independently, UV absorbance-modified comonomers; b is either 1 or 2; a, c, d, e, and f are each independently either 0 or 1, where a + c + d + f ≥ 1; L 1 is, -Z 1 A connectable comonomer linked to, where Z 1 These are chemoselective tags or acceptor chromophores; n is an integer between 1 and 10,000; m is an integer between 1 and 10,000; p is an integer between 1 and 100,000; and G 1 and G 2 Each is independently selected from terminal groups, π-conjugated segments, linkers, and linked specific-binding members, where G 1 and G 2 At least one of them is a linked specific binding member. In some cases of formula (VIII), F 1 is a fluorene comonomer. In the specific case of formula (VIII), F 1 It is a carbazole comonomer.

[0200] In certain embodiments of formula (VIII), the specific binding member is an antibody. In some cases of formula (VIII), the specific binding member is an antibody fragment or its binding derivative. In some cases of formula (VIII), the specific binding member is an antibody fragment or its binding derivative, selected from Fab fragments, F(ab')2 fragments, scFv, diabodies, and triabodies. In some cases of formula (VIII), the acceptor chromophore is selected from cyanine dyes, xanthene dyes, coumarin dyes, thiadin dyes, and acridine dyes. In certain cases of formula (VIII), the acceptor chromophore is DY 431, DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY Selected from 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 778, DY 782, DY 800, DY 831, Biotium CF 555, Cy3.5, and diethylaminocoumarin.

[0201] In some embodiments, the labeled specific binding member has the following structure:

[0202] [ka]

[0203] This is shown by, in the formula, R 1 Each of these is independently an alkyl group substituted with WSG or a branched group further substituted with two or more WSGs; The specific binding member is a biomolecule; and n is an integer between 1 and 100,000. In a particular embodiment, R 1 Each group is a branched group further substituted with two or more WSGs. In certain cases, the branched group is a substituted aralkyl group, such as a substituted benzyl group substituted with two PEG groups.

[0204] In a particular case of any one of the fluorene comonomers of formulas (I) to (VIII), R 1 or R 2 The side chain groups are benzyl groups substituted with one, two, or three PEG sites (e.g., -O(CH2CH2O)). n R'(where R' is H or alkyl, and n is 1-20, for example, 3-16, or 8-16). In a particular case of any one of the fluorene comonomers of formulas (II) to (XII), R 1 or R 2 Each side chain group is -O(CH2CH2O) n A benzyl group substituted with an R' group (for example, at the 2, 3, or 4 position), where R' is H or alkyl, and n is 1 to 20, for example 3 to 16, and n is 8 to 16, etc. In a particular case of any one of the fluorene comonomers of formulas (II) to (XII), R 1 or R 2 Each side chain group consists of two -O (CH2CH2O) groups. n A benzyl group substituted with an R' group (for example, at the 2,4-, 3,4-, or 3,5-position), where R' is H or alkyl, and n is independently 1 to 20, for example 3 to 16, and n is 8 to 16, etc. In a particular case of any one of the fluorene comonomers of formulas (II) to (XII), R 1 or R 2 Each side chain group consists of three -O(CH2CH2O) groups. n A benzyl group substituted with an R' group (for example, at the 2,4,6-, 2,4,5-, or 3,4,5- positions), where R' is independently H or alkyl, and n Each of these is independently 1 to 20, for example 3 to 16, and n is 8 to 16, etc. In a particular case of any one of the fluorene comonomers of formulas (I) to (VIII), R 1 or R 2 The side chain groups are lower alkyl groups each substituted with a trivalent branched group, each substituted with two PEG moieties (e.g., -CO-NR''2 or -O(CH2R'')2 trivalent branched group), where R'' is independently substituted with a PEG moiety (e.g., -O(CH2CH2O) n R'(where R' is H or alkyl, and n is 1-20, for example 3-16, for example n is 8-16)).

[0205] Alternatively, it is understood that any one of the polymer tandem dyes of formulas (I) to (VIII) can be represented by a formula that shows the molar percentage value of each comonomer in the polymer. For example, in some cases, any one of formulas (I) to (VIII) can be the following: G 1 -(M 1 ) v (F 1 ) x (M 2 ) y (L 1 ) z -G 2 G 1 -(M 1 ) v (F 1 ) x (M 2 ) y -G 2 G 1 -(F 1 ) x (M 2 ) y (L 1 ) z -G 2 G 1 -(F 1 ) x (M 2 ) y -G 2 It can be expressed by one of the following, where v, x, y, and z are the molar percentages of the comonomer in the conjugated polymer, and L 1 C 1 or Z 1 It can be connected to (for example, as described herein). In some cases of the formula, v is 1 mol% or more, for example, 5 mol% or more, 10 mol% or more, 15 mol% or more, 20 mol% or more, or 25 mol% or more. In some cases of the formula, v is 25 mol% or less, for example, 20 mol% or less, 15 mol% or less, 10 mol% or less, 8 mol% or less, 6 mol% or less, 5 mol% or less, 2 mol% or less, 1 mol% or less, or less than that. In some cases of the formula, x is 1 mol% or more, for example, 2 mol% or more, 3 mol% or more, 4 mol% or more, 5 mol% or more, 10 mol% or more, 15 mol% or more, 20 mol% or more, 25 mol% or more, 30 mol% or more, 35 mol% or more, 40 mol% or more, 45 mol% or more, 50 mol% or more, or more than that. In the specific case of the formula, x is in the range of 1 to 50 mol%, for example 5 to 25 mol% or 10 to 25 mol%; or for example 5 to 25 mol% or 10 to 25 mol%; or for example 1 to 25 mol%, 1 to 10 mol%, or 1 to 5 mol%. In some cases of the formula, z is 10 mol% or more, for example 15 mol% or more, 20 mol% or more, 25 mol% or more, 30 mol% or more, 35 mol% or more, 40 mol% or more, 45 mol% or more, 50 mol% or more, or greater than that. In some cases of the formula, z is 25 mol% or less, for example 20 mol% or less, 15 mol% or less, 10 mol% or less, 8 mol% or less, 6 mol% or less, 5 mol% or less, 2 mol% or less, 1 mol% or less, or less than that. In some cases of the formula, y is 1 mol% or more, for example 5 mol% or more, 10 mol% or more, 15 mol% or more, 20 mol% or more, or 25 mol% or more. In some cases of the formula, y is 25 mol% or less, for example 20 mol% or less, 15 mol% or less, 10 mol% or less, 8 mol% or less, 6 mol% or less, 5 mol% or less, 2 mol% or less, 1 mol% or less, or less.

[0206] In any of the structures and formulas shown herein, in the case of some subject polymer dyes, it is understood that the indicated end or end group may be located at the opposite end from the indicated end, for example, the end groups may be interchanged. In some embodiments of the polychromophores shown herein (e.g., formulas (I) to (VIII)), at least one end group (e.g., L, L)2 , G 1 , G 2 ,LZ) has the following structure 1~33:

[0207] [ka]

[0208] [ka]

[0209] [ka]

[0210] One of the following will be selected. * This is a site for covalent bonding to the unsaturated main chain; In the formula, R' is independent of H, halogen, and C1-C. 12 Alkyl, (C1~C 12 Alkyl)NH2, C2~C 12 Alkenes, 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 is; and Z 1 is -OH or -COOH; X 1 -NH2, -NHCOOH, -NHCOOC(CH3)3, -NHCO(C3~C 12 )Cycloalkyl(C1~C4)alkyl-N-maleimide; or -NHCO[CH2-CH2-O] s’ (CH2) s’ NH2(r' is an integer between 1 and 20; s' are each an independent integer between 1 and 20), (CH2)3(OCH2CH2)x’’ OCH3(x ’’ (These are independent integers from 0 to 50) or one or more halogens, hydroxyls, C1 to C 12 benzyl or (OCH2CH2) that is optionally substituted with an alkoxy. y’’ CH3(y ’’ (Each of these is an integer between 0 and 50 independently), and R' is different from R; k is 2, 4, 8, 12, or 24; R 15 Structure:

[0211] [ka]

[0212] Selected from l~u groups having the following properties. * This is a site for covalent bonding to the main chain.

[0213] In some embodiments of the polychromophores described herein (e.g., formulas (I) to (VIII)), at least one terminal group (e.g., L, L) 2 , G 1 , G 2 ,LZ) is structured as follows Construction:

[0214] [ka]

[0215] One of the following is selected, where r is an integer between 0 and 50 (e.g., 1 and 20); k is an integer between 0 and 50 (e.g., 1 and 20); R 1 This is as defined for any of the fluorene comonomers described herein; R 16 H, OH, NH2, -NH(CH2) r -NH2 and -NH(CH2) r Selected from COOH.

[0216] method As summarized above, aspects of the present invention include a method for evaluating a sample for the presence of a target sample. In some embodiments, the method is a step of (a) contacting the sample with a polymer dye conjugate that specifically binds to the target sample to produce a sample that has come into contact with a labeled composition, wherein the polymer dye conjugate is (i) a water-soluble light-harvesting polychromophore (e.g., as described herein) and a 6-5-6 condensed tricyclic comonomer (e.g., as described herein); and an ultraviolet absorbance-modified comonomer (e.g., as described herein), and violet The process comprises (ii) a water-soluble, light-harvesting, multichromophore having an external absorption maximum; and (ii) a specific binding member (e.g., as described herein); and (b) an assay of the sample that has come into contact with the labeled composition to determine whether a target sample is present in the sample, for the presence of a polymer dye conjugate-target sample binding complex.

[0217] A sample can be produced in contact with a labeled composition by contacting the sample with a polymer dye conjugate that specifically binds to the target sample using any of the following simple methods. The terms “polymer dye conjugate” and “labeled specific binding member” as used herein are used interchangeably. In some cases, when a target sample is present, the sample is contacted with the polymer dye conjugate under conditions that allow the specific binding member to specifically bind to the target sample. A suitable solution may be used to maintain the biological activity of the sample components and the specific binding member for the specific binding of the conjugate's specific binding member to the target sample. The solution can be an equilibrium salt solution, such as ordinary physiological saline, PBS, or Hanks' solution. Conveniently, it can be combined with a low-concentration, acceptable buffer (e.g., 5-25 mM) to add fetal bovine serum, human platelet lysate, or other factors. Convenient buffers include HEPES, phosphate buffer, and lactate buffer. Various culture media are commercially available, and different media such as dMEM, HBSS, dPBS, RPMI, and Iscove medium can be used depending on the properties of the target sample. Fetal bovine serum or human platelet lysate may be added as needed. The final components of the solution can be selected according to the components of the sample contained.

[0218] The temperature at which specific binding of the conjugate's specific binding member to the target sample occurs varies and, in some cases, can be in the range of 5–50°C, e.g., 10–40°C, 15–40°C, 20–40°C, e.g., 20°C, 25°C, 30°C, 35°C, or 37°C (e.g., the temperatures mentioned 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 sample. In specific cases, that temperature is 25°C, 30°C, 35°C, or 37°C. In specific cases, the specific binding member is an antibody or a fragment thereof, and the temperature at which specific binding occurs is room temperature (e.g., 25°C), 30°C, 35°C, or 37°C. A convenient incubation time for specific binding is selected to allow the desired amount of binding complex to form and, in some cases, can be 1 minute or more, e.g., 2 minutes or more, 10 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, or even 6 hours or more.

[0219] Any convenient specific binding member may be used in the conjugate. Examples of specific binding members include, but are not limited to, cell surface proteins of various cell types, and, but are not limited to, agents that specifically bind to stem cells, e.g., pluripotent stem cells, hematopoietic stem cells, T cells, T regulatory factor 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. Target cells include cells having convenient cell surface markers or antigens that can be captured by a convenient specific binding member conjugate. In some embodiments, target cells are selected from HIV-containing cells, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+) from whole blood, bone marrow, or umbilical cord blood. Convenient cell surface proteins or cell markers may be targeted for specific binding to polymer dye conjugates in the method of the subject. In some embodiments, target cells include cell surface markers selected from cell receptors and cell surface antigens. In some cases, target cells may be 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 / It may contain β, T cell receptor γ / δ, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminal), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71.

[0220] Convenient targets may be selected for evaluation using the subject method. Targets include, but are not limited to, nucleic acids such as RNA, DNA, PNA, CNA, HNA, LNA, or ANA molecules, proteins such as fusion proteins, modified proteins such as phosphorylated, glycosylated, ubiquitinated, SUMOylated, or acetylated proteins, or antibodies, peptides, aggregated biomolecules, cells, small molecules, vitamins, and drug molecules. As used herein, the term “target protein” means all members and fragments of the target family. Target proteins are any of the proteins of interest, such as therapeutic or diagnostic targets, but are not limited to: hormones, growth factors, receptors, enzymes, cytokines, bone inducers, colony-stimulating factors, and immunoglobulins. The term “target protein” is intended to encompass recombinant and synthetic molecules that can be produced or purchased using either a simple recombinant expression method or a simple synthesis method. In some embodiments, the polymer dye conjugate may be an antibody or antibody fragment. Any simple target sample that specifically binds to the antibody or antibody fragment of interest may be targeted in the method described herein.

[0221] In some embodiments, the target sample is associated with cells. In certain cases, the target sample is a cell surface marker of those cells. In certain cases, the cell surface marker is selected from cell receptors and cell surface antigens. In some cases, the target sample is an intracellular target, and the method further includes lysing the cells.

[0222] In some embodiments, the sample includes a heterogeneous cell population from which target cells are isolated. In some cases, the sample includes peripheral whole blood, peripheral whole blood with erythrocytes lysed before cell isolation, umbilical cord blood, bone marrow, density gradient purified peripheral blood mononuclear cells, or homogenized tissue. In some cases, the sample includes hematopoietic progenitor cells (e.g., CD34+ cells) in whole blood, bone marrow, or umbilical cord blood. In certain embodiments, the sample includes tumor cells in peripheral blood. In certain cases, the sample is a sample containing (or suspected to contain) viral cells (e.g., HIV).

[0223] Labeling-specific binding members are found to be used in the methods of the subject, for example, to label target cells, particles, targets, or specimens with polymer dyes or polymer tandem dyes. For example, labeling-specific binding members are found to be used in labeling cells processed in a flow cytometer (e.g., detected, analyzed, and / or sorted). Labeling-specific binding members may include, for example, antibodies that specifically bind to cell surface proteins of various cell types (e.g., as described herein). Labeling-specific binding members can be used to investigate various biological (e.g., cellular) properties or processes such as the cell cycle, cell proliferation, cell differentiation, DNA repair, T cell signaling, apoptosis, cell surface protein expression, and / or presentation. Labeling-specific binding members can be used in any application that includes (or may include) antibody-mediated labeling of cells, particles, or specimens.

[0224] In some embodiments, the polymer dye conjugate comprises a polymer tandem dye (e.g., as described herein). Thus, in some embodiments, the polymer dye conjugate further comprises an acceptor chromophore covalently linked to a multichromophore near its energy acceptor. In certain embodiments, the conjugate comprises a chromophore of formula (IX):

[0225] [ka]

[0226] This is shown by, in the formula, F 1 It is a 6-5-6 condensed tricyclic comonomer; M 1 and M 2 These are, independently, UV absorbance-modified comonomers; b is either 1 or 2; a, c, d, e, and f are each independently either 0 or 1, where a + c + d + f ≥ 1; L 1 is, -Z 1 A connectable comonomer linked to, where Z 1 These are chemoselective tags or acceptor chromophores; n is an integer between 1 and 10,000; m is an integer between 1 and 10,000; p is an integer between 1 and 100,000; and G 1 and G 2 Each is independently selected from terminal groups, π-conjugated segments, linkers, and linked specific binding members, G 1 and G 2 At least one of them is a linked specific binding member. In some cases of formula (IX), F 1 is a fluorene comonomer. In the specific case of formula (IX), F 1 It is a carbazole comonomer.

[0227] After contacting the sample with the polymer dye conjugate, any convenient method may be used to assay the sample that has come into contact with the resulting labeled composition for the presence of a polymer dye conjugate-target sample binding complex. The polymer dye conjugate-target sample binding complex, if present, is a binding complex formed by the specific binding of the conjugate's specific binding member to the target sample. The assay of the sample that has come into contact with the labeled composition may include the detection of a fluorescent signal from the binding complex, if present. Optionally, the assay may include a separation step, in which the target sample, if present, is separated from the sample. Target samples can be separated from other samples by various methods, such as immobilization on a support. Suitable assay methods include, but are not limited to, the use of specific binding member pairs such as avidin-biotin or hapten-anti-hapten antibody, and include simple methods and assay schemes. Suitable methods and assay schemes that may be adapted for use with the subject composition 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.

[0228] In certain embodiments, the method further comprises contacting the sample with a second specific binding member that specifically binds to the target sample. In certain cases, the second specific binding member is carrier-bound. The components of the method of the subject (e.g., the second specific binding member) can be immobilized using any convenient carrier. In certain cases, the carrier is particles such as magnetic particles. In some cases, the second specific binding member and the polymer dye conjugate can be isolated and, if present, isolated using any convenient method. A detectable sandwich complex is generated. In some embodiments, the method further includes analyzing the polymer dye conjugate-target sample binding complex, i.e., the fluorescently labeled target sample, by flow cytometry. Assaying for the presence of the polymer dye conjugate-target sample binding complex yields assay results (e.g., qualitative or quantitative assay data) that can be used to assess whether the target sample is present in the sample.

[0229] Any simple carrier can be used in the subject method. Suitable carriers include, but are not limited to, solid substrates, which may have various shapes, such as sheets, individual beads, or other structures like plates with holes; beads, polymers, particles, fiber meshes, hydrogels, porous matrices, pins, microarray surfaces, and chromatography carriers. In some cases, the carrier is selected from particles, planar solid substrates, fiber meshes, hydrogels, porous matrices, pins, microarray surfaces, and chromatography carriers. The carrier may be incorporated into a system providing cell isolation, assisted by simple methods such as manual syringes, centrifuges, or automated liquid handling systems. In some cases, the carrier is found in automated liquid handling systems for high-rate cell isolation, such as flow cytometers.

[0230] In some embodiments of this method, the separation step includes applying an external magnetic field to immobilize the magnetic particles. Any convenient magnetic source can be used as the external magnetic field source (e.g., a magnetic field gradient). In some cases, the external magnetic field is generated by a magnetic source, for example, a permanent magnet or an electromagnet. In some cases, the immobilization of the magnetic particles means that the magnetic particles accumulate near the surface closest to the magnetic field gradient source, i.e., the magnet.

[0231] The separation may further include one or more optional washing steps to remove unbound material of the sample from the carrier. Any simple washing method can be used; for example, the immobilized carrier can be washed with a biocompatible buffer that preserves the specific binding interaction between the polymer dye and the specific binding member. Separation of unbound material of the sample from the carrier and optional washing provide an enriched population of target cells from which unwanted cells and substances have been removed.

[0232] In certain embodiments, the method further includes detecting a labeled target. Detection of a labeled target may include exciting a multi-chromophore with one or more lasers and subsequently detecting fluorescence emission from a polymer dye using one or more optical detectors.

[0233] Methods for labeling target molecules are also provided. Subjective polymer dyes, such as tandem dyes, have been found to be used in a variety of labeling, separation, detection, and / or analysis methods. In some embodiments, the method comprises contacting a target molecule with a polymer dye to produce a labeled target molecule, wherein the polymer dye is a water-soluble, light-harvesting, multichromophore comprising a conjugated segment containing a fluorene comonomer and an ultraviolet absorbance-modified comonomer, comprising a multichromophore having an ultraviolet absorption maximum and a conjugated tag covalently linked to the target molecule.

[0234] In some embodiments, the polymer dye is a polymer tandem dye. Therefore, the polymer dye may further include an acceptor chromophore covalently linked to a multichromophore near its energy acceptor. In certain cases, the polymer dye itself is fluorescent. In some embodiments, the polymer dye is of formula (X):

[0235] [ka]

[0236] This is shown by, in the formula, F 1 It is a 6-5-6 condensed tricyclic comonomer; M 1 and M 2 These are, independently, UV absorbance-modified comonomers; b is either 1 or 2; a, c, d, e, and f are each independently either 0 or 1, where a + c + d + f ≥ 1; L 1 is a binding comonomer, where Z 1These are chemoselective tags or acceptor chromophores; n is an integer between 1 and 10,000; m is an integer between 0 and 10,000; p is an integer between 1 and 100,000; and G 1 and G 2 One of them is a terminal group, G 1 and G 2 The other of these is a conjugate tag. In a particular embodiment, Z 1 Z is an acceptor chromophore. In a particular embodiment, Z 1 is a chemoselective tag. In certain cases, e is 0. In some cases of equation (X), F 1 is a fluorene comonomer. In a specific case of formula (X), F 1 It is a carbazole comonomer.

[0237] As used herein, the term “conjugated tag” means a group comprising a chemoselective functional group (e.g., as described herein) that can be covalently linked to a compatible functional group of a target molecule after optional activation and / or deprotection. Any convenient conjugated tag may be used in the polymer dye of the subject to conjugate the dye to the target molecule of interest. In some embodiments, the conjugated tag comprises a terminal functional group selected from amino, carboxylic acid or its derivatives, thiol, hydroxyl, hydrazine, hydrazide, azide, alkyne, and protein-reactive groups (e.g., amino-reactive, thiol-reactive, hydroxyl-reactive, imidazolyl-reactive, or guanidinyl-reactive).

[0238] To covalently link the conjugated tag to the target molecule, any of the following simple methods or reagents can be adapted for use in the labeling method of the subject. Examples of target methods for labeling the target include, but are not limited to, the methods and reagents described in Hermanson, Bioconjugate Techniques, Third edition, Academic Press, 2013. The contact step may be carried out in aqueous solution. In some cases, the conjugated tag contains an amino functional group and the target molecule contains an activated ester functional group such as an NHS ester or sulfo-NHS ester, or vice versa. In specific cases, the conjugated tag contains a maleimide functional group and the target molecule contains a thiol functional group, or vice versa.

[0239] For labeling using the method described in the subject, any convenient target molecule can be selected. The target molecules are not limited to, but include nucleic acids such as RNA, DNA, PNA, CNA, HNA, LNA, or ANA molecules, proteins such as fusion proteins, phosphorylated molecules, and glycosylated molecules. Examples include modified proteins such as sylated, ubiquitinated, SUMOlated, or acetylated proteins, or antibodies, peptides, aggregated biomolecules, cells, small molecules, vitamins, and drug molecules. As used herein, the term “target protein” means all members of the target family and fragments thereof. A target protein may be any protein of interest, for example, a therapeutic or diagnostic target, but may also be a hormone, growth factor, receptor, enzyme, cytokine, bone-inducing factor, colony-stimulating factor, or immunoglobulin. The term “target protein” is intended to encompass recombinant and synthetic molecules that can be produced or purchased using simple recombinant expression methods or simple synthesis methods. In some embodiments, the target molecule is a specific binding member (e.g., as described herein). In certain 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 its binding derivative is selected from Fab fragments, F(ab')2 fragments, scFv, diabodies, and triabodies.

[0240] In some cases, this method includes a separation step in which the labeled target molecule is separated from the reaction mixture, such as excess reagent and unlabeled target. The target can be separated from the sample by various methods, such as immobilization on a support, precipitation, or chromatography.

[0241] In some cases, this method further includes the detection and / or analysis of labeled target molecules. In some cases, this method further includes the fluorescence detection of labeled target molecules. In combination with the method and composition of the subject, labeled target molecules can also be detected and / or analyzed by simple methods. Methods for analyzing the target of interest, which are found to be used in the method of the subject of interest, include, but are not limited to, flow cytometry, in-situ hybridization, enzyme immunosorbent assay (ELISA), Western blotting, magnetic cell separation assays, and fluorescent dye purification chromatography. Methods for detecting the target of interest include, but are not limited to, fluorescence spectroscopy, nucleic acid sequencing, fluorescence in-situ hybridization (FISH), protein mass spectrometry, and flow cytometry.

[0242] Detection can be achieved directly via a reporter molecule or indirectly through a secondary detection system. The latter is based on one or a combination of several different principles, but not limited to antibody-labeled anti-species antibodies and other forms of immunological or non-immunological crosslinking and signal amplification systems (e.g., biotin-streptavidin technology, protein-A and protein-G mediation technology, or nucleic acid probes / anti-nucleic acid probes, etc.). The label used for direct or indirect detection may be any of the detectable reported molecules. Suitable reporter molecules may be those known in the fields of immunocytochemistry, molecular biology, optics, fluorescence, and electron microscopy, immunophenotyping, cell sorting, flow cytometry, cell visualization, detection, enumeration, and / or signal output quantification. Target labels include, but are not limited to, fluorophores, luminescent labels, metal complexes, radioisotopes, biotin, streptavidin, enzymes, or other detection labels, as well as combinations of enzymes and luminogenic substrates. Target enzymes and their substrates include alkaline phosphatase, horseradish peroxidase, β-galactosidase, and luciferase. Multiple antibodies, both specific and / or nonspecific in nature, may be labeled and used simultaneously or sequentially to improve the detection, identification, and / or analysis of targets. Target labels include, but are not limited to, FITC (fluorescein isothiocyanate), AMCA (7-amino-4-methylcoumarin-3-acetic acid), Alexa Fluor 488, and A. Examples of stains that detect plexus include lexa Fluor 594, Alexa Fluor 350, DyLight 350, phycoerythrin, allophycocyanin, and nuclei, such as Hoechst 33342, LDS751, TO-PRO, and DAPI.

[0243] system Aspects of the present invention further include systems used for carrying out the subject method and composition. A sample analysis system may include a flow channel into which a sample and a labeled specific binding member are loaded. In some embodiments, the system is a flow cytometry system comprising a flow cytometer including a channel; a composition in the channel (the composition comprising a sample; and a labeled specific binding member (e.g., as described herein)). In some cases of this system, the labeled specific binding member comprises a water-soluble light-harvesting polychromophore (having an ultraviolet absorption maximum) comprising a conjugated segment containing a 6-5-6 condensed tricyclic comonomer and an ultraviolet absorbance-modified comonomer, and a specific binding member that specifically binds to a target sample and is covalently linked to the polychromophore. The polychromophore may be a polymer dye that is fluorescent itself. The polychromophore may be a polymer tandem dye. In certain cases, the labeled specific binding member further comprises an acceptor chromophore covalently linked to the polychromophore near its energy acceptor. In some embodiments, the labeled specific binding member comprises formula (XI):

[0244] [ka]

[0245] This is shown by, in the formula, F 1 It is a 6-5-6 condensed tricyclic comonomer; M 1 and M 2 These are, independently, UV absorbance-modified comonomers; b is either 1 or 2; a, c, d, e, and f are each independently either 0 or 1, where a + c + d + f ≥ 1; L 1 is, -Z 1 A connectable comonomer linked to, where Z 1 These are chemoselective tags or acceptor chromophores; n is an integer between 1 and 10,000; m is an integer between 1 and 10,000; p is an integer between 1 and 100,000; and G 1 and G 2 One of them is a terminal group, G 1 and G 2 The other is a linked specific binding member. In some cases of formula (XI), F 1 is a fluorene comonomer. In the specific case of formula (XI), F 1 It is a carbazole comonomer.

[0246] In certain embodiments of the system, the composition further comprises a second specific binding member that is carrier-bound and specifically binds to a target specimen. The carrier may include magnetic particles. Thus, in certain cases, the system is used to assemble the flow channel. This may also include a controllable external paramagnetic field configured for application in the region.

[0247] The sample may contain cells. In some cases, the sample is a cell-containing biological sample. In some cases, the sample is a labeled specific binding member that specifically binds to target cells. In certain cases, the target sample specifically bound by the specific binding member is a cell surface marker. In certain cases, the cell surface marker is selected from cell receptors and cell surface antigens.

[0248] In certain embodiments, the system may also include a light source configured to directly irradiate the assay region of the flow channel. The system may also include a detector configured to receive a signal from the assay region of the flow channel, 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 further signals.

[0249] In certain embodiments, the system may further include a computer-based system configured to detect the presence of a fluorescent signal. “Computer-based system” means hardware means, software means, and data storage means used to analyze the information of the present invention. The minimum hardware of the computer-based system of the present invention includes a central processing unit (CPU), input means, output means, and data storage means. Those skilled in the art will readily understand that any currently available computer-based system is suitable for use in the system of the subject. The data storage means may include any product containing the records of the information of the present invention as described above, or memory access means capable of accessing such product.

[0250] "Recording" data, programming, or other information on a computer-readable medium means the process of storing information using methods known in the art. Any convenient data storage structure may be selected based on the means used to access the stored information. Various data processing programs and formats can be used for storage, such as word processing text files, database formats, etc.

[0251] "Processor" means a combination of hardware and / or software that performs the functions required of it. For example, a processor as used herein may be a programmable digital microprocessor, such as one available in the form of an electronic controller, mainframe, server, or personal computer (desktop or portable). If the processor is programmable, appropriate programming can be communicated to the processor remotely or pre-stored in a computer program product (such as a storage medium readable by a portable or stationary computer, whether magnetic, optical, or solid-state based). For example, a magnetic medium or optical disk can hold programming that can be read by an appropriate reader that communicates with the respective processor at its corresponding station.

[0252] In addition to the sensor device and signal processing module, the system of the present invention may include, for example, many further components, such as data output devices, such as monitors and / or speakers, data input devices, such as interface ports, keyboards, etc., fluid processing components, power supplies, etc.

[0253] In certain embodiments, this system includes a flow cytometer. Examples of ilometers include, but are not limited to, the 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.

[0254] Other systems may also be found to be used in carrying out the subject method. In certain embodiments, the system may be a fluorometer or microscope loaded with a sample having a fluorescent composition of any of the embodiments described herein. The fluorometer or microscope may include a light source configured to directly illuminate the assay region of the flow channel. The fluorometer or microscope may also include a detector configured to receive a signal from the assay region of the flow channel, the signal being provided by the fluorescent composition.

[0255] kit Aspects of the present invention further include kits and compositions used to carry out the subject method. The compositions of the present invention may be included in the kit as reagents as starting materials or as reagents provided for use in the methodology described above, for example.

[0256] The kit comprises a water-soluble, light-harvesting, multichromophore having a UV absorption maximum, comprising a conjugated segment containing a 6-5-6 condensed tricyclic comonomer and an UV absorbance-modified comonomer, and may include one or more components selected from a polymer tandem dye, a fluorophore, a specific binding member, a specific binding member conjugate, a specific binding member bound to a carrier, cells, a carrier, a biocompatible aqueous elution buffer, and instructions for use. In some embodiments of the kit, the multichromophore is covalently linked to a specific binding member. In some cases, the specific binding member is an antibody. In certain cases, the specific binding member is an antibody fragment or its binding derivative. In certain cases, the antibody fragment or its binding derivative is selected from Fab fragments, F(ab')2 fragments, scFv, diabodies, and triabodies. The multichromophore may be a polymer dye that is fluorescent itself. The multichromophore may be a polymer tandem dye. In some cases, the multichromophore further includes an acceptor chromophore covalently linked to the multichromophore near its energy-receiving proximal point.

[0257] In certain embodiments, the kit is found to be used in evaluating a sample for the presence of target specimens, such as intracellular targets. Therefore, in some cases, the kit includes one or more components suitable for lysing cells. One or more further components of the kit may be provided in individual containers (e.g., individual tubes, bottles, wells in multi-well strips or plates).

[0258] In certain embodiments, the kit further comprises reagents for performing a flow cytometry assay. These reagents include, but are not limited to, buffers for reconstitution and dilution, buffers for contacting cell samples with a multichromophore, washing 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 combinations thereof or their buffers. Furthermore, the kit may include cell permeabilizing reagents, such as methanol, acetone, or washing agents (e.g., triton, NP-40, saponin, tween 20, digitonin, leucoperm, or any combination thereof or their buffers, as known to those skilled in the art. Other protein transport inhibitors, cell immobilization agents, and cell permeabilization agents are included in the subject kit.

[0259] The compositions in this kit may be provided in liquid form, such as in a suitable buffer. Alternatively, the compositions in this kit may be provided as dry compositions (e.g., lyophilized), and the kit may optionally include one or more buffers for reconstituting the dry compositions. In certain embodiments, the kit may include aliquots of the compositions provided in individual containers (individual tubes, bottles, wells in multiwell strips or plates).

[0260] Furthermore, one or more components may be combined in a single container, for example, in a glass or plastic vial, tube, or bottle. In certain cases, the kit may further include a container (e.g., a box, bag, insulated container, bottle, tube, etc.) in which all of the components (and their individual containers) are present. The kit may further include packaging, separate from or attached to the kit container, on which information about the kit is printed, the components of the kit, and / or instructions for using the kit.

[0261] In addition to the components described above, the subject kit may further include instructions for carrying out the subject method. These instructions may be present in the subject kit in various forms, one or more of which may be present in the kit. One possible form of these instructions is that the information is printed on a suitable medium or substrate, for example, one or more sheets of paper with the information printed on them included as an accompanying document in the kit package. Another means may be a computer-readable medium on which the information is recorded, such as a diskette, CD, DVD, or portable flash drive. Yet another possible means may be a website address that can be used via the internet to access the information remotely. Any of these convenient means may be present in the kit.

[0262] usefulness The compositions, methods, and systems described herein may be found in a variety of applications, such as diagnostic and research applications, where the labeling, detection, and / or analysis of a target is desired. Such applications include methodologies such as hemocytometry, microscopy, immunoassays (e.g., competitive or non-competitive), evaluation of free samples, and evaluation of receptor-binding ligands. The compositions, systems, and methods described herein may be useful in the analysis of many samples, including, but are not limited to, biological fluids, cell culture samples, and tissue samples. In certain embodiments, the compositions, systems, and methods described herein may be found in applications where the sample is detected in the sample if present, such as fluorescent labeling, for example, in fluorescence-activated cell sorting or analysis, immunoassays, and immunostaining. In certain cases, the compositions and methods are found in applications where the evaluation of the sample for the presence of a target sample is targeted.

[0263] Depending on the circumstances, the methods and compositions may be found for use in any assay format that involves the detection and / or analysis of a target from a sample, including but not limited to flow cytometry, in-situ hybridization, enzyme immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assays, and fluorescent dye purification chromatography. In specific cases, the methods and compositions may be found for use in any application that involves the fluorescent labeling of a target molecule. The compositions of the subject matter may be adapted for use in any convenient application where a pair of specifically binding members, such as biotin-streptavidin and hapten-anti-hapten antibody, is found for use in that application.

[0264] The following examples are provided as illustrative examples and are not intended to be limiting. [Examples]

[0265] experiment Example 1 A series of tandem dyes were prepared based on the core structure 1 shown below, which contains a series of bonded fluorophores. Figure 1 illustrates the fluorescence emission profiles of various polymer tandem dyes based on the multichromophore core structure 1 linked to various different acceptor chromophores.

[0266] [ka]

[0267] In the formula, the dye is Dyomics dye DY 431, DY 485XL, DY 610, DY 640, Cy3, Cy3.5, or diethylaminocoumarin (DEAC).

[0268] A second series of UV-absorbing polymer dyes were fabricated and characterized. Figure 2 illustrates the UV absorption spectra of various multichromophores, MC-1 to MC-5.

[0269] [ka]

[0270] Notwithstanding the attached clauses, the disclosures contained herein are also defined by the following clauses: A water-soluble, light-harvesting, multi-chromophore having a UV absorption maximum, comprising a conjugated segment containing a 1,6-5-6 condensed tricyclic comonomer and an UV absorbance-modified comonomer. 2. A multi-chromatophore as described in Clause 1, having an absorption maximum wavelength in the range of 300-400 nm and an emission maximum wavelength in the range of 375-900 nm. 3. A multichromophore according to either Section 1 or 2, which is a conjugated polymer containing 25% or more (by molar concentration) of UV absorbance-modified comonomers and 5 or more monomer repeating units. 4. Molar extinction coefficient 5 × 10 5 M -1 cm -1 Any one of the clauses 1 to 3 that has the above characteristics The multi-color group described. 5. A multi-chromophore having a quantum yield of 0.3 or more, as described in any one of the clauses 1 to 4. 6. Equation (I):

[0271] [ka]

[0272] (In the formula, F 1 It is a 6-5-6 condensed tricyclic comonomer; M 1 and M 2 each is independently an ultraviolet absorbance-modified comonomer; b is 1 or 2; a, c, d, e and f are each independently 0 or 1, where a+c+d+f≧1; L 1 This is a chemoselective tag-Z 1 A connectable comonomer containing; n is an integer from 1 to 10,000; m is 0 or an integer from 1 to 10,000; p is an integer from 1 to 100,000; G 1 and G 2 A polychromophore as described in any one of Clauses 1 to 5, each independently represented by a terminal group, a π-conjugated segment, a linker, and a linked specific binding member. 7. A multi-chromophore as described in Clause 6, where if e is 0, then d, f, and m are 0, and if e is 1, then d+f≦1 and m≧1. 8.Formula (II):

[0273] [ka]

[0274] The multicolor chromophore described in Article 6, as shown by... 9.Formula (III):

[0275] [ka]

[0276] The multicolor chromophore described in Article 6, as shown by... 10. b is 1 and a + c ≥ 1; G 1 is a terminal group; and G 2 A polychromophore as described in any one of Clauses 8 to 9, wherein the terminal group is a linker or a linked specific binding member. 11. A multicolor chromophore as described in any one of the clauses 8 to 10, where a + c = 1. 12.Formula (IV):

[0277] [ka]

[0278] The multicolor chromophore described in Article 6, as shown by... 13. b is 1; c is 0 or 1; e and f are each 0 or 1, where e + f ≥ 1; G 1 is a terminal group; and G 2 The polychromophore according to Clause 12, wherein is a terminal group, a linker, or a linked specific binding member. 14. A multicolor chromophore as described in any one of clauses 12-13, wherein c is 1. 15. A polychromophore according to any one of sections 1 to 14, wherein the ultraviolet absorbance-modified comonomer is an aryl or heteroaryl comonomer in which an aryl or heteroaryl comonomer is optionally substituted. 16. The polychromophore according to Clause 15, wherein the optionally substituted aryl or heteroaryl comonomer is selected from the group consisting of substituted or unsubstituted 1,4-phenyl, substituted or unsubstituted 1,3-phenyl, substituted or unsubstituted 4,4'-biphenyl, substituted or unsubstituted 2,5-pyridyl, and substituted or unsubstituted 2,6-pyridyl. 17. Aryl or heteroaryl comonomers that are optionally substituted are:

[0279] [ka]

[0280] One of the following is selected, and in the formula, Z 2 ~Z 5 These are independently either CR or N. , here, Z 2 ~Z 5 At least one of them is N; and R and R 11 ~R 16 Each of these is independently selected from the group consisting of hydrogen, halogen, cyano, alkoxy, substituted alkoxy, alkyl, and substituted alkyl, as described in Clause 16. 18. Aryl or heteroaryl comonomers that are optionally substituted are:

[0281] [ka]

[0282] A polychromophore as described in Clause 17, selected from one of the following, where n is 1 to 20 and R' is H or a lower alkyl group. 19.F 1 However, the structure:

[0283] [ka]

[0284] (In the formula, R 2 (Each of these is a substituted alkyl group containing a water-soluble group or a substituted aralkyl group containing a water-soluble group.) A multicolor chromophore as defined in any one of the clauses 6 to 18. 20L 1 A polychromophore as described in any one of the clauses 6 to 19, wherein the polychromophore is a fluorene comonomer. 21.L 1 However, the structure:

[0285] [ka]

[0286] (In the formula, R 3R is a substituted alkyl group containing a water-soluble group; 4 L 2 -Z 2 (L 2 It is a linker, and Z 2 (This is a chemoselective tag.) A multicolor chromophore as defined in any one of the clauses 6 to 20. A polymer tandem dye comprising a water-soluble, light-harvesting, multichromophore having a UV absorption maximum, and a conjugated segment containing a 22.6-5-6 condensed tricyclic comonomer and an UV absorbance-modified comonomer, and an acceptor chromophore covalently linked to the multichromophore near its energy-receiving proximal position. 23. The dye according to Clause 22, wherein the multichromophore has an absorption maximum wavelength in the range of 300-400 nm and an emission maximum wavelength in the range of 375-900 nm. 24. A dye according to any one of clauses 22 to 23, wherein ultraviolet absorbance-modified comonomers constitute 25% or more (by molar concentration) of the multichromophore; and the multichromophore is a conjugated polymer containing five or more repeating units.

[0287] 25. Multichromophore has a molar extinction coefficient of 5 × 10⁻⁶ 5 M -1 cm -1 A pigment as described in any one of the clauses 22 to 24, having the above characteristics. 26. A dye according to any one of the clauses 22 to 25, wherein the multichromophore has a quantum yield of 0.3 or more. 27. A dye according to any one of the clauses 22 to 26, wherein the acceptor chromophore is a fluorophore. 28. A dye as described in any one of the sections 22 to 26, wherein the acceptor chromophore is an inactivating agent. 29. The dye according to Clause 27, wherein the emission of the acceptor chromophore is 1.5 times or more when excited by a multichromophore compared to the direct excitation of the acceptor chromophore by incident light. 30. The multiple chromophore is given by formula (V):

[0288] [ka]

[0289] (In the formula, F 1 It is a 6-5-6 condensed tricyclic comonomer; M 1 and M 2 b is an ultraviolet absorbance-modified comonomer, each independently; b is 1 or 2; a, c, d, e, and f are each independently 0 or 1, where a+c+d+f≧1; e is 1 or 2; L 1 This is the Acceptor Chromogroup-C 1 A connectable comonomer linked to; n is an integer between 1 and 10,000; m is an integer between 1 and 10,000; p is an integer between 1 and 100,000; G 1 and G 2 Each of these is independently selected from the group consisting of terminal groups, π-conjugated segments, linkers, and linked specific-binding members. A pigment as indicated in any one of the sections 22 to 29 of the Articles. 31. The dye according to Clause 30, wherein e is 1, d + f ≤ 1, and m ≥ 1. 32. Multiple chromophores, formula (VI):

[0290] [ka]

[0291] The pigments described in Clause 30, as shown by... 33. Multiple chromophores, formula (VII):

[0292] [ka]

[0293] The pigments described in Clause 30, as shown by... 34. b is 1; c is 0 or 1; e is 1; f is 0 or 1; G 1 is a terminal group; and G 2The polychromophore according to Clause 33, wherein is a terminal group, a linker, or a linked specific binding member. 35. A pigment as described in any one of clauses 33 to 34, wherein c is 1. 36. The dye according to any one of sections 22 to 35, wherein the ultraviolet absorbance-modified comonomer is a substituted aryl or heteroaryl comonomer in which the comonomer is optionally substituted. 37. The dye according to Clause 36, wherein the optionally substituted aryl or heteroaryl comonomer is selected from the group consisting of substituted or unsubstituted 1,4-phenyl, substituted or unsubstituted 1,3-phenyl, substituted or unsubstituted 4,4'-biphenyl, substituted or unsubstituted 2,5-pyridyl, and substituted or unsubstituted 2,6-pyridyl. 38. Aryl or heteroaryl comonomers that are optionally substituted are:

[0294] [ka]

[0295] (In the formula, Z 2 ~Z 5 Each is independently either CR or N, and Z 2 ~Z 5 At least one of them is N; and R and R 11 ~R 16 Each of these is independently selected from the group consisting of hydrogen, halogen, cyano, alkoxy, substituted alkoxy, alkyl, and substituted alkyl. A pigment selected from one of the following, as described in any one of the sections 36-37. 39. Aryl or heteroaryl comonomers that are optionally substituted are:

[0296] [ka]

[0297] A dye as described in any one of clauses 36 to 38, selected from one of the formulas (wherein n is 1 to 20 and R' is H or a lower alkyl group). 40.F 1 However, the structure:

[0298] [ka]

[0299] (In the formula, R 1 These are, independently, alkyl, substituted alkyl, aralkyl, substituted aralkyl, PEG moiety, and -L. 1 -Selected from the group consisting of Z1, where L 1 It is a linker, and Z 1 (This is a chemoselective tag or WSG.) A pigment as specified in any one of the clauses 30 to 39, as indicated by [the relevant source]. 41.L 1 The dye is a fluorene comonomer as described in any one of the sections 30 to 40. 42.L 1 Structure:

[0300] [ka]

[0301] (In the formula, R 3 R is a substituent containing a water-soluble group; 4 L 2 -Z 2 (L 2 It is a linker, and Z 2 (is an acceptor chromophore) A pigment as indicated in any one of the clauses 30 to 41. 43.G 1 and G 2 At least one of the later ones is -L 3 -Z, where L 3 The dye according to any one of clauses 30 to 42, wherein is a linker and Z is a specific binding member. 44.G 1 and G 2 At least one of the later ones has the following structure:

[0302] [ka]

[0303] (wherein q is an integer between 0 and 1 to 12; L is an optional linker; and Z is a chemoselective tag or a specific binding member.) A pigment as specified in any one of the clauses 30 to 43, as indicated by [the relevant source]. 45. A dye described in any one of clauses 43-44, wherein Z is a biomolecule. 46. ​​A dye as described in any one of clauses 43-45, wherein Z is an antibody. 47. The dye according to any one of the clauses 43 to 45, wherein Z is an antibody fragment or a binding derivative thereof. 48. Antibody fragment or its binding derivative is Fab fragment, F(ab') ·BR>Q A dye according to Clause 47, selected from the group consisting of fragments, scFv, diabodies, and triabodies. 49.C 1 However, L is linked via an optional linker. 1 A pigment selected from the group consisting of cyanine pigments, xanthene pigments, coumarin pigments, thiadin pigments, and acridine pigments linked to the above, as described in any one of clauses 30 to 48.

[0304] 50. The dye according to Clause 49, wherein the linker is selected from the group consisting of alkyl, substituted alkyl, alkylamide, alkylamide-alkyl, and PEG moieties. 51. A dye according to any one of sections 22 to 50, wherein the acceptor chromophore is selected from the group consisting of DY 431, DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 778, DY 782, DY 800, DY 831, Biotium CF 555, Cy3.5, and diethylaminocoumarin. A labeled specific binding member comprising a conjugated segment containing a 52.6-5-6 condensed tricyclic comonomer and an ultraviolet absorbance-modified comonomer, having an ultraviolet absorption maximum, a water-soluble light-harvesting multichromophore, and a specific binding member covalently linked to the multichromophore. 53. A labeled specific binding member according to Clause 52, wherein the multi-chromophore has an absorption maximum wavelength in the range of 300-400 nm and an emission maximum wavelength in the range of 375-900 nm. 54. A labeled specific binding member according to any one of clauses 52 to 53, wherein the ultraviolet absorbance-modified comonomer accounts for 25% or more (by molar concentration) of the multichromophore; and the multichromophore is a conjugated polymer containing 5 or more repeating units.

[0305] 55. Multichromophore has a molar extinction coefficient of 1 × 10⁻⁶ 6 M -1 cm -1 A labeled specific binding member as described in any one of clauses 52 to 53, having the above characteristics. 56. A labeled specific binding member according to any one of the sections 52 to 55, wherein the multi-chromophore has a quantum yield of 0.3 or more. 57. A labeled specific binding member according to any one of the clauses 52 to 56, further comprising an acceptor chromophore covalently bound to a multichromophore near its energy acceptor. 58. A labeled specific binding member according to Clause 57, wherein the acceptor chromophore is a fluorophore. 59. A labeled specific binding member according to Clause 58, wherein the emission of the acceptor chromophore is 1.5 times or more when excited by a multichromophore compared to the direct excitation of the acceptor chromophore by incident light. 60. Multiple chromophores, formula (VIII):

[0306] [ka]

[0307] (In the formula, F 1 It is a 6-5-6 condensed tricyclic comonomer; M 1 and M 2 each is independently an ultraviolet absorbance-modified comonomer; b is 1 or 2; a, c, d, e and f are each independently 0 or 1, where a+c+d+f≧1; L 1 is, -Z 1 A connectable comonomer linked to; Z 1 is a chemoselective tag or acceptor chromophore; n is an integer between 1 and 10,000; m is an integer between 1 and 10,000; p is an integer between 1 and 100,000; G 1 and G 2 Each is independently selected from the group consisting of terminal groups, π-conjugated segments, linkers, and linked specific binding members, and G 1 and G 2 (At least one of them is a linked specific binding member.) A labeled specific binding member as described in any one of the sections 52 to 59, as shown by [the relevant authority].

[0308] 61. A labeled specific binding member as described in any one of clauses 52 to 60, wherein the specific binding member is an antibody. 62. A labeled specific binding member according to any one of the clauses 52 to 60, wherein the specific binding member is an antibody fragment or a binding derivative thereof. 63. A labeled specific binding member according to Clause 62, wherein the antibody fragment or its binding derivative is selected from the group consisting of Fab fragment, F(ab')2 fragment, scFv, diabody, and triabody. 64. A labeled specific binding member according to any one of the clauses 57 to 59, wherein the acceptor chromophore is selected from the group consisting of cyanine dyes, xanthene dyes, coumarin dyes, thiadin dyes, or acridine dyes. 65. A labeled specific binding member according to Clause 64, wherein the acceptor chromophore is selected from the group consisting of Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Alexa488, Alexa647, and Alexa700. 66. A method for evaluating the presence of a target sample, comprising: (a) a step of contacting the sample with a polymer dye conjugate that specifically binds to a target sample to produce a sample in contact with a labeled composition, wherein the polymer dye conjugate comprises (i) a water-soluble light-harvesting multichromophore comprising a conjugated segment containing a 6-5-6 condensed tricyclic comonomer and an ultraviolet absorbance-modified comonomer, and having an ultraviolet absorption maximum, and (ii) a specifically binding member; and (b) an assay of the sample in contact with the labeled composition to evaluate whether a target sample is present in the sample for the presence of a polymer dye conjugate-target sample binding complex. 67. The method according to clause 66, wherein the polymer dye conjugate further comprises an acceptor chromophore covalently linked to a multichromophore near its energy-receiving location. 68. The conjugate is given by equation (IX):

[0309] [ka]

[0310] (In the formula, F 1 It is a 6-5-6 condensed tricyclic comonomer; M 1 and M 2each is independently an ultraviolet absorbance-modified comonomer; b is 1 or 2; a, c, d, e and f are each independently 0 or 1, where a+c+d+f≧1; L 1 is, -Z 1 A connectable comonomer linked to; Z 1 is a chemoselective tag or acceptor chromophore; n is an integer between 1 and 10,000; m is an integer between 1 and 10,000; p is an integer between 1 and 100,000; G 1 and G 2 Each is independently selected from the group consisting of terminal groups, π-conjugated segments, linkers, and linked specific binding members, and G 1 and G 2 (At least one of them is a linked specific binding member.) The method as shown in either of the sections of Articles 66 and 67.

[0311] 69. The method according to any one of the clauses 66 to 68, further comprising contacting the sample with a second specific binding member that is carrier-bound and specifically binds to the target sample. 70. The method according to Clause 69, wherein the carrier comprises magnetic particles. 71. The method described in any one of the clauses 66-70, wherein the target sample is associated with cells. 72. The method according to clause 71, wherein the target sample is a cell surface marker of a cell. 73. The method according to Clause 72, wherein the cell surface marker is selected from the group consisting of cell receptors and cell surface antigens. 74. The method of Clause 73, wherein the target specimen is an intracellular target, and the method further comprises lysing cells.

[0312] 75. The method described in any one of the clauses 72 to 73, further comprising analyzing a fluorescently labeled target sample by flow cytometry. 76. A method for labeling a target molecule, comprising the step of contacting a target molecule with a polymer dye to produce a labeled target molecule, wherein the polymer dye comprises a conjugated segment containing a 6-5-6 condensed tricyclic comonomer and an ultraviolet absorbance-modified comonomer, and comprises a water-soluble light-harvesting multichromophore having an ultraviolet absorption maximum, and a conjugated tag covalently linked to the target molecule. 77. The method according to clause 76, further comprising fluorescently detecting a labeled target molecule. 78. The method according to any one of the provisions of 76 to 77, further comprising an acceptor chromophore covalently linked to a multichromophore near its energy-receiving location. 79. Polymer dyes, formula (X):

[0313] [ka]

[0314] (In the formula, F 1 It is a 6-5-6 condensed tricyclic comonomer; M 1 and M 2 each is independently an ultraviolet absorbance-modified comonomer; b is 1 or 2; a, c, d, e and f are each independently 0 or 1, where a+c+d+f≧1; L 1 Z is a binding comonomer; 1 is a chemoselective tag or acceptor chromophore; n is an integer from 1 to 10,000; m is 0 or an integer from 1 to 10,000; p is an integer from 1 to 100,000; G 1 and G 2 One of them is a terminal group, and G 1 and G 2 The other side is a conjugate tag. The method as shown in any one of the clauses 76-78. 80.Z 1 The method described in Article 79, wherein the acceptor chromophore is the acceptor chromophore.

[0315] 81. The method according to any one of the clauses 79 to 80, wherein the conjugated tag comprises a terminal functional group selected from amino, thiol, hydroxyl, hydrazine, hydrazide, azide, alkyne, and protein-reactive groups. 82. The method described in any one of the clauses 76 to 81, wherein the target molecule is a specific binding member. 83. The method according to clause 82, wherein the specific binding member is an antibody. 84. The method according to clause 82, wherein the specific binding member is an antibody fragment or a binding derivative thereof. 85. The method according to clause 84, wherein the antibody fragment or its binding derivative is selected from the group consisting of Fab fragment, F(ab')2 fragment, scFv, diabody, and triabody. 86. A flow cytometry system comprising a flow cytometer including a channel and a composition in the channel, wherein the composition comprises a sample and a labeled specific binding member, the labeled specific binding member comprising a conjugated segment containing a 6-5-6 condensed tricyclic comonomer and an ultraviolet absorbance modified comonomer, a water-soluble light-harvesting multichromophore having an ultraviolet absorption maximum, and a specific binding member that specifically binds to a target sample and is covalently linked to the multichromophore. 87. The system according to Clause 86, further comprising an acceptor chromophore in which a labeled specific binding member is covalently linked to a multichromophore near its energy acceptor. 88. The labeling-specific binding member is given by formula (XI):

[0316] [ka]

[0317] (In the formula, F 1 It is a 6-5-6 condensed tricyclic comonomer; M 1 and M 2 each is independently an ultraviolet absorbance-modified comonomer; b is 1 or 2; a, c, d, e and f are each independently 0 or 1, where a+c+d+f≧1; L1 is, -Z 1 A connectable comonomer linked to; Z 1 is a chemoselective tag or acceptor chromophore; n is an integer between 1 and 10,000; m is an integer between 1 and 10,000; p is an integer between 1 and 100,000; G 1 and G 2 One of them is a terminal group, and G 1 and G 2 The other party is a linked specific bonding member) as indicated by any one of the systems described in any one of the sections of Clauses 86 to 87. 89. The system according to any one of the clauses 86 to 88, wherein the composition further comprises a second specific binding member that is carrier-bound and specifically binds to a target specimen.

[0318] 90. The system according to Clause 89, wherein the carrier comprises magnetic particles. 91. A system described in any one of the sections 86-90, wherein the sample contains cells. 92. The system according to Clause 91, wherein the target sample is a cell surface marker of a cell. 93. The system according to Clause 92, wherein the cell surface marker is selected from the group consisting of cell receptors and cell surface antigens. A kit comprising a water-soluble light-harvesting multichromophore having an ultraviolet absorption maximum, comprising a conjugated segment containing a 94.6-5-6 condensed tricyclic comonomer and an ultraviolet absorbance-modified comonomer, and one or more components selected from the group consisting of polymer tandem dyes, fluorophores, specific binding members, specific binding member conjugates, carrier-bound specific binding members, cells, carriers, biocompatible aqueous elution buffers, and instructions for use.

[0319] 95. The kit according to Clause 94, wherein a multichromophore is covalently linked to a specific binding member. 96. The kit described in Clause 95, wherein the specific binding member is an antibody. 97. The kit according to Clause 95, wherein the specific binding member is an antibody fragment or a binding derivative thereof. 98. The kit according to Clause 97, wherein the antibody fragment or its binding derivative is selected from the group consisting of Fab fragment, F(ab')2 fragment, scFv, diabody, and triabody. 99. A kit as described in any one of clauses 94-98, further comprising a multichromophore covalently linked to the multichromophore at its energy-receiving proximal position.

[0320] Although the present invention has been described in detail with illustrations and examples to facilitate understanding, it will be readily apparent to those skilled in the art, in light of the teachings of the invention, that certain modifications and alterations thereof may be made without departing from the spirit or scope of the appended claims.

[0321] Therefore, the foregoing description merely illustrates the principles of the present invention. While not explicitly stated or shown herein, other provisions embodying the principles of the present invention and included within its spirit and scope may also be present. It will be understood that various configurations can be conceived by those skilled in the art. Furthermore, all embodiments and conditional statements described herein are intended primarily to facilitate understanding of the principles and concepts of the invention to which the inventors have contributed for the advancement of the art, and should be construed as not being limited to the embodiments and conditions thus explicitly stated. Moreover, all expressions herein that enumerate the principles, aspects, and embodiments of the invention, as well as specific embodiments thereof, are intended to encompass both their structural and functional equivalents. Furthermore, such equivalents are intended to encompass both currently known equivalents and equivalents to be developed in the future, i.e., any developed element that performs the same function regardless of its structure. Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied below.

Claims

1. 6-5-6 condensed tricyclic comonomers, It contains a conjugated segment that includes an ultraviolet absorbance modified comonomer, It has a maximum ultraviolet absorption capacity. Equation (I): 【Chemistry 1】 As shown by, F １ It is a 6-5-6 condensed tricyclic comonomer, b is either 1 or 2, e is either 0 or 1, a, c, d, and f are each independently either 0 or 1, where a + c + d + f ≥ 1. M １ and M ２ Each of these is independently the aforementioned UV absorbance modified comonomer, When e is 0, the UV absorbance modified comonomer is the following multiple comonomers 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 【Transformation 5】 【Transformation 6】 【Transformation 7】 【Transformation 8】 It is one of the following, n is 1 to 20, R' is an alkyl group or H containing 1 to 6 carbon atoms, R １１ ~R １４ Each of these is either hydrogen or a water-soluble group, R １１ ~R １４ At least one of them is a water-soluble group, When e is 1, the UV absorbance modified comonomer is 【Chemistry 9】 and R １１ ~R １４ is each hydrogen or a water-soluble group, and R １１ ~R １４ at least one of which is a water-soluble group L １ This is a chemoselective tag-Z. １ It is a binding comonomer containing, The aforementioned chemoselective tag-Z １ These are functional groups that can selectively react with other compatible functional groups to form covalent bonds, such as thiol groups, maleimide groups, iodoacetamide groups, amine groups, carboxylic acid groups, activated ester groups of carboxylic acids, or groups that can react with each other via Click chemistry. n is an integer between 1 and 10,000. m is an integer between 0 and 10,000. p is an integer between 1 and 100,000, and G １ and G ２ Each is independently selected from the group consisting of terminal groups, π-conjugated segments, linkers, and linked specific binding members, and when e is 0, G １ or G ２ These are linked specific binding members. Water-soluble, light-gathering, multi-chromophore.

2. Formulas (II) to (IV): 【Chemistry 10】 As shown by one of the following: The multi-chromophore described in claim 1.

3. The following 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 One of the following will be selected: R 1 is as follows: 【Chemistry 16】 As shown by The multi-chromophore described in claim 1.

4. L １ Structure: 【Chemistry 17】 This is shown by, in the formula, R ３ It is a substituted alkyl group containing a water-soluble group, R ４ is, L ２ -Z ２ And here, L ２ It is a linker, and Z ２ This is a chemoselective tag. The multi-chromophore according to claim 2.

5. A water-soluble light-gathering multi-chromophore according to any one of claims 1 to 4, It includes an acceptor chromophore covalently linked to the multichromophore near its energy-receiving location. Polymer tandem dye.

6. The polymer tandem dye is given by formula (V): [Chemistry 18] This is shown by, in the formula, F １ It is a 6-5-6 condensed tricyclic comonomer, M １ and M ２ These are, independently, UV absorbance-modified comonomers, b is either 1 or 2, a, c, d, and f are each independently either 0 or 1, where a + c + d + f ≥ 1, e is 1, L １ The acceptor chromophore-C １ It is a binding comonomer linked to, n is an integer between 1 and 10,000. m is an integer between 1 and 10,000. p is an integer between 1 and 100,000, and G １ and G ２ These are, independently, terminal groups, π-conjugated segments, linkers, and chains. Selected from a group consisting of specifically binding members, The dye according to claim 5.

7. L １ Structure: 【Chemistry 19】 This is shown by, in the formula, R ３ This is a substituent containing a water-soluble group, R ４ is, L ２ -Z ２ And here, L ２ It is a linker, and Z ２ This is the acceptor chromophore. The dye according to claim 6.

8. G １ and G ２ At least one of them is -L ３ -Z, and here, L ３ Z is a linker, and Z is a specific bonding member. The dye according to claim 6.

9. The dye according to claim 8, wherein Z is an antibody.

10. The dye according to claim 8, wherein Z is an antibody fragment.

11. C １ L is linked via an optional linker. １ Linked to it, selected from the group consisting of cyanine pigments, xanthene pigments, coumarin pigments, thiadin pigments, and acridine pigments, The dye according to claim 6.

12. A method for evaluating a sample for the presence of a target sample, (a) A step of bringing a polymer dye conjugate that specifically binds to the target sample into contact with the sample, wherein the polymer dye conjugate (i) A water-soluble light-gathering multi-chromophore according to any one of claims 1 to 4, (ii) Specific binding members and The process includes, where G1 or G2 is a linked specific bonding member, and (b) A step to evaluate whether the target sample is present in the sample by assaying the polymer dye conjugate-target sample binding complex for the presence of the polymer dye conjugate. A method that includes this.

13. The method according to claim 12, wherein the target sample is associated with cells.

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