Polymeric bodipy dyes and methods of use thereof

Abstract

Provided are polymeric BODIPY dyes including light-harvesting multichromophores comprising BODIPY units. In some embodiments, the dyes are polymeric tandem dyes including a light-harvesting multichromophore comprising BODIPY units and an acceptor chromophore covalently linked to the multichromophore near its energy acceptance. The polymeric tandem dyes can be covalently linked to a specific binding member. Also provided are methods of evaluating a sample for the presence of a target analyte and methods of labeling a target molecule using a composition including a polymeric tandem dye. Also provided are kits and systems for practicing the subject methods.

Description

[0001] Case Analysis

[0002] This application is a divisional application of Chinese patent application No. 201680006407.2, filed on July 19, 2017, entitled "Polymer BODIPY dye and method of use thereof".

[0003] Cross-references to related applications

[0004] Pursuant to 35 USC §119(e), this application claims priority to U.S. Provisional Patent Application Serial No. 62 / 132,446, filed March 12, 2015, the disclosure of which is incorporated herein by reference. Technical Field

[0005] This disclosure generally relates to polymeric BODIPY dyes comprising light-collecting polychromatic groups containing BODIPY units and methods of using them. Background Technology

[0006] Fluorescent dyes are compounds that emit (typically) light of different wavelengths when illuminated by light of wavelengths they absorb. Fluorescent dyes have a wide range of applications in biochemistry, biology, and medicine, such as in diagnostic kits, microscopy, or drug screening. A key feature of fluorescent dyes is that they allow users to select a suitable dye based on a number of parameters for the desired purpose. Parameters of interest include the maximum excitation wavelength, maximum emission wavelength, Stokes shift, extinction coefficient, fluorescence quantum yield, and fluorescence lifetime. Dyes can be selected based on the application of interest to, for example, allow excitation radiation to penetrate into the biological sample, minimize background fluorescence, and / or achieve a high signal-to-noise ratio.

[0007] Molecular recognition involves the specific binding of two molecules. Molecules that bind specifically to target biomolecules can be used in a variety of research and diagnostic applications, such as analyte labeling and separation, flow cytometry, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blotting, magnetic cell separation, and chromatography. Target biomolecules can be detected by labeling them with fluorescent dyes. Summary of the Invention

[0008] Polymer BODIPY dyes comprising a light-collecting polychromatophore containing a BODIPY unit are provided. In some embodiments, the dye is a polymer tandem dye comprising a light-collecting polychromatophore containing a BODIPY unit and an acceptor chromophore covalently linked to the polychromatophore in the vicinity of its energy receiver. The polymer tandem dye may be covalently linked to a specific binding member. Methods for evaluating samples in response to the presence of a target analyte and methods for labeling target molecules using compositions comprising polymer tandem dyes are also provided. Kits and systems for practicing the subject methods are also provided. Attached Figure Description

[0009] It should be understood that the following figures are for illustrative purposes only. The figures are not intended to limit the scope of this teaching in any way.

[0010] Figure 1 The absorption and emission of exemplary polymeric tandem dyes with multiple acceptor dyes attached to internal linker sites are shown. These structures do not have attachment-specific binding members.

[0011] Figure 2 The absorption and emission of exemplary polymer tandem dyes with multiple dye molecules attached to internal linker sites are shown. The absorbance of all solutions is 0.04 OD. Note that the polymers with attached acceptor chromophores exhibit significantly higher emission intensity compared to the individual polymers. These polymers do not have attachment-specific binding members.

[0012] definition

[0013] Before describing the exemplary embodiments in more detail, the following definitions are set forth in order to explain and define the meaning and scope of the terms used in this specification.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. 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, NY (1991) provide the general meanings of many terms used herein for those skilled in the art. However, for clarity and ease of reference, certain terms are defined as follows.

[0015] It must be noted that, as used herein and in the appended claims, the singular forms “a / an” and “the” include plural referents unless the context clearly indicates otherwise. For example, the term “a primer” refers to one or more primers, i.e., a single primer and multiple primers. It should also be noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as a preliminary basis for the use of exclusionary terms such as “solely,” “only,” etc., or the use of “negative” restrictions in conjunction with the description of such claim elements.

[0016] As used herein, the term "sample" refers to a material or mixture of materials (in some cases, in liquid form) containing one or more analytes of interest. In some embodiments, the term, used in its broadest sense, refers to any plant, animal, or bacterial material containing cells or producing cellular metabolites, such as tissues or fluids isolated from an individual (including, but not limited to, plasma, serum, cerebrospinal fluid, lymph, tears, saliva, and tissue sections) or tissues or fluids isolated from in vitro cell culture components, as well as samples from the environment. The term "sample" may also refer to "biological sample." As used herein, the term "biological sample" refers to a whole organism or a subset of its tissues, cells, or components (e.g., bodily fluids, including but not limited to blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen). "Biological sample" can also refer to homogenates, lysates, or extracts prepared from a whole organism or a subset, fraction, or part thereof of its tissues, cells, or components, including but not limited to plasma, serum, cerebrospinal fluid, lymph, external parts of skin, respiratory tract, intestinal and genitourinary tract, tears, saliva, breast milk, blood cells, tumors, and organs. In some embodiments, the sample has been taken from an animal or plant. Biological samples may include cells. The term "cell" is used in its conventional sense to refer to the basic structural unit of eukaryotes and prokaryotes that has at least a nucleus and a cell membrane. In some embodiments, cells include prokaryotic cells, such as those derived from bacteria. In other embodiments, cells include eukaryotic cells, such as those obtained from biological samples derived from animals, plants, or fungi.

[0017] As used herein, the terms “affinity” and “avidity” have the same meaning and may be used interchangeably. “Affinity” refers to the strength of the binding, and increased binding affinity is associated with lower Kd.

[0018] As used herein, the terms “determining,” “measuring,” and “assessing,” and “assaying,” are used interchangeably and include both quantitative and qualitative determinations.

[0019] As used herein, the terms “support binding” and “support-linked” are used interchangeably and refer to the portion (e.g., a specific binding member) covalently or nonvalently linked to a support of interest. A covalent link can involve a chemical reaction of two compatible functional groups (e.g., two chemically selective functional groups, electrophiles and nucleophiles, etc.) to form a covalent bond between the two portions of interest (e.g., the support and the specific binding member). In some cases, a nonvalent link can involve specific binding between two portions of interest (e.g., two affinity moieties, such as a hapten and an antibody, or a biotin moiety and streptavidin, etc.). In some cases, a nonvalent link can involve the absorption of a substrate.

[0020] As used herein, the term "biomolecule" refers to a class of naturally occurring molecules, organic molecules or macromolecules or derivatives thereof. Biomolecules are intended to include polypeptides (e.g., peptides, antibodies, or antibody fragments), polynucleotides, carbohydrates (e.g., sugars), and lipids. In some cases, biomolecules are specifically binding members (e.g., as described herein).

[0021] As used herein, the term "peptide" refers to a polymer of amino acids of any length, including peptides of 2-50 amino acids and polypeptides of more than 50 amino acids. The terms "peptide" and "protein" are used interchangeably herein. The term "peptide" includes polymers of encoded 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 has been replaced by a non-naturally occurring or synthetic backbone. Peptides can have any convenient length, such as 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, 50 or more amino acids, 100 or more amino acids, 300 or more amino acids, such as up to 500 or 1000 or more amino acids. A "peptide" can be 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, such as up to 50 amino acids. In some embodiments, the length of a peptide is between 5 and 30 amino acids.

[0022] As used herein, the term "separated" means the portion of interest that is at least 60% separated, at least 75% separated, at least 90% separated, at least 95% separated, at least 98% separated, and even at least 99% separated from other components associated with that portion before purification.

[0023] "Multiple" contains at least two members. In some cases, multiple can have 10 or more, such as 100 or more, 1000 or more, 10,000 or more, 100,000 or more, 10 6 One or more, 10 7 One or more, 10 8 One or more or 10 9 One or more members.

[0024] The numerical range includes the numbers that define the range.

[0025] As used herein, the term “separation” refers to the physical separation of two elements (e.g., by size or affinity) and the degradation of one element (while the other element remains intact).

[0026] As used herein, the term "specific binding" refers to the ability of a trapping agent (or the first member of a specific binding pair) to preferentially bind a particular analyte (or the second member of a specific binding pair) present in, for example, a homogeneous mixture of different analytes. In some instances, specific binding interactions will distinguish desired and undesired analytes in a sample with a specificity greater than 10 times (e.g., 100 times or more, or 1000 times or more) that against undesired analytes. In some cases, when specifically bound as a trapping agent / analyte complex, the affinity between the trapping agent and the analyte is at least 10. -8 M, at least 10 -9 M, such as up to 10 -10 M.

[0027] The method described herein includes multiple steps. Each step can be performed after a predetermined amount of time has elapsed between steps, as desired. Thus, the time between each step can be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more, and may include 5 hours or more. In some embodiments, each subsequent step is performed immediately after the completion of the preceding step. In other embodiments, a step may be performed after an incubation or waiting period following the completion of the preceding step, for example, after a waiting period of several minutes to overnight.

[0028] As used herein, the term "joint" or "link" refers to a connecting portion that links two groups and has a skeleton of 100 atoms or less in length. A joint or link can be a covalent bond connecting two groups or a chain of length between 1 and 100 atoms (e.g., a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, or more carbon atoms), wherein the joint can be linear, branched, cyclic, or a single atom. In some cases, a joint refers to a branched joint that links three or more groups. In some cases, one, two, three, four, or five or more carbon atoms in the joint skeleton may optionally be substituted with sulfur, nitrogen, or oxygen heteroatoms. The bonds between the skeleton atoms can be saturated or unsaturated, and in some cases, no more than one, two, or three unsaturated bonds may exist in the joint skeleton. The joint may include one or more substituent groups, such as alkyl, aryl, or alkenyl groups. The linker may include, but is not limited to, polyethylene glycol; ethers; thioethers; tertiary amines; alkyl groups (which may be linear or branched), such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), etc. The linker backbone may include cyclic groups, such as aryl, heterocyclic, or cycloalkyl groups, wherein two or more atoms of the cyclic group, such as two, three, or four atoms, are included in the backbone. The linker may be cleavable or non-cleavable.

[0029] As used herein, the terms “polyethylene oxide,” “PEO,” “polyethylene glycol,” “PEG,” and “PEG moiety” are used interchangeably and refer to components comprising the chemical formula --(CH2--CH2--O--). n- The polymeric group of the described chain or a derivative thereof. In some embodiments, "n" is 5000 or less, such as 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, such as 3 to 15 or 10 to 15. It should be understood that the PEG polymeric group can have any convenient length and can include multiple end groups and / or additional substituent groups, including but not limited to alkyl, aryl, hydroxyl, amino, acyl, acyloxy, and amide end groups and / or substituent groups. PEG groups that can be adapted for use in subject polychromatic compounds include those described by S. Zalipsky in “Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates”, Bioconjugate Chemistry 1995, 6(2), 150-165; and those described by Zhu et al. in “Water-Soluble Conjugated Polymers for Imaging, Diagnosis, and Therapy”, Chem. Rev., 2012, 112(8), pp 4687–4735.

[0030] As used herein, the terms “chemoselective functional group,” “chemoselective tag,” and “conjugated tag” are used interchangeably and refer to a functional group that can selectively react with another compatible functional group to form a covalent bond (in some cases, after optionally activating one of these functional groups). Chemoselective functional groups of interest include, but are not limited to, thiols and maleimides or iodoacetamides, amines and carboxylic acids or their reactive esters, and groups that can react with each other via click chemistry, such as azide and alkyne groups (e.g., cyclooctyne groups), as well as hydroxyl, hydrazide, hydrazine, aldehyde, ketone, azide, alkyne, phosphine, epoxide, etc. In some cases, the chemoselective functional group is a protected functional group that must be deprotected prior to covalent bonding. In some instances, the chemoselective functional group may be activated before or during covalent bonding with a compatible functional group.

[0031] As used herein, the term "alkyl" itself, or as part of another substituent, refers to a saturated, branched, or straight-chain monovalent hydrocarbon group obtained by removing a hydrogen atom from a single carbon atom of a parent alkane. Alkyl groups of interest include, but are not limited to, methyl; ethyl; propyl (e.g., propyl-1-yl or propyl-2-yl); and butyl (e.g., butyl-1-yl, butyl-2-yl, 2-methyl-propyl-1-yl, or 2-methyl-propyl-2-yl). In some embodiments, the alkyl group comprises from 1 to 20 carbon atoms. In some embodiments, the alkyl group comprises from 1 to 10 carbon atoms. In some embodiments, the alkyl group comprises from 1 to 6 carbon atoms, such as from 1 to 4 carbon atoms. By way of example, this term includes straight-chain and branched hydrocarbon groups, such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), tert-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0032] The term "substituted alkyl" refers to an alkyl group as defined herein, wherein one or more carbon atoms in the alkyl chain have been optionally replaced by heteroatoms (e.g., -O-, -N-, -S-), -S(O)n- (where n is 0 to 2), -NR- (where R is hydrogen or alkyl) and has 1 to 5 substituents selected from the group consisting of: alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amide, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxy, oxo, thione, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclicoxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, hydroxylamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl and NR a R b R' and R" can be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.

[0033] "Alynyl" refers to a straight-chain or branched monovalent hydrocarbon group having 2 to 6 carbon atoms, preferably 2 to 3 carbon atoms, and having at least 1, preferably 1 to 2, sites of triple bond unsaturation. Examples of such alkynyl groups include ethynyl (-C≡CH) and propynyl (-CH2C≡CH).

[0034] The term "substituted alkynyl" refers to an alkynyl group as defined herein, having 1 to 5 substituents selected from the following: alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, amide, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxyl, oxo, thion, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclic, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclic, hydroxylamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0035] "Aryl" itself, or as part of another substituent, refers to a monovalent aromatic hydrocarbon group obtained by removing a hydrogen atom from a single carbon atom in an aromatic ring system. Aryl groups of interest include, but are not limited to, groups derived from: anthracene, acenaphthene, phenanthrene, anthracene, azurite, benzene, etc. Aryl groups include benzo[a], fluoranthene, fluorene, hexaphenyl, hexalene, cyclohexanediene, as-indacene, s-indacene, indene, indene, naphthalene, octacene, octaphene, octalene, oleophane, pentyl-2,4-diene, pentylene, cyclopentadiene, pentaphene, perylene, phenanthracene, sepium, pyrene, pyranthrene, rubigin, triphenylene, trinaphthalene, etc. In some embodiments, the aryl group comprises from 6 to 20 carbon atoms. In some embodiments, the aryl group comprises from 6 to 12 carbon atoms. Examples of aryl groups are phenyl and naphthyl.

[0036] "Heteroaryl" itself, or as part of another substituent, refers to a monovalent heteroaryl obtained by removing a hydrogen atom from a single atom of a heteroaryl ring system. Interested heteroaryl groups include, but are not limited to, groups derived from: acridine, arsenol, carbazole, β-carboline, chromene, chromene, zoline, furan, imidazole, indazole, indole, dihydroindole, indene, isobenzofuran, isochromene, isoindole, isodihydroindole, isoquinoline, isothiazole, isoxazole, naphthidine, oxadiazole, oxazole, naphthalene-intercalated diazoxide, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrrolizine, quinazoline, quinoline, quinazine, quinoxaline, tetrazolium, thiadiazole, thiazolium, triazole, benzotriazole, thiophene, triazole, xanthan, benzodioxane, etc. In some embodiments, the heteroaryl group is a 5-20 member heteroaryl group. In some embodiments, the heteroaryl group is a 5- to 10-membered heteroaryl group. In some embodiments, the heteroaryl group is derived from those derived from: thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole, and pyrazine.

[0037] The terms “alkylaryl” or “aralkyl group” refer to the group -alkylene-aryl and substituted alkylene-aryl, wherein alkylene, substituted alkylene and aryl are defined herein.

[0038] "Alkoxy" refers to the group -O-alkyl, where alkyl is as defined herein. By example, alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, etc. The term "alkoxy" also refers to the groups alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and ynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl, and ynyl are as defined herein.

[0039] The term “substituted alkoxy” means a substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O- and substituted alkynyl-O-, wherein the substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.

[0040] "Alkylene" refers to a divalent aliphatic hydrocarbon group having preferably 1 to 6, more preferably 1 to 3, carbon atoms, being straight-chain or branched, and optionally interrupted by one or more groups selected from the following: -O-, -NR. 10 -、-NR 10 C(O)-、-C(O)NR 10 -etc. For example, this term includes methylene (-CH4) 2-), ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), isopropylene (-CH2CH(CH3)-), (-C(CH3)2CH2CH2-), (-C(CH3)2CH2C(O)-), (-C(CH3)2CH2C(O)NH-), (-CH(CH3)CH2-), etc. "Substituted alkylene" refers to an alkylene group in which one to three hydrogens are replaced by a substituent, as described for carbon in the following definition of "substituted".

[0041] "Substituted" refers to a group in which one or more hydrogen atoms are independently replaced by one or more substituents, either the same or different. Substituents of interest include, but are not limited to, alkylene dioxy groups (such as methylene dioxy), -M, and -R. 60 -O - =O, -OR 60 -SR 60 -S - =S, -NR 60 R 61 =NR 60 , -CF3, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -S(O)2O - -S(O)2OH, -S(O)2R 60 -OS(O)2O - -OS(O)2R 60 -P(O)(O) - )2、 -P(O)(OR 60 (O) - ), -OP(O)(OR 60 (OR) 61 -C(O)R 60 -C(S)R 60 -C(O)OR 60 -C(O)NR 60 R 61 -C(O)O - -C(S)OR 60 -NR 62 C(O)NR 60 R 61 -NR 62 C(S)NR 60 R 61 -NR 62 C(NR 63 )NR 60 R 61 and -C(NR) 62 )NR 60 R 61Where M is a halogen; R 60 R 61 R 62 and R 63 Independently, it is hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, or optionally R 60 and R 61 Together with the nitrogen atoms they are bonded to, they form a ring of cyclohexaalkyl or substituted cyclohexaalkyl; and R 64 and R 65 Independently, it is hydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, or optionally R 64 and R 65 Together with the nitrogen atoms they are bonded to, they form a ring of cyclohexaalkyl or a substituted cyclohexaalkyl. In some embodiments, the substituents include -M, -R. 60 =O, -OR 60 -SR 60 -S - =S, -NR 60 R 61 =NR 60 , -CF3, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -S(O)2R 60 -OS(O)2O - -OS(O)2R 60 -P(O)(O) - )2、 -P(O)(OR 60 (O) - ), -OP(O)(OR 60 (OR) 61 -C(O)R 60 -C(S)R 60 -C(O)OR 60 -C(O)NR 60 R 61 -C(O) - -NR 62 C(O)NR 60 R 61 In some embodiments, the substituents include -M and -R. 60 =O, -OR 60 -SR 60 -NR 60 R 61, -CF3, -CN, -NO2, -S(O)2R 60 -P(O)(OR) 60 (O) - ), -OP(O)(OR 60 (OR) 61 -C(O)R 60 -C(O)OR 60 -C(O)NR 60 R 61 -C(O) - In some embodiments, the substituents include -M and -R. 60 =O, -OR 60 -SR 60 -NR 60 R 61 , -CF3, -CN, -NO2, -S(O)2R 60 -OP(O)(OR) 60 (OR) 61 -C(O)R 60 -C(O)OR 60 -C(O)O - , where R 60 R 61 and R 62 As defined above. For example, the substituted group may have a methylenedioxy substituent or one, two, or three substituents selected from halogen atoms, (1-4C) alkyl groups, and (1-4C) alkoxy groups. When the substituted group is an aryl or heteroaryl group, this one or more substituents (e.g., as described herein) may be referred to as "one or more aryl substituents".

[0042] Other definitions of the term may appear throughout the specification. Detailed Implementation

[0043] As outlined above, polymer tandem dyes are provided. In some embodiments, the polymer tandem dye comprises a photosensitive polychromatophore containing a BODIPY unit and an acceptor chromatophore covalently linked to the polychromatophore in the vicinity of its energy receiver. The polymer tandem dye may be covalently linked to a specific binding member. Methods for evaluating samples in response to the presence of a target analyte and methods for labeling target molecules using compositions comprising polymer tandem dyes are also provided. Kits and systems for practicing the subject methods are also provided.

[0044] Before describing the various embodiments in more detail, it should be understood that the teachings of this disclosure are not limited to the specific embodiments described, as these can certainly be modified. It should also be understood that because the scope of this teaching will be limited only by the appended claims, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0045] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter in any way. While this instruction has been described in conjunction with various embodiments, it is not intended to limit this instruction to those embodiments. Rather, it will be understood by those skilled in the art that this instruction encompasses various alternatives, modifications, and equivalents.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the teachings herein, some example methods and materials will now be described.

[0047] References to any publication refer to its publication prior to the filing date and should not be construed as an admission that the present claims could not have been filed earlier than such publication due to prior invention. Furthermore, the date of the publication provided may differ from the actual publication date, which can be independently determined.

[0048] Upon reading this disclosure, it will be apparent to those skilled in the art that each embodiment in the individual embodiments described and illustrated herein has discontinuous components and features that can be readily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of this teaching. Any of the methods described may be performed in the order of the events narrated or in any other logically possible order.

[0049] All patents and publications mentioned herein, including all sequences disclosed within them, are expressly incorporated by reference.

[0050] In further describing the invention, polymer dyes and tandem dyes comprising receptor chromophores are first described in more detail. Next, conjugates comprising polymer dyes are described. Then, methods of interest for using compositions comprising the subject polymer tandem dyes are summarized. Systems and kits that can be used to practice the methods of the invention are also described.

[0051] Multi-color chromophore containing BODIPY unit

[0052] As outlined above, this disclosure provides polymeric BODIPY dyes. In some embodiments, the polymeric dye comprises a photosensitive polychromatophore containing a BODIPY unit. In some embodiments, the polychromatophore itself is fluorescent. In some instances, the polychromatophore is a polymeric tandem dye. Thus, in some embodiments, the polychromatophore additionally comprises an acceptor chromophore covalently linked to the polychromatophore in the vicinity of its energy receiver.

[0053] As used herein, the terms “light-collecting polychromatophore,” “polymer dye,” and “conjugated polymer” are used interchangeably and refer to a conjugated polymer having a structure capable of collecting light at a specific maximum absorption wavelength and converting it into emitted light at a longer maximum emission wavelength. In some cases, the light-collecting polychromatophore itself is fluorescent. Conjugated polymers (CPs) are characterized by a delocalized electronic structure and can have an effective conjugation length substantially shorter than the polymer chain length, as the backbone can contain a large number of closely spaced conjugated segments. In some cases, conjugated polymers are effective for light collection and provide optical amplification through Forster energy transfer to the acceptor. In some embodiments, the conjugated polymer comprises a plurality of first optically active units forming a conjugated system having first optically active units absorbing light to form an absorption wavelength of the excited state (e.g., as described herein). In some instances, polymer dyes comprise conjugated polymer segments or oligomeric structures that include n-conjugated repeating units that reduce the band gap.

[0054] As used herein, the term "unit" refers to a structural subunit of a polymer. The term "unit" is intended to include monomers, comonomers, coblocks, conjugated segments, repeating units, etc. A "repeating unit" is a subunit of a polymer defined by the minimum number of distinct structural features required to be considered a unit of monomers, such that when the unit is repeated n times, the resulting structure describes the polymer or its blocks. In some cases, a polymer may include two or more distinct repeating units, for example, when the polymer is a multiblock polymer, each block may define a distinct repeating unit. In some cases, the repeating unit of a polymer comprises a single monomer group. In some instances, the repeating unit of a polymer comprises two or more monomer groups, i.e., comonomer groups, such as two, three, four, or more comonomer groups. As used herein, the terms "comonomer" or "comonomer group" refer to a structural unit of a polymer, which itself may be part of the repeating unit of the polymer. In some embodiments, a conjugated polymer comprises a block copolymer composed of polymeric monomer blocks. In this case, the block copolymer may be described as having distinct repeating units, each corresponding to a different coblock of the polymer. In some cases, the polymer is a diblock copolymer containing two distinct coblocks. In this context, the polymer can be described as comprising coblocks, wherein each coblock may consist of one, two, three or more comonomers.

[0055] As used herein, the term "BODIPY unit" refers to a multichromophore structural subunit that includes chromophores having the following boron-dipyrrole methylene (BODIPY) core structure:

[0056]

[0057] Each R is independently selected from the group consisting of: F, OH, H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkynyl, and substituted alkynyl. The BODIPY core structure can be connected to adjacent units of the polychromatophore at any convenient location within the core structure and can be further optionally substituted. In some instances, the BODIPY unit is conjugated with adjacent units of the polymer. In some embodiments, the BODIPY unit defines a repeating unit. In some embodiments, the BODIPY unit defines a comonomer as part of a repeating unit. Any convenient BODIPY-containing structure can be adapted for use in the subject polychromatophore that is the BODIPY unit. Structures containing BODIPY of interest include, but are not limited to, those BODIPY dyes and derivatives described by Loudet and Burgess in “BODIPY Dyes and Their Derivatives: Syntheses and Spectroscopic Properties”, Chem. Rev. 2007, 107(11):4891–4932.

[0058] In some embodiments, the BODIPY unit is described by the following structure:

[0059]

[0060] in:

[0061] R 1 R 2 R 3 and R 4 Each is independently selected from H, alkyl, or substituted alkyl;

[0062] R 5 Selected from alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, wherein R 5 Optionally replaced by water-solubilizing groups; and

[0063] Each R is selected from the group consisting of: F, OH, H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkynyl and substituted alkynyl.

[0064] Any convenient light-gathering multi-chromophore can be adapted to include the BODIPY unit. Interesting photochromic polychromatic groups that can be modified to include BODIPY units include, but are not limited to, those dyes described below, namely, those published by Gaylord et al. in U.S. Publications 20040142344, 20080293164, 20080064042, 20100136702, 20110256549, 20120028828, 20120252986 and 20130190193 and U.S. Patents 8,575,303 and 8,802450, the disclosures of which are incorporated herein by reference in their entirety; and Gaylord et al., J.Am.Chem.Soc., 2001, 123(26), pp 6417–6418 [Gaylord et al., Journal of the American Chemical Society, 2001, 123(26), pp 6417–6418]; Feng et al. al., Chem. Soc. Rev., 2010, 39, 2411-2419 [Feng et al., Journal of the American Chemical Society, 2010, 39, 2411-2419]; and Traina et al., J. Am. Chem. Soc., 2011, 133(32), pp 12600–12607 [Traina et al., Journal of the American Chemical Society, 2011, 133(32), pp 12600–12607], the disclosures of which are incorporated herein by reference in their entirety.

[0065] The polychromatophore can be water-soluble. Any convenient water-solubilizing group can be included in the polychromatophore to provide increased water solubility of the dye. While the increase in solubility may vary, in some instances, the increase (compared to compounds without this one or more WSGs) is 2-fold or more, such as 5-fold, 10-fold, 25-fold, 50-fold, 100-fold or more. The term "water-solubilizing group" (WSG) refers to a group that is well solubilized in an aqueous environment (e.g., under physiological conditions) and imparts improved water solubility to the molecule to which it is attached. In some embodiments, the WSG increases the solubility of the polychromatophore in a predominantly aqueous solution compared to a polychromatophore lacking a WSG. The water-soluble group can be any convenient hydrophilic group that is well solubilized in an aqueous environment. In some cases, the hydrophilic water-soluble group is charged, such as positively or negatively charged. In some cases, the hydrophilic water-soluble group is a neutral hydrophilic group. In some embodiments, the WSG is a hydrophilic polymer, such as polyethylene glycol, cellulose, chitosan, or derivatives thereof. Water-soluble groups of interest include, but are not limited to, carboxylates, phosphonates, phosphates, sulfonates, sulfates, sulfinates, sulfonates, sulfonium, esters, polyethylene glycol (PEG) and modified PEG, hydroxyl groups, amines, ammonium, guanidinium, pyridinium, polyamines and sulfonium, polyols, linear or cyclic sugars, primary amines, secondary amines, tertiary or quaternary amines and polyamines, phosphonate groups, hypophosphonate groups, ascorbate groups, and glycols (including polyethers, -COOM′, -SO3M′, -PO3M′, -NR3). + Y′, (CH2CH2O) p R) and mixtures thereof, wherein Y' can be any halogen, sulfate, sulfonate or oxyanion, p can be 1 to 500, each R can be independently H or alkyl (such as methyl), and M' can be a cationic counterion or hydrogen, --(CH2CH2O). yy CH2CH2XR yy --(CH2CH2O) yy CH2CH2X--、 --X(CH2CH2O) yy CH2CH2--, glycol and polyethylene glycol, wherein yy is selected from 1 to 1000, and X is selected from O, S and NR. ZZ And R ZZ and R YY Independently selected from H and C 1-3 alkyl.

[0066] Multiple WSGs can be included at a single position within the subject polychromatophore via a branching connector. Any convenient branching connector can be utilized to provide connection to multiple WSGs. Branching connectors of interest include, but are not limited to, tertiary amino groups (e.g., where N is a branching atom), amino acid residues, substituted aryl groups, substituted heteroaryl groups, substituted heterocyclic groups, dendritic groups, etc. In some embodiments, the branching connector is an aralkyl substituent further disubstituted by one or more water-soluble groups. Thus, in some cases, the branching connector group is a substituent of the polychromatophore that links the polychromatophore to two or more water-soluble groups. In some cases, incorporating multiple WSGs via a branching connector imparts desired solubility to the polychromatophore. In some embodiments, the polychromatophore comprises one or more substituents selected from the group consisting of alkyl, aralkyl, and heterocyclic groups, each further substituted by a water-solubilizing group. In some cases, the WSG is a hydrophilic polymer group, such as polyethylene glycol (PEG) (e.g., PEG of 2-20 units).

[0067] Polychromatograms can have any convenient length. In some cases, the specific number of monosomal repeating units or segments of a polychromatogram can fall within the range of 2 to 500,000, such as 2 to 100,000, 2 to 30,000, 2 to 10,000, 2 to 3,000, or 2 to 1,000 units or segments, or such as 5 to 100,000, 10 to 100,000, 100 to 100,000, 200 to 100,000, or 500 to 50,000 units or segments. In some instances, the specific number of monoclonal repeating units or segments of the polychromatogram 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.

[0068] Multichromophores can have any convenient molecular weight (MW). In some cases, the MW of a multichromophore can be expressed as the average molecular weight. In some instances, polymer dyes have average molecular weights ranging from 500 to 500,000, such as from 1,000 to 100,000, from 2,000 to 100,000, from 10,000 to 100,000, or even from 50,000 to 100,000.

[0069] In some embodiments, the BODIPY unit constitutes 25% or more of the multi-chromophore molar concentration, such as 30% or more, 40% or more, 45% or more, 50% or more, 60% or more, 70% or more, or even more of the multi-chromophore molar concentration. In this case, the multi-chromophore may include 5 or more repeating units, such as 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more, 500 or more, 1000 or more, 10,000 or more, or even more repeating units. In this case, the polychromatic group may include 5 or more comonomer units, such as 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more, 500 or more, 1000 or more, 10,000 or more, or even more comonomer units.

[0070] Thematic polychromatic groups can possess one or more desirable spectral properties, such as a specific maximum absorption wavelength, a specific maximum emission wavelength, extinction coefficient, quantum yield, narrow-band spectral characteristics, low-energy absorption bands, etc.

[0071] In some embodiments, the multi-chromophore has a narrow-band spectral characteristic. A narrow-band spectral characteristic refers to an absorbance or emission spectrum with a full width at half maximum (FWHM) of 50 nm or less, with a peak centered at 500 nm or greater. In some embodiments, the dye has a low-energy absorption band with a bandwidth of 200 nm or less, such as 150 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, 50 nm or less, 40 nm or less, 30 nm or less, 20 nm or less, or even less. In some cases, the bandwidth is determined by measuring the full width at half maximum (FWHM). In some embodiments, the dye has a low-energy absorption band with a bandwidth of 50 nm or less.

[0072] In some embodiments, the polychromatin has a maximum absorption wavelength in the range of 300 to 900 nm, such as 350 to 850 nm, 350 to 600 nm, 360 to 500 nm, 370 to 500 nm, 380 to 500 nm, 390 to 500 nm, or 400 to 500 nm. Specific examples of the absorption maxima of interest include, but are not limited to, 590 nm, 630 nm, 650 nm, 680 nm, and 750 nm. In some embodiments, the polychromatin has a maximum absorption wavelength of 590 nm ± 5 nm, 630 nm ± 5 nm, 650 nm ± 5 nm, 680 nm ± 5 nm, or 750 nm ± 5 nm. In some embodiments, the polychromatin has a maximum emission wavelength in the range of 300 to 900 nm, such as 350 to 850 nm, 350 to 600 nm, 360 to 500 nm, 370 to 500 nm, 380 to 500 nm, 390 to 500 nm, or 400 to 500 nm. Specific examples of the emission maxima of interest include, but are not limited to, 605 nm, 650 nm, 680 nm, 700 nm, and 805 nm. In some embodiments, the polychromatin has a maximum emission wavelength of 605 nm ± 5 nm, 650 nm ± 5 nm, 680 nm ± 5 nm, 700 nm ± 5 nm, or 805 nm ± 5 nm.

[0073] In some instances, the polychromatic chromatids have 5x10 5 cm -1 M -1 Or a larger extinction coefficient, such as 6x10 5 cm -1 M -1 Or larger, 7x10 5 cm -1 M -1 Or larger, 8x10 5 cm -1 M -1 Or larger, 9x10 5 cm -1 M -1 Or larger, such as 1 x 10 6 cm -1 M -1 Or larger, 1.5x10 6 cm -1 M -1 Or larger, 2x10 6 cm -1 M -1 Or larger, 2.5x10 6 cm -1 M -1 Or larger, 3x10 6 cm -1M -1 Or larger, 4x10 6 cm -1 M -1 Or larger, 5x10 6 cm -1 M -1 Or larger, 6x10 6 cm -1 M -1 Or larger, 7x10 6 cm -1 M -1 Or larger, or 8x10 6 cm -1 M -1 Or larger. In this case, the polychromatogram can have 5 or more repeating units, such as 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or even more repeating units. In some embodiments, the polychromatogram has 5 x 10 5 M -1 cm -1 Or a larger molar extinction coefficient. In some embodiments, the polychromatic chromophores have a molar extinction coefficient of 1x10⁻⁶. 6 M -1 cm -1 Or a larger molar extinction coefficient.

[0074] In some instances, the polychromatin has a length of 40,000 cm. -1 M -1 / Comonomer or a larger extinction coefficient, such as 45,000 cm⁻¹ -1 M -1 / Comonomer or larger, 50,000 cm -1 M -1 / Comonomer or larger, 55,000cm -1 M -1 / Comonomer or larger, 60,000 cm -1 M -1 / Comonomer or larger, 70,000 cm -1 M -1 / Comonomer or larger, 80,000 cm -1 M -1 / Comonomer or larger, 90,000 cm -1 M -1 / Comonomer or larger, 100,000 cm -1 M -1 The comonomers are either larger or even larger. In some instances, the polychromatophores have a diameter of 40,000 cm⁻¹. -1 M -1 / Extinction coefficient of repeating unit or larger, such as 45,000 cm⁻¹ -1 M -1 / Repeating unit or larger, 50,000cm -1 M -1 / Repeating unit or larger, 55,000cm -1 M -1 / Repeating unit or larger, 60,000cm -1 M -1 / Repeating unit or larger, 70,000cm -1 M -1 / Repeating unit or larger, 80,000 cm -1 M -1 / Repeating unit or larger, 90,000cm -1 M -1 / Repeating unit or larger, 100,000cm -1 M -1 / Repeating unit or larger, 100,000cm -1 M -1 / Repeating unit or larger, 120,000 cm -1 M -1 The repeating unit may be larger, or even larger. In some instances, the extinction coefficient described herein is the average extinction coefficient. In some instances, the repeating unit of the polychromatic chromophore may include a single monomer, two comonomers, or three or more comonomers.

[0075] In some embodiments, the polychromatoid has a quantum yield of 0.05 or greater, such as 0.1 or greater, 0.15 or greater, 0.2 or greater, 0.25 or greater, 0.3 or greater, 0.35 or greater, 0.4 or greater, 0.45 or greater, 0.5 or greater, 0.6 or greater, 0.7 or greater, or even greater. In some cases, the polychromatoid has a quantum yield of 0.1 or greater. In some instances, the polychromatoid has a quantum yield of 0.3 or greater.

[0076] It should be understood that, in some cases, the polychromatophore may include coblocks (e.g., n and m coblocks). The subject polychromatophore may include any convenient linear arrangement of n and m coblocks of various lengths throughout the structure of the polymer. Furthermore, the polychromatophore may include any convenient arrangement of comonomers within these n and / or m coblocks. In the preparation of the subject polychromatophore, various polymer synthesis methods can be used to prepare the comonomers and coblocks of interest. It should be understood that, in some cases, the polymerization method can produce compositions comprising a conjugated polymer community, which includes some variation with respect to a specific length and / or terminal groups (e.g., end groups) present in each CP of the community. The chemical formulas depicted herein may refer to a single compound or a community or subgroup of polymeric compounds.

[0077] In some embodiments, the polychromatin comprises a conjugated segment containing BODIPY as described by chemical formula (I):

[0078]

[0079] in:

[0080] B is a BODIPY unit (e.g., as described herein);

[0081] M is a π-conjugated comonomer;

[0082] Each L is a terminal group; and

[0083] n is an integer from 1 to 100,000. In some instances of chemical formula (I), each L is independently selected from the group consisting of: end group, π-conjugated segment, linker and the specific binding member to which it is attached.

[0084] Any convenient π-conjugated comonomer can be used in the subject polychromatic group. As used herein, the term "π-conjugated comonomer" refers to any convenient monomeric subunit of the polymer that is π-conjugated with adjacent groups along the polymer backbone (i.e., π electrons are delocalized across adjacent units). In some embodiments of formula (I), M is selected from fused 6-5-6 tricyclic comonomers, fluorene comonomers, phenylene-vinylene comonomers, phenylene-ethynylene comonomers, carbazole comonomers, C2-C 12 Alkyne comonomers, aryl-ethynyl comonomers, heteroaryl-ethynyl comonomers, aryl comonomers, and heteroaryl comonomers. In some embodiments of Formula (I), M is a phenylene-vinylene comonomer. In some instances of Formula (I), M is a phenylene-ethynyl comonomer. In some cases of Formula (I), M is a carbazole comonomer. In some instances of Formula (I), M is C2-C12 Alkyne comonomer. In some cases of Formula (I), M is an arylene-ethynylene comonomer. In some embodiments of Formula (I), M is a heteroarylene-ethynylene comonomer. In some instances of Formula (I), M is an arylene comonomer. In some cases of Formula (I), M is a heteroarylene comonomer.

[0085] In some embodiments of chemical formula (I), M is a fused 6-5-6 tricyclic comonomer. A fused 6-5-6 tricyclic comonomer is a comonomer comprising a tricyclic aromatic group having three fused rings, having a configuration of 6-5-6, i.e., two benzo[a] rings fused to a central 5-membered ring. The 5-membered ring can be a carbocyclic or heterocyclic ring and may additionally include side-chain substituents on the ring atoms that are not fused to the benzo[a] ring. In some instances, the fused 6-5-6 tricyclic comonomer is described by the following structure:

[0086]

[0087] in:

[0088] Z is –C(R) 1 )2– or –N(R 1 )–;

[0089] Each R is independently H or one or more aryl substituents; and

[0090] Each R 1 Independently selected from the group consisting of: alkyl, substituted alkyl, aralkyl, substituted aralkyl, PEG moiety, and -L 1 -Z 1 L 1 It's a connector, and Z 1 This is a chemoselective tag (e.g., a tag including chemoselective functional groups) or WSG. As used in any of the chemical formulas described herein, * indicates a site for covalent attachment to the unsaturated backbone or terminal group of a conjugated polymer. In some embodiments, when Z is –N(R 1 When Z is –C(R), the fused 6-5-6 tricyclic comonomer is a carbazole comonomer. Any convenient carbazole comonomer can be used in the subject polychromatic group. In some embodiments, when Z is –C(R) 1 When )2–, the fused 6-5-6 tricyclic comonomer is a fluorene comonomer. Any convenient fluorene comonomer can be used in the subject polychromatic group. In some embodiments of formula (I), M is a carbazole comonomer. In some instances of the fused 6-5-6 tricyclic comonomer, each R 1Selected from benzyl groups substituted by one, two or more PEG moieties or alkyl groups substituted by two or more PEG moieties.

[0091] In some embodiments of formula (I), M is a fluorene comonomer. The fluorene comonomer is an aromatic group having a 9H-fluorene core structure, which is substituted at position 9 by one or more convenient side-chain substituents. In some cases, the fluorene comonomer is a 9,9-disubstituted fluorene. The fluorene comonomer can be conjugated with adjacent polymer backbone groups at any convenient position of the fluorene core structure, such as any two convenient positions selected from positions 1-8 (see the numbering scheme below). In some embodiments, the fluorene core structure is linked to adjacent groups of the polymer backbone via positions 2- and 7- (see the numbering scheme below). In some embodiments, the fluorene comonomer is described by the following structure:

[0092]

[0093] Each R 1 Independently selected from alkyl, substituted alkyl, aralkyl, substituted aralkyl, PEG moiety and -L 1 -Z 1 L 1 It's a connector, and Z 1 It is a chemiselective label (e.g., a label that includes chemiselective functional groups) or WSG. In some instances of fluorene comonomers, each R 1 Selected from benzyl groups substituted with one, two, or more PEG moieties, or alkyl groups substituted with two or more PEG moieties. In some cases, Z 1 This includes functional groups that can be used to covalently link polychromatophores to receptor chromatophores (e.g., as described herein). In some instances, Z... 1 This includes amino groups used for covalent attachment to the acceptor chromophore. In some instances, Z... 1 This includes a carboxylic acid group or a derivative thereof for covalent attachment to the acceptor chromophore. In some embodiments, L 1 It is connected to two or more Z 1 Branching heads of groups (e.g., WSG). In some instances, the fluorene comonomer is further supported by R groups located at one, two, or more positions selected from positions 1, 3, 4, 5, 6, and 8. 5 and / or R 6 Substituent substitution, where R 5 and R 6 Independently selected from water-solubilizing groups (WSG) and aryl substituents (e.g., as described herein).

[0094] In some instances, fluorene comonomers are described by the following structures:

[0095]

[0096] Each R 2 It is an alkyl group substituted with a water-soluble group, or a branched head linked to two or more water-soluble groups (e.g., PEG-disubstituted benzyl or PEG-substituted alkyl). In some instances of fluorene comonomers, each R 2 It is composed of one, two, or three PEG moieties (e.g., -O(CH2CH2O)). n R', where R' is H or alkyl, and n is 1-20 (e.g., 3-16, such as 8-16) substituted benzyl groups. In some instances of fluorene comonomers, each R 2 It is controlled by a -O (CH2CH2O) n The R' group is substituted (e.g., at the 2, 3, or 4 position) with a benzyl group, wherein R' is H or an alkyl group, and n is 1-20, for example 3-16, such as when n is 8-16. In some instances of fluorene comonomers, each R... 2 It is formed by two -O (CH2CH2O) n The R' group is substituted (e.g., at the 2,4-, 3,4-, or 3,5-position) with a benzyl group, wherein each R' is independently H or alkyl, and each n is independently 1-20, for example 3-16, such as when n is 8-16. In some instances of fluorene comonomers, each R... 2 It is formed by three -O (CH2CH2O) n The R' group is substituted (e.g., at the 2,4,6-, 2,4,5-, or 3,4,5- position) with a benzyl group, wherein each R' is independently H or alkyl, and each n is independently 1-20, e.g., 3-16, such as when n is 8-16. In certain instances of fluorene comonomers, each R... 2 It is a lower alkyl group substituted with a trivalent branched group, each branched group being replaced by two PEG moieties (e.g., -CO-NR”2 or -O(CH2R”)2 trivalent branched groups), wherein each R” is independently a PEG moiety (e.g., -O(CH2CH2O)). n R', where R' is H or an alkyl group, and n is 1-20, for example 3-16 (e.g., n is 8-16). In some embodiments, the fluorene comonomer is described by the following structure:

[0097]

[0098] Where R 3 It is an alkyl group substituted with a water-soluble group (e.g., an alkyl group substituted with PEG), and R 4 It is L1 -Z 2 L 1 It's a connector, and Z 2 This is a chemoselective tag (e.g., for conjugation with the receptor chromophore) or the receptor chromophore. The subject polychromophore can include any convenient chemoselective functional group, including but not limited to carboxylic acids, reactive esters (e.g., NHS or sulfonyl-NHS esters), amino, hydroxyl, thiols, maleimides, iodoacetyl, hydrazide, hydrazine, aldehydes, ketones, azide, alkynes, phosphine, epoxides, etc. In some cases, a chemoselective tag is used to covalently link any convenient part (e.g., the receptor chromophore) to the polychromophore. In some instances, Z 2 This includes amino groups used for covalent attachment to the acceptor chromophore. In some instances, Z... 2 This includes carboxylic acid groups or derivatives thereof used for covalent attachment to acceptor chromophores. In some instances of fluorene comonomers, R 3 It is a lower alkyl group substituted with a trivalent branched group, each branched group being replaced by two PEG moieties (e.g., -CO-NR”2 or -O(CH2R”)2 trivalent branched groups), where each R” is a PEG moiety (e.g., -O(CH2CH2O)). n R', where R' is H or an alkyl group, and n is 1-20, for example 3-16, such as when n is 8-16).

[0099] In some instances, fluorene comonomers are described by the following structures:

[0100]

[0101] in:

[0102] R 3 It is a substituent containing a water-solubilizing group (e.g., as described herein);

[0103] R 4 It is L 1 -Z 2 L 1 It's a connector, and Z 2 It is a chemically selective tag (e.g., used for conjugation with the receptor chromophore) or a receptor chromophore; and

[0104] R 5 and R 6 Independently selected from H, water-solubilizing groups, and aryl substituents (e.g., alkyl, substituted alkyl, alkoxy, substituted alkoxy, halogen, or nitro). In some examples of fluorene comonomers, R 3It is a lower alkyl group substituted with a trivalent branched group, each branched group being replaced by two PEG moieties (e.g., -CO-NR”2 or -O(CH2R”)2 trivalent branched groups), where each R” is a PEG moiety (e.g., -O(CH2CH2O)). n R', where R' is H or an alkyl group, and n is 1-20, for example 3-16, such as when n is 8-16).

[0105] Any convenient terminal group can be used at the end of the polychromatophore. Terminal groups of interest include, but are not limited to, end-capping groups, π-conjugated segments, linkers, and attached specific binding members. In some embodiments, the end-capping group is a monovalent group conjugated to the backbone of the polychromatophore after polymerization. In some instances, the end-capping group is an aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, or substituted alkyl. In some embodiments, the end-capping group is replaced by a linker and / or conjugated tag that can attach any convenient portion (such as a specific binding member). In some cases, the terminal group is a group derived from the monomer used in the polymerization method, such as a group capable of undergoing further conjugation, such as a halogen (e.g., Br), borate, or borate ester. In some instances, the terminal group is a π-conjugated segment. As used herein, a π-conjugated segment refers to any convenient additional segment of a conjugated polymer in which the polychromatophore can be conjugated (i.e., allowing π electrons to delocalize across adjacent units). In some embodiments, the terminal unit is a connector, such as a connector including a functional group adapted to conjugate with a specific binding moiety. It should be understood that the connector and conjugation tag located at the end of the polychromatophore can be selected to be orthogonal to any other connector and chemiselective tag that may be present on the side chain of the polychromatophore. As used herein, the terms chemiselective tag and conjugation tag are used interchangeably and refer to any convenient group including the functional group of interest (e.g., the chemiselective functional group as described herein). In some embodiments, an amino functional group or a derivative thereof is included in the terminal group (e.g., G...). 1 and / or G 2 At position Z, and the carboxylic acid functional group or its derivative is included. 1 In some embodiments, a carboxylic acid functional group or a derivative thereof is included in the terminal group (e.g., G). 1 and / or G 2 At position Z, and an amino functional group or its derivative is included. 1 Place.

[0106] Polymer Tandem Dyes

[0107] As outlined above, this disclosure provides polymer tandem dyes comprising a light-collecting polychromatophore containing a BODIPY unit. Any of the light-collecting polychromatophores containing a BODIPY unit described herein can be used in the subject polymer tandem dyes. In some embodiments, the polymer tandem dye comprises a light-collecting polychromatophore containing a BODIPY unit and an acceptor chromophore covalently linked to the polychromatophore in the vicinity of its energy receiver. In some embodiments, the light-collecting polychromatophore is water-soluble.

[0108] Polymer tandem dyes comprise two covalently linked parts: a donor photosensitive polychromatophore (e.g., as described herein) and an acceptor chromophore. As used herein, the term "acceptor chromophore" refers to a light-absorbing molecule capable of receiving or absorbing energy transferred from the polychromatophore. In some cases, the acceptor chromophore can both emit light of the energy received from the polychromatophore and dissipate that energy as heat. It should be understood that, unless otherwise specified, in the structures and chemical formulas described herein, the designation "dye" refers to "acceptor chromophore." In some instances, the acceptor chromophore is a quencher. As used herein, the term "quencher" refers to an acceptor chromophore that absorbs energy from the polychromatophore and dissipates that energy as heat without emitting light. In some instances, the acceptor chromophore is a fluorescent dye. In some embodiments, the polymer tandem dye can be excited at the wavelength of maximum absorption of the donor polychromatophore and can emit light at the emission wavelength of the acceptor chromophore. In some cases, the photosensitive polychromatophore can transfer energy to acceptor chromophore species near the energy receiver. Energy transfer mechanisms include, for example, resonant energy transfer (e.g., Forster (or fluorescence) resonant energy transfer, FRET), quantum charge exchange (Dexter energy transfer), etc. In some instances, these energy transfer mechanisms are relatively short-range; that is, the close proximity of the photocollector chromophore system to the acceptor chromophore provides efficient energy transfer. In some instances, under conditions of efficient energy transfer, when the number of chromophores in the photocollector chromophore system is large, emission from the acceptor chromophore is amplified; that is, emission from the signal transducer chromophore is stronger when the incident light (“pump light”) is at a wavelength absorbed by the photocollector chromophore compared to when the signal transducer chromophore is directly excited by the pump light.

[0109] In some cases, "effective" energy transfer means that 5% or more (e.g., 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, or even more) of the collected energy is transferred to the acceptor. In some instances, when the acceptor chromophore is a fluorescent dye, the term effective energy transfer can refer to a fluorescence quantum yield of 0.05 or greater, such as 0.1 or greater, 0.2 or greater, 0.3 or greater, 0.4 or greater, 0.5 or greater, or even greater. "Amplification" means that the signal from the acceptor chromophore when excited by the collecting chromophore is 1.5 times or greater compared to direct excitation by incident light of equivalent intensity, such as 2.0 times or greater, 2.5 times or greater, 3 times or greater, 4 times or greater, 5 times or greater, 6 times or greater, or greater, 8 times or greater, 10 times or greater, or even greater. The signal can be measured using any convenient method. In some cases, 1.5 times or greater signal refers to the intensity of the emitted light. In some cases, a signal strength of 1.5 times or greater refers to an increased signal-to-noise ratio. In certain embodiments of polymeric tandem dyes, the emission of the receptor chromophore is 1.5 times or more greater when excited by multiple chromophores compared to when the receptor chromophore is directly excited by incident light.

[0110] In some instances, polymer tandem dyes have 5x10 5 cm -1 M -1 Or a larger extinction coefficient, such as 6x10 5 cm - 1 M -1 Or larger, 7x10 5 cm -1 M -1 Or larger, 8x10 5 cm -1 M -1 Or larger, 9x10 5 cm -1 M -1 Or larger, such as 1x10 6 cm -1 M -1 Or larger, 1.5x10 6 cm -1 M -1 Or larger, 2x10 6 cm -1 M -1 Or larger, 2.5x10 6 cm -1 M -1 Or larger, 3x10 6 cm -1 M -1 Or larger, 4x10 6 cm-1 M -1 Or larger, 5x10 6 cm -1 M -1 Or larger, 6x10 6 cm -1 M -1 Or larger, 7x10 6 cm -1 M -1 Or larger, or 8x10 6 cm -1 M -1 Or larger. In some embodiments, the polymer tandem dye has 5x10 5 M -1 cm -1 Or a larger molar extinction coefficient. In some embodiments, the polymer tandem dye has a molar extinction coefficient of 1x10⁻⁶. 6 M -1 cm -1 Or a larger molar extinction coefficient.

[0111] In some embodiments, the polymer tandem dye has a quantum yield of 0.05 or greater, such as 0.10 or greater, 0.15 or greater, 0.20 or greater, 0.25 or greater, 0.30 or greater, 0.35 or greater, such as 0.40 or greater, 0.45 or greater, 0.5 or greater, or even greater. In some cases, the polymer tandem dye has a quantum yield of 0.1 or greater. In some instances, the polymer tandem dye has a quantum yield of 0.3 or greater.

[0112] Any convenient fluorescent dye can be used as the acceptor chromophore in the subject polymer tandem dye. The terms "fluorescent dye" and "fluorophore" are used interchangeably herein. In some embodiments, the acceptor chromophore is anthocyanin dye, xanthan dye, coumarin dye, thiazine dye, or acridine dye. Fluorescent dyes of interest include, but are not limited to, fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP (polydoxyphylla-chlorophyll a protein), PerCP-Cy5.5, JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350, Alexa Fluor 430, and 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. Lanthanide chelates of interest include, but are not limited to, europium chelates, terbium chelates, and samarium chelates. In some embodiments, polymer tandem dyes comprise polymer dyes linked to acceptor fluorophores selected from Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Alexa488, Alexa 647, and Alexa 700.In some embodiments, the polymer tandem dye comprises a polymer dye linked to an acceptor fluorophore selected from Dyomics dyes (such as DY 431, DY 485XL, DY 500XL, DY530, DY 610, DY 633, DY 640, DY 651, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 754, DY 778, DY 782, DY 800, or DY 831), Biotium CF 555, Cy 3.5, and diethylaminocoumarin.

[0113] In some embodiments of polymeric tandem dyes, the ratio of the comonomer lacking the acceptor chromophore to the comonomer including the linked acceptor chromophore is in the range of 40:1 to 3:1, such as in the range of 20:1 to 3:1, 10:1 to 3:1, 9:1 to 3:1, 5:1 to 3:1 or 4:1 to 3:1, or in the range of 20:1 to 4:1, 20:1 to 5:1, 20:1 to 9:1 or 20:1 to 10:1.

[0114] In some instances, polymer tandem dyes are described by chemical formula (II):

[0115]

[0116] in:

[0117] B 1 and B 2 Each is an independent BODIPY unit;

[0118] Each M 1 and each M 2 It is an independent conjugated comonomer;

[0119] a, b, c, d, e, and f are each independently 0, 1, or 2, where b + e ≥ 1;

[0120] n and m are independently 0 or integers from 1 to 100,000, where n+m≥1;

[0121] p is an integer from 1 to 100,000; and

[0122] Each L 2 It is an independent terminal group.

[0123] In some instances of chemical formula (II), each L 2Independently selected from end groups, π-conjugated segments, linkers, and the specific binding members to which they are attached. In some embodiments of formula (II), when b is 0, a and c are each 1; when e is 0, d and f are each 1; when b is 1, a + c ≥ 1; and when e is 1, d + f ≥ 1. In some embodiments of formula (II), b is 1, and e is 0. In some embodiments of formula (II), b is 0, and e is 1. In some embodiments of formula (II), b is 1, and e is 1. In some embodiments of formula (II), b is 2, and e is 0. In some embodiments of formula (II), b is 0, and e is 2. In some embodiments of formula (II), b is 2. In some embodiments of formula (II), e is 2.

[0124] In some instances of chemical formula (II), B 1 B 2 M 1 and M 2 At least one of them includes –L 1 -C 1 L 1 It is an optional connector, and C 1 It is a receptor chromophore. In some instances of chemical formula (II), B 1 and B 2 At least one of them includes –L 1 -C 1 In some instances of chemical formula (II), M 1 and M 2 At least one of them includes –L 1 -C 1 In some cases of chemical formula (II), M 1 Including –L 1 -C 1 And M 2 Not included. In some cases of chemical formula (II), M 2 Including –L 1 -C 1 And M 1 Not included. In some cases of chemical formula (II), B 1 Including –L 1 -C 1 And B 2 Not included. In some instances of chemical formula (II), B 2 Including –L 1 -C 1 And B 1 Not included.

[0125] In some embodiments of formula (II), a is 0. In some instances of formula (II), a is 1. In some embodiments of formula (II), c is 0. In some instances of formula (II), c is 1. In some instances of formula (II), a is 1 and c is 0. In some instances of formula (II), a is 0 and c is 1. In some cases of formula (II), d is 0. In some instances of formula (II), d is 1. In some embodiments of formula (II), f is 0. In some instances of formula (II), f is 1. In some instances of formula (II), d is 1 and f is 0. In some instances of formula (II), d is 0 and f is 1.

[0126] In some instances, polymer tandem dyes are described by chemical formula (III):

[0127]

[0128] Among them B 1 B 2 M 1 M 2 , n, m, p and each L 2 It is as defined for chemical formula (II), and each L 1 Independently, it is an optional connector, and each C 1 It is an independent receptor chromophore. In some cases of chemical formula (III), each M 1 and each M 2 Independently, they are fluorene comonomers optionally substituted with water-soluble groups. In some cases of formula (III), each M 1 and each M 2 Independently, it is a fused 6-5-6 tricyclic comonomer, such as a fluorene comonomer or a carbazole comonomer optionally substituted with a water-soluble group. In some embodiments of formula (III), the ratio of n to m is in the range of 20:1 to 3:1, such as in the range of 10:1 to 3:1, 9:1 to 3:1, 5:1 to 3:1 or 4:1 to 3:1, or in the range of 20:1 to 4:1, 20:1 to 5:1, 20:1 to 9:1 or 20:1 to 10:1.

[0129] In some embodiments, the polymer tandem dye is described by chemical formula (IV):

[0130]

[0131] Among them B 1 M 2 , n, m, p and each L 2As defined for chemical formula (II), each L 1 It is an optional connector, and each C 1 It is a receptor chromophore. In some embodiments of formula (IV), each M 2 Independently, they are fluorene comonomers optionally substituted with water-soluble groups. In some cases of formula (IV), each M 2 Independently, it is a fused 6-5-6 tricyclic comonomer, such as a fluorene comonomer or a carbazole comonomer optionally substituted with a water-soluble group. In some instances of formula (IV), the ratio of n to m is in the range of 20:1 to 3:1, such as in the range of 10:1 to 3:1, 9:1 to 3:1, 5:1 to 3:1 or 4:1 to 3:1, or in the range of 20:1 to 4:1, 20:1 to 5:1, 20:1 to 9:1 or 20:1 to 10:1.

[0132] In some embodiments, the polymer tandem dye is described by chemical formula (V):

[0133]

[0134] Among them B 1 B 2 M 2 , n, m, p and each L 2 It is as defined for chemical formula (II), and each L 2 It is an optional connector, and each C 1 It is a receptor chromophore. In some embodiments of chemical formula (V), each M 2 Independently, it is a fluorene comonomer optionally substituted with a water-soluble group. In some cases of chemical formula (V), each M 2 Independently, it is a fused 6-5-6 tricyclic comonomer, such as a fluorene comonomer or a carbazole comonomer optionally substituted with a water-soluble group. In some instances of formula (V), the ratio of n to m is in the range of 20:1 to 3:1, such as in the range of 10:1 to 3:1, 9:1 to 3:1, 5:1 to 3:1 or 4:1 to 3:1, or in the range of 20:1 to 4:1, 20:1 to 5:1, 20:1 to 9:1 or 20:1 to 10:1.

[0135] In some embodiments, the polymer tandem dye is described by chemical formula (VI):

[0136]

[0137] Among them B 1 M 1 M 2 , n, m, p and each L2 As defined for chemical formula (II), each L 1 It is an optional connector, and each C 1 It is a receptor chromophore. In some embodiments of chemical formula (VI), each M 1 It is a carbazole comonomer, and each M 2 It is a fluorene comonomer, which is optionally replaced by a water-solubilizing group. In some cases of formula (VI), each M 1 and each M 2 Independently, it is a fused 6-5-6 tricyclic comonomer, such as a fluorene comonomer or a carbazole comonomer optionally substituted with a water-soluble group. In some instances of formula (VI), the ratio of n to m is in the range of 20:1 to 3:1, such as in the range of 10:1 to 3:1, 9:1 to 3:1, 5:1 to 3:1 or 4:1 to 3:1, or in the range of 20:1 to 4:1, 20:1 to 5:1, 20:1 to 9:1 or 20:1 to 10:1.

[0138] In some embodiments of chemical formulas (II) to (VI), B 1 and B 2 Each is independently described by the following structure:

[0139]

[0140] Where R 6 It is an aryl, heteroaryl, or linker optionally substituted with one or more water-solubilizing groups (WSG). In some instances, R 6 This connects the BODIPY core structure to the branch headers of two or more WSGs. In some cases, R... 6 It is an aryl or heteroaryl moiety further replaced by one, two or more WSGs via an optional connector. In some embodiments, R 6 It is a phenyl group substituted with one, two, three or more hydrophilic polymer substituents (e.g., PEG or modified PEG substituents). In some instances, R 6 It is composed of one, two, or three PEG moieties (e.g., -O(CH2CH2O)). n R', where R' is H or an alkyl group, and n is 1-20, for example 3-16 (e.g., 8-16 substituted phenyl groups). In some instances, R... 6 It is controlled by a -O (CH2CH2O) n The phenyl group is substituted with an R' group (e.g., at the 2, 3, or 4 position), wherein R' is H or an alkyl group, and n is 1-20, for example 3-16, such as when n is 8-16. In some instances, R... 6It is formed by two -O (CH2CH2O) n R' groups are substituted (e.g., at the 2,4-, 3,4-, or 3,5-positions) with phenyl groups, wherein each R' is independently H or an alkyl group, and each n is independently 1-20, for example 3-16, such as n being 8-16. In some instances, R 6 It is formed by three -O (CH2CH2O) n R' group substituted (e.g., at the 2,4,6-, 2,4,5-, or 3,4,5- positions) phenyl groups, wherein each R' is independently H or alkyl, and each n is independently 1-20, e.g. 3-16, such as n being 8-16.

[0141] In some instances of chemical formulas (II) to (VI), B 1 and B 2 Each is independently described by the following structure:

[0142]

[0143] Each R' is independently selected from H and alkyl groups; and p is 0 or an integer from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some instances, each R' is methyl. In some cases, each p is 3.

[0144] In some embodiments of chemical formulas (II) to (VI), at least one L 2 The group is -L 3 -Z, where L 3 It is a connector, and Z is a specific binding member (e.g., as described herein). In some embodiments of chemical formulas (II) to (VI), at least one L 2 Yes -L 3 -Z, where L 3 Z is a connector (e.g., as described herein), and Z is a chemically selective label (e.g., as described herein). In some instances, Z is selected from carboxylic acids, active esters (e.g., N-hydroxysuccinimide ester (NHS) or sulfonyl-NHS), amino groups, maleimides, iodoacetyl groups, and thiols. In some embodiments of formulas (II) to (VI), at least one L 2 The functional group is described by the following structure:

[0145] *-Ar-LZ

[0146] Where Ar is a π-conjugated aryl or heteroaryl group, L is a linker, and Z is a chemically selective tag or specifically binding member. In some embodiments of formulas (II) to (VI), at least one L 2 The group is described by one of the following structures:

[0147]

[0148] Where q is 0 or an integer from 1 to 12; L is an optional connector; and Z is a chemically selective tag or specifically binding member. In some embodiments of formulas (II) to (VI), at least one L 2 The functional group is described by the following structure:

[0149]

[0150] Where q is 0 or an integer from 1 to 12; L is an optional linker; and Z is a chemoselective tag or specific binding member. In some instances, -NH-LZ includes an amide bond to a chemoselective tag or specific binding member. In some embodiments, Z is a specific binding member as a biomolecule. In some instances, Z is an antibody. In some instances, Z is an antibody fragment or a binding derivative thereof. In some cases, the antibody fragment or a binding derivative thereof is selected from Fab fragments, F(ab′)2 fragments, scFv, biantibodies, and triantibodies.

[0151] In some embodiments of chemical formulas (II) to (VI), C 1 Selected from cyanide dyes, xanthan dyes, coumarin dyes, thiazine dyes, and acridine dyes. In some cases, the linker is selected from alkyl, substituted alkyl, alkyl-amide, alkyl-amide-alkyl, and PEG moiety. In some embodiments of formulas (II) to (VI), the acceptor chromophore C 1 Selected from DY431, DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701, DY 704, DY730, DY 731, DY 732, DY 734, DY 752, DY 778, DY 782, DY 800, DY 831, Biotium CF 555, Cy3.5, and diethylaminocoumarin. In some instances of formulas (II) to (VI), the receptor chromophore is selected from Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Alexa488, Alexa647, and Alexa700.

[0152] In some embodiments, the polymer tandem dye is described by chemical formula (VII):

[0153]

[0154] in:

[0155] Each q and r is an independent integer from 1 to 20;

[0156] Each R' is independently hydrogen or alkyl (e.g., methyl);

[0157] Each R 1 Independently, it is an alkyl, aryl, heteroaryl, or linker that is optionally substituted with one, two, or more water-solubilizing groups (WSG) (e.g., PEG-disubstituted benzyl or PEG-substituted alkyl);

[0158] Dyes are receptor chromophores;

[0159] n, m, p, and L 2 It is as defined for chemical formula (II); and

[0160] L is the linker, and Z is a chemically selective tag or specifically binding member. In some cases for formula (VII), L is –O-(CH2). n -NH-, where n is 2-12, such as n being 4. In some instances, each R 1 Described by the following structure:

[0161]

[0162] Each q is an independent integer from 2 to 20, and each R' is independently hydrogen, alkyl, or a substituted alkyl group. In some cases, each R' is methyl. In some cases, each q is 2. In some cases, each q is 3. In some cases, each q is 4. In some cases, each q is 5. In some cases, each q is 6. In some cases, each q is 7. In some cases, each q is 8. In some cases, each q is 9. In some cases, each q is 10. In some cases, each q is 11. In some instances, L 2 It is an end base.

[0163] In some embodiments, the polymer tandem dye is described by chemical formula (VIII):

[0164]

[0165] in:

[0166] Each q and r is an independent integer from 1 to 20;

[0167] Each R' is independently hydrogen, alkyl, or substituted alkyl;

[0168] n, m, p, and L 2 It is as defined for chemical formula (II);

[0169] Each R 1Independently, it is an alkyl, aryl, heteroaryl, or linker that is optionally substituted with one, two, or more water-solubilizing groups (WSG) (e.g., PEG-disubstituted benzyl or PEG-substituted alkyl);

[0170] Dyes are receptor chromophores;

[0171] L is the linker, and Z is a chemically selective tag or specifically binding member. In some cases, each R' is a methyl group. In certain cases of formula (VIII), LZ is –O-(CH2). n -NH2, where n is 2-12 (e.g., n is 2, 3, or 4). In some cases of formula (VIII), L is –O-(CH2). n -NH-, where n is 2-12, such as when n is 4, and Z is a specifically bound member (e.g., a biomolecule). In some cases of formula (VIII), LZ is –(CH2). n -NH2, where n is 2-12 (e.g., n is 2, 3, or 4). In some cases of formula (VIII), L is –(CH2). n -NH-, and Z is a specific binding member (e.g., a biomolecule), where n is 2-12 (e.g., n is 2, 3, or 4). It should be understood that a dye group of formula (VIII) linked to a fluorene comonomer via a -CONH- dye bond can alternatively be linked via an -NHCO- dye bond. In this alternative depiction of formula (VIII), L can be –(CH2). n -CO-, where n is 2-12 (e.g., n is 2, 3, or 4). In some instances, each R 1 Described by the following structure:

[0172]

[0173] Each q is an independent integer from 2 to 20, and each R' is independently hydrogen, alkyl, or a substituted alkyl group. In some cases, each R' is methyl. In some cases, each q is 2. In some cases, each q is 3. In some cases, each q is 4. In some cases, each q is 5. In some cases, each q is 6. In some cases, each q is 7. In some cases, each q is 8. In some cases, each q is 9. In some cases, each q is 10. In some cases, each q is 11. In some instances, L 2 It is an end base.

[0174] In some embodiments, the polymer tandem dye is described by chemical formula (IX):

[0175]

[0176] in:

[0177] Each q is an independent integer from 1 to 20;

[0178] Each R' is independently hydrogen, alkyl, or substituted alkyl;

[0179] Each R 1 Independently, it is an alkyl, aryl, heteroaryl, or linker that is optionally substituted with one, two, or more water-solubilizing groups (WSG) (e.g., PEG-disubstituted benzyl or PEG-substituted alkyl);

[0180] Dyes are receptor chromophores;

[0181] n, m, p, and L 2 It is as defined in chemical formula (II);

[0182] L is the linker, and Z is a chemically selective tag or specifically binding member. In some cases, each R' is a methyl group. In certain cases for formula (IX), L is –O-(CH2). n -NH-, where n is 2-12, such as n being 4. In some instances, each R 1 Described by the following structure:

[0183]

[0184] Each q is an independent integer from 2 to 20, and each R' is independently hydrogen, alkyl, or a substituted alkyl group. In some cases, each R' is methyl. In some cases, each q is 2. In some cases, each q is 3. In some cases, each q is 4. In some cases, each q is 5. In some cases, each q is 6. In some cases, each q is 7. In some cases, each q is 8. In some cases, each q is 9. In some cases, each q is 10. In some cases, each q is 11. In some instances, L 2 It is an end base.

[0185] In some embodiments, the polymer tandem dye is described by chemical formula (X):

[0186]

[0187] in:

[0188] Each PEGn is independently derived from 1-20 units of PEG or modified PEG;

[0189] Each R 1Independently, it is an alkyl, aryl, heteroaryl, or linker that is optionally substituted with one, two, or more water-solubilizing groups (WSG) (e.g., PEG-disubstituted benzyl or PEG-substituted alkyl);

[0190] Dyes are receptor chromophores;

[0191] n, m, p, and L 2 It is as defined in chemical formula (II);

[0192] Each L is a linker, and Z is a chemically selective tag or specifically binding member. In some cases of chemical formula (X), L is –O-(CH2). n -NH-, where n is 2-12, such as n being 4. In some instances of chemical formula (X), R 1 It is an alkyl group. In some embodiments of chemical formula (X), the ratio of n to m is in the range of 20:1 to 3:1, such as 15:1 to 4:1, 10:1 to 4:1 or 9:1 to 5:1.

[0193] In some embodiments, the polymer tandem dye is described by chemical formula (XI):

[0194]

[0195] in:

[0196] Each q is an independent integer from 1 to 20;

[0197] Each R' is independently hydrogen, alkyl, or substituted alkyl;

[0198] Dyes are receptor chromophores;

[0199] n, m, p, and L 2 It is as defined in chemical formula (II);

[0200] Each L is a linker, and Z is a chemically selective tag or specifically binding member. In some cases of chemical formula (XI), L is –O-(CH2). n -NH-, where n is 2-12, such as n being 4. In some cases of chemical formula (XI), L is -(CH2). n -CONH-, where n is 1-12, such as n is 1.

[0201] In some embodiments, the polymer tandem dye is described by chemical formula (XII):

[0202]

[0203] in:

[0204] Each q is an independent integer from 1 to 20;

[0205] Each R' is independently hydrogen, alkyl, or substituted alkyl;

[0206] Dyes are receptor chromophores;

[0207] n, m, p, and L 2 It is as defined in chemical formula (II);

[0208] Each L is a linker, and Z is a chemically selective tag or specifically binding member. In some cases of formula (XII), L is –O-(CH2). n -NH-, where n is 2-12, such as n being 4. In some cases of chemical formula (XII), L is -(CH2). n -CONH-, where n is 1-12, such as n is 1.

[0209] In certain instances of fluorene comonomers having any one of the chemical formulas (II)-(XII), each R 1 or R 2 The side chain group is surrounded by one, two, or three PEG moieties (e.g., -O(CH2CH2O)). n R', where R' is H or alkyl, and n is 1-20, for example 3-16 (e.g., n is 8-16) substituted benzyl groups. In certain examples of fluorene comonomers having any one of the chemical formulas (II)-(XII), each R 1 or R 2 The side chain group is surrounded by a -O(CH) group. 2 CH2O) n R' group substituted (e.g., at position 2, 3, or 4) a benzyl group, wherein R' is H or alkyl, and n is 1-20, for example 3-16, such as when n is 8-16. In certain examples of fluorene comonomers having any one of the chemical formulas (II)-(XII), each R 1 or R 2 The side chain group is formed by two -O groups (CH2CH2O). n The R' group is substituted (e.g., at the 2,4-, 3,4-, or 3,5-position) with a benzyl group, wherein each R' is independently H or alkyl, and each n is independently 1-20, for example 3-16, such as when n is 8-16. In certain examples of fluorene comonomers having any one of the chemical formulas (II)-(XII), each R 1 or R 2 The side chain group is surrounded by three -O(CH) groups. 2 CH2O) nThe R' group is substituted (e.g., at the 2,4,6-, 2,4,5-, or 3,4,5- position) with a benzyl group, wherein each R' is independently H or an alkyl group, and each n is independently 1-20, for example 3-16, such as when n is 8-16. In certain examples of fluorene comonomers having any one of the chemical formulas (II)-(XII), each R 1 or R 2 The side chain group is a lower alkyl group substituted with a trivalent branched group, each branched group being substituted by two PEG moieties (e.g., -CO-NR”2 or -O(CH2R”)2 trivalent branched groups), wherein each R” is independently a PEG moiety (e.g., -O(CH2CH2O)). n R', where R' is H or an alkyl group, and n is 1-20, for example 3-16, such as when n is 8-16).

[0210] It should be understood that polymeric tandem dyes having any of the chemical formulas (I) to (XII) can alternatively be represented by a chemical formula indicating the mol% value of each comonomer in the polymer. For example, in some cases, any of the chemical formulas (II) to (XII) can be represented by one of the following chemical formulas:

[0211] L 2 -(B 1 ) x (M 1 ) y (M 2 -L 1 -C 1 ) z -L 2

[0212] L 2 -(B 1 ) x (M 2 ) y (B 2 -L 1 -C 1 ) z -L 2

[0213] L 2 -(B 1 ) x (M 2 ) y (M 2 -L 1 -C 1 ) z -L 2

[0214] L 2 -(B1 ) x (M 2 ) y (M 1 -L 1 -C 1 ) z -L 2

[0215] Where x, y, and z are the mol% values ​​of the copolymer monomers in the conjugated polymer. In some instances of this chemical formula, x is 1 mol% or greater, such as 2 mol% or greater, 3 mol% or greater, 4 mol% or greater, 5 mol% or greater, 10 mol% or greater, 15 mol% or greater, 20 mol% or greater, 25 mol% or greater, 30 mol% or greater, 35 mol% or greater, 40 mol% or greater, 45 mol% or greater, 50 mol% or greater, or even greater. In some instances of this chemical formula, x ranges from 1 mol% to 50 mol%, such as from 5 mol% to 25 mol% or from 10 mol% to 25 mol%; or from 5 mol% to 25 mol% or from 10 mol% to 25 mol%; or from 1 mol% to 25 mol%, from 1 mol% to 10 mol%, or from 1 mol% to 5 mol%. In some instances of this chemical formula, z is 10 mol% or greater, such as 15 mol% or greater, 20 mol% or greater, 25 mol% or greater, 30 mol% or greater, 35 mol% or greater, 40 mol% or greater, 45 mol% or greater, 50 mol% or greater, or even greater. In some instances of this chemical formula, z is 25 mol% or less, such as 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 even less. In some instances of this chemical formula, y is 1 mol% or greater, such as 5 mol% or greater, 10 mol% or greater, 15 mol% or greater, 20 mol% or greater, or 25 mol% or greater. In some instances of this chemical formula, y is 25 mol% or less, such as 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 even less.

[0216] With respect to any structures and chemical formulas depicted herein, it should be understood that in some cases of the subject polymer tandem dyes, the depicted terminal groups or end groups may be located at opposite ends of those shown, for example, these terminal groups may be interchangeable. In some embodiments of the polychromatic groups described herein (e.g., chemical formulas (I)-(XII)), at least one terminal group (e.g., L, L...) 2 G 1 G 2 LZ) is selected from one of the following structures 1-33:

[0217]

[0218]

[0219]

[0220] * = Sites used for covalent attachment to an unsaturated backbone;

[0221] Where R' is independently H, halogen, C1-C 12 Alkyl, (C1-C 12 Alkyl)NH2, C2-C 12 Olefins, C2-C 12 Alkynes, C3-C 12 cycloalkyl, C1-C 12 Haloalkyl, C2-C 18 (Miscellaneous) aryl, C2-C 18 (Hetero)arylamino, —[CH2—CH2] r′ —Z 1 、or (C1-C 12 )alkoxy-X 1 ; and Z 1 It is -OH or -COOH; X 1 It is —NH2, —NHCOOH, —NHCOOC(CH3)3, —NHCO(C3-C12)cycloalkyl(C1-C4)alkyl-N-maleimide; or —NHCO[CH2—CH2—O] s' (CH2) s' NH2; r' is an integer from 1 to 20; and each s' is independently an integer from 1 to 20, (CH2)3(OCH2CH2) x″ OCH3 (where x" is an integer independently from 0 to 50), or optionally by one or more halogens, hydroxyl groups, C1-C 12 Alkyl group or (OCH2CH2) y″CH3-substituted benzyl groups (where each y” is an independent integer from 0 to 50), and R' is different from R; where k is 2, 4, 8, 12 or 24; where R 15 Selected from groups lu having the following structures:

[0222]

[0223] * = Sites used for covalent attachment to the backbone.

[0224] In some embodiments of the polychromatic groups described herein (e.g., chemical formulas (I)-(XII)), at least one terminal group (e.g., L, L...) 2 G 1 G 2 LZ) is selected from one of the following structures:

[0225]

[0226] Where r is 0 or an integer from 1 to 50 (e.g., 1-20); k is 0 or an integer from 1 to 50 (e.g., 1-20); R 1 It is as defined for any fluorene comonomer described herein; and R 16 Selected from H, OH, NH2, -NH(CH2)r-NH2, and -NH(CH2). r COOH.

[0227] Labeled specific binding members

[0228] This disclosure includes labeled specific binding members. Labeled specific binding members are conjugates of a polychromatic folium (e.g., as described herein) comprising the subject BODIPY unit and the specific binding member. The polychromatic folium may be a polymer dye. The polychromatic folium may be a polymer tandem dye. The specific binding member and the polychromatic folium may be conjugated to each other (e.g., covalently linked) via an optional linker, through any convenient location of the polychromatic folium.

[0229] As used herein, the term "specifically binding member" refers to one member of a pair of molecules that are specific to binding with each other. One member of this pair of molecules may have a region on its surface or in its cavity that specifically binds to a region on the surface or in the cavity of the other member of the pair of molecules. Thus, the members of this pair have the property of specifically binding with each other to form a binding complex. In some embodiments, the affinity between the specifically binding members in the binding complex is characterized by 10 -6 M or smaller K d (Dissociation constant), such as 10 -7 M or smaller, including 10-8 M or smaller, such as 10 -9 M or smaller, 10 -10 M or smaller, 10 -11 M or smaller, 10 -12 M or smaller, 10 -13 M or smaller, 10 -14 M or smaller, including 10 -15 M or smaller. In some embodiments, the specific binding member binds specifically with high affinity. High affinity means that the binding member binds specifically with an apparent affinity characterized by 10 x 10-10. -9 M or smaller apparent K d , such as 1x10 -9 M or smaller, 3x10 -10 M or smaller, 1x10 -10 M or smaller, 3x10 -11 M or smaller, 1x10 -11 M or smaller, 3x10 -12 M or smaller or 1x10 -12 M or smaller.

[0230] As used herein, the term "protein-specific" refers to a portion consisting of amino acid residues (e.g., a specific binding member). A portion of a protein may be a polypeptide. In some embodiments, the specific binding member is protein-specific. In some cases, the specific binding member of a protein is an antibody. In some embodiments, the specific binding member of a protein is an antibody fragment, such as a binding fragment of an antibody that specifically binds to a polymer dye. As used herein, the terms "antibody" and "antibody molecule" are used interchangeably and refer to a protein consisting of one or more polypeptides encoded by substantially all or part of recognized immunoglobulin genes. Recognized immunoglobulin genes, for example in humans, include κ(k), λ(I), and heavy chain loci, which together constitute numerous variable region genes, and constant region genes μ(u), δ(d), γ(g), σ(e), and α(a), respectively, encoding IgM, IgD, IgG, IgE, and IgA isotypes. The variable region of an immunoglobulin light or heavy chain consists of a "framework" region (FR) interrupted by three hypervariable regions (also called "complementarity-determining regions" or "CDRs"). The scope of the frame region and CDR has been precisely defined (see “Sequences of Proteins of Immunological Interest,” E. Kabat et al., U.S. Department of Health and Human Services, (1991)). All antibody amino acid sequences discussed here are numbered according to the Kabat system. The sequences of the frame regions of different light or heavy chains are relatively conserved within species. The antibody frame region, which is the combined frame region of the constitutive light and heavy chains, is used to locate and align the CDR. The CDR is primarily responsible for binding to the epitopes of the antigen.

[0231] The term antibody is intended to include full-length antibodies and may refer to natural, engineered, or recombinant antibodies from any organism for experimental, therapeutic, or other purposes 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, generated by modifying whole antibodies or by de novo synthesis using recombinant DNA technology. Antibodies may be monoclonal or polyclonal and may have other specific activities against cells (e.g., antagonists, agonists, neutralizing antibodies, inhibitory antibodies, or stimulating antibodies). It should be understood that antibodies may have additional conserved amino acid substitutions that have substantially no effect on antigen binding or other antibody function.

[0232] In some embodiments, the specific binding member is an antibody. In some embodiments, the specific binding member is a Fab fragment, an F(ab')2 fragment, scFv, a biantibody, or a triantibody. In some cases, the specific binding member is a mouse antibody or a binding fragment thereof. In some instances, the specific binding member is a recombinant antibody or a binding fragment thereof.

[0233] In some embodiments, the labeled specific binding member comprises: a polymeric tandem dye comprising a light-collecting polychromatophore containing a BODIPY unit; an acceptor chromophore covalently linked to the polychromatophore in the vicinity of its energy receiver; and a specific binding member covalently linked to the polychromatophore. In some instances of the labeled specific binding member, the light-collecting polychromatophore is water-soluble. In some instances of the labeled specific binding member, the dye has narrow-band spectral characteristics. In some instances of the labeled specific binding member, the dye has a low-energy absorption band with a bandwidth of 100 nm or less, such as 50 nm or less. In some instances of the labeled specific binding member, the polychromatophore has a 5 x 10⁻⁶ ohm² / 40⁻¹² ... 5 M -1 cm -1 Or a larger (e.g., as described herein) molar extinction coefficient. In some cases of labeled specific binding members, the polychromatophore has a quantum yield of 0.05 or greater (e.g., as described herein). In some embodiments, the labeled specific binding member further includes an acceptor chromophore covalently linked to the polychromatophore in the vicinity of its energy receiver, for example, the polychromatophore being a polymer tandem dye. In some embodiments of the labeled specific binding member, the ratio of the dye's acceptor chromophore to the polychromophore repeating unit is in the range of 1:40 to 1:4, such as a ratio in the range of 1:20 to 1:4, 1:10 to 1:4, 1:9 to 1:4, 1:8 to 1:4, 1:7 to 1:4, 1:6 to 1:4, or 1:5 to 1:4, or such a ratio in the range of 1:40 to 1:5, 1:40 to 1:6, 1:40 to 1:7, 1:40 to 1:8, 1:40 to 1:9, 1:40 to 1:10, or 1:40 to 1:20.

[0234] In some cases, the receptor chromophore is a fluorophore. In some embodiments of the labeled specific binding member, the receptor chromophore emitted 1.5 times or more (e.g., 2.0 times or more, 2.5 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, 10 times or more, or even more) when excited by multiple chromophores compared to when the receptor chromophore was directly excited by incident light.

[0235] In some instances of labeled, specifically binding members, polychromatin comprises a conjugated segment containing BODIPY as described by chemical formula (I):

[0236]

[0237] Where B is a BODIPY unit; M is a π-conjugated comonomer; each L is independently selected from end groups, π-conjugated segments, linkers, and the specific binding members to which they are attached; and n is an integer from 1 to 100,000. In some embodiments of formula (I), the BODIPY unit is described by the following structure:

[0238]

[0239] Where: R 1 R 2 R 3 and R 4 Each is independently selected from H, alkyl, and substituted alkyl; R 5 Selected from alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl, wherein R 5 Optionally substituted with a water-solubilizing group; and each R is selected from F, OH, H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkynyl, and substituted alkynyl. In some embodiments of formula (I), M is selected from fluorene comonomers, phenylene-vinylene comonomers, phenylene-ethynylene comonomers, carbazole comonomers, C2-C 12 Alkyne copolymer monomers, arylene-ethynylene copolymer monomers, heteroarylene-ethynylene copolymer monomers, arylene copolymer monomers and heteroarylene copolymer monomers.

[0240] In some embodiments of the labeled, specifically binding member, the polychromatin is described by chemical formula (II):

[0241]

[0242] Wherein: B1 and B2 are each independently a BODIPY unit; each M1 and each M2 are independently π-conjugated comonomers; a, b, c, d, e, and f are each independently 0, 1, or 2, where b+e≥1; n and m are independently 0 to 100,000, where n+m≥1; and one L2 group is a terminal group (G1), and the other L2 group is the specifically bound member (e.g., LZ). In some embodiments of formula (II), at least one of B1, B2, M1, and M2 includes –L1-C1, where L1 is an optional linker, and C1 is an acceptor chromophore.

[0243] In some embodiments of Formula (II), the attached specific binding member is an antibody. In some instances of Formula (II), the attached specific binding member is an antibody fragment or a binding derivative thereof. In some cases of Formula (II), the attached specific binding member is an antibody fragment or a binding derivative thereof selected from Fab fragments, F(ab')2 fragments, scFv, biantibodies, and triantibodies. In some instances of Formula (II), the receptor chromophore is selected from anthocyanin dyes, xanthan dyes, coumarin dyes, thiazine dyes, and acridine dyes. In some instances of formula (II), the receptor chromophore is selected from DY 431, DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 778, DY 782, DY 800, DY 831, Biotium CF 555, Cy3.5, and diethylaminocoumarin. In some instances of formula (II), the receptor chromophore is selected from Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Alexa488, Alexa647, and Alexa700.

[0244] In some embodiments, the labeled specific binding member is a polymeric tandem dye having chemical formula (III), wherein one of the L 2 The group is the specific binding member to which it is attached. In some embodiments, the labeled specific binding member is a polymer tandem dye having the chemical formula (IV), wherein one of the L groups is an L group. 2 The group is the specific binding member to which it is attached. In some embodiments, the labeled specific binding member is a polymer tandem dye having the chemical formula (V), where L 2 The group is the specific binding member to which it is attached. In some embodiments, the labeled specific binding member is a polymer tandem dye having the chemical formula (VI), wherein one of the L groups is a polymer dye having the chemical formula (VI). 2 A group is a specific binding member to which it is attached.

[0245] In some embodiments, the labeled specific binding member is a polymer tandem dye having chemical formula (VII), where Z is the specific binding member. In some embodiments, the labeled specific binding member is a polymer tandem dye having chemical formula (VIII), where Z is the specific binding member. In some embodiments, the labeled specific binding member is a polymer tandem dye having chemical formula (IX), where Z is the specific binding member. In some embodiments, the labeled specific binding member is a polymer tandem dye having chemical formula (X), where Z is the specific binding member. In some embodiments, the labeled specific binding member is a polymer tandem dye having chemical formula (XI), where Z is the specific binding member. In some embodiments, the labeled specific binding member is a polymer tandem dye having chemical formula (XII), where Z is the specific binding member.

[0246] In some embodiments, the labeled specific binding member is described by the following structure:

[0247]

[0248] In some embodiments, the labeled specific binding member is described by the following structure:

[0249]

[0250] In some embodiments, the labeled specific binding member is described by the following structure:

[0251]

[0252] In some embodiments, the labeled specific binding member is described by the following structure:

[0253]

[0254] In some embodiments, the labeled specific binding member is described by the following structure:

[0255] In some embodiments, the labeled specific binding member is described by the following structure:

[0256]

[0257] Polymer tandem dye precursors having any of the structures shown above are also provided, comprising terminal amino functional groups suitable for conjugation with "biomolecules". The structure of such polymer tandem dye precursors can be represented by replacing the "biomolecule" groups depicted in the above structures with "H". In some examples of the structures depicted above, the attached "dye" is a attached fluorescent dye.

[0258] method

[0259] As outlined above, aspects of the present invention include methods for evaluating a sample in response to the presence of a target analyte. In some embodiments, the method includes: (a) contacting the sample with a polymer dye conjugate that specifically binds the target analyte to produce a labeled composition-contacted sample; and (b) measuring the labeled composition-contacted sample in response to the presence of a polymer dye conjugate-target analyte binding complex to assess the presence of the target analyte in the sample. In some embodiments of the method, the polymer dye conjugate includes: (i) a photosensitive polychromatophore comprising a BODIPY unit (e.g., as described herein); (ii) an acceptor chromophore covalently linked to the polychromatophore in the vicinity of its energy receiver; and (iii) a specifically binding member (e.g., as described herein).

[0260] Any convenient method can be used to contact the sample with a polymer dye conjugate that specifically binds to the target analyte to produce a sample in which the labeled composition has been contacted. As used herein, the terms "polymer dye conjugate" and "labeled specific binding member" are used interchangeably. In some instances, the sample is contacted with the polymer dye conjugate under conditions where the specific binding member specifically binds to the target analyte (if present). For the specific binding member of the conjugate to specifically bind to the target analyte, an appropriate solution that maintains the bioactivity of both the sample components and the specific binding member can be used. This solution can be a balanced salt solution, such as physiological saline, PBS, Hank's balanced salt solution, etc., conveniently supplemented with fetal bovine serum, human platelet lysate, or other factors, at low concentrations, such as an acceptable buffer from 5–25 mM. Convenient buffers include HEPES, phosphate buffer, lactate buffer, etc. Various culture media are commercially available and can be used depending on the nature of the target analyte; these media include dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., in some cases supplemented with fetal bovine serum or human platelet lysate. The final composition of the solution can be selected based on the sample components included.

[0261] The temperature at which the specific binding member of the conjugate specifically binds to the target analyte can vary and, in some instances, can be in the range of 5°C to 50°C, such as 10°C to 40°C, 15°C to 40°C, 20°C to 40°C, for example 20°C, 25°C, 30°C, 35°C, or 37°C (e.g., as described above). In some instances, the temperature at which specific binding occurs is chosen to be compatible with the bioactivity of the specific binding member and / or the target analyte. In some instances, this temperature is 25°C, 30°C, 35°C, or 37°C. In some cases, the specific binding member is an antibody or a fragment thereof and the temperature at which specific binding occurs is room temperature (e.g., 25°C), 30°C, 35°C, or 37°C. Any convenient incubation time can be chosen for specific binding to allow the formation of a desired amount of binding complex, and in some instances can be 1 minute (min) or more, such as 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.

[0262] Any convenient specific binding member can be utilized in the polymer dye conjugate. Specific binding members of interest include, but are not limited to, those that specifically bind to cell surface proteins of various cell types, including but not limited to stem cells (e.g., pluripotent stem cells, hematopoietic stem cells), T cells, regulatory T cells, dendritic cells, B cells (e.g., memory B cells, antigen-specific B cells), granulocytes, leukemia cells, lymphoma cells, viral cells (e.g., HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythrocytes. Target cells of interest include cells with convenient cell surface markers or antigens that can be captured by the 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+) derived from whole blood, bone marrow, or umbilical cord blood. In the subject method, any convenient cell surface protein or cell marker can be targeted to the specifically binding polymer dye conjugate. In some embodiments, target cells include cell surface markers selected from cell receptors and cell surface antigens. In some cases, target cells may include cell surface antigens such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor α / β, T cell receptor γ / δ, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminal), SSEA-3, TRA-1-60 antigen, disalivary ganglioside GD2, and CD71.

[0263] Any convenient target can be selected for evaluation using the topic approach. Targets of interest include, but are not limited to, nucleic acid molecules such as RNA, DNA, PNA, CNA, HNA, LNA, or ANA; proteins such as fusion proteins, modified proteins (e.g., 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” refers to all members of a target family and fragments thereof. Target proteins can be any protein of interest, such as therapeutic or diagnostic targets, including but not limited to: hormones, growth factors, receptors, enzymes, cytokines, osteogenic inducing factors, colony-stimulating factors, and immunoglobulins. The term “target protein” is intended to include recombinant and synthetic molecules that can be prepared or commercially available using any convenient recombinant expression method or any convenient synthetic method. In some embodiments, the polymer dye conjugate includes an antibody or antibody fragment. Any convenient target analyte can specifically bind to the antibody or antibody fragment of interest in the topic approach.

[0264] In some embodiments, the target analyte is cell-related. In some instances, the target analyte is a cell surface marker. In some cases, the cell surface marker is selected from cell receptors and cell surface antigens. In some instances, the target analyte is an intracellular target, and the method further includes lysing the cells. In some instances, the method further includes extracting proteins from the cells. Any convenient methods and reagents may be used for cell lysis. Methods and reagents of interest include those described at www.piercenet.com / method / traditional-methods-cell-lysis for cell lysis and protein extraction.

[0265] In some embodiments, the sample may include a heterogeneous cell population from which target cells have been isolated. In some instances, the sample includes peripheral whole blood, peripheral whole blood from which red blood cells have been lysed prior to cell separation, cord blood, bone marrow, density gradient purified peripheral blood mononuclear cells, or homogenate tissue. In some cases, the sample includes hematopoietic progenitor cells (e.g., CD34+ cells) in whole blood, bone marrow, or cord blood. In some embodiments, the sample includes tumor cells in peripheral blood. In some instances, the sample is a sample that includes (or is suspected of including) viral cells (e.g., HIV).

[0266] Subject-labeled specific binding members can be used in subject-specific methods, such as labeling target cells, particles, targets, or analytes with polymer dyes or polymer tandem dyes. For example, in flow cytometry, labeled specific binding members can be used to label cells to be treated (e.g., detected, analyzed, and / or sorted). Labeled specific binding members can include antibodies that specifically bind to cell surface proteins of various cell types (e.g., as described herein). Labeled specific binding members can be used to study a variety of biological (e.g., cellular) properties or processes, such as cell cycle, cell proliferation, cell differentiation, DNA repair, T cell signaling, apoptosis, cell surface protein expression and / or presentation, etc. Labeled specific binding members can be used in any application of antibody-mediated labeling that includes (or may include) cells, particles, or analytes.

[0267] 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 polychromophore in the vicinity of its energy receiver. In some embodiments, the conjugate is described by chemical formula (II):

[0268]

[0269] Among them B 1 and B 2 Each is an independent BODIPY unit; each M 1 and each M 2 Each of the following is an independent π-conjugated comonomer; a, b, c, d, e, and f are each independently 0, 1, or 2, where b + e ≥ 1; n and m are independently 0 to 100,000, where n + m ≥ 1; and an L 2 The group is the terminal group (G) 1 ), and another L 2 The group is the specific binding member to which it is attached. In some embodiments, B 1 B 2 M 1 and M 2 At least one of them includes –L 1 -C 1 L 1 It is an optional connector, and C 1 It is a receptor chromophore.

[0270] Once the sample has been contacted with the polymer dye conjugate, the resulting labeled composition-contaminated sample can be measured for the presence of the polymer dye conjugate-target analyte binding complex using any convenient method. The polymer dye conjugate-target analyte binding complex is a binding complex that is generated when a specific binding member of the conjugate specifically binds to the target analyte (if present). Measurement of the labeled composition-contaminated sample may include detecting a fluorescence signal from the binding complex (if present). In some cases, the measurement includes a separation step in which the target analyte (if present) is separated from the sample. A variety of methods can be used to separate the target analyte from the sample, for example, by immobilization on a support. Assays of interest include, but are not limited to, any convenient method and assay form where the specific binding member pair of interest is, for example, the use of avidin-biotin or hapten-anti-hapten antibody. Methods and assay forms of interest adaptable to the subject composition include, but are not limited to, flow cytometry, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blotting, magnetic cell separation assay, and fluorescent dye purification chromatography.

[0271] In some embodiments, the method further includes contacting the sample with a second specifically binding member that specifically binds to the target analyte. In some instances, the second specifically binding member is bound by a support. Any convenient support can be used to immobilize components of the main method (e.g., the second specifically binding member). In some instances, the support is a particle, such as a magnetic particle. In some instances, the second specifically binding member and the polymer dye conjugate form a sandwich complex, which can be separated and detected (if present) using any convenient method. In some embodiments, the method further includes analyzing the polymer dye conjugate-target analyte binding complex, i.e., the fluorescently labeled target analyte, by flow cytometry. Determining the presence of the polymer dye conjugate-target analyte binding complex can provide assay results (e.g., qualitative or quantitative assay data) that can be used to assess the presence of the target analyte in the sample.

[0272] Any convenient support can be used in the subject method. Supports of interest include, but are not limited to: solid matrices, wherein the matrix can have various configurations, such as sheets, beads, or other structures, such as perforated plates; beads, polymers, particles, fibrous meshes, hydrogels, porous matrices, needles, microarray surfaces, chromatographic supports, etc. In some instances, the support is selected from particles, planar solid matrices, fibrous meshes, hydrogels, porous matrices, needles, microarray surfaces, and chromatographic supports. The support can be incorporated into systems that provide cell separation assisted by any convenient method, such as manually operated syringes, centrifuges, or automated liquid handling systems. In some cases, the support can be used in automated liquid handling systems for high-throughput cell separation, such as flow cytometers.

[0273] In some embodiments of this method, the separation step includes applying an external magnetic field to immobilize the magnetic particles. Any convenient magnet can be used as the source of the external magnetic field (e.g., a magnetic field gradient). In some cases, the external magnetic field is generated by a magnetic source, such as a permanent magnet or an electromagnet. In some cases, immobilizing the magnetic particles means that the magnetic particles accumulate near the surface closest to the source of the magnetic field gradient (i.e., the magnet).

[0274] Separation may additionally include one or more optional washing steps to remove unbound material of the sample from the support. Any convenient washing method can be used, such as washing the immobilized support with a biocompatible buffer that preserves the specific binding interactions between the polymer dye and the specific binding member. Separation from the support and optional washing of the unbound material of the sample provides rich clusters of target cells, in which unwanted cells and material can be removed.

[0275] In some embodiments, the method further includes detecting a labeled target. Detecting 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.

[0276] Methods for labeling target molecules are also provided. Thematic polymer dyes (including tandem dyes) can be used in a variety of labeling, separation, detection, and / or analytical methods. In some embodiments, the method includes contacting a target molecule with a polymer tandem dye to produce a labeled target molecule, wherein the polymer tandem dye comprises: a photosensitive polychromatophore containing a BODIPY unit (e.g., as described herein); and a conjugated tag. In some instances, the polymer dye is itself fluorescent. In some embodiments, the polymer dye is a polymer tandem dye. Thus, in some cases, the polymer dye additionally includes an acceptor chromophore covalently linked to the polychromatophore in the vicinity of its energy receiver. As used herein, the term "labeled target molecule" refers to a target molecule covalently linked to a thematic polychromatophore.

[0277] In some embodiments, the polymer dye is described by chemical formula (II):

[0278]

[0279] Among them: B 1 and B 2 Each is an independent BODIPY unit; each M 1 and each M 2 Each of the following is an independent π-conjugated comonomer; a, b, c, d, e, and f are each independently 0, 1, or 2, where b + e ≥ 1; n and m are independently 0 to 100,000, where n + m ≥ 1; and an L 2 The group is the terminal group (G) 1 ), and another L 2 The group is a conjugated tag. In some instances of formula (II), B 1 B 2 M 1 and M 2 At least one of them includes –L 1 -C 1 L 1 It is an optional connector, and C 1 It is a receptor chromophore.

[0280] As used herein, the term "conjugated tag" refers to a group comprising a chemically selective functional group (e.g., as described herein) that can be covalently linked to a compatible functional group of the target molecule upon optional activation and / or deprotection. Any convenient conjugated tag can be utilized in the subject polymer dyes to conjugate the dye with the target molecule of interest. In some embodiments, the conjugated tag comprises a terminal functional group selected from amino, carboxylic acids or derivatives thereof, thiols, hydroxyl, hydrazine, acylhydrazine, azide, alkyne, and protein-reactive groups (e.g., amino-reactive, thiol-reactive, hydroxyl-reactive, imidazole-reactive, or guanidinyl-reactive).

[0281] Any convenient methods and reagents can be adapted for subject labeling methods to covalently link conjugated tags to target molecules. Methods of interest for tagging targets include, but are not limited to, those described in Hermanson, Bioconjugate Techniques, Third Edition, Academic Press, 2013. The contact step can be performed in aqueous solution. In some instances, the conjugated tag comprises an amino functional group, and the target molecule comprises an activated ester functional group, such as an NHS ester or a sulfonated NHS ester, or vice versa. In some instances, the conjugated tag comprises a maleimide functional group, and the target molecule comprises a thiol functional group, or vice versa.

[0282] Any convenient target molecule can be selected for labeling using a topical approach. Target molecules of interest include, but are not limited to, nucleic acid molecules such as RNA, DNA, PNA, CNA, HNA, LNA, or ANA; proteins such as fusion proteins, modified proteins (e.g., 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” refers to all members of a target family and fragments thereof. Target proteins can be any protein of interest, such as therapeutic or diagnostic targets, including but not limited to: hormones, growth factors, receptors, enzymes, cytokines, osteogenic inducing factors, colony-stimulating factors, and immunoglobulins. The term “target protein” is intended to include recombinant and synthetic molecules that can be prepared or commercially available using any convenient recombinant expression method or synthetic method. In some embodiments, the target molecule is a specifically binding member (e.g., as described herein). In some instances, the specifically binding member is an antibody. In some instances, the specifically binding member is an antibody fragment or a binding derivative thereof. In some cases, the antibody fragment or its binding derivative is selected from Fab fragments, F(ab')2 fragments, scFv, biantibodies, and triantibodies.

[0283] In some cases, the method includes a separation step in which the labeled target molecule is separated from the reaction mixture (e.g., excess reagent or unlabeled target). A variety of methods can be used to separate the target from the sample, such as immobilization on a support, precipitation, chromatography, etc.

[0284] In some instances, the method further includes the detection and / or analysis of labeled target molecules. In some instances, the method further includes the fluorescence detection of labeled target molecules. By combining the subject method and the composition, any convenient method can be used to detect and / or analyze labeled target molecules. Methods available in the subject method for analyzing targets of interest include, but are not limited to, flow cytometry, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blotting, magnetic cell separation assay, and fluorescent dye purification chromatography. Detection methods of interest include, but are not limited to, fluorescence spectroscopy, nucleic acid sequencing, fluorescence in situ hybridization (FISH), protein mass spectrometry, flow cytometry, etc.

[0285] Detection can be achieved directly via reporter molecules or indirectly via secondary detection systems. The latter can be based on any or a combination of several different principles, including but not limited to antibody-labeled anti-species antibodies and other forms of immunological or non-immune bridging and signal amplification systems (e.g., biotin-streptavidin technology, protein-A and protein-G mediated technologies, or nucleic acid probes / anti-nucleic acid probes, etc.). The label used for direct or indirect detection can be any detectable reporter molecule. Suitable reporter molecules can be those known in the fields of immunocytochemistry, molecular biology, light, fluorescence and electron microscopy, cell immunophenotyping, cell sorting, flow cytometry, cell visualization, detection, enumeration and / or signal output quantification. Labels of interest include, but are not limited to, fluorophores, luminescent labels, metal complexes, radioisotopes, biotin, streptavidin, enzymes or other detection labels and combinations of labels (e.g., enzymes and luminescent substrates). Enzymes of interest and their substrates include alkaline phosphatase, horseradish peroxidase, β-galactosidase, and luciferase, etc. More than one antibody with specific and / or nonspecific properties can be labeled and used simultaneously or sequentially to enhance target detection, identification, and / or analysis. Labels of interest include, but are not limited to, FITC (fluorescein isothiocyanate), AMCA (7-amino-4-methylcoumarin-3-acetic acid), Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 350, DyLight350, phycoerythrin, allophycocyanin, and staining agents for nuclear detection (such as Hoechst 33342, LDS751, TO-PRO, and DAPI).

[0286] system

[0287] The invention further includes systems for use in practical subject methods and compositions. A sample analysis system may include a flow channel loaded with a sample and a labeled specific binding member. In some embodiments, the system is a flow cytometry system comprising: a flow cytometer including a flow path; a composition in the flow path, wherein the composition comprises: a sample; and a labeled specific binding member (e.g., as described herein). In some instances of the system, the labeled specific binding member comprises a light-gathering polychromatophore containing a BODIPY unit; and a specific binding member that specifically binds to a target analyte and is covalently linked to the polychromatophore. The polychromatophore may be a polymeric dye that is fluorescent itself. The polychromatophore may be a polymeric tandem dye. In some instances, the labeled specific binding member further comprises a receptor chromophore covalently linked to the polychromatophore in the vicinity of its energy receiver. In some embodiments, the labeled specific binding member is described by chemical formula (II):

[0288]

[0289] Among them: B 1 and B 2 Each is an independent BODIPY unit; each M 1 and each M 2 Each of the following is an independent π-conjugated comonomer; a, b, c, d, e, and f are each independently 0, 1, or 2, where b + e ≥ 1; n and m are independently 0 to 100,000, where n + m ≥ 1; and an L 2 The group is the terminal group (G) 1 ), and another L 2 The group is a conjugated tag. In some instances of formula (II), B 1 B 2 M 1 and M 2 At least one of them includes –L 1 -C 1 L 1 It is an optional connector, and C 1 It is a receptor chromophore.

[0290] In some embodiments of the system, the composition further includes a second specific binding member that is bound by the support and specifically binds the target analyte. In some cases, the support comprises magnetic particles. Thus, in certain instances, the system may also include a controllable external paramagnetic field configured to be applied to the measurement region of the flow channel.

[0291] Samples may include cells. In some cases, the sample is a biological sample containing cells. In some instances, the sample includes a labeled, specifically binding member that binds specifically to target cells. In some instances, the target analyte specifically bound by the specific binding member is a cell surface marker. In some cases, the cell surface marker is selected from cell receptors and cell surface antigens.

[0292] In some aspects, the system may also include a light source configured to direct light to a measurement region of the flow channel. The system may include a detector configured to receive a signal from the measurement region of the flow channel, wherein the signal is provided by the fluorescent composition. Optionally further, the sample analysis system may include one or more additional detectors and / or light sources for detecting one or more additional signals.

[0293] In some aspects, the system may additionally include a computer-based system configured to detect the presence of a fluorescence signal. "Computer-based system" refers to hardware devices, software devices, and data storage devices used for analyzing information of the present invention. The minimum hardware of the computer-based system of the present invention includes a central processing unit (CPU), input devices, output devices, and data storage devices. Those skilled in the art will readily understand that any of the currently available computer-based systems is suitable for use in the subject system. The data storage device may include any product containing: a record of the information of the present invention as described above, or a storage access device that can access such a product.

[0294] For the purpose of "recording" data, programmed information or other information on a computer-readable medium refers to the process of storing information using any such methods known in the art. Based on the means used to access the stored information, any convenient data storage structure can be chosen. Various data processing programs and formats can be used for storage, such as text processing files, database formats, etc.

[0295] The term "processor" refers to any combination of hardware and / or software that will perform the functions required to do so. For example, any processor herein can be a programmable digital microprocessor, as is available in the form of an electronic controller, mainframe, server, or personal computer (desktop or portable). Where the processor is programmable, suitable programming can be transferred to the processor from a remote location or pre-stored in a computer program product (such as a portable or fixed computer-readable storage medium, whether magnetic, optical, or solid-state based). For example, magnetic media or optical discs can carry the programming and can be read by a suitable reader connected to each processor at its respective base station.

[0296] In addition to sensor devices and signal processing modules, for example, as described above, the system of the present invention may include a number of other components, such as data output devices (e.g., monitors and / or speakers), data input devices (e.g., interface ports, keyboards, etc.), fluid handling components, power supplies, etc.

[0297] In some aspects, the system includes a flow cytometer. Flow cytometers of interest include, but are not limited to, those devices described in the following 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.

[0298] Other systems may be used in the practical subject method. In some aspects, the system may be a fluorometer or microscope loaded with a sample having a fluorescent composition of any of the embodiments discussed herein. The fluorometer or microscope may include a light source configured to direct light to a measurement region of a flow channel. The fluorometer or microscope may also include a detector configured to receive a signal from a measurement region of the flow channel, wherein the signal is provided by the fluorescent composition.

[0299] Reagent test kit

[0300] The invention further includes kits for use in practical subject methods and compositions. The compositions of the invention can be included as reagents in kits, as starting materials, or provided for use, for example, in the methods described above.

[0301] The kit may include a photosensitive polychromatic chromophore containing a BODIPY unit (e.g., as described above); and one or more components selected from polymer tandem dyes, fluorophores, specific binding members, specific binding member conjugates, support-bound specific binding members, cells, supports, biocompatible aqueous elution buffers, and instructions for use. In some embodiments of the kit, the polychromatic chromophore is covalently linked to a specific binding member. In some instances, the specific binding member is an antibody. In some instances, the specific binding member is an antibody fragment or a binding derivative thereof. In some cases, the antibody fragment or a binding derivative thereof is selected from Fab fragments, F(ab')2 fragments, scFv, biantibodies, and triantibodies. The polychromatic chromophore may be a polymeric dye that is fluorescent itself. The polychromatic chromophore may be a polymer tandem dye. In some cases, the polychromatic chromophore additionally includes a receptor chromophore covalently linked to the polychromatic chromophore in the vicinity of its energy receiver.

[0302] In some embodiments, the kit can be used to evaluate samples for the presence of a target analyte, such as an intracellular target. Thus, in some instances, the kit includes one or more components suitable for lysing cells. One or more additional components of the kit may be provided in separate containers (e.g., separate tubes, bottles, or individual wells in a multi-well strip or plate).

[0303] In some respects, the kit additionally includes reagents for performing flow cytometry assays. Reagents of interest include, but are not limited to, buffers for remodeling and dilution, buffers for contacting cell samples with multiple chromophores, wash buffers, control cells, control beads, fluorescent beads for flow cytometry calibration, and combinations thereof. The kit may also include one or more cell fixation reagents, such as paraformaldehyde, glutaraldehyde, methanol, acetone, formalin, or any combination thereof or buffers thereof. Additionally, the kit may include cell permeabilization reagents, such as methanol, acetone, or detergents (e.g., Triton, NP-40, saponins, Tween 20, digitalis saponins, leucoperm), or any combination thereof or buffers thereof. Other protein transport inhibitors, cell fixation reagents, and cell permeabilization reagents familiar to those skilled in the art are within the scope of this subject kit.

[0304] The kit composition can be provided in a liquid composition, such as any suitable buffer. Alternatively, the kit composition can be provided as a dry composition (e.g., lyophilized), and the kit may optionally include one or more buffers for reconstituted the dry composition. In some aspects, the kit may include aliquots of the composition provided in separate containers (e.g., separate tubes, bottles, or separate wells in a multi-well strip or plate).

[0305] In addition, one or more components may be incorporated into a single container, such as a glass or plastic vial, tube, or bottle. In some instances, the kit may additionally include a container (e.g., a box, bag, insulated container, bottle, tube, etc.) containing all components (and their individual containers). The kit may also additionally include packaging separate from or attached to the kit container and printed with information about the kit, its components, and / or instructions for use.

[0306] In addition to the components described above, the subject kit may also include instructions for practicing these subject methods. These instructions may be in various forms, one or more of these forms, within the subject kit. One form of these instructions is as information printed on a suitable medium or substrate (e.g., one or more sheets of paper on which information is printed), printed in the kit packaging, printed in packaging inserts, etc. Another form is as computer-readable media on which information is already recorded, such as disks, CDs, DVDs, portable flash drives, etc. Yet another form is as a URL where the information can be accessed remotely via the Internet. Any convenient means may be included in these kits.

[0307] practicality

[0308] The compositions, methods, and systems described herein can be used in a variety of applications, including diagnostic and research applications, where the labeled detection and / or analysis of targets of interest is desirable. Such applications include methodologies such as flow cytometry, microscopy, immunoassays (e.g., competitive or non-competitive), evaluation of free analytes, evaluation of receptor-bound ligands, etc. The compositions, systems, and methods described herein can be used to analyze any of a variety of samples, including but not limited to biofluids, cell culture samples, and tissue samples. In some aspects, the compositions, systems, and methods described herein can be used for methods that use fluorescent labeling (in areas such as fluorescently activated cell sorting or analysis, immunoassays, immunostaining, etc.) to detect analytes in a sample (if present). In some instances, these compositions and methods can be used for applications where the evaluation of a sample in response to the presence of a target analyte is of interest.

[0309] In some cases, these methods and compositions can be used for any assay of interest in detecting and / or analyzing targets from a sample, including but not limited to flow cytometry, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assay, and fluorescent dye purification chromatography. In some instances, these methods and compositions can be used for any application of interest in fluorescently labeled target molecules. The subject compositions can be adapted for any convenient application that may use specific binding member pairs (such as biotin-streptavidin and hapten-anti-hapten antibody).

[0310] The following examples are provided by way of explanation rather than by way of limitation.

[0311] experiment

[0312] Example 1

[0313] Based on the core structure 1 described below, a series of tandem dyes were prepared, including a series of linked fluorophores, DY633, DY651, DY682 and DY752.

[0314]

[0315] Figure 1 Absorption and emission of polymeric tandem dyes based on a core structure 1 with multiple acceptor dyes (e.g., dyes DY 633, DY 651, DY 682, and DY 752) attached to internal linker sites are shown. These structures do not have specific binding members attached.

[0316] Based on the core structure 1 described above, a second series of tandem dyes were prepared, including a series of acceptor dye fluorophores linked at internal linker sites, namely DY 633, DY 654, DY 682, DY 754 and DY 752. Figure 2 The fluorescence of these polymer tandem dyes based on structure 1, which has multiple dye molecules attached to internal linker sites, is shown. The absorbance of all solutions is 0.04 OD. Note that the polymers with attached acceptor chromophores exhibit significantly higher emission intensity compared to the individual polymers. These polymers do not have attachment-specific binding members.

[0317] The quantum yield of the tandem pair was brighter than that of the core polymer alone. For structure 1, a series of acceptor chromophores were attached, and the resulting tandem pair was compared spectroscopically with a polymer without a second chromophore. All prepared solutions exhibited the same optical density for an excitation wavelength of 562 nm. Figure 2 As can be seen, the peak height and peak area of ​​the tandem pair (peaks 2-6) are both greater than those emitted from the underlying polymer (peak 1). In this way, extensive prototyping of increasingly bright underlying polymers is unnecessary, and the brightness of the tandem pair is limited, mainly due to the quantum yield of the acceptor.

[0318] Although there are additional terms, the disclosures presented herein are further limited by the following terms:

[0319] 1. A polymer tandem dye comprising: a photosensitive polychromatophore containing a BODIPY unit; and an acceptor chromatophore covalently linked to the polychromatophore in the vicinity of its energy receiver.

[0320] 2. The dye as described in Clause 1, wherein the dye is water-soluble.

[0321] 3. The dye according to any one of the preceding clauses, wherein the dye has narrow-band spectral characteristics.

[0322] 4. The dye according to any one of the preceding clauses, wherein the dye has a low-energy absorption band with a bandwidth of 100 nm or less.

[0323] 5. The dye according to any one of the preceding clauses, wherein the dye has a density of 5x10 5 M -1 cm -1 Or a larger molar extinction coefficient.

[0324] 6. The dye according to any one of the preceding clauses, wherein the ratio of the acceptor chromophore to the polychromatic repeating unit of the dye is in the range of 1:40 to 1:4.

[0325] 7. The dye according to any one of the preceding clauses, wherein the acceptor chromophore is a fluorophore.

[0326] 8. The dye according to any one of clauses 1-6, wherein the receptor chromophore is a quencher.

[0327] 9. The dye according to Clause 7, wherein the receptor chromophore emits 1.5 times or more when excited by the multi-chromophore compared to when the receptor chromophore is directly excited by incident light.

[0328] 10. The dye as described in Clause 8, wherein the dye has a quantum yield of 0.05 or greater.

[0329] 11. The dye according to any one of the preceding clauses, wherein the polychromatin comprises a conjugated segment containing BODIPY as described by chemical formula (I):

[0330]

[0331] in:

[0332] B stands for BODIPY unit;

[0333] M is a π-conjugated comonomer;

[0334] Each L is independently selected from the end group, π-conjugated segment, linker, and the specific binding member to which it is attached; and

[0335] n is an integer from 1 to 100,000.

[0336] 12. The dye according to any one of the preceding clauses, wherein the BODIPY unit is described by the following structure:

[0337]

[0338] in:

[0339] R 1 R 2R 3 and R 4 Each is independently selected from H, alkyl, and substituted alkyl;

[0340] R 5 Selected from alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, wherein R 5 Optionally replaced by water-solubilizing groups; and

[0341] Each R is selected from F, OH, H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkynyl and substituted alkynyl.

[0342] 13. The dye according to Clause 11, wherein M is selected from fluorene comonomers, phenylene-vinylene comonomers, phenylene-ethynylene comonomers, carbazole comonomers, C2-C 12 Alkyne copolymer monomers, arylene-ethynylene copolymer monomers, heteroarylene-ethynylene copolymer monomers, arylene copolymer monomers and heteroarylene copolymer monomers.

[0343] 14. The dye according to any one of the preceding clauses, wherein the polychromatic group is described by chemical formula (II):

[0344]

[0345] in:

[0346] B 1 and B 2 Each is an independent BODIPY unit;

[0347] Each M 1 and each M 2 It is an independent π-conjugated comonomer;

[0348] a, b, c, d, e, and f are each independently 0, 1, or 2, where b + e ≥ 1;

[0349] n and m are independently 0 or integers from 1 to 100,000, where n+m≥1;

[0350] p is an integer from 1 to 100,000; and

[0351] Each L 2 Independently selected from end groups, π-conjugated segments, linkers, and the specific binding members to which they are attached.

[0352] 15. The dye as described in Clause 14, wherein:

[0353] When b is 0, a and c are both 1;

[0354] When e is 0, d and f are both 1;

[0355] When b is 1, a + c ≥ 1; and

[0356] When e is 1, d+f≥1.

[0357] 16. The dye according to any one of clauses 14-15, wherein B 1 B 2 M 1 and M 2 At least one of them includes –L 1 -C 1 L 1 It is an optional connector, and C 1 It is a receptor chromophore.

[0358] 17. The dye according to any one of clauses 14-16, wherein the dye is described by chemical formula (III):

[0359]

[0360] Each L 1 It is an optional connector, and each C 1 It is a receptor chromophore.

[0361] 18. The dye as described in Clause 17, wherein each M 1 and each M 2 Independently, it is a fluorene comonomer that is optionally substituted with a water-solubilizing group.

[0362] 19. The dye according to any one of clauses 17-18, wherein the ratio of n to m is in the range of 20:1 to 3:1.

[0363] 20. The dye according to any one of clauses 14-16, wherein the dye is described by chemical formula (IV):

[0364]

[0365] Each L 1 It is an optional connector, and each C 1 It is a receptor chromophore.

[0366] 21. The dye as described in Clause 20, wherein each M 1 Independently, it is a fluorene comonomer that is optionally substituted with a water-solubilizing group.

[0367] 22. The dye according to any one of clauses 20-21, wherein the ratio of n to m is in the range of 20:1 to 3:1.

[0368] 23. The dye according to any one of clauses 14-16, wherein the dye is described by chemical formula (V):

[0369]

[0370] Each L 2 It is an optional connector, and each C 1 It is a receptor chromophore.

[0371] 24. The dye as described in Clause 23, wherein each M 2 It is a fluorene comonomer that is optionally replaced by a water-solubilizing group.

[0372] 25. The dye according to any one of clauses 23-24, wherein the ratio of n to m is in the range of 20:1 to 3:1.

[0373] 26. The dye according to any one of clauses 14-16, wherein the dye is described by chemical formula (VI):

[0374]

[0375] Each L 1 It is an optional connector, and each C 1 It is a receptor chromophore.

[0376] 27. The dye as described in Clause 26, wherein each M 1 It is a carbazole comonomer, and each M 2 It is a fluorene copolymer monomer.

[0377] 28. The dye according to any one of clauses 26-27, wherein the ratio of n to m is in the range of 20:1 to 3:1.

[0378] 29. The dye according to any one of clauses 14-28, wherein B 1 and B 2 Each is independently described by the following structure:

[0379]

[0380] Where R 6 It is an aryl or heteroaryl group that is optionally substituted with one or more water-solubilizing groups.

[0381] 30. The dye according to any one of clauses 14-29, wherein B 1 and B 2 Each is independently described by the following structure:

[0382]

[0383] in:

[0384] Each R' is independently selected from H and alkyl groups; and

[0385] p is 0 or an integer from 1 to 12.

[0386] 31. The dye according to any one of clauses 14-30, wherein at least one L 2 Yes -L 3 -Z, where L 3 It is a connector, and Z is a specific binding member.

[0387] 32. The dye according to any one of clauses 14-31, wherein at least one L 2 Described by the following structure:

[0388]

[0389] in:

[0390] q is an integer from 1 to 12; and

[0391] Z is a specific binding member.

[0392] 33. The dye according to any one of clauses 31-32, wherein Z is a biomolecule.

[0393] 34. The dye according to any one of clauses 31-33, wherein Z is an antibody.

[0394] 35. The dye according to any one of clauses 31-33, wherein Z is an antibody fragment or a binding derivative thereof.

[0395] 36. The dye according to Clause 35, wherein the antibody fragment or its binding derivative is selected from Fab fragments, F(ab')2 fragments, scFv, biantibodies, and triantibodies.

[0396] 37. The dye according to any one of clauses 16-36, wherein: L 1 It is an alkyl, substituted alkyl, alkyl-amide, alkyl-amide-alkyl, or PEG moiety; and C 1 It is selected from cyanine dyes, xanthan dyes, coumarin dyes, thiazine dyes and acridine dyes.

[0397] 38. The dye according to any one of the preceding clauses, wherein the receptor chromophore is selected from Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Alexa488, Alexa647 and Alexa700.

[0398] 39. The dye according to any one of clauses 14-38, wherein each M1 and M 2 Each is independently selected from fluorene comonomers, carbazole comonomers, vinylene comonomers, arylene-ethynylene comonomers, and arylene-vinylene comonomers, which are optionally substituted with water-soluble groups.

[0399] 40. The dye according to any one of clauses 14-39, wherein each M 1 and M 2 Each is independently selected from phenylene-ethynylene comonomers and ethynylene comonomers that are optionally substituted with water-soluble groups.

[0400] 41. The dye according to any one of clauses 14-39, wherein each M 1 and M 2 Each is independently described by the following structure:

[0401]

[0402] in:

[0403] Each R 7 Independently selected from alkyl, substituted alkyl, aralkyl, substituted aralkyl, PEG moiety and -L 1 -C 1 .

[0404] 42. The dye according to any one of clauses 14-39, wherein:

[0405] M 1 Described by the following structure:

[0406]

[0407] Each R 8 It is a substituted aralkyl group including a water-solubilizing group; and

[0408] M 2 Described by the following structure:

[0409]

[0410] Where R 9 It is a substituted alkyl group including a water-solubilizing group, and R 10 Yes –L 1 -C 1 .

[0411] 43. A labeled, specifically binding member, comprising:

[0412] Polymer tandem dye, the polymer tandem dye comprising:

[0413] A light-collecting multi-chromophore containing a BODIPY unit;

[0414] The receptor chromophore is covalently linked to the polychromophore near its energy receiver; and

[0415] A specific binding member covalently linked to the polychromatin.

[0416] 44. The labeled specific binding member as described in Clause 43, wherein the light-collecting polychromatic group is water-soluble.

[0417] 45. A labeled specific binding member according to any one of clauses 43-44, wherein the dye has narrow-band spectral characteristics.

[0418] 46. ​​A labeled specific binding member according to any one of clauses 43-45, wherein the dye has a low-energy absorption band with a bandwidth of 100 nm or less.

[0419] 47. The labeled specific binding member according to any one of clauses 43-46, wherein the dye has a 5x10 5 M -1 cm -1 Or a larger molar extinction coefficient.

[0420] 48. The labeled specific binding member according to any one of clauses 43-47, wherein the ratio of the receptor chromophore to the polychromophore repeating unit of the dye is in the range of 1:40 to 1:4.

[0421] 49. The labeled specific binding member according to any one of clauses 43-48, wherein the receptor chromophore is a fluorophore.

[0422] 50. The labeled specific binding member as described in Clause 49, wherein the receptor chromophore emits 1.5 times or more when excited by the multi-chromophore compared to when the receptor chromophore is directly excited by incident light.

[0423] 51. The labeled specific binding member as described in Clause 49, wherein the dye has a quantum yield of 0.05 or greater.

[0424] 52. The labeled specific binding member according to any one of clauses 43-51, wherein the polychromatin comprises a conjugated segment containing BODIPY as described by chemical formula (I):

[0425]

[0426]

[0427] in:

[0428] B stands for BODIPY unit;

[0429] M is a π-conjugated comonomer;

[0430] Each L is independently selected from the end group, π-conjugated segment, linker, and the specific binding member to which it is attached; and

[0431] n is an integer from 1 to 100,000.

[0432] 53. The labeled specific binding member according to any one of clauses 43-52, wherein the BODIPY unit is described by the following structure:

[0433]

[0434] in:

[0435] R 1 R 2 R 3 and R 4 Each is independently selected from H, alkyl, and substituted alkyl;

[0436] R 5 Selected from alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl, wherein R 5 Optionally replaced by water-solubilizing groups; and

[0437] Each R is selected from F, OH, H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkynyl and substituted alkynyl.

[0438] 54. The labeled specific binding member according to any one of clauses 52-53, wherein M is selected from fluorene comonomers, phenylene-vinylene comonomers, phenylene-ethynylene comonomers, carbazole comonomers, C2-C 12 Alkyne copolymer monomers, arylene-ethynylene copolymer monomers, heteroarylene-ethynylene copolymer monomers, arylene copolymer monomers and heteroarylene copolymer monomers.

[0439] 55. The labeled specific binding member according to any one of clauses 43-54, wherein the polychromatin is described by chemical formula (II):

[0440]

[0441]

[0442] in:

[0443] B 1 and B2 Each is an independent BODIPY unit;

[0444] Each M 1 and each M 2 It is an independent π-conjugated comonomer;

[0445] a, b, c, d, e, and f are each independently 0, 1, or 2, where b + e ≥ 1;

[0446] n and m are independently from 0 to 100,000, where n + m ≥ 1; and

[0447] An L 2 The group is the terminal group (G) 1 ), and another L 2 A group is a specific binding member to which it is attached.

[0448] 56. The labeled specific binding member as described in Clause 55, wherein B 1 B 2 M 1 and M 2 At least one of them includes –L 1 -C 1 L 1 It is an optional connector, and C 1 It is a receptor chromophore.

[0449] 57. A labeled specific binding member according to any one of clauses 43-56, wherein the specific binding member is an antibody.

[0450] 58. The labeled specific binding member according to any one of clauses 43-56, wherein the specific binding member is an antibody fragment or a binding derivative thereof.

[0451] 59. The labeled specific binding member as described in Clause 58, wherein the antibody fragment or its binding derivative is selected from Fab fragments, F(ab')2 fragments, scFv, biantibodies, and triantibodies.

[0452] 60. The labeled specific binding member according to any one of clauses 43-59, wherein the receptor chromophore is selected from cyanine dyes, xanthan dyes, coumarin dyes, thiazine dyes, and acridine dyes.

[0453] 61. The labeled specific binding member according to any one of clauses 43-50, wherein the receptor chromophore is selected from Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Alexa488, Alexa647 and Alexa700.

[0454] 62. A method for evaluating a sample in response to the presence of a target analyte, the method comprising:

[0455] (a) Contacting the sample with a polymer tandem dye conjugate that specifically binds the target analyte to produce a labeled composition-contacted sample, wherein the polymer tandem dye conjugate comprises:

[0456] (i) A light-collecting polychromatic group containing a BODIPY unit;

[0457] (ii) the receptor chromophore covalently linked to multiple chromophores near its energy receptor; and

[0458] (iii) Specifically binding members; and

[0459] (b) To assess the presence of the target analyte in a sample that has been in contact with the labeled composition, in order to determine the presence of the target analyte in the sample.

[0460] 63. The method according to Clause 62, wherein the polychromatin is described by chemical formula (II):

[0461]

[0462] in:

[0463] B 1 and B 2 Each is an independent BODIPY unit;

[0464] Each M 1 and each M 2 It is an independent π-conjugated comonomer;

[0465] a, b, c, d, e, and f are each independently 0, 1, or 2, where b + e ≥ 1;

[0466] n and m are independently from 0 to 100,000, where n + m ≥ 1; and

[0467] An L 2 The group is the terminal group (G) 1 ), and another L 2 A group is a specific binding member to which it is attached.

[0468] 64. The method described according to Clause 63, wherein B 1 B 2 M 1 and M 2 At least one of them includes –L 1 -C 1 L 1 It is an optional connector, and C1 It is a receptor chromophore.

[0469] 65. The method according to any one of clauses 62-64, the method further comprising contacting the sample with a second specific binding member that binds to the support and specifically binds the target analyte.

[0470] 66. The method according to Clause 65, wherein the support comprises magnetic particles.

[0471] 67. The method according to any one of clauses 62-66, wherein the target analyte is cell-related.

[0472] 68. The method according to Clause 67, wherein the target analyte is a cell surface marker of the cell.

[0473] 69. The method according to Clause 68, wherein the cell surface marker is selected from cell receptors and cell surface antigens.

[0474] 70. The method according to Clause 67, wherein the target analyte is an intracellular target, and the method further includes lysing the cell.

[0475] 71. The method according to any one of clauses 62-69, wherein the method further comprises analyzing the fluorescently labeled target analyte by flow cytometry.

[0476] 72. A method for labeling a target molecule, the method comprising:

[0477] The target molecule is contacted with a polymer tandem dye to generate a labeled target molecule, wherein the polymer tandem dye comprises:

[0478] A light-collecting multi-chromophore containing a BODIPY unit;

[0479] The receptor chromophore is covalently linked to the polychromophore near its energy receiver; and

[0480] Conjugate tags.

[0481] 73. The method according to Clause 72, the method further comprising fluorescence detection of the labeled target molecule.

[0482] 74. The method according to any one of clauses 72-73, wherein the polychromatin is described by chemical formula (II):

[0483]

[0484] in:

[0485] B 1 and B 2 Each is an independent BODIPY unit;

[0486] Each M 1 and each M 2 It is an independent π-conjugated comonomer;

[0487] a, b, c, d, e, and f are each independently 0, 1, or 2, where b + e ≥ 1;

[0488] n and m are independently from 0 to 100,000, where n + m ≥ 1; and

[0489] An L 2 The group is the terminal group (G) 1 ), and another L 2 The group is the conjugated tag.

[0490] 75. The method described in Clause 74, wherein B 1 B 2 M 1 and M 2 At least one of them includes –L 1 -C 1 L 1 It is an optional connector, and C 1 It is a receptor chromophore.

[0491] 76. The method according to any one of clauses 72-75, wherein the conjugated tag comprises a terminal functional group selected from amino, thiol, hydroxyl, hydrazine, acylhydrazine, azide, alkyne and protein reactive groups.

[0492] 77. The method according to any one of clauses 72-76, wherein the target molecule is a specifically binding member.

[0493] 78. The method according to Clause 77, wherein the specific binding member is an antibody.

[0494] 79. The method according to Clause 77, wherein the specific binding member is an antibody fragment or a binding derivative thereof.

[0495] 80. The method according to Clause 79, wherein the antibody fragment or its binding derivative is selected from Fab fragments, F(ab')2 fragments, scFv, biantibodies, and triantibodies.

[0496] 81. A flow cytometry system, comprising:

[0497] A flow cytometer, the flow cytometer including a flow path;

[0498] The composition in the flow path, wherein the composition comprises:

[0499] Samples; and

[0500] The labeled specific binding member includes:

[0501] A light-collecting multi-chromophore containing a BODIPY unit;

[0502] The receptor chromophore is covalently linked to the polychromophore near its energy receiver; and

[0503] A specific binding member that specifically binds to the target analyte and is covalently linked to the polychromatin.

[0504] 82. The system according to Clause 81, wherein the polychromatin is described by chemical formula (II):

[0505]

[0506] in:

[0507] B 1 and B 2 Each is an independent BODIPY unit;

[0508] Each M 1 and each M 2 It is an independent π-conjugated comonomer;

[0509] a, b, c, d, e, and f are each independently 0, 1, or 2, where b + e ≥ 1;

[0510] n and m are independently from 0 to 100,000, where n+m≥1;

[0511] An L 2 The group is the terminal group (G) 1 ), and another L 2 The group is a connector that links the specific binding member.

[0512] 83. The system as described in Clause 82, wherein B 1 B 2 M 1 and M 2 At least one of them includes –L 1 -C 1 L 1 It is an optional connector, and C 1 It is a receptor chromophore.

[0513] 84. The system according to any one of clauses 81-83, wherein the composition further comprises a second specific binding member that is bound by the support and specifically binds the target analyte.

[0514] 85. The system according to Clause 84, wherein the support comprises magnetic particles.

[0515] 86. The system according to any one of clauses 81-85, wherein the sample comprises cells.

[0516] 87. The system according to Clause 86, wherein the target analyte is a cell surface marker of the cell.

[0517] 88. The system according to Clause 87, wherein the cell surface marker is selected from cell receptors and cell surface antigens.

[0518] 89. A reagent kit comprising:

[0519] Polymer tandem dye, the polymer tandem dye comprising:

[0520] A light-collecting multi-chromophore containing a BODIPY unit;

[0521] The receptor chromophore is covalently linked to the polychromophore near its energy receiver; and

[0522] It is selected from one or more components of polymer dyes, fluorophores, specific binding members, specific binding member conjugates, cells, supports, biocompatible aqueous elution buffers, and instructions for use.

[0523] 90. The kit according to Clause 89, wherein the polychromatin is covalently linked to a specific binding member.

[0524] 91. The kit according to Clause 90, wherein the specific binding member is an antibody.

[0525] 92. The kit according to Clause 90, wherein the specific binding member is an antibody fragment or a binding derivative thereof.

[0526] 93. The kit according to Clause 92, wherein the antibody fragment or its binding derivative is selected from Fab fragments, F(ab')2 fragments, scFv, biantibodies, and triantibodies.

[0527] Although the invention has been described in considerable detail by way of illustration and example for the purpose of clarity, it will be readily apparent to those skilled in the art, based on the teachings of the invention, that certain changes and modifications may be made therein without departing from the spirit or scope of the appended claims.

[0528] Therefore, the foregoing only illustrates the principles of the invention. It should be understood that those skilled in the art will be able to design different arrangements, which, while not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all instances and conditional language described herein are primarily intended to help the reader understand the principles and concepts of the invention contributed by the inventors to advance the field, and are to be considered as not being limited to these specific instances and conditions. Moreover, all statements herein referring to the principles, aspects, and embodiments of the invention, together with their specific examples, are intended to cover both their structural and functional equivalents. Additionally, such equivalents are contemplated to include both currently known equivalents and equivalents to be developed in the future, i.e., any element developed that performs the same function regardless of structure. Therefore, the scope of the invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied below.

Claims

1. A polymer tandem dye, described by chemical formula (III), (IV), (V) or (VI): in, Each B 1 And each B 2 Independently described by the following structure in: R 1 R 2 R 3 and R 4 Each is independently selected from H and alkyl groups; R 5 Choose from the group consisting of: alkyl, aryl, and heteroaryl. Each R is selected from the group consisting of the following: F, OH, H, alkyl, aryl, heteroaryl, alkoxy, and alkynyl. Each L 1 It is an optional group that connects two groups and has a skeleton of 100 atoms or less in length; Each C 1 It is capable of emitting light from sources containing B 1 and / or B 2 The energy received by the structure can also be dissipated as heat by the group; each L 2 It is a group that connects two groups and has a skeleton of 100 atoms or less in length; Each M 1 and each M 2 It is an independent π-conjugated comonomer; n and m are independent integers from 1 to 100,000; and p is an integer from 1 to 100,000.

2. The dye according to claim 1, wherein the polymer tandem dye is described by chemical formula (III).

3. The dye according to claim 1, wherein the polymer tandem dye is described by chemical formula (IV).

4. The dye according to claim 1, wherein the polymer tandem dye is described by chemical formula (V).

5. The dye according to claim 1, wherein the polymer tandem dye is described by chemical formula (VI).

6. The dye according to any one of claims 1-5, wherein B 1 and B 2 Each is independently described by the following structure: Where R 6 It is aryl or heteroaryl.

7. The dye according to any one of claims 1-5, wherein at least one L 2 Described by the following structure: Where q is an integer from 1 to 12; Z is a specific binding member.

8. The dye according to any one of claims 1-5, wherein: L 1 It is an alkyl, alkyl-amide, or alkyl-amide-alkyl moiety; and C 1 Selected from the following group, which consists of: cyanide dyes, xanthan dyes, coumarin dyes, thiazine dyes and acridine dyes.

9. The dye according to any one of claims 1-5, wherein: Each M 1 and M 2 Each is independently selected from fluorene comonomers, carbazole comonomers, vinylene comonomers, aryl-ethynylene comonomers, and aryl-vinylene comonomers.

10. The dye according to claim 9, wherein: Each M 1 and M 2 Each is an independent fluorene comonomer.

11. The dye according to claim 1, wherein [B 1 -M 2 ] n -[M 1 (L 1 )-M 2 ] m The group has the following structure: in, *For L 2 The points of connection, and Where, ** is related to C 1 The points that are connected.

12. The dye according to claim 11, wherein: C 1 Choose the group consisting of DY 633, DY 651, DY 682 and DY 752.

13. A labeled specific binding member, the labeled specific binding member comprising: Polymer tandem dyes according to any one of claims 1-12; as well as A specific binding member covalently linked to the polymer tandem dye, which specifically binds to a particular analyte or a second member of a specific binding pair.

14. A method for evaluating a sample in response to the presence of a target analyte, the method comprising: (a) The sample is contacted with a polymer tandem dye conjugate that specifically binds the target analyte to produce a labeled composition-contacted sample, wherein the polymer tandem dye conjugate includes the labeled specifically binding member as described in claim 13; and (b) To assess the presence of the target analyte in a sample that has been in contact with the labeled composition, in order to determine the presence of the target analyte in the sample.

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