Photoactive macromolecules and uses thereof

Abstract

To provide water soluble photoactive macromolecular complexes and methods for detecting an analyte in a sample by using a binding agent conjugated to a water soluble photoactive macromolecule.SOLUTION: The present invention provides water soluble photoactive macromolecular complexes and methods for detecting an analyte in a sample by using a binding agent conjugated to a water soluble photoactive macromolecule. In one embodiment, the present invention relates to complexes and methods for detecting an analyte in a sample.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 323,444, filed April 15, 2016, the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to a conjugate and a method for detecting an analyte in a sample. [Background technology]

[0003] Water-soluble fluorescent polymers can be used in a variety of biological applications by generating signals that can be monitored in real time, providing a simple and rapid method for detecting biological targets and events.

[0004] The brightness of a dye is the overall contribution from the extinction coefficient (ε, a measure of the amount of light absorbed at a particular wavelength) and the fluorescence quantum yield (Φ, a measure of the light emitted in the form of radiation from the singlet excited state). Many of the reported organic purple dyes, such as coumarins, BODIPYs, cyanines, and squaraines, are single molecules and have fluorescence luminances ranging from 10,000 to 70,000 M at 405 nm. -1 cm -1 They exhibit relatively small extinction coefficients in the range of 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.79, 0.79, 0.79, 0.79, 0.

[0005] However, many of the polymer dyes reported to date are highly hydrophobic and are used in materials applications such as light-emitting diodes and solar cells. Therefore, many polymer dyes are not useful under aqueous conditions due to poor solubility, brightness, and spectral breadth. Only a few reports have addressed water-soluble fluorescent polymers for biological applications that are excitable with 405 nm and 355 nm lasers. Therefore, the identification of novel polymer cores is needed to expand the collection of water-soluble polymer dyes for biological applications, including those for analyte detection.

[0006] The present invention addresses these and other disadvantages of prior art conjugates and methods for detecting analytes in a sample. Summary of the Invention [Means for solving the problem]

[0007] The present invention generally provides novel water-soluble fluorescent polymers and methods for detecting analytes in a sample using a conjugate comprising a fluorescent polymer conjugated to a binding agent.

[0008] In a first embodiment, the present invention provides a water-soluble fluorescent polymer having the structure of Formula I, [ka] During the ceremony, each X is independently selected from the group consisting of C and Si; Each Y is a bond, CR 1 R 2 , and SiR 1 R 2 are independently selected from the group consisting of When Y is a bond, X is directly attached to both rings; Each R 1are polyethylene glycol (PEG), ammonium alkyl salts, ammonium alkyloxy salts, ammonium oligoether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligoether salts, sulfonamide oligoethers, and [ka] are independently selected from the group consisting of Each R 2 is H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, and [ka] are independently selected from the group consisting of Each R 3 is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and PEG; each Z is independently selected from the group consisting of C, O, and N; Each Q is a bond, NH, or NR 4 and CH2, Each M is an electron-rich linker unit that can independently modify the polymer bandgap and is uniformly or randomly distributed along the polymer backbone, and [ka] are each independently selected from the group consisting of: During the ceremony, Each R4 is a non-ionic side chain capable of imparting solubility in water of greater than 10 mg / mL, and is selected from halogen, hydroxyl, C1-C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, C3-C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 Alkoxy, C2-C 18 (Hetero)aryloxy, C2-C 18 (hetero)arylamino, each x' is independently an integer from 0 to 20, and each y' is independently an integer from 0 to 50 (CH2) x’ (OCH2-CH2) y’ OCH3 and C2~C 18 (hetero)aryl groups; Each optional linker, L, is an aryl or heteroaryl group uniformly or randomly distributed along the polymer backbone and can be linked to another substrate, an acceptor dye, substituted with one or more pendant chains terminated with a functional group selected from the group consisting of amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and protected groups thereof, for conjugation to a molecule or binding agent; each G 1 and G 2 is hydrogen, halogen, alkyne, optionally substituted aryl, any optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, boronate-substituted aryl, boronate ester-substituted aryl, boronate ester, boronic acid, optionally substituted dihydrophenanthrene (DHP), optionally substituted fluorene, aryl or heteroaryl substituted with one or more pendant chains terminated with a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and protected groups thereof for conjugation to a substrate or a binder; a, c, and d define the mole % of each unit in the structure, and each of these units can be uniformly or irregularly repeated, a is 10-100% mole %, c is 0-90% mole %, and each d is 0-25% mole %; each b is independently 0 or 1; m is an integer from 1 to about 10,000; Each n is independently an integer from 1 to 20, providing a water-soluble fluorescent polymer.

[0009] In some cases, the polymer has a structure of Formula II [ka] .

[0010] In some cases, the polymer has a structure of Formula III: [ka] In the formula, each f is independently an integer of 0 to 50, and each R 5 H, C1~C 12 Alkyl, C2~C 12 Alkenes, C2-C12 Alkynes, C3-C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 Alkoxy, C2-C 18 (Hetero)aryloxy, C2-C 18 (Hetero)arylamino and C1-C 12 alkoxy is independently selected from the group consisting of:

[0011] In some cases, the polymer has a structure of Formula IV [ka] .

[0012] In some cases, the polymer has a structure of Formula V [ka] .

[0013] In some cases, the polymer is a copolymer and has the structure of Formula VI: [ka] In the formula, g and a are both mol % of 10 to 100%.

[0014] In some cases, the polymer is a copolymer and has the structure of Formula VII: [ka] In the formula, each g and a are both 10 to 100% by mole, each f is independently an integer of 0 to 50, and each R 5 H, C1~C 12 Alkyl, C2-C 12 Alkenes, C2-C 12Alkynes, C3-C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 Alkoxy, C2-C 18 (Hetero)aryloxy, C2-C 18 (Hetero)arylamino and C1-C 12 alkoxy is independently selected from the group consisting of:

[0015] In some cases, the polymer is a copolymer having the structure of Formula VIII [ka] .

[0016] In some cases, the polymer is a copolymer and has the structure of Formula IX [ka] .

[0017] In some embodiments, L is [ka] are each independently selected from the group consisting of: During the ceremony, Each R 6 is one or more halogens, hydroxyl, C1-C 12 Alkoxy, or (OCH2CH2) f H, OH, SH, NHCOO-t-butyl optionally substituted with OCH3, (CH2) n COOH, (CH2) n COOCH3, (CH2) n NH2, (CH2) n NH-(CH2) n -CH3, (CH2) n NHCOOH, (CH2) n NHCO-(CH2) n -CO-(CH2)n -CH3, (CH2) n NHCOO-(CH2) n -CH3, (CH2) n NHCOOC(CH3)3, (CH2) n NHCO(C3~C 12 ) cycloalkyl, (CH2) n NHCO(CH2CH2O) f , (CH2) n NHCO(CH2) n COOH, (CH2) n NHCO(CH2) n COO(CH2) n CH3, (CH2) n (OCH2CH2) f OCH3, N-maleimide, halogen, C2-C 12 Alkenes, C2-C 12 Alkynes, C3-C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 (Hetero)aryl, C1-C 12 independently selected from the group consisting of (hetero)arylamino, and benzyl; each f is independently an integer from 0 to 50; Each n is independently an integer from 1 to 20.

[0018] In some embodiments, G 1 and G 2 are each independently selected from the group consisting of optionally substituted dihydrophenanthrene (DHP), optionally substituted fluorene, aryl substituted with one or more pendant chains terminated with a functional group, and heteroaryl substituted with one or more pendant chains terminated with a functional group.

[0019] In some embodiments, G 1 and G 2 teeth, [ka] are each independently selected from the group consisting of: During the ceremony, Each R 6 is one or more halogens, hydroxyl, C1-C 12 Alkoxy, or (OCH2CH2) f H, OH, SH, NHCOO-t-butyl optionally substituted with OCH3, (CH2) n COOH, (CH2) n COOCH3, (CH2) n NH2, (CH2) n NH-(CH2) n -CH3, (CH2) n NHCOOH, (CH2) n NHCO-(CH2) n -CO-(CH2) n -CH3, (CH2) n NHCOO-(CH2) n -CH3, (CH2) n NHCOOC(CH3)3, (CH2) n NHCO(C3~C 12 ) cycloalkyl, (CH2) n NHCO(CH2CH2O) f , (CH2) n NHCO(CH2) n COOH, (CH2) n NHCO(CH2) n COO(CH2) n CH3, (CH2) n (OCH2CH2) f OCH3, N-maleimide, halogen, C2-C 12 Alkenes, C2-C 12 Alkynes, C3-C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 (Hetero)aryl, C1-C 12 independently selected from the group consisting of (hetero)arylamino, and benzyl; each f is independently an integer from 0 to 50; Each n is independently an integer from 1 to 20.

[0020] In some embodiments, the present invention provides a method for detecting an analyte in a sample, comprising: providing a sample suspected of containing the analyte; contacting the sample with a binding agent conjugated to a water-soluble polymer having the structure of Formula I, [ka] During the ceremony, each X is independently selected from the group consisting of C and Si; Each Y is a bond, CR 1 R 2 , and SiR 1 R 2 are independently selected from the group consisting of When Y is a bond, X is directly attached to both rings; Each R 1 are ammonium alkyl salts, ammonium alkyloxy salts, ammonium oligoether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligoether salts, sulfonamide oligoethers, and [ka] are independently selected from the group consisting of Each R 2 is H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, and [ka] are independently selected from the group consisting of Each R 3is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and PEG; each Z is independently selected from the group consisting of C, O, and N; Each Q is a bond, NH, or NR 4 and CH2, Each M is an electron-rich linker unit that can independently modify the polymer bandgap and is uniformly or randomly distributed along the polymer backbone, and [ka] are each independently selected from the group consisting of: During the ceremony, Each R 4 is a non-ionic side chain capable of imparting solubility in water of greater than 10 mg / mL, and is selected from halogen, hydroxyl, C1-C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, C3-C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 Alkoxy, C2-C 18 (Hetero)aryloxy, C2-C 18 (hetero)arylamino, each x' is independently an integer from 0 to 20, and each y' is independently an integer from 0 to 50 (CH2) x’ (OCH2-CH2) y’ OCH3 and C2~C 18 (hetero)aryl groups; each optional linker, L, is an aryl or heteroaryl group uniformly or randomly distributed along the polymer backbone and is substituted with one or more pendant chains terminated with a functional group selected from the group consisting of amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and protected groups thereof, for conjugation to another substrate, acceptor dye, molecule, or binder; G 1 and G 2 are each independently selected from the group consisting of hydrogen, halogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, boronate-substituted aryl, boronate ester-substituted aryl, boronate ester, boronic acid, optionally substituted dihydrophenanthrene (DHP), optionally substituted fluorene, aryl or heteroaryl substituted with one or more pendant chains terminated with a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and protected groups thereof for conjugation to a substrate or binder; a, c, and d define the mole % of each unit in the structure, each of which can be uniformly or irregularly repeated, a being 10-100% mole % and c being 0-90% mole %; Each d is a mole percent of 0 to 25%; each b is independently 0 or 1; m is an integer from 1 to about 10,000; each n is independently an integer from 1 to 20; The binding agent is capable of interacting with an analyte or target-associated biomolecule, providing a method.

[0021] In some embodiments, the method further includes applying a light source to the sample that is capable of exciting the polymer and detecting whether light is emitted from the conjugated polymer conjugate.

[0022] In some embodiments, the binding agent is a protein, peptide, affinity ligand, antibody, antibody fragment, sugar, lipid, nucleic acid, or aptamer, hi some embodiments, the binding agent is an antibody.

[0023] In some embodiments, the method is configured for flow cytometry. In some embodiments, the binding agent is bound to a substrate. In some embodiments, the analyte is a protein expressed on a cell surface.

[0024] In some embodiments, the method is configured as an immunoassay. In some embodiments, the method further comprises providing an additional binding agent for simultaneously detecting an additional analyte. [Brief explanation of the drawings]

[0025] [Figure 1] 1 shows a comparison of the fluorescence emission spectra of fluorene (FF), dihydrophenanthrene (DD), and fluorene-DHP (DF) polymers.

[0026] [Figure 2] The absorption spectra of both the FF and DD polymers are shown. The graph shows the absorption of the DD polymer (black curve) at 390 and 410 nm, while the FF (grey curve) polymer shows a maximum at approximately 401 nm. The samples were measured at different concentrations.

[0027] [Figure 3] Flow cytometry analysis of lysed whole blood stained with the new polymer-labeled anti-human CD4 and Pacific Blue-labeled CD4. The positive signal intensity of the polymer dye was approximately 5-fold greater than that of Pacific Blue.

[0028] [Figure 4] The polymers of the present invention, when conjugated to antibodies and the like, are shown to have certain physical and chemical properties of absorption, fluorescence, brightness, molecular weight, polydispersity, and dye-to-protein ratio. Preferred ranges for these parameters are shown in this table.

[0029] [Figure 5] The excitation and emission spectra of the tandem polymers are shown. Excitation was performed at the polymer maximum (405 nm), and emission was observed from various acceptor dyes attached to the backbone: dye 1 - FITC, dye 2 - Cy3B, and dye 3 - Cy55. DETAILED DESCRIPTION OF THE INVENTION

[0030] I. Overview The present invention provides novel water-soluble fluorescent polymers and methods for detecting analytes in a sample using a conjugate comprising a fluorescent polymer conjugated to a binding agent. The water-soluble conjugated polymers of the present invention demonstrate significantly increased brightness compared to other dyes. II. Definition

[0031] The abbreviations used herein have their conventional meaning within the chemical biology field.

[0032] As used herein, the term "ammonium" refers to a compound of the formula NHR3 + wherein each R group is independently hydrogen or a substituted or unsubstituted alkyl, aryl, aralkyl, or alkoxy group. Preferably, each R group is hydrogen.

[0033] As used herein, "oligoether" is understood to mean an oligomer containing structural repeat units with ether functionality. As used herein, "oligomer" is understood to mean a molecule containing one or more distinguishable structural repeat units of the same or different formula.

[0034] The term "sulfonate functional group" or "sulfonate," as used herein, refers to both the free sulfonate anion (-S(=O)2O-) and its salts. Thus, the term sulfonate encompasses sulfonate salts such as sodium, lithium, potassium, and ammonium sulfonates.

[0035] The term "sulfonate," as used herein, refers to a group of formula -SO2NR-, where R is hydrogen, alkyl, or aryl.

[0036] As used herein, the term "alkyl" refers to a linear or branched saturated aliphatic radical having the specified number of carbon atoms. For example, C1-C6 alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, and the like. Other alkyl groups include, but are not limited to, heptyl, octyl, nonyl, decyl, and the like. Alkyl can contain any number of carbons: 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6, and 5-6. Alkyl groups are typically monovalent but may be divalent, such as when the alkyl group links two moieties together.

[0037] The term "cycloalkyl" as used herein refers to a saturated or partially saturated monocyclic, fused bicyclic, or bridged polycyclic ring assembly containing 3 to 12 ring atoms, or a monocyclic ring with a specified number of atoms, including, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentyl, and cyclopentyl. Bicyclic and polycyclic rings include, for example, norbornane, decahydronaphthalene, and adamantane. For example, C 3~8 Cycloalkyl includes cyclopropyl, cyclobutyl, and cyclopentyl. , cyclopentyl, cyclopentyl, and norbornane.

[0038] The term "haloalkyl" as used herein refers to an alkyl as defined above in which some or all of the hydrogen atoms have been replaced with halogen atoms. Halogen (halo) preferably refers to chloride or fluoride, but may also be bromide or iodide. For example, haloalkyl includes trifluoromethyl, flouromethyl, 1,2,3,4,5-pentafluorophenyl, etc. The term "perfluoro" defines a compound or radical having at least two available hydrogens replaced with fluorine. For example, perfluorophenyl refers to 1,2,3,4,5-perfluorophenyl, perfluoromethane refers to 1,1,1-trifluoromethyl, and perfluoromethoxy refers to 1,1,1-trifluoromethoxy.

[0039] As used herein, the term "halogen" refers to fluorine, chlorine, bromine and iodine.

[0040] The term "alkoxy" as used herein refers to an alkyl group, as defined above, having an oxygen atom connecting the alkyl group to its point of attachment. Alkoxy groups include, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, and the like. Alkoxy groups can be further substituted with various substituents as described herein. For example, alkoxy groups can be substituted with halogens to form "halo-alkoxy" groups.

[0041] As used herein, the term "alkene" refers to either a straight-chain or branched hydrocarbon having at least one double bond. Examples of alkene groups include, but are not limited to, vinyl, propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl. Alkene groups are typically monovalent but may be divalent, such as when the alkene group links two moieties together.

[0042] The term "alkyne" as used herein refers to either a straight-chain or branched hydrocarbon having at least one triple bond. Examples of alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl. Alkynyl groups are typically monovalent but may be divalent, such as when the alkynyl group links two moieties together.

[0043] The term "aryl" as used herein refers to a monocyclic or fused bicyclic, tricyclic or higher aromatic ring assembly containing 6 to 16 ring carbon atoms. For example, aryl can be phenyl, benzyl or naphthyl, preferably phenyl. "Arylene" means a divalent radical derived from an aryl group. The aryl group can be mono-, di- or trisubstituted with one, two or three radicals selected from alkyl, alkoxy, aryl, hydroxy, halogen, cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy and oxy-C2-C3-alkylene; All of these are optionally further substituted, for example as defined above, or as 1- or 2-naphthyl, or 1- or 2-phenanthrenyl. Alkylenedioxy is a divalent substituent attached to two adjacent carbon atoms of phenyl, for example, methylenedioxy or ethylenedioxy. Oxy-C2-C3-alkylene is also a divalent substituent attached to two adjacent carbon atoms of phenyl, for example, oxyethylene or oxypropylene. An example of oxy-C2-C3-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

[0044] Preferred as aryl is naphthyl, phenyl or phenyl mono- or di-substituted by alkoxy, phenyl, halogen, alkyl or trifluoromethyl, especially phenyl or phenyl mono- or di-substituted by alkoxy, halogen or trifluoromethyl, and especially phenyl.

[0045] The term "aryloxy" as used herein refers to an O-aryl group, where aryl is as defined above. An aryloxy group can be unsubstituted or substituted with one or two suitable substituents. The term "phenoxy" refers to an aryloxy group in which the aryl moiety is a phenyl ring. The term "heteroaryloxy" as used herein means an -O-heteroaryl group, where heteroaryl is as defined below. The term "(hetero)aryloxy" is used to indicate that the moiety is either an aryloxy group or a heteroaryloxy group.

[0046] The term "polyethylene glycol" or "PEG" as used herein refers to a group of biocompatible, water-soluble linear polymers based on ethylene glycol monomer units.

[0047] The term "heteroaryl" as used herein refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, wherein 1 to 4 of the ring atoms are heteroatoms N, O, or S. For example, heteroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any other radical substituted, particularly mono- or di-substituted, by, for example, alkyl, nitro, or halogen. Pyridyl refers to 2-, 3-, or 4-pyridyl, advantageously 2- or 3-pyridyl. Thienyl refers to 2- or 3-thienyl. Quinolinyl preferably refers to 2-, 3-, or 4-quinolinyl. Isoquinolinyl preferably represents 1-, 3-, or 4-isoquinolinyl. Benzopyranyl and benzothiopyranyl preferably represent 3-benzopyranyl or 3-benzothiopyranyl, respectively. Thiazolyl preferably represents 2- or 4-thiazolyl, most preferably 4-thiazolyl. Triazolyl preferably represents 1-, 2-, or 5-(1,2,4-triazolyl). Tetrazolyl preferably represents 5-tetrazolyl.

[0048] Preferably, heteroaryl is pyridyl, indolyl, quinolinyl, pyrrolyl, thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl, benzothienyl, oxazolyl, indazolyl, or any substituted, especially mono- or di-substituted, radical.

[0049] Similarly, substituents on the aryl and heteroaryl groups are varied and include, in numbers ranging from 0 to the total number of open valences on the aromatic ring system, -halogen, -OR', -OC(O)R', -NR'R", -SR', -R', -CN, -NO2, -CO2R', -CONR'R", - are selected from C(O)R', -OC(O)NR'R", -NR"C(O)R', -NR"C(O)R', -NR'-C(O)NR"R"', -NH-C(NH)=NH, -NR'C(NH)=NH, -NH-C(NH)=NR', -S(O)R', -S(O)R', -S(O)NR'R", -N, -CH(Ph), perfluoro(C1-C4)alkoxy, and perfluoro(C1-C4)alkyl, where R', R" and R"' are independently selected from hydrogen, (C1-C8)alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4)alkyl, and (unsubstituted aryl)oxy-(C1-C4)alkyl.

[0050] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring are of the formula -TC(O)-(CH2) q -U-, where T and U are independently -NH-, -O-, -CH-, or a single bond, and q is an integer from 0 to 2. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may be of the formula -A-(CH) r A and B may optionally be replaced with a substituent of the formula -B-, where A and B are independently -CH2-, -O-, -NH-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'-, or a single bond, and r is an integer from 1 to 3. One of the single bonds in the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may be replaced with a group of the formula -(CH2) s -X-(CH2) t where s and t are independently integers from 0 to 3, and X is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. The substituent R' in -NR'- and -S(O)2NR'- is selected from hydrogen or unsubstituted (C1-C6) alkyl.

[0051] The term "(hetero)arylamino" as used herein refers to an amine radical substituted with an aryl group (e.g., -NH-aryl). Arylamino may also be an aryl radical substituted with an amine group (e.g., -aryl-NH). Arylamino may be substituted or unsubstituted.

[0052] The term "amine" as used herein refers to an alkyl group, as defined herein, having one or more amino groups. The amino groups may be primary, secondary, or tertiary. The alkylamine may be further substituted with a hydroxy group. Amines useful in the present invention include, but are not limited to, ethylamine, propylamine, isopropylamine, ethylenediamine, and ethanolamine. The amino group may be at the omega position of the alkyl group, which may link the alkylamine to the point of attachment with the remainder of the compound, or may link at least two carbon atoms of the alkyl group together. One of ordinary skill in the art will recognize that other alkylamines are useful in the present invention.

[0053] The term "carbamate," as used herein, refers to a functional group having the structure -NR"COR', where R' and R" are independently selected from hydrogen, (C1-C8) alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4) alkyl, and (unsubstituted aryl)oxy-(C1-C4) alkyl. Examples of carbamates include t-Boc, Fmoc, benzyloxy-carbonyl, alloc, methyl carbamate, ethyl carbamate, 9-(2-sulfb)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, Tbfmoc, Climoc, Bimoc, DBD-Tmoc, Bsmoc, Troc, Teoc, 2-phenylethyl carbamate, Adpoc, 2-chloroethyl carbamate, l,l-dimethyl-2-haloethyl carbamate, DB-t-BOC, TCBOC, Bpoc, t-Bumeoc, Pyoc, Bnpeoc, V-(2-pivaloylamino)-1,1-dimethylethyl carbamate, and NpSSPeoc.

[0054] The term "carboxylate" as used herein refers to the conjugate base of a carboxylic acid, which can generally be represented by the formula RCOO. For example, "magnesium carboxylate" refers to the magnesium salt of a carboxylic acid.

[0055] The term "activated ester" as used herein refers to a carboxyl-activating group used to facilitate the facile condensation of the free amino group and the carboxyl group of an amino acid derivative. Descriptions of these carboxyl-activating groups can be found in general textbooks of peptide chemistry, such as KD Kopple, "Peptides and Amino Acids," W.A. Benjamin, Inc., New York, 1966, pp. 50-51, and E. Schroder and K. Lubke, "The Peptides," Vol. 1, Academic Press, New York, 1965, pp. 77-128.

[0056] The terms "hydrazine" and "hydrazide" refer to compounds containing single-bonded nitrogens, one of which is a primary amine functionality.

[0057] The term "aldehyde" as used herein refers to a chemical compound having a -CHO group.

[0058] The term "thiol" as used herein refers to a compound containing a functional group consisting of a sulfur-hydrogen bond. The general chemical structure of a thiol functional group is R-SH, where R represents an alkyl, alkene, aryl, or other carbon-containing group.

[0059] As used herein, the term "silyl" refers to Si(R z ) 3, wherein each R z are independently alkylaryl or other carbon-containing groups.

[0060] As used herein, the term "diazonium salt" refers to a compound of the structure R-N2 + X - where R can be any organic residue (e.g., alkyl or aryl) and X is an inorganic or organic anion (e.g., a halogen).

[0061] The term "triflate," also referred to as trifluoromethanesulfonate, is a group having the formula CF3SO3.

[0062] The term "boronic acid" as used herein refers to the structure -B(OH). It will be understood by those skilled in the art that boronic acids may be present as boronate esters at various stages in the synthesis of quenchers. Boronic acid is intended to include such esters. As used herein, the term "boronic acid ester" or "boronate ester" refers to the structure -B(Z 1 )(Z 2 ) moiety, wherein Z 1 and Z 2together form a moiety in which the atom attached to the boron in each instance is an oxygen atom. In some embodiments, the boronic ester moiety is a 5-membered ring. In some other embodiments, the boronic ester moiety is a 6-membered ring. In some other embodiments, the boronic ester moiety is a mixture of 5- and 6-membered rings. III. Composition polymer

[0063] The compounds of the present invention include water-soluble fluorescent polymers having the structures of Formulas I-XIII. In some embodiments, the polymers of the present invention utilize dihydrophenanthrene (DHP), fluorene, and combinations of DHP and fluorene monomers, as shown in Formula I. [ka] .

[0064] The polymer conjugates of the present invention may contain units capable of modifying the polymer bandgap, distributed uniformly or randomly along the polymer backbone. These units are represented as M in Formula I. The polymer conjugates of the present invention may also contain linkers, represented as L in Formula I. Each optional linker, L, is an aryl or heteroaryl group distributed uniformly or randomly along the polymer backbone and is substituted with one or more pendant chains terminated with a functional group selected from the group consisting of amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and protected versions thereof, for conjugation to a substrate or binder.

[0065] The polymer conjugates of the present invention also contain, in Formula I, each G 1 and G 2and wherein these units are each independently selected from the group consisting of hydrogen, halogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, boronate-substituted aryl, boronate ester-substituted aryl, boronate ester, boronic acid, optionally substituted dihydrophenanthrene (DHP), optionally substituted fluorene, aryl or heteroaryl substituted with one or more pendant chains terminated with a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and these protected groups for conjugation to a substrate or binder.

[0066] In some cases, the polymer has a structure of Formula II [ka] .

[0067] In some cases, the polymer has a structure of Formula IV [ka] .

[0068] In some cases, the polymer has a structure of Formula V [ka] .

[0069] In some cases, the polymer is a copolymer and has the structure of Formula VI [ka] .

[0070] In some cases, the polymer is a copolymer and has the structure of Formula VII [ka] .

[0071] In some embodiments, the polymer has an acceptor dye attached to the backbone that allows for excitation of the polymer backbone and monitoring the emission of the acceptor dye attached to the backbone upon energy transfer. Acceptor dyes useful in the present invention include FITC, CY3B, Cy55, Alexa 488, Texas red, Cy5, Cy7, Alexa 750, and 800CW. For example, polymers with acceptor dyes of the present invention include: [ka] Examples include: monomer

[0072] Monomers of the present invention include dihydrophenanthrene (DHP) monomers and fluorene-based monomers. For example, monomers of the present invention include: [ka] Examples include: Both ends of these monomers are independently or both halogen atoms, boronic esters or acids, silyl, diazonium salts, triflates, acetyloxy, sulfonates, or phosphates capable of undergoing Pd or nickel salt catalyzed polymerization reactions. 1 are independently side chains that can confer solubility in water / buffer, and each R 1are ammonium alkyl salts, ammonium alkyloxy salts, ammonium oligoether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligoether salts, sulfonamide oligoethers, and [ka] and each R is independently selected from the group consisting of 2 is H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, and [ka] and each R is independently selected from the group consisting of 3 is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and PEG; each Z is independently selected from the group consisting of C, O, and N; and each Q is a bond, NH, NR 4 and CH2, wherein each R 5 H, C1~C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, C3-C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 Alkoxy, C2-C 18 (Hetero)aryloxy, C2-C 18 (Hetero)arylamino and C1-C 12 alkoxy is independently selected from the group consisting of:

[0073] In some embodiments, the monomers of the present invention also include cross-linking monomers. For example, cross-linking monomers of the present invention include: [ka] Examples include: synthesis

[0074] The DHP monomers of the present invention can be made as shown below. [ka]

[0075] For example, 2,7-dibromo-trans-9,10-dihydrophenanthrene-9,10-diol (DHP-OH) can be prepared as follows: In a 2000 L Erlenmeyer flask, approximately 26 g of NaBH4 is added to a stirring water-ethanol mixture (120 mL + 780 mL). To this solution, approximately 24 g of 2,7-dibromophenanthrene, 9,10-dione is added in small portions over a short period (5 minutes). The reaction mixture is stirred for one day. The color of the solution changes from orange-red to pale yellow to white by the end of the reaction. The reaction is stopped, and the reaction mixture is neutralized with dilute hydrochloric acid. After neutralization, the white precipitate is filtered and washed with excess water. The resulting white precipitate is washed with very cold (<-15°C) ethanol (100 mL) and methanol (100 mL).

[0076] DHP-OSO3H can be prepared as follows. In a two-neck round-bottom flask, DHP-OH (3.6 g) and 18C6 (500) were dissolved in 120 mL of THF. The solution was purged with nitrogen (20 min), and NaH (2 g) was added while continuing the nitrogen purge. The color of the solution changed from colorless to light pink, dark pink, brown, and dark green over 10-15 min. In a separate RB, 12 g of 1,3 propane sultone was dissolved in 20 mL of THF and purged with nitrogen. The sultone solution was added via a dropping funnel over 20-30 min. The reaction mixture was stirred at room temperature for 4-5 hours. The solvent was evaporated and the precipitate was dissolved in water. Acetone was added to obtain a white precipitate of DPS in the form of its disodium salt. The precipitate was filtered, redissolved in a minimum amount of water, neutralized with HCl, and precipitated again in acetone. Repeated precipitation (2-3 times) followed by centrifugation yielded DPS as a white solid.

[0077] DHP-OSO2Cl can be prepared as follows: 5 g of DHP-OSO3H was placed in a round-bottom flask and mixed with 25 mL of DMF. Approximately 10 mL of SOCl2 was added dropwise, and the mixture was stirred overnight. The next morning, the reaction mixture was poured into 200 mL of water, and the precipitate was filtered and dried.

[0078] DHP-sulfonamide PEG can be prepared as follows: DHP-OSO2Cl was mixed with 2.2 equivalents of PEG amine in a dichloromethane / TEA mixture. After a 3-hour sonication reaction, the crude product was extracted in dichloromethane followed by column chromatography (silica gel, MeOH-CHCl3).

[0079] The diboronic acid ester of DHP-sulfonamidoPEG can be prepared as follows: The dibromo compound was mixed with DMSO under nitrogen, and 3 equivalents of bis-pinacolatodiboron was added. The reagent was reacted with 12 equivalents of potassium acetate and 4 equivalents of Pd(dppf)Cl catalyst at 80°C for 5 hours. The reaction mixture was cooled and extracted with CHCl / water. The organic layer was concentrated and purified by column chromatography (silica gel, MeOH-CHCl).

[0080] Similarly, fluorene monomers of the present invention can be made as described below. For example, FL-OSO3H can be prepared as follows: 5 g of fluorene was mixed with 70 g of DMSO in a two-neck round-bottom flask. The solution was purged with nitrogen (20 minutes), and 50% NaOH (12 equivalents) was added while continuing the nitrogen purge. The solution color changed from colorless to dark brown. Propane sultone (3 equivalents) was weighed and dissolved in DMSO. This was added dropwise to the fluorene reaction mixture over a 5-minute period. The reaction was stirred at room temperature for 4-5 hours. The solvent was evaporated, and the precipitate was dissolved in water. Acetone was added to yield a white precipitate of DPS in the form of the disodium salt. The precipitate was filtered, redissolved in a minimum amount of water, neutralized with HCl, and precipitated again in acetone. Repeated precipitation (2-3 times) followed by centrifugation yielded FL-OSO3H as a white solid.

[0081] FL-OSO2Cl can be prepared as follows: 5 g of FL-OSO3H was placed in a round-bottom flask and mixed with 25 mL of DMF. Approximately 10 mL of SOCl2 was added dropwise, and the mixture was stirred overnight. The next morning, the reaction mixture was poured into 200 mL of water, and the precipitate was filtered and dried.

[0082] FL-sulfonamide PEG can be prepared as follows: FL-OSO2Cl was mixed with 2.2 equivalents of PEG amine in a dichloromethane / TEA mixture. After a 3-hour sonication reaction, the crude product was extracted in dichloromethane, followed by column chromatography (silica gel, MeOH-CHCl3).

[0083] The diboronic ester of FL-sulfonamide PEG can be prepared as follows: The dibromo compound was mixed with DMSO under nitrogen, and 3 equivalents of bis-pinacolatodiboron was added. The reagent was reacted with 12 equivalents of potassium acetate and 4 equivalents of Pd(dppf)Cl catalyst at 80°C for 5 hours. The reaction mixture was cooled and extracted with CHCl / water. The organic layer was concentrated and purified by column chromatography (silica gel, MeOH-CHCl). polymerization

[0084] The compounds described in the above embodiments may be made using procedures known in the art. In some embodiments, fluorescent polymers can be made from dihydrophenanthrene (DHP) monomers coupled with electron-rich linker units. In some embodiments, bright polymer dyes can be made from fluorene monomers coupled with electron-rich linker units. In some embodiments, bright polymer dyes can be made from a combination of DHP and fluorene monomers coupled with electron-rich linker units.

[0085] Generally, polymerizing the above-described monomer units can be accomplished using polymerization techniques known to those skilled in the art or using methods known in the art in combination with the methods described herein. For example, synthesis of diboronic ester derivatives from dihalide monomers can be achieved by Suzuki coupling with bis(pinacolato)diboron. [ka] Similarly, polymerization can also be achieved by Suzuki coupling. [ka] J 1 and J 2 are independently H, Br, B(OH)2, or a boronic ester.

[0086] For example, polymerization can proceed as follows: In a round-bottom flask, both the bromo and boronic acid monomers were placed in a (DMF-water) mixture and purged with nitrogen for 10 minutes. Approximately 20 equivalents of CsF and 10% Pd(OAc)2 were mixed under nitrogen and heated to 80°C. Polymerization was monitored using UV-Vis spectroscopy and SEC chromatography. An end-capping agent (selected from G1) containing an appropriate functional group was then added to the reaction mixture, followed 3 hours later by the addition of a second end-capping agent (selected from G2). After the reaction, the crude reaction mixture was evaporated and passed through a gel filtration column to separate small organic molecules and low MW oligomers. - was removed. Terminal Protection Unit

[0087] Linkers and capping units can be conjugated to the polymer backbone of the present invention by similar mechanisms as described above. For example, bromoester and boronic ester capping units can be used to append one or both ends of a polymer. Using both bromoester and boronic ester capping units will append both ends of the polymer. Using only one form, i.e., either the bromoester or boronic ester capping unit, will append only the end terminated with its corresponding complement, which can be used in symmetric polymerizations to statistically modify only one end of the polymer. In asymmetric polymers, this approach is used to chemically ensure that the polymer is modified only at a single chain end. Capping units can also be appended asymmetrically by first reacting a bromoester capping unit with a polymer bearing a Y end, followed by reacting the polymer with a boronic ester capping unit.

[0088] For example, the endcapping agent of the present invention can be prepared as shown below. [ka] Binder

[0089] A "binding agent" of the present invention can be any molecule or complex of molecules that can specifically bind to a target analyte. Binding agents of the present invention include, for example, proteins, small organic molecules, carbohydrates (including polysaccharides), oligonucleotides, polynucleotides, lipids, affinity ligands, antibodies, antibody fragments, aptamers, and the like. In some embodiments, a binding agent is an antibody or fragment thereof. Specific binding, in the context of the present invention, refers to a binding reaction that is determinative of the presence of a target analyte in the presence of a heterogeneous population. Thus, under designated assay conditions, a designated binding agent preferentially binds to a particular protein or isoform of a particular protein and does not bind in significant amounts to other proteins or isoforms present in the sample.

[0090] When the binding agents are antibodies, they may be monoclonal or polyclonal antibodies. As used herein, the term antibody refers to immunoglobulins and immunoglobulins. (Ig) refers to an immunologically active portion of a molecule. Such antibodies include, but are not limited to, polyclonal, monoclonal, monospecific polyclonal antibodies, antibody mimetics, chimeric, single chain, Fab, Fab' and F(ab')2 fragments, Fv, and an Fab expression library. Complex

[0091] Generally, the fluorescent polymers of the present invention can be conjugated to binding agents using techniques known to those skilled in the art or using methods known in the art in combination with the methods described herein. [ka]

[0092] For example, the preparation of polymeric NHS ester can proceed as follows: 5 mg of polymer is placed in a clean vial and dissolved in 1 mL of dry CH3CN. 15 mg of TSTU is added and stirred for an additional 2 minutes. 100 μL of DIPEA is added, the cap is sealed with parafilm, and stirring is continued overnight. The organic solvent in the reaction mixture is then evaporated, and the crude NHS is dissolved in approximately 750 μL of 1x BBS buffer (pH 8.8) by brief vortexing and transferred to a Zeba column with a 40K MWCO. The sample is spun down at 2200 RPM for 2 minutes, and the polymeric NHS is used immediately.

[0093] Conjugation of polymeric NHS to CD4 can proceed as follows: Spin down polymeric NHS in 1x BBS (~800 µL), add to 0.6 mg of CD4, and mix with 100 µL of 0.5 M borate buffer (pH 9.0). Vortex quickly for 30 seconds and let mix in a Coulter mixer for 3-4 hours.

[0094] Purification of the conjugate using a Histrap HP column can proceed as follows: Approach 1: After the crude reaction, purify the conjugate using a Histrap HP column. Load the sample using 1x PBS buffer and collect the unbound fraction. This can be done using 20 CV of buffer. Then, change the buffer to wash out the unbound fraction, which contains both the conjugate and free antibody. This can be done using 10 CV of running 1x PBS containing 0.25 M imidazole.

[0095] Approach 2: Hitrap SP Sepharose FF column. Equilibrate the column and load the sample using 20 mM citrate buffer pH 3.5, collecting the unbound fraction. This can be done using 20 CV of buffer. Then, change the buffer to elute the unbound fraction, which contains both the conjugate and free antibody. This can be done using 20 CV of 20 mM Tris buffer pH 8.5. Approach 3: Load the crude conjugate into a tangential flow filtration system equipped with a 300K MWCO membrane. Wash the conjugate with 1x PBS until the filtrate shows no absorbance at 405 nm. Then, concentrate the compound.

[0096] Purification of the conjugate by SEC column can proceed as follows: 1x PBS Load the crude conjugate containing the free antibody onto a size-exclusion column using a centrifuge. Pool the tubes after checking the absorption spectrum and concentrate with an Amicon Ultra-15 with a 30 KDa MWCO centrifugal concentrator. IV. Methods for Detecting Analytes overview

[0097] The present invention provides a method for detecting an analyte in a sample, comprising providing a sample suspected of containing the analyte and providing a conjugated polymer conjugate comprising a binding agent linked to a water-soluble conjugated polymer. The binding agent is capable of interacting with the analyte. A light source capable of exciting the polymer is applied to the sample, and light emitted from the conjugated polymer conjugate is detected. In a typical assay, the fluorescent polymers of the present invention can be excited with light having a wavelength of about 395 nm to about 415 nm. The emitted light is typically about 415 nm to about 475 nm. Alternatively, the excitation light can have a wavelength of about 340 nm to about 370 nm, and the emitted light is about 390 nm to about 420 nm. sample

[0098] The sample in the methods of the present invention can be, for example, blood, bone marrow, spleen cells, lymph node cells, bone marrow aspirate (or any cells obtained from bone marrow), urine (washing), serum, saliva, cerebrospinal fluid, urine, amniotic fluid, interstitial fluid, feces, mucus, or tissue (e.g., tumor sample, isolated tissue, isolated solid tumor). In certain embodiments, the sample is a blood sample. In some embodiments, the blood sample is whole blood. Standard clinical procedures can be used to obtain whole blood from a subject. In some embodiments, the sample is one or more cellular subsets of whole blood (e.g., erythrocytes, leukocytes, lymphocytes (e.g., T cells, B cells, or NK cells), phagocytes, monocytes, macrophages, granulocytes, basophils, neutrophils, eosinophils, platelets, or any cells with one or more detectable markers). In some embodiments, the sample can be derived from a cell culture.

[0099] The subject can be a human (e.g., a patient suffering from a disease), a commercially significant mammal, such as a monkey, cow, or horse. Samples can also be obtained from household pets, including, for example, dogs or cats. In some embodiments, the subject is a laboratory animal used as an animal model of disease or for drug screening, such as a mouse, rat, rabbit, or guinea pig. specimen

[0100] "Analyte" as used herein refers to a substance, e.g., a molecule, whose abundance / concentration is determined by some analytical procedure. For example, in the present invention, an analyte can be a protein, peptide, nucleic acid, lipid, carbohydrate, or small molecule.

[0101] Target analytes can be, for example, nucleic acids (DNA, RNA, mRNA, tRNA, or rRNA), peptides, polypeptides, proteins, lipids, ions, monosaccharides, oligosaccharides, polysaccharides, lipoproteins, glycoproteins, glycolipids, or fragments thereof. In some embodiments, target analytes can be proteins, such as structural microfilament, microtubule, and intermediate filament proteins, organelle-specific markers, proteasomes, transmembrane proteins, surface receptors, nuclear pore proteins, protein / peptide translocases, protein folding chaperones, signaling scaffolds, and ion channels. Proteins can be activatable proteins or proteins that are differentially expressed or activated in diseased or abnormal cells, including, but not limited to, transcription factors, DNA and / or RNA binding and modifying proteins, nuclear transport and export receptors, and regulators of apoptosis or survival. Assay

[0102] Assay systems utilizing binding agents and fluorescent labels to quantify bound molecules are well known, and examples of such systems include flow cytometers, scanning cytometers, imaging cytometers, fluorescence microscopes, and confocal fluorescence microscopes.

[0103] In some embodiments, flow cytometry is used to detect fluorescence. Many devices suitable for this purpose are available and known to those skilled in the art. Examples include the BCI Navios, Gallios, Aquios, and CytoFLEX flow cytometers.

[0104] In another embodiment, the assay is an immunoassay. Examples of immunoassays useful in the present invention include, but are not limited to, fluoroluminescence assays (FLA). The assay can also be performed on a protein array.

[0105] When the binding agent is an antibody, an antibody assay or a multi-antibody sandwich assay can also be used. Sandwich assays refer to the use of sequential recognition events that build layers of different binding agents and signaling elements to signal the presence of a particular analyte. Examples of sandwich assays are disclosed in U.S. Patent No. 4,486,530 and the references cited therein. V. Working Examples Example 1: Preparation of DHP polymer conjugate [ka]

[0106] Method 1: In a round-bottom flask, both dibromo DHP and diboronic acid DHP monomers (1:1) were placed in a DMF-water mixture and purged with nitrogen for 10 minutes. Approximately 20 equivalents of CsF and 10% Pd(OAc)2 were mixed under nitrogen and heated to 80°C. Polymerization was monitored using UV-Vis spectroscopy and SEC chromatography. An end-capping agent (selected from G1) containing the appropriate functional group was then added to the reaction mixture, followed 3 hours later by the addition of a second end-capping agent (selected from G2). After the reaction, the crude reaction mixture was evaporated and passed through a gel filtration column to remove small organic molecules and low-MW oligomers. The crude polymer was then passed through a tangential flow filtration system equipped with a 100K MWCO membrane. It was washed with 20% ethanol until the filtrate showed low absorbance.

[0107] Method 2: Alternatively, polymerization can be achieved by self-polymerizing the bromo-boronic ester of the DHP molecule. In a round-bottom flask, the DHP bromo-boronic ester was placed in a (DMF-water) mixture and purged with nitrogen for 10 minutes. Under nitrogen, approximately 10 equivalents of CsF and 5% Pd(OAc)2 were mixed and heated to 80°C. UV-Vis spectroscopy and SE Polymerization was monitored using C chromatography. An endcapping agent (selected from G1) containing an appropriate functional group was then added to the reaction mixture, followed 3 hours later by the addition of a second endcapping agent (selected from G2). After the reaction, the crude reaction mixture was evaporated and passed through a gel filtration column to remove small organic molecules and low-MW oligomers. The crude polymer was then passed through a tangential flow filtration system equipped with a 100K MWCO membrane. It was washed with 20% ethanol until the filtrate showed low absorbance.

[0108] Method 3: In a round-bottom flask, 10 equivalents of both dibromodihydrophenanthrene and diboronic acid dihydrophanenthrene monomers (1:1) were added. The polymer was dissolved in a 4:1 THF-water mixture containing 100% K2CO3 and 3% Pd(PPh3)4. The reaction mixture was placed in a Schlenk line and degassed using three freeze-pump cycles. It was then heated to 80 °C under nitrogen with vigorous stirring for 18 h. An end-capping agent (selected from G1) containing the appropriate functional group was then added to the reaction mixture via cannula under excess nitrogen pressure, followed 3 h later by the addition of a second end-capping agent (selected from G2). After the reaction, the crude reaction mixture was evaporated and passed through a gel filtration column to remove small organic molecules and low-MW oligomers. The crude polymer was then passed through a tangential flow filtration system equipped with a 100K MWCO membrane. It was washed with 20% ethanol until the filtrate showed low absorption.

[0109] Method 4: Alternatively, polymerization can be achieved by the self-polymerization of a bromo-boronic acid ester of a dihydrophenanthrene molecule. Dihydrophenanthrene bromo-boronic acid ester was dissolved in a 4:1 THF-water mixture containing 10 equivalents of K2CO3 and 3% Pd(PPh3)4 in a round-bottom flask. The reaction mixture was charged to a Schlenk line, degassed using three freeze-pump cycles, and then heated to 80 °C under nitrogen with vigorous stirring for 18 hours. An end-capping agent (selected from G1) containing the appropriate functional group was then added to the reaction mixture via cannula under excess nitrogen pressure, followed 3 hours later by the addition of a second end-capping agent (selected from G2). After the reaction, the crude reaction mixture was evaporated and passed through a gel filtration column to remove small organic molecules and low-MW oligomers. The crude polymer was then passed through a tangential flow filtration system equipped with a 100K MWCO membrane. This was washed with 20% ethanol until the absorbance of the filtrate was low. Example 2: Preparation of fluorene-DHP copolymer conjugate [ka]

[0110] Method 1: In a round-bottom flask, both dibromoDHP and diboronic acid fluorene monomers (1:1) were placed in a DMF-water mixture and purged with nitrogen for 10 minutes. Approximately 20 equivalents of CsF and 10% Pd(OAc)2 were mixed under nitrogen and heated to 80°C. Polymerization was monitored using UV-Vis spectroscopy and SEC chromatography. An end-capping agent (selected from G1) containing the appropriate functional group was then added to the reaction mixture, followed 3 hours later by the addition of a second end-capping agent (selected from G2). After the reaction, the crude reaction mixture was evaporated and passed through a gel filtration column to remove small organic molecules and low-MW oligomers. The crude polymer was then filtered through a tangential flow filtration system equipped with a 100K MWCO membrane. This was washed with 20% ethanol until the absorbance of the filtrate was reduced.

[0111] Method 2: In a round-bottom flask, both dibromofluorene monomer and diboronic acid DHP monomer (1:1) were placed in a DMF-water mixture and purged with nitrogen for 10 minutes. Approximately 20 equivalents of CsF and 10% Pd(OAc)2 were mixed under nitrogen and heated to 80°C. Polymerization was monitored using UV-Vis spectroscopy and SEC chromatography. An end-capping agent (selected from G1) containing the appropriate functional group was then added to the reaction mixture, followed 3 hours later by the addition of a second end-capping agent (selected from G2). After the reaction, the crude reaction mixture was evaporated and passed through a gel filtration column to remove small organic molecules and low-MW oligomers. The crude polymer was then passed through a tangential flow filtration system equipped with a 100K MWCO membrane. It was washed with 20% ethanol until the filtrate showed low absorbance.

[0112] Method 3: In a round-bottom flask, both dibromodihydrophenanthrene and diboronic acid fluorene monomers (1:1) were dissolved in a 4:1 THF-water mixture containing 10 equivalents of K2CO3 and 3% Pd(PPh3)4. The reaction mixture was charged to a Schlenk line, degassed using three freeze-pump cycles, and then heated to 80 °C under nitrogen with vigorous stirring for 18 h. An end-capping agent (selected from G1) containing the appropriate functional group was then added to the reaction mixture via cannula under excess nitrogen pressure, followed 3 h later by the addition of a second end-capping agent (selected from G2). After the reaction, the crude reaction mixture was evaporated and passed through a gel filtration column to remove small organic molecules and low-MW oligomers. The crude polymer was then passed through a tangential flow filtration system equipped with a 100K MWCO membrane. It was washed with 20% ethanol until the filtrate showed low absorption.

[0113] Method 4: In a round-bottom flask, dibromofluorene monomer and diboronic acid dihydrophenanthrene monomer (1:1) were dissolved in a THF-water (4:1) mixture containing 10 equivalents of K2CO3 and 3% Pd(PPh3)4. The reaction mixture was charged to a Schlenk line, degassed using three freeze-pump cycles, and then heated to 80 °C under nitrogen with vigorous stirring for 18 h. An end-capping agent (selected from G1) containing the appropriate functional group was then added to the reaction mixture via cannula under excess nitrogen pressure, followed 3 h later by the addition of a second end-capping agent (selected from G2). After the reaction, the crude reaction mixture was evaporated and passed through a gel filtration column to remove small organic molecules and low-MW oligomers. The crude polymer was then passed through a tangential flow filtration system equipped with a 100K MWCO membrane. It was washed with 20% ethanol until the filtrate showed low absorption. Example 3 Comparison of Fluorescence Emission Spectra

[0114] A comparison of the fluorescence emission spectra of fluorene (Fl-Fl), dihydrophenanthrene (DHP-DHP), and fluorene-DHP (DHP-Fl) polymers was performed. The DHP-containing polymers show a significant difference in their fluorescence maxima, which are 426–428 nm, while the fluorene-based polymers show a maximum at 421 nm (Figure 1). Example 4 Comparison of absorption spectra

[0115] The absorption spectra of both fluorene (Fl-Fl) and dihydrophenanthrene (DHP-DHP) polymers were measured. The graph shows the absorption of the DHP-DHP polymer (black curve) at 390 and 410 nm, while the Fl-Fl (gray curve) polymer exhibits a maximum at approximately 400 nm. The samples were measured at different concentrations (Figure 2). Example 5 CD4 Signal-to-Noise Ratio

[0116] New polymer-labeled anti-human CD4 and Pacific Blue-labeled anti-human CD4 Flow cytometry analysis of lysed whole blood stained with CD4 was performed, and the positive signal intensity of the polymeric dye was approximately 5-fold greater than that of Pacific Blue (Figure 3). Example 6

[0117] The polymers of the present invention, when conjugated to antibodies and the like, have been found to have certain physical and chemical properties of absorption, fluorescence, brightness, molecular weight, polydispersity, and dye-to-protein ratio. Preferred ranges for these parameters are shown in the table in Figure 4.

[0118] The excitation and emission spectra of the tandem polymers were measured: excitation was at the polymer maximum (405 nm), and emission was observed from the various acceptor dyes attached to the backbone (Figure 5).

[0119] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes therein will be suggested to those skilled in the art and are to be included within the spirit and scope of this application and within the scope of the appended claims. All publications, patents, and patent applications mentioned herein are incorporated herein by reference in their entirety for all purposes.

[0120] According to a preferred embodiment of the present invention, for example, the following is provided: (Section 1) A water-soluble fluorescent polymer having the structure of Formula I: [ka] During the ceremony, each X is independently selected from the group consisting of C and Si; Each Y is a bond, CR 1 R 2 , and SiR 1 R 2 are independently selected from the group consisting of When Y is a bond, X is directly attached to both rings; Each R 1are polyethylene glycol (PEG), ammonium alkyl salts, ammonium alkyloxy salts, ammonium oligoether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligoether salts, sulfonamide oligoethers, and [ka] are independently selected from the group consisting of Each R 2 is H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG group, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salts, sulfonamide oligoethers, and [ka] are independently selected from the group consisting of Each R 3 are independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and a PEG group; each Z is independently selected from the group consisting of C, O, and N; Each Q is a bond, NH, or NR 4 and CH2, Each M is an electron-rich linker unit that can independently modify the polymer bandgap and is uniformly or randomly distributed along the polymer backbone, and [ka] are each independently selected from the group consisting of: During the ceremony, Each R 4 is a non-ionic surfactant that can provide solubility in water of greater than 10 mg / mL side chain and halogen, hydroxyl, C1-C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, C3-C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 Alkoxy, C2-C 18 (Hetero)aryloxy, C2-C 18 (hetero)arylamino, each x' is independently an integer from 0 to 20, and each y' is independently an integer from 0 to 50 (CH2) x’ (OCH2-CH2) y’ OCH3 and C2~C 18 (hetero)aryl groups; each optional linker, L, is an aryl or heteroaryl group uniformly or randomly distributed along the polymer backbone and substituted with one or more pendant chains terminated with a functional group selected from the group consisting of amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and protected groups thereof, for linkage to another substrate, acceptor dye, molecule, or binder; G 1 and G 2are each independently selected from the group consisting of hydrogen, halogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, boronate-substituted aryl, boronate ester-substituted aryl, boronate ester, boronic acid, optionally substituted dihydrophenanthrene (DHP), optionally substituted fluorene, aryl or heteroaryl substituted with one or more pendant chains terminated with a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and protected groups thereof for linkage to a substrate or binder; a, c, and d define the mole % of each unit in said structure, which may each be uniformly or irregularly repeated, where a is 10-100% mole %, c is 0-90% mole %, and each d is 0-25% mole %; each b is independently 0 or 1; m is an integer from 1 to about 10,000; each n is independently an integer from 1 to 20; (Section 2) Item 1, wherein the polymer has a structure of formula II. [ka] . (Section 3) the polymer has the structure of Formula III, [ka] During the ceremony, Each f is independently an integer from 0 to 50, and each R 5 H, C1~C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, C3-C 12 Cycloalkyl, C1-C 12Haloalkyl, C1-C 12 Alkoxy, C2-C 18 (Hetero)aryloxy, C2-C 18 (Hetero)arylamino and C1-C 12 Item 1. The polymer according to item 1, wherein the alkoxy group is independently selected from the group consisting of: (Section 4) Item 1, wherein the polymer has a structure of formula IV. [ka] . (Section 5) Item 1, wherein the polymer has a structure of formula V. [ka] . (Section 6) the polymer is a copolymer and has the structure of Formula VI: [ka] Item 2. The polymer according to item 1, wherein g and a are both in mole percent of 10 to 100%. (Section 7) the polymer is a copolymer and has the structure of Formula VII: [ka] During the ceremony, Each of g and a is a mole percent of 10 to 100%; Each f is independently an integer from 0 to 50, and each R 5 H, C1~C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, C3-C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 Alkoxy, C2-C 18 (Hetero)aryloxy, C2-C 18 (Hetero)arylamino and C1-C12 Item 1. The polymer according to item 1, wherein the alkoxy group is independently selected from the group consisting of: (Section 8) Item 1, wherein the polymer is a copolymer and has a structure of formula VIII: [ka] . (Section 9) Item 1, wherein the polymer is a copolymer and has a structure of formula IX: [ka] . (Section 10) L, [ka] are each independently selected from the group consisting of: During the ceremony, Each R 6 is one or more halogens, hydroxyl, C1-C 12 Alkoxy, or (OCH2CH2) f H, OH, SH, NHCOO-t-butyl optionally substituted with OCH3, (CH2) n COOH, (CH2) n COOCH3, (CH2) n NH2, (CH2) n NH-(CH2) n -CH3, (CH2) n NHCOOH, (CH2) n NHCO-(CH2) n -CO-(CH2) n -CH3, (CH2) n NHCOO-(CH2) n -CH3, (CH2) n NHCOOC(CH3)3, (CH2) n NHCO(C3~C 12 ) cycloalkyl, (CH2) n NHCO(CH2CH2O) f , (CH2) nNHCO(CH2) n COOH, (CH2) n NHCO(CH2) n COO(CH2) n CH3, (CH2) n (OCH2CH2) f OCH3, N-maleimide, halogen, C2-C 12 Alkenes, C2-C 12 Alkynes, C3-C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 (Hetero)aryl, C1-C 12 (hetero)arylamino, and and benzyl; each f is independently an integer from 0 to 50; Item 1. The polymer according to item 1, wherein each n is independently an integer of 1 to 20. (Section 11) G 1 and G 2 are each independently selected from the group consisting of optionally substituted dihydrophenanthrene (DHP), optionally substituted fluorene, aryl substituted with one or more pendant chains terminated with functional groups, and heteroaryl substituted with one or more pendant chains terminated with functional groups. (Section 12) G 1 and G 2 but, [ka] are each independently selected from the group consisting of: During the ceremony, Each R 6 is one or more halogens, hydroxyl, C1-C 12 Alkoxy, or (OCH2CH2) f H, OH, SH, NHCOO-t-butyl optionally substituted with OCH3, (CH2) n COOH, (CH2) n COOCH3, (CH2) n NH2, (CH2)n NH-(CH2) n -CH3, (CH2) n NHCOOH, (CH2) n NHCO-(CH2) n -CO-(CH2) n -CH3, (CH2) n NHCOO-(CH2) n -CH3, (CH2) n NHCOOC(CH3)3, (CH2) n NHCO(C3~C 12 ) cycloalkyl, (CH2) n NHCO(CH2CH2O) f , (CH2) n NHCO(CH2) n COOH, (CH2) n NHCO(CH2) n COO(CH2) n CH3, (CH2) n (OCH2CH2) f OCH3, N-maleimide, halogen, C2-C 12 Alkenes, C2-C 12 Alkynes, C3-C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 (Hetero)aryl, C1-C 12 independently selected from the group consisting of (hetero)arylamino, and benzyl; each f is independently an integer from 0 to 50; Item 1. The polymer according to item 1, wherein each n is independently an integer of 1 to 20. (Section 13) Item 1, further comprising a binder linked to the polymer. (Section 14) Item 14. The polymer according to item 13, wherein the binding agent is an antibody. (Section 15) 1. A method for detecting an analyte in a sample, comprising: providing a sample suspected of containing said analyte; contacting the sample with a binder linked to a water-soluble polymer having the structure of Formula I, [ka] During the ceremony, each X is independently selected from the group consisting of C and Si; Each Y is a bond, CR 1 R 2 , and SiR 1 R 2 are independently selected from the group consisting of When Y is a bond, X is directly attached to both rings; Each R 1 are PEG, ammonium alkyl salts, ammonium alkyloxy salts, ammonium oligoether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligoether salts, sulfonamide oligoethers, and [ka] are independently selected from the group consisting of Each R 2 is H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG group, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, and [ka] are independently selected from the group consisting of Each R 3 are independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and a PEG group; each Z is independently selected from the group consisting of C, O, and N; Each Q is a bond, NH, or NR 4 and CH2, Each M is an electron-rich linker unit that can independently modify the polymer bandgap and is uniformly or randomly distributed along the polymer backbone, and [ka] are each independently selected from the group consisting of: During the ceremony, Each R 4 is a non-ionic side chain capable of imparting solubility in water of greater than 10 mg / mL, and is selected from halogen, hydroxyl, C1-C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, C3-C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 Alkoxy, C2-C 18 (Hetero)aryloxy, C2-C 18 (hetero)arylamino, each x' is independently an integer from 0 to 20, and each y' is independently an integer from 0 to 50 (CH2) x’ (OCH2-CH2) y’ OCH3 and C2~C 18 (hetero)aryl groups; each optional linker, L, is an aryl or heteroaryl group uniformly or randomly distributed along the polymer backbone and substituted with one or more pendant chains terminated with a functional group selected from the group consisting of amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and protected groups thereof, for linkage to another substrate, acceptor dye, molecule, or binder; G 1 and G 2 is hydrogen, halogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, each independently selected from the group consisting of an aryl or heteroaryl substituted with one or more pendant chains terminated with a functional group selected from triflate, acetyloxy, azide, sulfonate, phosphate, boronate-substituted aryl, boronate ester-substituted aryl, boronate ester, boronic acid, optionally substituted dihydrophenanthrene (DHP), optionally substituted fluorene, amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and protected groups thereof for linkage to a substrate or binder; a, c, and d define the mole % of each unit in said structure, which may each be uniformly or irregularly repeated, where a is 10-100% mole %, c is 0-90% mole %, and each d is 0-25% mole %; each b is independently 0 or 1; m is an integer from 1 to about 10,000; each n is independently an integer from 1 to 20; The method, wherein said binding agent is capable of interacting with said analyte or target-associated biomolecule. (Section 16) Item 16. The method according to item 15, wherein the binding agent is a protein, a peptide, an affinity ligand, an antibody, an antibody fragment, a sugar, a lipid, a nucleic acid, or an aptamer. (Section 17) 16. The method according to claim 15, wherein the binding agent is an antibody. (Section 18) 18. The method according to paragraph 17, wherein the method is configured for flow cytometry. (Section 19) 18. The method according to claim 17, wherein the binding agent is bound to a substrate. (Section 20) Item 18. The method according to item 17, wherein the specimen is a protein expressed on the cell surface. (Section 21) Item 18. The method according to item 17, wherein the method is configured as an immunoassay. (Section 22) 18. The assay method of claim 17, further comprising providing an additional binding agent for simultaneously detecting an additional analyte.

Claims

1. 1. A water-soluble fluorescent polymer having the structure of Formula I: 【Chemical 1】 During the ceremony, each X is independently selected from the group consisting of C and Si; each Y is a bond, where X is directly attached to both rings; Each R １ is a sulfonamide oligoether, and 【Chemistry 2】 are independently selected from the group consisting of Each R ２ is H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG group, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, and 【Chemistry 3】 are independently selected from the group consisting of Each R ３ are independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and PEG groups; Each Z is CH ２ , O, and NH; Each Q is a bond, NH, NR ４ , and C.H. ２ are independently selected from the group consisting of Each M is independently an electron-rich linker unit that can modify the polymer bandgap and is uniformly or randomly distributed along the polymer backbone, and 【Chemistry 4】 are each independently selected from the group consisting of: During the ceremony, Each R ４ is a non-ionic side chain that can confer solubility in water of greater than 10 mg / mL, and is selected from halogen, hydroxyl, C １ ~C １２ Alkyl, C ２ ~C １２ Alkene, C ２ ~C １２ Alkyne, C ３ ~C １２ Cycloalkyl, C １ ~C １２ Haloalkyl, C １ ~C １２ Alkoxy, C ２ ~C １８ (hetero)aryloxy, C ２ ~C １８ (hetero)arylamino, each x' is independently an integer from 0 to 20, and each y' is independently an integer from 0 to 50 (CH ２ ) ｘ’ (OCH ２ -CH ２ ) ｙ’ OCH ３ , and C ２ ~C １８ (hetero)aryl groups; each optional linker, L, is an aryl or heteroaryl group uniformly or randomly distributed along the polymer backbone and is substituted with one or more pendant chains terminated with a functional group selected from the group consisting of amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and protected groups thereof, for linkage to another substrate, receptor dye, or binder; G １ and G ２ are each independently selected from the group consisting of hydrogen, halogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, boronate-substituted aryl, boronate ester-substituted aryl, boronate ester, boronic acid, optionally substituted dihydrophenanthrene (DHP), optionally substituted fluorene, aryl or heteroaryl substituted with one or more pendant chains terminated with a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and protected groups thereof for linkage to a substrate or binder; a, c, and d define the mole % of each unit within said structure, which may each be uniformly or irregularly repeated, with a being 10-100% mole %, c being 0-90% mole %, and each d being 0-25% mole %; each b is independently 0 or 1; m is an integer from 1 to 10,000; A water-soluble fluorescent polymer, wherein each n is independently an integer from 1 to 20.

2. 2. The polymer of claim 1, wherein X is C.

3. The polymer of claim 1 , wherein the polymer has the structure of Formula II: 【Chemistry 5】 。

4. L, 【Chemistry 13】 are each independently selected from the group consisting of: During the ceremony, Each R ６ represents one or more halogens, hydroxyl, C １ ~C １２ Alkoxy, or (OCH ２ CH ２ ) ｆ OCH ３ H, OH, SH, NHCOO-t-butyl, optionally substituted with (CH ２ ) ｎ COOH, (CH ２ ) ｎ COOCH ３ , (CH ２ ) ｎ NH ２ , (CH ２ ) ｎ NH-(CH ２ ) ｎ -CH ３ , (CH ２ ) ｎ NHCOOH, (CH ２ ) ｎ NHCO-(CH ２ ) ｎ -CO-(CH ２ ) ｎ -CH ３ , (CH ２ ) ｎ NHCOO-(CH ２ ) ｎ -CH ３ , (CH ２ ) ｎ NHCOOC (CH ３ ) ３ , (CH ２ ) ｎ NHCO(C ３ ~C １２ ) cycloalkyl, (CH ２ ) ｎ NHCO(CH ２ ) ｎ COOH, (CH ２ ) ｎ NHCO(CH ２ ) ｎ COO (CH ２ ) ｎ CH ３ , (CH ２ ) ｎ (OCH ２ CH ２ ) ｆ OCH ３ , N-maleimide, halogen, C ２ ~C １２ Alkene, C ２ ~C １２ Alkyne, C ３ ~C １２ Cycloalkyl, C １ ~C １２ Haloalkyl, C １ ~C １２ (hetero)aryl, C １ ~C １２ independently selected from the group consisting of (hetero)arylamino, and benzyl; each f is independently an integer from 0 to 50; 2. The polymer of claim 1, wherein each n is independently an integer from 1 to 20.

5. G １ and G ２ are each independently selected from the group consisting of halogen-substituted aryl, boronic acid-substituted aryl, boronic ester-substituted aryl, aryl or heteroaryl substituted with one or more pendant chains terminated with a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and protected groups thereof, optionally linked to a substrate or a binder.

6. G １ and G ２ are each independently selected from the group consisting of optionally substituted dihydrophenanthrene (DHP), optionally substituted fluorene, aryl substituted with one or more pendant chains terminated with functional groups, and heteroaryl substituted with one or more pendant chains terminated with functional groups.

7. G １ and G ２ but, 【Chemistry 14】 are each independently selected from the group consisting of: During the ceremony, Each R ６ represents one or more halogens, hydroxyl, C １ ~C １２ Alkoxy, or (OCH ２ CH ２ ) ｆ OCH ３ H, OH, SH, NHCOO-t-butyl, optionally substituted with (CH ２ ) ｎ COOH, (CH ２ ) ｎ COOCH ３ , (CH ２ ) ｎ NH ２ , (CH ２ ) ｎ NH-(CH ２ ) ｎ -CH ３ , (CH ２ ) ｎ NHCOOH, (CH ２ ) ｎ NHCO-(CH ２ ) ｎ -CO-(CH ２ ) ｎ -CH ３ , (CH ２ ) ｎ NHCOO-(CH ２ ) ｎ -CH ３ , (CH ２ ) ｎ NHCOOC (CH ３ ) ３ , (CH ２ ) ｎ NHCO(C ３ ~C １２ ) cycloalkyl, (CH ２ ) ｎ NHCO(CH ２ ) ｎ COOH, (CH ２ ) ｎ NHCO(CH ２ ) ｎ COO (CH ２ ) ｎ CH ３ , (CH ２ ) ｎ (OCH ２ CH ２ ) ｆ OCH ３ , N-maleimide, halogen, C ２ ~C １２ Alkene, C ２ ~C １２ Alkyne, C ３ ~C １２ Cycloalkyl, C １ ~C １２ Haloalkyl, C １ ~C １２ (hetero)aryl, C １ ~C １２ independently selected from the group consisting of (hetero)arylamino, and benzyl; each f is independently an integer from 0 to 50; 2. The polymer of claim 1, wherein each n is independently an integer from 1 to 20.

8. The polymer of claim 1 further comprising a linking agent linked to the polymer.

9. The polymer of claim 8 , wherein the binding agent is an antibody.

10. A polymer according to any one of claims 1 to 7, comprising an acceptor dye linked to a linker L of the polymer.

11. The polymer of claim 10 further comprising a linking agent linked to the polymer.

12. 1. A method for detecting an analyte in a sample, comprising: providing a sample suspected of containing said analyte; A binder linked to a water-soluble polymer according to any one of claims 1 to 7 and 10. contacting the sample, The method, wherein said binding agent is capable of interacting with said analyte or target-associated biomolecule.

13. 13. The method of claim 12, wherein the binding agent is a protein, peptide, affinity ligand, antibody, antibody fragment, sugar, lipid, nucleic acid, or aptamer.

14. The method of claim 12 , wherein the binding agent is an antibody.

15. 15. The method of claim 14, wherein the method is configured for flow cytometry.

16. The method of claim 14 , wherein the binding agent is bound to a substrate.

17. 15. The method of claim 14, wherein the analyte is a protein expressed on the surface of a cell.

18. 15. The method of claim 14, wherein the method is configured as an immunoassay.

19. 15. The method of claim 14, wherein the method further comprises providing an additional binding agent for simultaneously detecting an additional analyte.

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