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 a fluorescence yield ranging from 10,000 to 70,000 M at 405 nm. -1 cm -1 The extinction coefficients of the chromophores are relatively small, ranging from 0.1 to 0.2. Molecules with multiple chromophores have been shown to exhibit larger ε values ​​due to the overall contributions from the various chromophores. There are various reports based on dendrimer and polymer backbone approaches, where a single molecule contains multiple chromophores.

[0005] However, most of the polymer dyes reported so far are highly hydrophobic and are used in materials applications such as light-emitting diodes and solar cells. For this reason, many polymer dyes are not useful under aqueous conditions due to poor solubility, brightness, and spectral breadth. Only a few reports have dealt with 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 for the detection of analytes.

[0006] The present invention addresses these and other disadvantages of prior art conjugates and methods for detecting an analyte 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 complex comprising a fluorescent polymer conjugated to a binding agent.

[0008] In a first embodiment, the present invention provides a water-soluble fluorescent polymer having a 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 glycols (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 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 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 C.H. 2 are independently selected from the group consisting of Each M is independently an electron rich linker unit capable of modifying the polymer band gap, 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, C 1 ~C 12 Alkyl, C 2 ~C 12 Alkenes, 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 that is uniformly or randomly distributed along the polymer backbone and can be linked to another substrate, a receptor dye, for conjugation to a molecule or a binding agent, is substituted with one or more pendant chains terminated with a functional group selected from the group consisting of amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and protected groups thereof; each G 1 and G 2 is hydrogen, halogen, alkyne, optionally substituted aryl, any optionally 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 optionally substituted dihydrophenanthrene (DHP), optionally 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 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 from 0 to 50, and each R 5 , H, C 1 ~C 12 Alkyl, C 2 ~C12 Alkenes, 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, and C 1 ~C 12 alkoxy.

[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 in a mole percentage ranging from 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 is a mole percent of 10 to 100%, each f is independently an integer of 0 to 50, and each R 5 , H, C 1 ~C 12 Alkyl, C 2 ~C 12 Alkenes, 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, and C 1 ~C 12 alkoxy.

[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, C 1 ~C 12 Alkoxy, or (OCH2 CH 2 ) f OCH 3 H, OH, SH, NHCOO-t-butyl, (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 CH 2 O) f , (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 Alkenes, 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; 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, C 1 ~C 12 Alkoxy, or (OCH 2 CH 2 ) f OCH 3 H, OH, SH, NHCOO-t-butyl, (CH 2 ) n COOH, (CH2 ) 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 CH 2 O) f , (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 Alkenes, C 2 ~C 12 Alkyne, C 3 ~C12 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; 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 2is H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, 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 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 C.H. 2 are independently selected from the group consisting of Each M is independently an electron rich linker unit capable of modifying the polymer band gap, 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, C 1 ~C 12 Alkyl, C 2 ~C 12 Alkenes, C 2 ~C 12 Alkyne, C 3 ~C 12 Cycloalkyl, C 1 ~C 12 Haloalkyl, C1 ~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 amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and protected groups thereof, for conjugation to another substrate, receptor 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 is a mole % of 10-100%, c is a mole % of 0-90%, Each d is a mole percentage between 0 and 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 capable of exciting the polymer to the sample 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 the 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 description 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] [Diagram 2] The absorption spectra of both 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 under different concentrations.

[0027] [Diagram 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 Pacific Blue.

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

[0029] [Diagram 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 the various acceptor dyes attached to the backbone: dye 1 - FITC, dye 2 - Cy3B, dye 3 - Cy55. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] I. Overview The present invention provides novel water-soluble fluorescent polymers and methods for detecting analytes in a sample using a complex comprising a fluorescent polymer conjugated to a binder. 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 arts.

[0032] As used herein, the term "ammonium" refers to a compound of the formula NHR 3 + 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 having ether functionality. As used herein, "oligomer" is understood to mean a molecule containing one or more identifiable structural repeat units of the same or different formula.

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

[0035] As used herein, the term "sulfonate" refers to a group of the formula -SO 2 It refers to the group NR--, 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, C 1 ~C 6Alkyl groups include, but are 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 groups can contain any number of carbons, from 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6, and 5 to 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 hydrogen atoms are replaced by halogen atoms. Halogen (halo) preferably represents chloride or fluoride, but may also be bromide or iodide. For example, haloalkyl includes trifluoromethyl, flouromethyl, 1,2,3,4,5-pentafluoro-phenyl, etc. The term "perfluoro" defines a compound or radical having at least two available hydrogens replaced by 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, with 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 a variety of substituents as described herein. For example, alkoxy groups can be substituted with halogens to form "halo-alkoxy" groups.

[0041] The term "alkene" as used herein refers to any straight 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 any straight 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" refers to a divalent radical derived from an aryl group. Aryl groups include alkyl, alkoxy, aryl, hydroxy, halogen, cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy and oxy-C. 2 -C 3-alkylene, which may be mono-, di- or tri-substituted by one, two or three radicals selected from 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-C 2 -C 3 -Alkylene is also a divalent substituent attached to two adjacent carbon atoms of phenyl, such as oxyethylene or oxypropylene. 2 -C 3 An example of an -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. The aryloxy group may be unsubstituted or substituted with one or two suitable substituents. The term "phenoxy" refers to an aryloxy group where 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, with 1 to 4 of the ring atoms being heteroatoms N, O or S, respectively. 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, in particular mono- or di-substituted, for example by alkyl, nitro or halogen. Pyridyl represents 2-, 3- or 4-pyridyl, advantageously 2- or 3-pyridyl. Thienyl represents 2- or 3-thienyl. Quinolinyl preferably represents 2-, 3- or 4-quinolinyl. Isoquinolinyl preferably represents 1-, 3- or 4-isoquinolinyl. Benzopyranyl, benzothiopyranyl preferably represent 3-benzopyranyl or 3-benzothiopyranyl, respectively. Thiazolyl preferably represents 2- or 4-thiazolyl, most preferably 4-thiazolyl. Triazolyl is preferably 1-, 2- or 5-(1,2,4-triazolyl). Tetrazolyl is preferably 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, in particular mono- or di-substituted, radical.

[0049] Similarly, the substituents on the aryl and heteroaryl groups are varied and include -halogen, -OR', -OC(O)R', -NR'R", -SR', -R', -CN, -NO, in numbers ranging from 0 to the total number of open valences on the aromatic ring system. 2 , -CO2 R',-CONR'R",- C(O)R', -OC(O)NR'R", -NR"C(O)R', -NR"C(O) 2 R',, -NR'-C(O)NR"R"', -NH-C(NH 2 )=NH, -NR'C(NH 2 )=NH, -NH-C(NH 2 )=NR', -S(O)R', -S(O) 2 R', -S(O) 2 NR'R", -N 3 , -CH(Ph) 2 , perfluoro(C 1 ~C 4 ) alkoxy, and perfluoro (C 1 ~C 4 ) alkyl, and R', R" and R"' are selected from hydrogen, (C 1 ~C 8 ) alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C 1 ~C 4 ) alkyl, and (unsubstituted aryl)oxy-(C 1 ~C 4 ) alkyl.

[0050] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring are of the formula -TC(O)-(CH 2 ) q T and U are optionally substituted with a substituent of -U-, where T and U are independently -NH-, -O-, -CH 2 - 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 are of the formula -A-(CH 2 ) r A and B are optionally substituted with a -B- substituent, and A and B are independently -CH 2 -, -O-, -NH-, -S-, -S(O)-, -S(O) 2 -, -S(O) 2NR'- or a single bond, where r is an integer from 1 to 3. One of the single bonds of 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 of the formula -(CH 2 ) s -X-(CH 2 ) t -, s and t are independently an integer from 0 to 3, and X is -O-, -NR'-, -S-, -S(O)-, -S(O) 2 - or -S(O) 2 -NR'- and -S(O) 2 The substituent R' in NR'- is hydrogen or an unsubstituted (C 1 ~C 6 ) alkyl.

[0051] The term "(hetero)arylamino" as used herein refers to an amine radical substituted with an aryl group (e.g., -NH-aryl). Arylamino also refers to an amine radical substituted with an amine group (e.g., -aryl-NH 2 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 attachment point with the remainder of the compound, or may link at least two carbon atoms of the alkyl group together. Those skilled in the art will appreciate that other alkylamines are useful in the present invention.

[0053] The term "carbamate" as used herein refers to a compound of the structure -NR"CO 2 R′ and R″ are hydrogen, (C 1 ~C 8 ) alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C 1 ~C 4 ) alkyl, and (unsubstituted aryl)oxy-(C 1 ~C 4 ) 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, 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", WA 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 that has a --CHO group.

[0058] The term "thiol" as used herein refers to a compound that contains 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 In the formula, each R z is independently alkylaryl or other carbon-containing group.

[0060] As used herein, the term "diazonium salt" refers to a compound having the structure RN 2 + 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" is also referred to as trifluoromethanesulfonate and has the formula CF 3 SO 3 It is a group having the formula:

[0062] The term "boronic acid" as used herein refers to a compound of the structure -B(OH) 2 It will be appreciated by those skilled in the art that boronic acids may be present as boronate esters at various stages in the synthesis of the quencher. Boronic acid is intended to include such esters. As used herein, the term "boronic acid ester" or "boronate ester" refers to -B(Z 1 )(Z 2 ) moiety, 1 and Z 2 together 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 band gap, distributed uniformly or randomly along the polymer backbone. These units are represented in formula I as M. The polymer conjugates of the present invention may also contain linkers, represented in formula I as L. Each optional linker L is an aryl or heteroaryl group distributed uniformly or randomly along the polymer backbone and substituted with one or more pendant chains terminated with a functional group selected from the group consisting of amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and protected groups thereof, for conjugation to substrates or binders.

[0065] The polymer conjugates of the present invention also include those having formula I, 1 and G 2 and each of these units is 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 due to 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 1 are 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 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 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; and each Q is a bond, NH, NR 4 and C.H. 2 and each R is independently selected from the group consisting of 5 , H, C 1 ~C 12 Alkyl, C 2 ~C 12 Alkenes, 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, and C 1 ~C 12 alkoxy.

[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 an Erlenmeyer flask (2000 L), add approximately 26 g of NaBH 4 is added to a stirring water-ethanol mixture (120 mL + 780 mL). To this solution, about 24 g of 2,7-dibromophenanthrene,9,10-dione is added in small portions but quickly (in 5 min). The reaction mixture is stirred for 1 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-OSO 3 H can be prepared as follows. In a 2-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 in 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 to DHP-OH (3.6 g) by dropping funnel over 20-30 min. The HP-OH solution was added. 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 obtain a white precipitate of DPS in the form of the disodium salt. The precipitate was filtered, redissolved in water (minimal amount), neutralized with HCl, and precipitated again in acetone. Repeated precipitation (2-3 times) followed by centrifugation yields DPS as a white solid.

[0077] DHP-OSO 2 Cl can be prepared as follows: 5 g of DHP-OSO 3 H was placed in a round-bottom flask and mixed with 25 mL of DMF. 2was 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: 2 Cl was mixed with 2.2 equivalents of PEG amine in a dichloromethane / TEA mixture. After 3 hours of sonication reaction, the crude product was extracted in dichloromethane followed by column chromatography (silica gel, MeOH-CHCl 3 ) was carried out.

[0079] The diboronic ester of DHP-sulfonamide PEG can be prepared as follows: The dibromo compound was mixed with DMSO under nitrogen, to which was added 3 equivalents of bis-pinacolatodiboron. The reagent was combined with 12 equivalents of potassium acetate and 4 equivalents of Pd(dppf)Cl. 2 The catalyst was reacted at 80°C for 5 hours. The reaction mixture was cooled and extracted with CHCl3 / water. Column chromatography (silica gel, MeOH-CHCl 3 The organic layer was concentrated and purified by HCl.

[0080] Similarly, the fluorene monomers of the present invention can be made as described below. For example, FL-OSO 3 H 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 min) and 50% NaOH (12 eq.) was added while continuing the nitrogen purge. The color of the solution changed from colorless to dark brown. Propane sultone (3 eq.) was weighed and dissolved in DMSO. This was added dropwise over 5 min to the fluorene reaction mixture. The reaction was stirred at room temperature for 4-5 h. The solvent was evaporated and the precipitate was dissolved in water. Acetone was added to give a white precipitate of DPS in the form of the disodium salt. The precipitate was filtered, redissolved in water (minimal amount), neutralized with HCl, and precipitated again in acetone. Repeated precipitation (2-3 times) followed by centrifugation gave FL-OSO as a white solid. 3 H is obtained.

[0081] FL-OSO 2 Cl can be prepared as follows: 5 g of FL-OSO 3 H was placed in a round-bottom flask and mixed with 25 mL of DMF. 2 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: 2 Cl was mixed with 2.2 equivalents of PEG amine in a dichloromethane / TEA mixture. After 3 hours of sonication reaction, the crude product was extracted in dichloromethane followed by column chromatography (silica gel, MeOH-CHCl 3 ) was carried out.

[0083] The diboronic ester of FL-sulfonamide PEG can be prepared as follows: The dibromo compound was mixed with DMSO under nitrogen, to which was added 3 equivalents of bis-pinacolatodiboron. The reagent was mixed with 12 equivalents of potassium acetate and 4 equivalents of Pd(dppf)Cl. 2 The catalyst was reacted at 80°C for 5 hours. The reaction mixture was cooled and CHCl 3 Extraction was performed with 100 mL of MeOH / water. Column chromatography (silica gel, MeOH-CHCl 3 The organic layer was concentrated and purified by HCl. polymerization

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

[0085] Generally, polymerizing the above-mentioned monomer units can be accomplished using polymerization techniques known to those of skill in the art or methods known in the art in combination with the methods described herein. For example, synthesis of diboronic ester derivatives from dihalide monomers can be accomplished 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, the polymerization can proceed as follows: In a round bottom flask, both bromo and boronic acid monomers were placed in a (DMF-water) mix and purged with nitrogen for 10 minutes. Under nitrogen, approximately 20 equivalents of CsF and 10% Pd(OAc)2 were mixed and heated at 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 isolate 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, the bromo ester and boronic ester capping units can be used to append one or both ends of the polymer. Utilizing both the bromo ester and boronic ester capping units will append both ends of the polymer. Utilizing only one form, i.e., either the bromo ester or boronic ester capping units, 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 the case of 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 added asymmetrically by first reacting a bromo capping unit with a polymer with 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 made 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, the binding agent is an antibody or a 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 other 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 the 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 Fragment, Fv, and Fab expression libraries are included. Complex

[0091] In general, the fluorescent polymers of the present invention can be conjugated to binding agents using techniques known to those of skill 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 esters can proceed as follows: 5 mg of polymer is placed in a clean vial and dissolved in 1 mL of dry CH 3Dissolve in CN. Add 15mg TSTU to this and stir for another 2 minutes. Add 100μL DIPEA to this and continue stirring overnight with the cap sealed with parafilm. The organic solvent in the reaction mixture is then evaporated and the crude NHS is dissolved in about 750μL 1x BBS buffer (pH 8.8) by brief vortexing and transferred to a Zeba column 40K MWCO. Spin down the sample at 2200RPM for 2 minutes and use this polymeric NHS immediately.

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

[0094] Purification of the conjugate by 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 the unbound fraction, which has both the conjugate and free antibody. This can be done using 10 CV of running 1x PBS with 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 and collect the unbound fraction. This can be done using 20 CV of buffer. Then change the buffer to elute the unbound fraction with both 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 100% HPLC-MS / MS column. Pool the tubes after checking the absorption spectrum and concentrate with an Amicon Ultra-15 with 30 KDa MWCO centrifugal concentrator. IV. Methods of Detecting Analytes overview

[0097] The present invention provides a method for detecting an analyte in a sample, the method 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 are excitable 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 method 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 cell subsets of whole blood (e.g., red blood cells, white blood cells, 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 from a cell culture.

[0099] The subject may be a human (e.g., a patient suffering from a disease), a commercially significant mammal, such as a monkey, cow, or horse. Samples may 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, a peptide, a nucleic acid, a lipid, a carbohydrate, or a small molecule.

[0101] A target analyte may be, for example, a nucleic acid (DNA, RNA, mRNA, tRNA, or rRNA), a peptide, a polypeptide, a protein, a lipid, an ion, a monosaccharide, an oligosaccharide, a polysaccharide, a lipoprotein, a glycoprotein, a glycolipid, or a fragment thereof. In some embodiments, a target analyte is a protein, 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. A protein may be an activatable protein or a protein that is differentially expressed or activated in diseased or abnormal cells, such as, 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 quantitate bound molecules are well known, and examples of such systems include flow cytometers, scanning cytometers, imaging cytometers, fluorescent microscopes, and confocal fluorescent 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 other embodiments, the assay is an immunoassay. Examples of immunoassays useful in the present invention include, but are not limited to, fluoroluminescence assay (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 up 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 in 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. Under nitrogen, about 20 equivalents of CsF and 10% Pd(OAc)2 were mixed and heated at 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 using 20% ​​ethanol until the filtrate had low absorption.

[0107] Method 2: Alternatively, polymerization can be achieved by self-polymerization of 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 The mixture was mixed and heated at 80° C. UV-Vis spectroscopy and SE The 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 had low absorption.

[0108] Method 3: In a round bottom flask, 10 equivalents of both dibromodihydrophenanthrene and diboronic acid dihydrophanenthrene monomers (1:1) were added. K 2 CO 3 and 3% Pd(PPh 3 ) 4 The reaction mixture was placed in a THF-water (4:1) mixture containing and dissolved. The reaction mixture was charged to a Schlenk line and degassed with three freeze-pump cycles, 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. It was washed with 20% ethanol until the filtrate had low absorption.

[0109] Method 4: Alternatively, polymerization can be achieved by self-polymerization of the bromo-boronic ester of the dihydrophenanthrene molecule. In a round-bottom flask, the dihydrophenanthrene bromoboronic ester is dissolved in 10 equivalents of K 2 CO 3 and 3% Pd(PPh 3 ) 4The reaction mixture was placed in a THF-water (4:1) mixture containing and dissolved. The reaction mixture was charged to a Schlenk line and degassed with three freeze-pump cycles, 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. It was washed with 20% ethanol until the filtrate had low absorption. Example 2: Preparation of Fluorene-DHP Copolymer Conjugate [ka]

[0110] Method 1: In a round bottom flask, both dibromo DHP and diboronic acid fluorene monomers (1:1) were placed in a (DMF-water) mixture and purged with nitrogen for 10 minutes. Under nitrogen, about 20 equivalents of CsF and 10% Pd(OAc)2 were mixed and heated at 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 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 and diboronic acid DHP monomers (1:1) were placed in a (DMF-water) mixture and purged with nitrogen for 10 minutes. Under nitrogen, about 20 equivalents of CsF and 10% Pd(OAc)2 were mixed and heated at 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 using 20% ​​ethanol until the filtrate had low absorption.

[0112] Method 3: In a round-bottom flask, both dibromodihydrophenanthrene and diboronic acid fluorene monomers (1:1) were mixed with 10 equivalents of K 2 CO 3 and 3% Pd(PPh 3 ) 4 The reaction mixture was placed in a THF-water (4:1) mixture containing and dissolved. The reaction mixture was charged to a Schlenk line and degassed with three freeze-pump cycles, 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. It was washed with 20% ethanol until the filtrate had low absorption.

[0113] Method 4: In a round-bottom flask, dibromofluorene monomer and dihydrophenanthrene diboronate monomer (1:1) were added to 10 equivalents of K 2 CO 3and 3% Pd(PPh3)4 in a THF-water (4:1) mixture and dissolved. The reaction mixture was charged to a Schlenk line and degassed with three freeze-pump cycles, 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. It was washed with 20% ethanol until the filtrate had 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 between 426 and 428 nm, while the fluorene-based polymer shows 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 (grey curve) polymer shows a maximum at approximately 400 nm. The samples were measured under different concentrations (Figure 2). Example 5 CD4 signal-to-noise ratio

[0116] Anti-human CD4 labeled with a new polymer and Pacific Blue labeled Flow cytometric 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 invention, when conjugated to antibodies and the like, have been found to have consistent physical and chemical properties of absorption, fluorescence, brightness, molecular weight, polydispersity, dye-to-protein ratio, and preferred ranges for these parameters are shown in the table of FIG.

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

[0119] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various improvements or modifications therein will be suggested to one 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 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 1 are polyethylene glycols (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 is 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 C.H. 2 are independently selected from the group consisting of Each M is independently an electron rich linker unit capable of modifying the polymer band gap, 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 capable of imparting solubility in water of greater than 10 mg / mL. side chain and halogen, hydroxyl, C 1 ~C 12 Alkyl, C 2 ~C 12 Alkenes, C 2 ~C 12 Alkyne, C3 ~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 substituted with one or more pendant chains terminated with a functional group selected from the group consisting of amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and protected groups thereof, for linkage to another substrate, receptor 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 from 10 to 100% mole %, c is from 0 to 90% mole %, and each d is from 0 to 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) The polymer according to 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, C 1 ~C 12 Alkyl, C 2 ~C 12 Alkenes, C 2 ~C 12 Alkyne, C3 ~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, and C 1 ~C 12 Item 2. The polymer according to item 1, wherein the aryl group is independently selected from the group consisting of alkoxy. (Section 4) The polymer according to claim 1, wherein the polymer has a structure of formula IV. [ka] . (Section 5) The polymer according to 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] 2. The polymer according to item 1, wherein g and a are both in a mole percentage 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 percentage ranging from 10 to 100%; Each f is independently an integer from 0 to 50, and each R 5 , H, C 1 ~C 12 Alkyl, C 2 ~C 12 Alkenes, C 2 ~C 12 Alkyne, C3 ~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, and C 1 ~C 12 Item 2. The polymer according to item 1, wherein the aryl group is independently selected from the group consisting of alkoxy. (Section 8) Item 1, wherein the polymer is a copolymer and has a structure of formula VIII: [ka] . (Section 9) The polymer according to 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, C 1 ~C 12 Alkoxy, or (OCH 2 CH 2 ) f OCH 3 Optionally substituted H, OH, SH, NHCOO-t-butyl, (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 CH 2 O) f , (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 Alkenes, 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 (Hetero)arylamino, and and benzyl; each f is independently an integer from 0 to 50; 2. 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 an optionally substituted dihydrophenanthrene (DHP), an optionally substituted fluorene, an aryl substituted with one or more pendant chains terminated with a functional group, and a heteroaryl substituted with one or more pendant chains terminated with a functional group. (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, C 1 ~C 12 Alkoxy, or (OCH 2 CH 2 ) f OCH 3 Optionally substituted H, OH, SH, NHCOO-t-butyl, (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 CH 2 O) f , (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 Alkenes, 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 according to item 1, wherein each n is independently an integer of 1 to 20. (Section 13) 2. The polymer of claim 1, further comprising a linking agent linked to the polymer. (Section 14) 14. The polymer according to claim 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 is 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 C.H. 2 are independently selected from the group consisting of Each M is independently an electron rich linker unit capable of modifying the polymer band gap, 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, C 1 ~C 12 Alkyl, C 2 ~C 12 Alkenes, 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 substituted with one or more pendant chains terminated with a functional group selected from the group consisting of amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and protected groups thereof, for linkage to another substrate, receptor 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 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 from 10 to 100% mole %, c is from 0 to 90% mole %, and each d is from 0 to 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 of claim 15, wherein the binding agent is an antibody. (Section 18) 18. The method according to claim 17, wherein the method is configured for flow cytometry. (Section 19) 18. The method of claim 17, wherein the binding agent is bound to a substrate. (Section 20) 18. The method according to claim 17, wherein the specimen is a protein expressed on the cell surface. (Section 21) 18. The method according to claim 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

【Claim 1】 Photoactivity.

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