UV-absorbing polymer, composition and use thereof

Abstract

The present disclosure provides a method for detecting an analyte in a sample by using a UV-absorbing polymer dye and a binding partner conjugated to the UV-absorbing polymer dye. A composition is provided that includes a UV-absorbing polymer dye, a UV-absorbing tandem dye, or a quenched UV polymer dye. The present disclosure provides novel UV-excitable (e.g., 355 nm) polymer dyes, polymer tandem dyes, polymer dye conjugates, and polymer tandem dye conjugates. The present disclosure also provides a method for detecting an analyte in a sample using the polymer dyes and polymer dye conjugates, for example, by flow cytometry. A composition is also provided that includes a UV polymer dye, a UV polymer tandem dye, a UV polymer conjugate, and / or a UV polymer tandem dye conjugate.

Description

[Technical Field]

[0001] This application claims the interests and priority of U.S. Provisional Patent Application No. 63 / 183,862 filed on May 4, 2021, and U.S. Provisional Patent Application No. 63 / 306,946 filed on February 4, 2022, which were filed as PCT International Patent Applications on May 3, 2022, and which are incorporated herein by reference in their entirety. [Background technology]

[0002] background Polymers that absorb ultraviolet ("UV") light 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.

[0003] However, many of the UV-absorbing polymers reported to date are highly hydrophobic. Many UV-absorbing polymer dyes are unusable under aqueous conditions due to poor solubility, low brightness, and spectral broadening. Therefore, the availability of UV-absorbing polymer dyes for biological applications, including the detection of analytes, remains insufficient. [Overview of the Initiative] [Means for solving the problem]

[0004] Summary of this disclosure This disclosure provides novel UV-excitable (e.g., 355 nm) polymer dyes, polymer tandem dyes, polymer dye conjugates, and polymer tandem dye conjugates. The disclosure also provides a method for detecting analytes in a sample using polymer dyes and polymer dye conjugates, for example, by flow cytometry. Compositions comprising UV polymer dyes, UV polymer tandem dyes, UV polymer conjugates, and / or UV polymer tandem dye conjugates are also provided.

[0005] The present disclosure provides a UV-absorbing polymer having the structure of Formula I: [Chemical Formula] [wherein each X is independently selected from the group consisting of C and Si, and each Y is a bond, CR , R 2 , CHR 1 , CHR 2 , SiHR 2 , SiHR 1 and SiR 1 R 2 independently selected from the group consisting of, and when Y is a bond, X is directly bonded to both rings, and each R 1 is a water-solubilizing moiety, a linker moiety, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, (hetero)aryloxy, (hetero)arylamino, aryl, heteroaryl, polyethylene glycol (PEG) group, carboxylic acid, alkylammonium salt, alkyloxyammonium salt, oligoetherammonium salt, alkylsulfonate, alkoxysulfonate, sulfonamido oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamido, [Chemical Formula] [Chemical Formula] independently selected from the group consisting of, and each R 2This includes the water-soluble portion, the linker portion, H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, heteroaryl, (hetero)arylamino, PEG group, sulfonamide-PEG, phosphoramide-PEG, alkylammonium salt, alkyloxyammonium salt, oligoetherammonium salt, alkyl sulfonate, alkoxysulfonate, oligoethersulfonate, sulfonamide oligoether, sulfonamide, sulfinamide, phosphoamidate, phosphinamide [ka] Independently selected from the group consisting of, each R 3 Z is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, water-soluble moiety, and PEG group, and each Z is CH2, CHR 4 , O, NH and NR 4 Independently selected from the group consisting of, each Q is a combination of NH, NR 4 , C1~C 12 Alkylene, CHR 4 and CH2 are independently selected from the group, and each R 4 H, PEG group, water-soluble portion, linker portion, chromophore, linked chromophore, functional group, linked functional group, substrate, linked substrate, binding partner, linked binding partner, quenching portion, L 2 -E, halogen, hydroxyl, C1~C 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylamino, (CH2) x’ (OCH2-CH2) y’ Ure 9 (R9 (where C1-C8 alkyl is, each x' is an independent integer between 0 and 20, and each y' is an independent integer between 0 and 50), Z-(CH2) n -SO2-QR 3 , C2~C 18 (Hetero)aryl groups, amides, amines, carbamates, carboxylic acids, carboxylic acid esters, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazones, azides, aldehydes, thiols and their protected forms are independently selected from the group, each W 1 This is independently a water-soluble portion, L 1 , L 2 and L 3 Each is an independently selected linker moiety, and each E is independently selected from the group consisting of a chromophore, linked chromophore, functional moiety, linked functional moiety, substrate, linked substrate, binding partner, and linked binding partner, and each R 7 H, hydroxyl, C1-C 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylamino, C2~C 12 Carboxylic acids, C2-C 12 Carboxylic acid esters and -OC1~C 12 Independently selected from the group consisting of hydroxyl, R 1 , R 2 , R 3 or R 4 At least one of them includes a water-soluble portion, and each M 1 R is further substituted as needed. 4 and / or arylene substituted with trifluoromethyl, further substituted as needed, R 4and / or heteroarylenes substituted with trifluoromethyl, further substituted as needed, R 4 Independently selected from the group consisting of 9,10-dihydrophenanthrene substituted with and / or trifluoromethyl, and binaphthyl substituted as needed, each M 2 R is further substituted as needed. 4 and / or arylene substituted with trifluoromethyl, further substituted as needed, R 4 and / or heteroarylenes substituted with trifluoromethyl, further substituted as needed, R 4 Independently selected from the group consisting of 9,10-dihydrophenanthrene substituted with and / or trifluoromethyl, and binaphthyl substituted as needed, M 2 M 1 It has a different structure, M 2 and M 1 L is distributed evenly or randomly along the polymer backbone, and each linker L as needed is an aryl or heteroaryl group distributed evenly or randomly along the polymer backbone, and L is substituted as needed, G 1 and G 2 [where a, c, d, and e are independently selected from the group consisting of unmodified and modified polymer ends, which are conjugated to E as needed, and a, c, d, and e define the mol% of each unit in the structure which can be repeated evenly or randomly along the polymer backbone, where a is 10 to 100% (mol%), c is >0 to 90% (mol%), each d is 0 to 90% (mol%), each e is 0 to 25% (mol%), each b is independently 0 or 1, each f is independently an integer from 0 to 50, m is an integer from 1 to about 10,000, each n is independently an integer from 1 to 20, s is 1 or 2, and t is 0, 1, 2, or 3].

[0006] UV-absorbing polymer dyes having the structure of formula I may have a near-ultraviolet excitation spectrum and / or absorption maximum in the range of about 300 nm to about 400 nm or about 350 nm to about 400 nm. Near-UV-absorbing polymer dyes having the structure of formula I may be water-soluble polymers. In some cases, the UV-absorbing polymer contains at least one water-soluble group.

[0007] The units in the UV-absorbing polymer structure represented by Formula I can occur in any preferred order within the polymer backbone, including random orders such as the same or different orders as shown in Formula I. 1 and M 2 The units can be randomly distributed at alternating positions throughout the polymer backbone. 1 and M 2 The units can be present in the UV-absorbing polymer in any preferred molar ratio to one another. Each L may be independently substituted with one or more pendant chains terminated with functional groups selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and their protected groups, for conjugation to another substrate, acceptor dye, molecule, or binding partner.

[0008] In various embodiments, this disclosure relates to Formula I: [ka] The present invention provides a UV-absorbing polymer having the following structure. Each X is independently selected from C and Si. Each Y is bonded to CR. 1 R 2 , CHR1, CHR2 and SiR 1 R 2 Selected independently from each other, if Y is a bond, then X is directly bonded to both rings. 1is selected independently from polyethylene glycol (PEG), PEG group, alkylammonium salt, alkyloxyammonium salt, oligoetherammonium salt, alkylsulfonate, alkoxysulfonate, sulfonamido oligoether, -Z-(CH2) n -SO2-Q-R 3 、-Z-(CH2) n -SO2-NH-R 3 and -Z-(CH2) n -SO2-N(R 4 )-R 3 Each R<000​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylamino, (CH2) x’ (OCH2-CH2) y’ OCH3 (each x' is an independent integer between 0 and 20, and each y' is an independent integer between 0 and 50), -Z-(CH2) n -SO2-QR 3 and C2~C 18 The (hetero)aryl group is independently selected. Each modification unit M 1 and M 2 This can be independently selected from arylene or heteroarylene, which allows for modification of the polymer's band cap. 1 R is further substituted as needed. 4 and / or arylene substituted with trifluoromethyl, further substituted as needed, R 4 and / or heteroarylenes substituted with trifluoromethyl, further substituted as needed, R 4 9,10-dihydrophenanthrene substituted with trifluoromethyl and / or binaphthyl, which may be substituted as needed, are independently selected. 2 R is further substituted as needed. 4 and / or arylene substituted with trifluoromethyl, further substituted as needed, R 4 and / or heteroarylenes substituted with trifluoromethyl, further substituted as needed, R 4 Independently selected from 9,10-dihydrophenanthrene substituted with trifluoromethyl and / or binaphthyl substituted as needed, M 2 M 1It has a different structure. Each linker L is an aryl or heteroaryl group evenly or randomly distributed along the polymer backbone. Each L can be replaced by one or more pendant chains terminated with functional groups selected from, for example, amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols and their protected groups, for conjugation to another substrate, acceptor dye, molecule or binding partner.

[0009] G 1 and G 2 These are independently selected from the group consisting of unmodified polymer ends and modified polymer ends. In some examples, the variable element G 1 and G 2 Each of these may be independently selected from hydrogen, halogens, alkynes, halogen-substituted aryls, silyls, diazonium salts, triflates, acetyloxys, azides, sulfonates, phosphates, boronic acid-substituted aryls, boronic acid ester-substituted aryls, boronic acid esters, boronic acids, optionally substituted aryls, optionally substituted heteroaryls, optionally substituted dihydrophenanthrene (DHP), or optionally substituted fluorenes, wherein the optionally substituted aryls, heteroaryls, fluorenes, or DHPs may be substituted by one or more pendant chains terminated with functional groups selected from, for example, amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxylsuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and their protected groups, for conjugation to a substrate or binding partner.

[0010] Variables a, c, d, and e define the mol% of each unit in the structure, which can be repeated evenly or randomly along the polymer backbone, respectively, where a is 10–100% (mol%), c is >0–90% (mol%), each d is 0–90% (mol%), and each e is 0–25% (mol%). Each b is independently 0 or 1. Variable m is an integer from 1 to approximately 10,000. Each n is an integer from 1 to 20.

[0011] In some examples, M 1 R is further substituted as needed. 4 and / or arylenes mono-, d-, t-, or tetra-substituted with trifluoromethyl; further substituted as needed, R 4 and / or heteroarylenes mono-, d-, t-, or tetra-substituted with trifluoromethyl; further substituted as needed, R 4 9,10-dihydrophenanthrenes mono-, d-, t- or tetra-substituted with trifluoromethyl; and may be independently selected from binaphthyl, which may be substituted as needed. In some examples, each M 2 R is further substituted as needed. 4 and / or arylenes mono-, d-, t-, or tetra-substituted with trifluoromethyl; further substituted as needed, R 4 and / or heteroarylenes mono-, d-, t-, or tetra-substituted with trifluoromethyl; further substituted as needed, R 4 9,10-dihydrophenanthrenes mono-, d-, t- or tetra-substituted with trifluoromethyl; and may be independently selected from binaphthyl, as needed. In some examples, M 2 M 1 It has a different structure.

[0012] Each linker section is L, L 1 , L 2 and L 3 They may be independently selected from the group consisting of the following.

[0013] In some examples, the connecting part L 1 , L 2 and L 3 Independently, but not limited to, covalent bonds, C 1~8 The linker may be an alkylene or a 2- to 8-membered heteroalkylene. In some embodiments, the linker is a single atom, linear, branched, or cyclic portion. In some embodiments, the linker is a chain of main chain atoms (e.g., carbon atoms) between 2 and 100 in length, such as main chain atoms between 2 and 50 in length, or main chain atoms between 2 and 20 in length. In certain cases, one, two, three, four, or five, or more carbon atoms in the linker main chain may be replaced as needed by sulfur, nitrogen, or oxygen. The bonds between the main chain atoms may be saturated or unsaturated, and typically there are one, two, or three or fewer unsaturated bonds in the linker main chain. The linker may contain one or more substituents (e.g., alkyl or aryl groups). The linker may, non-limitingly, contain oligo(ethylene glycol); ether; thioether; tertiary amine; and alkylene groups (i.e., divalent alkyl radicals), which may be linear or branched. The linker backbone may contain cyclic groups, such as divalent aryl radicals, divalent heterocyclic radicals, or divalent cycloalkyl radicals, in which case two or more atoms of the cyclic group, for example, two, three, or four atoms, are included in the backbone.

[0014] In some examples, L 1 This includes sulfonamides, sulfonimides, saltum, disulfinamides, amides, phosphonamides, phosphoamidates, phosphinamides, selenoonamide, seleninamde, or secondary amines. In some embodiments, L 1 This includes sulfonamides, amides, phosphoamides, or secondary amines. In some cases, L 1 L 2 -E is the linker section terminated as needed. In some cases, L 2 This is linear or branched saturated or unsaturated C 1~30Contains an alkylene group, C 1~30 One or more carbon atoms in the alkylene group are O, S, NR a It is replaced as needed and independently by C 1~30 Grouping of two or more adjacent carbon atoms in an alkylene is, as needed and independently, -NR a (CO)- or -(CO)NR a - is replaced by each R a H and C 1~6 Selected independently of alkyl, each R a H and C 1~6 It is selected independently of alkyl.

[0015] In some examples, L 2 The linker moiety is optionally terminated with a functional group moiety selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and their protected groups, for conjugation to a chromophore, substrate, or binding partner.

[0016] In some examples, L 3 Covalent bond, C 1~8 Alkylenes, 2-8 member heteroalkylenes (e.g., divalent alkoxylinkers such as -O-alkyl), C 3~8 Cycloalkylene, C 6~10 Arirenes, 5-12 member heteroarirenes, 5-12 member heterocyclylenes, amines, -NHC(O)L a -, -C(O)NHL a -, -C(O)L a -and selected from the group consisting of combinations thereof, L a C 1~8 The material is selected from the group consisting of alkylenes and heteroalkylenes with 2 to 8 members.

[0017] In some cases, L 1 , L 2 and L 3Together, the following: [ka] [In the formula, R 8 R 4 , a hydrogen or amine protecting group, L 1a This is the linker section. Forms L 1a Covalent bond, C 1~8 Alkylene, C 1~8 Alkoxy, 2-8 member heteroalkylenes (e.g., divalent alkoxylinkers), C 3~8 Cycloalkylene, C 6~10 Arirene, 5-12 member heteroarirene, 5-12 member heterocycline, -NHC(O)L a -, -C(O)NHL a -, -C(O)L a - and combinations thereof are selected from the group. In some embodiments, L 1a Covalent bond, C 1~8 Alkylene, 2-8 member heteroalkylene, -NHC(O)L a -, -C(O)NHL a -and -C(O)L a - Selected from the group consisting of these.

[0018] In some examples, L 3 is the first L 1 Part (or the first L) 1a The first connection point to the part, the second L 1 Part (or the second L) 1a A trivalent arylalkyl moiety having a second bonding site to the (mole) and a third bonding site to the A monomer. For example, some embodiments of the present disclosure are of formula XIII: [ka] [In the formula, L 3a Covalent bond, C 1~8 Alkylene, 2-8 member heteroalkylene, -NHC(O)La -, -C(O)NHL a -and -C(O)L a - Selected from the group consisting of, L 1a C 1~8 Alkylene or 2-8 member heteroalkylene, W 1 This is the water-soluble portion, The wavy lines indicate the binding points to monomer A, and each of E and L 2 [These are, independently, as described above in this specification.] The present invention provides conjugated polymers having two or more E groups, such as chromophores bonded as shown in [reference].

[0019] In some examples, each E is an independently selected chromophore (e.g., and an independently selected fluorophore). In some embodiments, all E moieties in the polymer have the same fluorophore structure. In some embodiments, all E moieties in the polymer have different fluorophore structures.

[0020] In some examples, W 1 The water-soluble moiety is selected from ethylene glycol, PEG group, carboxyl group (including carboxylic acids and carboxylates, but not limited to these), polyvinyl alcohol, glycol, peptide, polyphosphate, polyalcohol, sulfonate, phosphonate, boronate, amine, ammonium, sulfonium, phosphonium, alcohol, polyol, oxazoline, zwitterionic derivative, carbohydrate, nucleotide, polynucleotide, substituted PEG group, substituted carboxyl group (including substituted carboxylic acids and substituted carboxylates, but not limited to these), substituted glycol, substituted peptide, substituted polyphosphate, substituted polyalcohol, substituted sulfonate, substituted phosphonate, substituted boronate, substituted amine, substituted ammonium, substituted sulfonium, substituted phosphonium, alcohol, substituted zwitterionic derivative, substituted carbohydrate, substituted nucleotide, substituted polynucleotide, or a combination thereof.

[0021] In some cases, W1 It contains one or more ethylene glycol monomers. In some cases, W 1 It contains a PEG group.

[0022] In some examples, variable element G 1 and G 2 Each of these may be independently selected from hydrogen, halogens, alkynes, halogen-substituted aryls, silyls, diazonium salts, triflates, acetyloxys, azides, sulfonates, phosphates, boronic acid-substituted aryls, boronic acid ester-substituted aryls, boronic acid esters, boronic acids, optionally substituted aryls, optionally substituted heteroaryls, optionally substituted dihydrophenanthrene (DHP), or optionally substituted fluorenes, wherein the optionally substituted aryls, heteroaryls, fluorenes, or DHPs may be substituted by one or more pendant chains terminated with functional groups selected from, for example, amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxylsuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and their protected groups, for conjugation to a substrate or binding partner.

[0023] In some cases, each L as needed, [ka] [ka] [In the formula, Each R 6 H, OH, SH, NHCOO-t-butyl, (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 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 (hetero)aryl, C1~C 12 (hetero)arylamino, optionally substituted benzyl, halogen, hydroxyl, C1-C 12 Alkoxy, (OCH2CH2) f OCH3, [ka] Independently selected from the group consisting of, W 1 This is the water-soluble portion, R 3 , R 4 , R 7 [Z, Q, f, n, s, and t are each of the above.] This is a linker portion that is independently selected from the group consisting of the following.

[0024] In some examples, this disclosure is based on formula XIV: [ka] [In the formula, R 2 , R 3 , G 1 , G 2 Each of L, Q, X, Y, Z, a, b, c, e, n, and m is independently as described herein. Each R 4’ These are F, Cl, -CH3, -CF3 and -(OCH2CH2) f Ure 9 Selected independently from each R 4’’ These are F, Cl, -CH3, -CF3 and -(OCH2CH2) f Ure 9 Selected independently from, R 9 [where is a C1-C8 alkyl group, f is 0-50 or 10-20, each o is an integer independently selected from 1, 2, 3 or 4, and each p is an integer independently selected from 1, 2, 3 or 4] The present invention provides a UV-absorbing polymer. The units in the polymer structure represented by formula XIV can exist in any preferred or random order, such as the same or different order, within the polymer main chain, as shown in formula XIV. 1 and M 2 The units can be randomly distributed at alternating positions throughout the polymer backbone. In some examples, UV-absorbing polymers according to formula XIV have near-ultraviolet excitation spectra and / or absorption maxima in the range of 300 nm to 400 nm or 350 nm to 400 nm.

[0025] In some cases, the present disclosure provides copolymers comprising the structure of formula (I) as previously defined.

[0026] In some embodiments, the disclosure provides a polymer tandem dye comprising a UV-absorbing polymer dye having the structure of formula (I) as previously defined, and a signal transduction chromophore covalently bonded to the UV-absorbing polymer dye near its energy receptor.

[0027] In some cases, the disclosure provides labeled binding partners, including a UV-absorbing polymer dye having the structure of formula (I) as previously defined, and a binding partner covalently bonded to the UV-absorbing polymer dye.

[0028] In various embodiments, the Disclosure provides a method for detecting an analyte in a sample. The method includes the step of bringing a sample suspected to contain an analyte into contact with a binding partner capable of interacting with the analyte, which is conjugated to a UV-absorbing polymer (polymer conjugate) according to the Disclosure. In some cases, the UV-absorbing polymer conjugate is made of formula I: [ka] [In the formula, X, Y, G 1 , G 2 , R 1 , R 2 M 1 M 2 L, a, b, c, d, and e are each individually defined in this disclosure. It includes the structure.

[0029] In some cases, X is selected independently from C and Si, and each Y is a bond, CHR1, CHR2, CR 1 R 2 and SiR 1 R 2 Selected independently from each other, if Y is a bond, then X is directly bonded to both rings. 1 This is polyethylene glycol (PEG), PEG group, alkylammonium salt, alkyloxyammonium salt, oligoetherammonium salt, alkyl sulfonate, alkoxysulfonate, sulfonamide oligoether, linked chromophore, -Z-(CH2) n -SO2-QR 3 ,-Z-(CH2) n -SO2-NH-R 3 and -Z-(CH2) n -SO2-N(R 4 )-R 3Selected independently from each R. 2 H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG group, alkylammonium salt, alkyloxyammonium salt, oligoetherammonium salt, alkyl sulfonate, alkoxysulfonate, oligoethersulfonate, sulfonamide oligoether, and -Z-(CH2) n -SO2-QR 3 Selected independently from each R. 3 Z is independently selected from H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and PEG groups. Each Z is CH2, CHR 4 , O, NH and NR 4 Selected independently from each other. Each Q is a combination of NH and NR. 4 , CHR 4 , C1~C 12 Selected independently from alkylene and CH2. Each R 4 These are chromophores, halogens, hydroxyls, and C1-C 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylamino, (CH2) x’ (OCH2-CH2) y’ OCH3 (each x' is an independent integer between 0 and 20, and each y' is an independent integer between 0 and 50), -Z-(CH2) n -SO2-QR 3 and C2~C 18 The (hetero)aryl group is independently selected. Each modification unit M 1 and M 2 This can be independently selected from arylene or heteroarylene, which allows for modification of the polymer's band cap.1 R is further substituted as needed. 4 and / or arylene substituted with trifluoromethyl, further substituted as needed, R 4 and / or heteroarylenes substituted with trifluoromethyl, further substituted as needed, R 4 9,10-dihydrophenanthrene substituted with trifluoromethyl and / or binaphthyl, which may be substituted as needed, are independently selected. 2 R is further substituted as needed. 4 and / or arylene substituted with trifluoromethyl, further substituted as needed, R 4 and / or heteroarylenes substituted with trifluoromethyl, further substituted as needed, R 4 Independently selected from 9,10-dihydrophenanthrene substituted with trifluoromethyl and / or binaphthyl substituted as needed, M 2 M 1 It has a different structure. Each linker L is an aryl or heteroaryl group evenly or randomly distributed along the polymer backbone and is substituted by one or more pendant chains terminated with functional groups selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols and their protected groups, for conjugation to another substrate, acceptor dye, molecule, or binding partner.

[0030] In some examples, variable element G 1 and G 2Each of these may be independently selected from hydrogen, halogens, alkynes, halogen-substituted aryls, silyls, diazonium salts, triflates, acetyloxys, azides, sulfonates, phosphates, boronic acid-substituted aryls, boronic acid ester-substituted aryls, boronic acid esters, boronic acids, optionally substituted aryls, optionally substituted heteroaryls, optionally substituted dihydrophenanthrene (DHP), or optionally substituted fluorenes, wherein the optionally substituted aryls, heteroaryls, fluorenes, or DHPs may be substituted by one or more pendant chains terminated with functional groups selected from, for example, amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxylsuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and their protected groups, for conjugation to a substrate or binding partner.

[0031] Variables a, c, d, and e define the mol% of each unit in the structure, which can be repeated uniformly or randomly along the polymer backbone, respectively, where a is 10–100% (mol%), c is >0–90% (mol%), each d is 0–90% (mol%), and each e is 0–25% (mol%). Each b is independently 0 or 1. Variable m is an integer from 1 to approximately 10,000. Each n is an integer from 1 to 20. The binding partners can interact with the analyte or target-binding biomolecule.

[0032] The UV-absorbing polymers of this disclosure can exhibit good absorption in the near-ultraviolet (UV) region of the spectrum (e.g., 350-400 nm or 355-375 nm), for example at approximately 355 nm, without excitation at approximately 405 nm or reduced excitation at approximately 405 nm, compared to other polymers that exhibit absorption at approximately 355 nm. This reduces / eliminates spillover in the pacific blue channel (450 ± 25 nm). The UV-absorbing polymers of this disclosure can have good absorption at 355 nm and 375 nm and can therefore be used in instruments equipped with 355 nm and 375 nm lasers.

[0033] This disclosure provides compositions comprising a UV-absorbing polymer dye, a UV-absorbing tandem polymer dye, or a quenched UV polymer dye and a nonionic surfactant for reducing or preventing nonspecific interactions between polymer dye conjugates. The UV-absorbing polymer dye, UV-absorbing tandem polymer dye, or quenched UV polymer dye may be a polymer dye conjugate. The UV-absorbing polymer dye, UV-absorbing tandem polymer dye, or quenched UV polymer dye may be a water-soluble UV-absorbing polymer dye. The UV-absorbing polymer dye, UV-absorbing tandem polymer dye, or quenched UV polymer dye may be a polymer dye according to this disclosure.

[0034] A kit comprising the composition according to this disclosure is also provided, comprising a container containing the composition and, optionally, at least one or more fluorescent polymer dye conjugates. The kit may contain the composition in one container and at least one or more fluorescent polymer dye conjugates in another container.

[0035] The drawings are generally illustrative examples and are not intended to limit the various embodiments of this disclosure. [Brief explanation of the drawing]

[0036] [Figure 1]Figure 1 illustrates a comparison of the signal-to-noise ratio in a 405 nm channel of different lots (B, C, D) of the UV-absorbing polymer according to this disclosure, conjugated with a CD4 antibody, compared to a competitive BUV395-CD4 antibody conjugate (A) (Becton Dickinson Biosciences) after laser excitation at 355 nm. The UV polymer dye conjugate according to this disclosure shows an improvement in the signal-to-noise ratio compared to the competing product.

[0037] [Figure 2] Figure 2 illustrates a comparison of the signal-to-noise ratio in the 740 / 40 nm channel of the UV polymer-DY704 tandem dye CD4 antibody conjugate of the present invention, according to various aspects of this disclosure, compared with a competitive BUV737-CD4 conjugate after laser excitation at 355 nm.

[0038] [Figure 3] Figure 3 shows flow cytometry (FCA) dot plots of stained and dissolved blood samples in the presence of various concentrations of UV-absorbing polymer, with or without 1% PF-68. The upper left panel shows blood only, without additives; the upper right panel shows blood with 10 ug of quenched polymer added; the four panels in the center column (from left to right) show blood with 2.5 ug, 5 ug, 10 ug, and 20 ug of UV-absorbing polymer added; and the four panels at the bottom (from left to right) show blood with 1% PF-68 and 2.5 ug, 5 ug, 10 ug, and 20 ug of UV-absorbing polymer added. Monocyte MFI values ​​show that UV polymers with or without 1% PF-68 (2.5 ug to 20 ug / test) did not show strong nonspecific binding to cells compared to control cells (blood only panel, upper left).

[0039] [Figure 4]Figure 4 shows FCA dot plots illustrating the effects of UV-absorbing polymers and other components of the staining buffer composition in stained and lysed human whole blood samples containing a mixture of two polymer dye conjugates according to this disclosure: CD20-UV excitable polymer dye (UVEPD) and CD4-UV408. The Y-axis represents the FL1 channel, and the X-axis represents the UV405 channel. Cells stained in the presence of the combined UV polymer (5–20 μg / test) + 1% PF-68 (the three lower panels) showed less spillover than the control, including samples without buffer (upper left panel), 1% PF-68 (upper center panel), and UV polymer alone (upper right panel). The values ​​in each panel represent the MFI values.

[0040] [Figure 5] Figure 5 shows an FCA dot plot illustrating the effects of UV-absorbing polymers and other components of the staining buffer composition on stained and lysed human whole blood samples containing mixtures of two types of polymer dye conjugates: CD20-SN v428 (Beckman Coulter Life Sciences) and CD4-BV650 (Becton Dickinson Biosciences). The Y-axis represents V450-PB channels, and the X-axis represents V660 channels. Cells stained in the presence of the combined UV polymer (5–20 μg / test) + 1% PF-68 (lower left, lower center, and lower right panels) showed better separation than the control, including samples without buffer (upper left), 1% PF-68 (upper center), and UV polymer alone (10 ug / test; upper right). The values ​​in each panel represent the MFI values.

[0041] [Figure 6]Figure 6 shows FCA dot plots of stained and lysed whole blood cells illustrating the effect of various concentrations of nonionic surfactant (0.1–1% PF-68 / test) in the presence of 10 ug of the UV polymer according to this disclosure in a staining buffer composition containing a mixture of two polymer dye conjugates: CD20-SN v428 (Beckman Coulter Life Sciences) and CD4-BV650 (Becton Dickinson Biosciences). The Y-axis represents V450-PB channels, and the X-axis represents V660 channels. Cells stained in the presence of the combined UV polymer (10 ug / test) + various concentrations (0.1–1% / test) of PF-68 (lower left, lower center, and lower right panels) showed better separation than the control, including samples without buffer (upper left panel), 1% PF-68 (upper center panel), and UV polymer alone (10 ug / test; upper right panel). The values ​​in each panel represent the MFI values.

[0042] [Figure 7] Figure 7 shows the emission spectra of the three quenched UV polymers according to this disclosure after excitation at 355 nm. The quenching agent-to-polymer ratio (D / P) was determined to be 2.5, 5, and 10. The quantum yields (QY) at 405 nm for quenched polymer 1 (D / P=2.5), quenched polymer 2 (D / P=5.0), and quenched polymer 3 (D / P=10) are 0.072, 0.030, and 0.003, respectively. The quantum yield for the non-quenched polymer is 0.739.

[0043] [Figure 8]Figure 8 shows an FCA dot plot of blood cells stained and lysed using a mixture of two different commercially available polymer dye conjugates: CD20-SN v428 (Beckman Coulter Life Sciences) and CD4-BV650 (BD Biosciences). The Y-axis represents V450-PB channels, and the X-axis represents V660 channels. Stained cells containing no additives (lower left panel), containing 1% PF-68 (upper left panel), containing a combined 10ug of UV polymer + 1% PF-68 (center left panel), containing quenched polymer 1 (QY=0.072) at 5ug, 10ug, or 20ug / test with 1% PF-68 (second upper, center, and lower panels from the left, respectively), containing quenched polymer 2 (QY=0.03) at 5ug, 10ug, or 20ug / test with 1% PF-68 (second upper, center, and lower panels from the right, respectively), and containing quenched polymer 3 (QY=0.003) at 5ug, 10ug, or 20ug / test with 1% PF-68 (upper right, center, and lower panels, respectively). Cells stained in the presence of a test staining buffer composition containing combined UV polymer (10 μg / test) + 1% PF-68 (left center panel), or cells stained in the presence of quenched polymers 1, 2, and 3 at 5 ug, 10 ug, or 20 ug / test with 1% PF-68 (right three columns, upper, center, and lower panels, respectively), each showed better separation than the control without additives (left lower panel). The values ​​in each panel represent the MFI values.

[0044] [Figure 9]Figure 9 shows FCA dot plots of cells stained and lysed with a mixture of CD4-BV650 (BD Biosciences) and CD19-SNv428 (Beckman Coulter Life Sciences). The Y-axis represents V450-PB channels, and the X-axis represents V660 channels. Cells stained with no buffer (left panel), 0.1% PF-68 (second panel from the left), 0.5% PF-68 (second panel from the right), and 1% PF-68 (weight / volume) (right panel). The presence of various concentrations of PF-68 (0.1–1% weight / volume) is associated with a reduction in nonspecific interactions in the mixture, as evidenced by the improved separation compared to the absence of PF-68.

[0045] [Figure 10] Figure 10 shows an FCA dot plot of cells stained and lysed with a mixture of two polymer dye conjugates: CD4-violet excitable polymer dye 1 (CD4-VEPD1) and CD19-violet excitable polymer dye 2 (CD19-VEPD2) (both from Beckman Coulter Life Sciences). The Y-axis represents FL3 channels and the X-axis represents FL2 channels. Cells were stained without buffer (top panel), with a combined staining buffer (UV polymer composition + PF-68), and with Empigen at various concentrations (0, 0.03%, 0.05%, and 0.07%) (center panel: left, second from the left, second from the right, and right, respectively). The presence of various concentrations of Empigen in the staining buffer did not affect the performance of the staining buffer. Lower panel: The MFI values ​​of monocytes show that nonspecific binding to cells is reduced when various concentrations of Empigen are added to the staining buffer (lower panel: left to right) compared to the control sample (lower left panel).

[0046] [Figure 11]Figure 11 shows an exemplary partial scheme for preparing a UV-absorbing polymer according to the present disclosure, showing that two different modification units M1 (e.g., difluorophenylene) and M2 (e.g., trifluorophenylene) are randomly distributed at alternating positions relative to repeating DHP units. [Modes for carrying out the invention]

[0047] Detailed explanation Hereafter, certain embodiments of the disclosed subject matter will be described in detail, with some examples illustrated in the accompanying drawings. The disclosed subject matter is described in relation to the enumerated claims, but it should be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0048] Throughout this specification, values ​​expressed in range form should be interpreted flexibly to include not only the numerical values ​​explicitly listed as limits to the range, but also all the individual numerical values ​​or subranges contained within that range, as if each numerical value and subrange were explicitly listed. For example, the range "approximately 0.1% to approximately 5%" or "approximately 0.1% to approximately 5%" should be interpreted not simply as approximately 0.1% to approximately 5%, but also as including the individual values ​​(e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, and 3.3% to 4.4%) within the indicated range. The statement "approximately X to Y" has the same meaning as "approximately X to approximately Y" unless otherwise specified. Similarly, the statement "approximately X, Y, or approximately Z" has the same meaning as "approximately X, approximately Y, or approximately Z" unless otherwise specified.

[0049] In this specification, the terms “a,” “an,” or “the” are used to mean one or more unless the context specifically indicates otherwise. The term “or” is used to mean non-exclusive “or” unless otherwise specified. The phrases “at least one of A and B” or “at least one of A or B” are synonymous with “A, B, or A and B.” Furthermore, it should be understood that any expressions or technical terms used herein and not specifically defined are for illustrative purposes only and not for limiting purposes. Any use of item headings is intended to aid in the reading of this specification and should not be construed as limiting. Information relating to an item heading may appear within or outside that particular item. All publications, patents, and patent documents referenced herein are incorporated herein by reference in whole, as if each were incorporated by reference individually. In the event of any inconsistency between usage in this specification and in any document so as to be incorporated by reference, the usage in the incorporated reference should be considered to supplement the usage in this specification, and in the event of an irreconcilable conflict, the usage in this specification shall prevail.

[0050] In the methods described herein, the acts may be performed in any order without departing from the principles of this disclosure, unless a temporal or operational order is explicitly enumerated. Furthermore, certain acts may be performed concurrently unless explicitly stated in the claim language that they are performed separately. For example, the claimed act of performing X and the claimed act of performing Y may be performed concurrently within a single operation, and the resulting process is within the literal scope of the claimed process.

[0051] The term “about” as used herein means a value or range that may allow variation of, for example, within 10%, 5%, or 1% of the specified value or specified boundary value of the range, including the specified value or range. The term “substantially” as used herein means the majority or most of, such as at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999%, or higher, or at 100%. The term "substantially unincorporated," as used herein, can mean having nothing, or having such a small amount of material that the amount of material present does not affect the material properties of the composition containing the material, i.e., about 0% to about 5% by weight, or about 0% to about 1% by weight, or about 5% by weight or less, or about 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01% by weight or less, or about 0% by weight being the material.

[0052] In this specification, the terms “part” and “base” shall be used interchangeably.

[0053] Unless otherwise specified, the term "room temperature" refers to a temperature between 18 and 27 degrees Celsius.

[0054] Unless otherwise specified, the term "weight percentage" or "weight %" refers to weight percentage per volume.

[0055] The terms "ready-to-use reagent," "ready-to-use reagent composition," "working-grade concentrated reagent," and "working-grade concentrated reagent composition" refer to, for example, staining buffer compositions prepared at a working concentration of approximately 1× suitable for use in a mixture of polymer dye conjugates for staining biological samples for flow cytometry analysis (FCA).

[0056] The term "protecting group" (also called "protected group") refers to a reversibly formed derivative of a functional group present in a molecule, which is attached to reduce its reactivity. As a result, the protected functional group does not react under the synthetic conditions to which the molecule is provided. Typical amine protecting groups can include carbamates such as tert-butyloxycarbonyl (Boc), benzyloxycarbamate (CBz), or fluorenylmethyloxycarbonyl (Fmoc) protecting groups.

[0057] The expression "concentrated staining buffer" or "concentrated staining buffer composition" refers to a staining buffer composition prepared with a concentration factor of approximately 10x for dilution using a diluent such as biological buffer or water, to obtain a working concentrated staining buffer composition useful for reducing nonspecific polymer interactions in multicolor panels when staining biological samples for flow cytometry analysis. A concentrated staining buffer composition can be prepared and stable at concentrations from 1x to at least 10x, or at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x higher than a working concentrated staining buffer composition. A working concentrated staining buffer composition is stable for at least 3 months, 6 months, 9 months, or at least 12 months, or longer, from the date of manufacture when stored in its original unopened container at temperatures within the range of 2–40°C, 2–30°C, or 2–8°C. The concentrated staining buffer composition is stable for at least 3 months, 6 months, 9 months, 12 months, or at least 18 months or longer from the date of manufacture, when stored in its original unopened container at temperatures within the range of 2-40°C, 2-30°C, or 2-8°C.

[0058] The acronym "SN" stands for SuperNova (trademark).

[0059] The acronym "SSC" refers to lateral scattering.

[0060] The term "WBC" refers to white blood cells.

[0061] The term "quantum yield" (QY)(Φ) or "fluorescence quantum yield" refers to the ratio of the number of emitted photons to the number of absorbed photons. Quantum yield is independent of instrument settings and describes how efficiently a fluorophore converts excitation energy into fluorescence. Experimentally, relative fluorescence quantum yield can be determined by measuring the fluorescence of a fluorophore with a known quantum yield using the same experimental parameters (excitation wavelength, slit width, photomultiplier tube voltage, etc.) as the test dye. Quantum yield can be determined by any method known in the art. For example, QY can be determined at a selected excitation wavelength using a fluorescence spectrophotometer or fluorescence spectrometer according to the manufacturer's instructions. For example, quantum yield (QY) can be determined using a Shimadzu Rf-6000 fluorescence spectrophotometer by measuring the emission intensity at a predetermined wavelength (nm) from a diluted PBS solution of a staining buffer having absorbance at a specific excitation wavelength, under specified conditions (e.g., excitation slit 1.5, emission slit 3.0, 1 cm quartz cuvette). The quantum yield can be calculated, for example, by comparing the intensity measured from the sample with the intensity measured from a standard dye solution under identical experimental conditions. In some embodiments, QY can be determined, for example, according to Lawson-Wood et al., Application Note - Fluorescence Spectroscopy, Determination of relative fluorescence quantum yield using the FL5600 fluorescence spectrometer, 2018, PerkinElmer, Inc. The selected excitation wavelength may be, for example, 355 nm. In some embodiments, the QY of the quenched polymer may be compared with that of a pro-fluorescent polymer that does not contain the quenched moiety.

[0062] The term "substrate," as used herein, refers to a reagent, medium, surface, substance, or material on which a molecule is bound internally or to its surface, or on which a reaction may occur. Substrates can have a variety of configurations, such as solids, fibrous materials, or gels. Substrates include, but are not limited to, solid substrates, such as solid supports including particles (e.g., magnetic particles), beads, sheets, plates with wells, fibrous meshes, hydrogels, porous matrices, pins, microarray surfaces, and chromatography supports.

[0063] The term “binding partner,” as used herein, refers to one of a pair of molecules that have binding specificity to each other (e.g., as well as to antibodies and analytes). A binding partner specifically binds to other molecules to form a binding complex. Any polymer dye or polymer tandem dye described herein can be conjugated to the binding partner at any convenient position on the dye and the binding partner. For example, the binding partner can be conjugated to the polymer dye (G) via a functional group. 1 or G 2 ) can be conjugated to the terminal group on the above.

[0064] The terms “nonspecific interaction” or “nonspecific binding,” as used herein, generally refer to any binding not caused by specific binding, and more specifically, to the binding of polymer dye conjugates by means other than the specific binding of the binding partner to the target analyte. Nonspecific binding can be due to several factors, including the hydrophobicity of the polymer, immunocompounds, charged proteins, and antibody-interfering proteins that may be present in the staining buffer or biological sample. One type of nonspecific binding is polymer-polymer interaction, which can occur between one or more, or two or more, fluorescent polymer dye conjugates. Nonspecific binding in a test staining buffer composition can be evaluated, for example, by comparing an FCA dot plot of a mixture of multicolor fluorescent polymer dye conjugates in a biological sample with an FCA dot plot of individual monocolor fluorescent polymer dye conjugates in the same sample, according to the method provided herein, or by determining the R ratio. For example, if the solution is efficient at inhibiting polymer-polymer interactions through nonspecific binding, each cell population appears to be sufficiently offset, as well as stained using the monochromatic conjugates used individually. In contrast, if the solution is inefficient, the populations will appear disaligned and tilted in the flow cytometry analysis dot plot.

[0065] An alternative method for measuring the efficiency of the staining buffer compositions according to this disclosure for reducing nonspecific binding such as polymer-polymer interactions is to use MFI of the negative and positive populations of the conjugate, both when they are used individually and when they are used in combination.

[0066] The term "organic group," as used herein, refers to any carbon-containing functional group. Examples include oxygen-containing groups such as alkoxy groups, aryloxy groups, aralkyloxy groups, and oxo(carbonyl) groups; carboxyl groups, including carboxylic acids, carboxylates, and carboxylic acid esters; sulfur-containing groups such as alkyl sulfide groups and aryl sulfide groups; and other heteroatom-containing groups. Non-limiting examples of organic groups include OR, OOR, OC(O)N(R)2, CN, CF3, OCF3, R, C(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, O(O)R, C(O)N(R)2, O(O)N(R)2, C(S)N(R)2, (CH2) 0~2 N(R)C(O)R, (CH2) 0~2 N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N( R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, C(=NOR)R, and substituted or unsubstituted (C1~C 100 ) contains hydrocarbyl, where R can be hydrogen (in examples containing other carbon atoms) or a carbon-based portion, which may be substituted or unsubstituted.

[0067] The term "substituted," as used herein in relation to a molecule or organic group as defined herein, means a state in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms.

[0068] The terms “functional group” or “substituent” as used herein refer to a group that may be substituted or is substituted on a molecule or organic group. Examples of substituents or functional groups include, but are not limited to, halogens (e.g., F, Cl, Br, and I), oxygen atoms in groups such as hydroxyl groups, alkoxy groups, aryloxy groups, aralkyloxy groups, and oxo(carbonyl) groups, sulfur atoms in groups such as carboxyl groups including carboxylic acids, carboxylates, and carboxylic acid esters, thiol groups, alkyl sulfide groups and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups, nitrogen atoms in groups such as amines, hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines, as well as other heteroatoms in various other groups. Non-limiting examples of substituents that can bond to the substituted carbon (or other) atom include F, Cl, Br, I, OR, OCO(O)N(R)2, CN, NO, NO2, ONO2, azide, CF3, OCF3, R, O(oxo), S(thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OCO(O)R, C(O)N(R)2, OCO(O)N(R)2, C(S)N(R)2, (CH2) 0~2 N(R)C(O)R, (CH2) 0~2 N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)con(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, and C(=NOR)R contain, where R can be a hydrogen or carbon-based part, for example, R is hydrogen, (C1~C 100) can be hydrocarbyl, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl or heteroarylalkyl, or two R groups bonded to a nitrogen atom or adjacent nitrogen atoms can combine with the nitrogen atom(s) to form a heterocyclyl. Examples of functional groups include, but are not limited to, amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols and their protected groups for conjugation to another substrate, acceptor dye, molecule or binding partner.

[0069] The term “alkyl” as used herein refers to linear and branched alkyl groups, as well as cycloalkyl groups, having 1 to 40 carbon atoms, 1 to about 20 carbon atoms, 1 to 12 carbon atoms, or, in some embodiments, 1 to 8 carbon atoms. Examples of linear alkyl groups include those having 1 to 8 carbon atoms, such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. As used herein, the term “alkyl” encompasses n-alkyl, isoalkyl, and anteisoalkyl groups, as well as other branched forms of alkyl. Typical substituted alkyl groups may be substituted once or more times with any of the groups listed herein, such as amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.

[0070] The term “alkenyl,” as used herein, refers to linear, branched, and cyclic alkyl groups as defined herein, except that at least one double bond is present between two carbon atoms. Thus, alkenyl groups have 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12 carbon atoms, or in some embodiments, 2 to 8 carbon atoms. Examples include, but are not limited to, vinyl, -CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl.

[0071] As used herein, the term "alkynyl" refers to linear and branched alkyl groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have 2 to 40 carbon atoms, 2 to about 20 carbon atoms, or 2 to 12 carbon atoms, or in some embodiments, 2 to 8 carbon atoms. Examples include, but are not limited to, -C≡CH, -C≡C(CH3), -C≡C(CH2CH3), -CH2C≡CH, -CH2C≡C(CH3), and -CH2C≡C(CH2CH3).

[0072] The term “acyl,” as used herein, refers to a group containing a carbonyl moiety, bonded via a carbonyl carbon atom. The carbonyl carbon atom may be bonded to a hydrogen atom to form a “formyl” group, or to another carbon atom, which can be part of alkyl, aryl, aralkylcycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl, etc. An acyl group may contain 0 to about 12, 0 to about 20, or 0 to about 40 additional carbon atoms bonded to the carbonyl group. An acyl group may contain double or triple bonds within the scope of its meaning as herein. The acryloyl group is an example of an acyl group. An acyl group may also contain heteroatoms within the scope of its meaning as herein. The nicotinoyl group (pyridyl-3-carbonyl) is an example of an acyl group within the scope of its meaning as herein. Other examples include acetyl, benzoyl, phenylacetyl, pyridylacetyl, cinnamoyl, and acryloyl groups. When a group containing a carbon atom bonded to a carbonyl carbon atom also contains a halogen, the group is called a "haloacyl" group. An example is the trifluoroacetyl group.

[0073] The term "cycloalkyl," as used herein, refers to cyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups, but is not limited to those listed below. In some embodiments, cycloalkyl groups may have 3 to about 8 to 12 ring members, while in other embodiments, the number of ring carbon atoms is in the range of 3 to 4, 5, 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups such as norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and calenyl groups, but is not limited to those listed below, as well as fused rings such as dekalinyl, but is not limited to those listed below. Cycloalkyl groups also include rings substituted with linear or branched alkyl groups as defined herein. Typical substituted cycloalkyl groups may be monosubstituted or, without limitation, 2,2-, 2,3-, 2,4-, 2,5-, or 2,6-disubstituted cyclohexyl groups, or monosubstituted, disubstituted, or trisubstituted norbornyl or cycloheptyl groups, which may be substituted with, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. The term "cycloalkenyl" alone or in combination refers to a cyclic alkenyl group.

[0074] The term "aryl," as used herein, refers to a cyclic aromatic hydrocarbon group that does not contain heteroatoms in the ring. That is, aryl groups include, but are not limited to, phenyl, benzyl, azlenyl, heptarenyl, biphenyl, indacenyl, fluorenyl, phenantrenyl, triphenylenyl, pyrenyl, naphthacenyl, crisenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, the aryl group contains about 6 to about 14 carbon atoms in the ring portion of the group. The aryl group may be unsubstituted or substituted as defined herein. Typical substituted aryl groups are monosubstituted or may be more than once substituted, such as a phenyl or benzyl group substituted at one or more of the 2, 3, 4, 5, or 6 positions of the phenyl ring or benzyl, or a naphthyl group substituted at one or more of the 2-8 positions. For example, a benzyl group that is optionally substituted may contain halogens, hydroxyls, or C1-C 12 Alkoxy, PEG group, (OCH2CH2) f OCH3, [ka] This may be replaced as needed.

[0075] The term "arirene," as used herein, refers to a cyclic aromatic hydrocarbon group that does not contain heteroatoms in the ring and is a divalent group derived from an aryl group by the removal of hydrogen atoms from two ring carbon atoms. In some embodiments, the arirene may be phenylene, dihydrophenanthrene, fluorene, or binaphthyl group. In some examples, the arirene may be 9,10-dihydrophenanthrene. In some examples, the arirene is phenylene. In some examples, the arirene is 1,4-phenylene. In some examples, the arirene is 1,3-phenylene. In some examples, the heteroarirene may be carbazole or oxepine. In some examples, the arirene is not a biphenyl group. In some examples, the arirene is not a sulfonyldibenzene group. In some cases, the arirene may be optionally substituted. For example, optionally substituted arirenes may be halogens, hydroxyls, C1-C 12 Alkoxy, PEG group, (OCH2CH2) f OCH3, [ka] It may be replaced by this.

[0076] The term "aralkyl," as used herein, refers to an alkyl group as defined herein, in which a hydrogen or carbon bond of the alkyl group is replaced by a bond to an aryl group as defined herein. Typical aralkyl groups include benzyl and phenylethyl groups, as well as condensed (cycloalkylaryl) alkyl groups such as 4-ethyl-indanyl. An aralkenyl group is an alkenyl group as defined herein, in which a hydrogen or carbon bond of the alkyl group is replaced by a bond to an aryl group as defined herein.

[0077] The term "heteroaryl," as used herein, refers to a monocyclic, fused bicyclic, or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, in which case 1 to 4 of the ring atoms are heteroatoms such as N, O, or S. For example, heteroaryls include pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imadozolyl, thienyl, or any other radical that is monosubstituted or disubstituted, particularly those substituted by alkyl, nitro, or halogen. Pyridyl represents 2-, 3-, or 4-pyridyl, such as 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. Benzopyranil and benzothiopyranil preferably represent 3-benzopyranil or 3-benzothiopyranil, respectively. Thiazolyl preferably represents 2- or 4-thiazolyl, for example, 4-thiazolyl. Triazolyl preferably is 1-,2-, or 5-(1,2,4-triazolyl). Tetrazolyl preferably is 5-tetrazolyl.

[0078] Preferably, the heteroaryl is any of the following substituted radicals: pyridyl, indolyl, quinonyl, pyrrolyl, thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl, benzothienyl, oxazoyl (oxazoy1), indazolyl, or monosubstituted or disubstituted.

[0079] Substituents of aryl and heteroaryl groups are in the range of 0 to the total number of free valencies on the aromatic ring system, including -halogen, -OR', -OC(O)R', -NR'R'', -SR', -R', -CN, -NO2, -CO2R', -CONR'R'', -C(O)R', -OC(O)NR'R'', -NR''C(O)R', -NR''C(O)2R', -NR'-C(O)NR''R'', -NH-C(CH2)=NH, -NR'C(NH2)=NH, and -NH-C(NH2)= The following can be selected from NR', -S(O)R', -S(O)2R', -S(O)2NR'R'', -N3, -CH(Ph)2, 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.

[0080] Two substituents on adjacent atoms of an aryl ring or heteroaryl ring may, if necessary, be of the formula -TC(O)-(CH2) q It may be replaced by a substituent of -U-, where T and U are independently -NH-, -O-, -CH2- or a single bond, and q is an integer between 0 and 2. Alternatively, two substituents on adjacent atoms of an aryl ring or heteroaryl ring may be of the formula -A-(CH2) r -B- may be replaced as needed by substituents, where A and B are independently -CH2-, -O-, -NH-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'- or single bonds, and r is an integer from 1 to 3. One of the single bonds in the new ring thus formed may be replaced as needed by a double bond. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may be of the formula -(CH2) s -X-(CH2) tThe substituents R' in -NR'- and -S(O)2NR'- are selected from hydrogen or unsubstituted (C1-C6) alkyl groups.

[0081] The term "(hetero)arylamino," as used herein, refers to an amine radical substituted with an aryl group (e.g., -NH-aryl). An arylamino may also be an aryl radical substituted with an amine group (e.g., -aryl-NH2). An arylamino may be substituted or unsubstituted.

[0082] The term "alkoxy," as used herein, refers to an oxygen atom bonded to an alkyl group, including cycloalkyl groups, as defined herein. Examples of linear alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentyloxy, and hexyloxy. Examples of branched alkoxy groups include, but are not limited to, isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, and isohexyloxy. Examples of cyclic alkoxy groups include, but are not limited to, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy. An alkoxy group may contain about 1 to about 12, about 1 to about 20, or about 1 to about 40 carbon atoms bonded to an oxygen atom, and may further contain double or triple bonds, and may also contain heteroatoms. For example, an allyloxy group or a methoxyethoxy group is also an alkoxy group within the meaning of this specification, such as a methylenedioxy group in which two adjacent atoms of a certain structure are substituted by methylenedioxy groups.

[0083] The term "amine," as used herein, refers to primary, secondary, and tertiary amines having, for example, formula N(group)3, where each group can independently be H or non-H such as alkyl or aryl. Amines include, but are not limited to, R-NH2, e.g., alkylamines, arylamines, alkylarylamines; R2NH (each R independently selected) (e.g., dialkylamines, diarylamines, aralkylamines, heterocyclylamines), and R3N (each R independently selected) (e.g., trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines). The term "amine," as used herein, also includes ammonium ions.

[0084] The term "amino group" as used herein refers to the forms -NH2, -NHR, -NR2, and -NR3. + (Each R is selected independently), and their respective protonation forms (Non-protonable -NR3) + This refers to substituents (excluding ). Therefore, any compound substituted with an amino group can be considered an amine. Within the scope of the meaning of this specification, “amino group” can be a primary, secondary, tertiary, or quaternary amino group. The “alkylamino” group includes monoalkylamino, dialkylamino, and trialkylamino groups.

[0085] The term "carbamate," as used herein, refers to a functional group having the structure -NR''CO2R', where R' and R'' are independently selected from hydrogen, (C1-C8) alkyl and heteroalkyl groups, unsubstituted aryl and heteroaryl groups, (unsubstituted aryl)-(C1-C4) alkyl groups, and (unsubstituted aryl)oxy-(C1-C4) alkyl groups. Examples of carbamates include t-Boc, Fmoc, benzyloxycarboxyl, alloc, methyl carbamate, ethyl carbamate, 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, Tbfmoc, Climoc, Bimoc, DBD-Tmoc, Bsmoc, Troc, Teoc, 2-phenylethyl carbamate, Adpoc, 2-chloroethyl carbamate, 1,1-dimethyl-2-haloethyl carbamate, DB-t-BOC, TCBOC, Bpoc, t-Bumeoc, Pyoc, Bnpeoc, N-2-(pivaloylamino)-1,1-dimethylethyl carbamate, and NpSSPeoc.

[0086] The terms “halo,” “halogen,” or “halide” group, as used herein, mean a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, either by themselves or as part of another substituent, unless otherwise specified.

[0087] The term "haloalkyl" as used herein includes monohaloalkyl groups, polyhaloalkyl groups (where all halo atoms may be the same or different), and perhaloalkyl groups (where all hydrogen atoms are replaced by halogen atoms such as fluoro). Examples of haloalkyl groups include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3-difluoropropyl, and perfluorobutyl.

[0088] As used herein, "oligoether" means an oligomer containing repeating units of a structure having an ether functional group. As used herein, "oligomer" means one or more repeating units of a identifiable structure, or a molecule containing the same or different formulas.

[0089] As used herein, "sulfonate functional group" or "sulfonate" refers to the free sulfonic acid anion (-S(=O)2O - This refers to both sodium sulfonate and its salts. Therefore, the term sulfonate encompasses sulfonates such as sodium sulfonate, lithium sulfonate, potassium sulfonate, and ammonium sulfonate.

[0090] As used herein, the terms "sulfonamide" or "sulfonamide" refer to compounds of the formula -SO2NHR- or -SO2N(R 4 ) refers to the R- group, where R is not limited to but can be hydrogen, alkyl, aryl, water-soluble moiety, PEG group, linker group, or carboxylic acid group.

[0091] To increase water solubility, a water-soluble moiety may be included in the polymer dye. The increase in solubility can vary, but in some cases, the increase compared to a polymer dye without a water-soluble moiety can be at least twice as high, for example, five times, ten times, twenty-five times, fifty times, one hundred times, or even higher.

[0092] The term "water-solubilizing moiety" refers to a group that, in an aqueous environment, such as under physiological conditions, is well solvated and results in improved water solubility for the molecule to which it is bound. The water-solubilizing moiety can be any suitable hydrophilic group that is well solvated in an aqueous environment. In some cases, the hydrophilic water-solubilizing group is charged, for example, positively or negatively charged. In certain cases, the hydrophilic water-solubilizing group is a neutral hydrophilic group. In some embodiments, the water-solubilizing moiety is a hydrophilic polymer, such as polyethylene glycol, cellulose, chitosan, or derivatives thereof. The water-soluble portion may include, but is not limited to, carboxylates, phosphonates, phosphates, sulfonates, sulfates, sulfinates, sulfonium, esters, sulfonamides, polyethylene glycol (PEG), modified PEG, hydroxyl, amines, ammonium, guanidinium, pyridinium, polyamines and sulfonium, polyalcohols, linear or cyclic sugars, primary, secondary, tertiary or quaternary amines and polyamines, phosphonate groups, phosphinate groups, ascorbate groups, and glycols. In some embodiments, the water-soluble portion is a PEG group.

[0093] The term "water-soluble UV-absorbing polymer dye" refers to UV-absorbing polymer dyes, tandem dyes, quenched dyes, or conjugates thereof that exhibit solubility in water at room temperature of 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, or 50 mg / mL, or greater than 1-250 mg / mL, 2-200 mg / mL, 3-150 mg / mL, 4-125 mg / mL, 5-100 mg / mL, 7-70 mg / mL, or 10-50 mg / mL.

[0094] As used herein, the terms "polyethylene glycol," "poly(ethylene glycol)," or "PEG" refer to the formula -(CH2-CH2-O-) n- Refers to a family of biocompatible, water-soluble linear polymers based on ethylene glycol monomer units as described by or their derivatives. The PEGn moiety may be used as the water-soluble moiety. The water-soluble moiety may impart to the molecule it is bound to a solubility in water at room temperature of at least 1 mg / mL, at least 5 mg / mL, at least 10 mg / mL, at least 20 mg / mL, at least 30 mg / mL, at least 40 mg / mL or at least 50 mg / mL, or 1-250 mg / mL, 2-200 mg / mL, 3-150 mg / mL, 4-125 mg / mL, 5-100 mg / mL, 7-70 mg / mL or 10-50 mg / mL. In some embodiments, "n" is less than 1000, less than 500, less than 200, less than 100, less than 50, less than 40, less than 30, less than 20, or less than 15 (e.g., 3-15 or 10-15). The PEG polymer group may be of any convenient length and is understood to include a variety of terminal groups and / or further substituents, including, but not limited to, alkyl, aryl, hydroxyl, alkoxy, alkanol, -OCH3, -O-C1-C4 alkyl, amino, acyl, carboxylic acid, carboxylic acid ester, acyloxy, and amide terminals and / or substituents. The number after "PEG" refers to the average molecular weight.

[0095] The term "Mw" refers to weight-average molecular weight, and "Mn" refers to number-average molecular weight. The average molecular weight of a polymer can be determined by any suitable method. For example, the average molecular weight of a polymer can be determined by light scattering techniques or size exclusion chromatography. Gel permeation chromatography can be used to determine the number-average molecular weight and weight-average molecular weight of a polymer.

[0096] The term "crosstalk index" refers to the percentage of the residual absorbance of a UV polymer dye at 405 nm relative to its absorbance at 355 nm, which are the excitation wavelengths of violet and UV lasers, respectively. The calculation of the crosstalk index, derived from the absorption spectrum, can be performed by obtaining the absorption spectrum of a filtered (0.22u) solution of the polymer dye in PBS. The absorbances at wavelengths 355 nm and 405 nm are recorded. The ratio of the absorbance at 405 nm to that at 355 nm is measured; this ratio provides a reasonable indication of the total leakage that can be predicted in the pacific blue channel when excited by a violet laser, in the case of a conjugate made from this polymer.

[0097] As used herein, the term "carboxylate" refers to the conjugate base of a carboxylic acid, which can generally be represented by the formula RCOO. For example, the term "magnesium carboxylate" refers to the magnesium salt of a carboxylic acid.

[0098] As used herein, the term “activated ester” refers to a carboxyl activating group used in peptide chemistry to facilitate the condensation of a carboxyl group with the free amino group of an amino acid derivative. Descriptions of these carboxyl activating groups can be found in general textbooks on peptide chemistry, for example, 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.

[0099] The terms "hydrazine" and "hydrazide" refer to compounds containing a single nitrogen atom, one of which is a primary amine functional group.

[0100] As used herein, the term "aldehyde" refers to a chemical compound having a -CHO group.

[0101] The term "thiol," as used herein, refers to a compound containing a functional group composed of a sulfur-hydrogen bond. The common chemical structure of a thiol functional group is R-SH, where R represents an alkyl, alkene, aryl, or other group of carbon-containing atoms.

[0102] The term "silyl" as used herein refers to Si(R z ) pointing to 3 、 Each R z These are independently alkyl, aryl, or other carbon-containing atom groups.

[0103] The term "diazonium salt" as used herein refers to R-N2 + X - This refers to a group of an organic compound having the structure, where R can be any organic residue (e.g., alkyl or aryl), and X can be an inorganic or organic anion (e.g., halogen).

[0104] The term "trifluromethanesulfonate," also known as "trifluromethanesulfonate," refers to a group with the formula CF3SO3.

[0105] The term "boronic acid" as used herein refers to the structure -B(OH)2. It is recognized by those skilled in the art that boronic acids can exist as boronic acid esters at various stages of synthesis. Boronic acids are intended to include such esters. The term "boronic acid ester" or "boronate ester" as used herein refers to -B(Z 1 )(Z 2 This refers to a chemical compound that contains the Z portion. 1 and Z 2Together, they form a moiety in which, in each case, the atom bonded to boron is an oxygen atom. The boronic acid ester moiety may be a five-membered ring, a six-membered ring, or a mixture of five-membered and six-membered rings.

[0106] The terms "hydrocarbon" or "hydrocarbyl," as used herein, refer to a molecule or functional group containing carbon and hydrogen atoms. The terms may also refer to a molecule or functional group that typically contains both carbon and hydrogen atoms, but all hydrogen atoms are substituted by other functional groups. The term "hydrocarbyl" refers to a functional group derived from a linear, branched, or cyclic hydrocarbon and may be alkyl, alkenyl, alkynyl, aryl, cycloalkyl, acyl, or any combination thereof. A hydrocarbyl group is (C a ~C b ) can be expressed as hydrocarbyl, where a and b are integers and mean having any of the number of carbon atoms from a to b. For example, (C1~C4) hydrocarbyl means that the hydrocarbyl group can be methyl (C1), ethyl (C2), propyl (C3), or butyl (C4), and (C0~C b )Hydrocarbyl means that, in certain embodiments, the hydrocarbyl group is absent. The hydrocarbylene group is a diradical hydrocarbon, for example, a hydrocarbon bonded at two positions. UV-absorbing polymer

[0107] In various embodiments, the present disclosure provides a UV-absorbing polymer having the structure of formula I: [ka] [In the formula, each X is independently selected from the group consisting of C and Si, and each Y is a bond, CR 1 R 2 , CHR 1 , CHR 2 SiHR 2 SiHR 1 and SiR 1 R 2Independently selected from the group consisting of, if Y is a bond, then X is directly bonded to both rings, and each R 1 This includes water-soluble moieties, alkyl groups, alkenes, alkynes, cycloalkyl groups, haloalkyl groups, (hetero)aryloxy groups, (hetero)arylamino groups, aryl groups, heteroaryl groups, polyethylene glycol (PEG) groups, carboxylic acids, alkylammonium salts, alkyloxyammonium salts, oligoetherammonium salts, alkyl sulfonates, alkoxysulfonates, sulfonamide oligoethers, sulfonamides, sulfinamides, phosphoamides, phosphineamides, [ka] [ka] Independently selected from the group consisting of, each R 2 This includes the water-soluble portion, the linker portion, H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, heteroaryl, (hetero)arylamino, PEG group, sulfonamide-PEG, phosphoramide-PEG, alkylammonium salt, alkyloxyammonium salt, oligoetherammonium salt, alkyl sulfonate, alkoxysulfonate, oligoethersulfonate, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamide, phosphineamide, [ka] Independently selected from the group consisting of, each R 3 Z is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, water-soluble moiety, and PEG group, and each Z is CH2, CHR 4 , O, NH and NR 4 Independently selected from the group consisting of, each Q is a combination of NH, NR4 , C1~C 12 Alkylene, CHR 4 and CH2 are independently selected from the group, and each R 4 H, PEG group, water-soluble portion, linker portion, chromophore, linked chromophore, functional group, linked functional group, substrate, linked substrate, binding partner, linked binding partner, quenching portion, L 2 -E, halogen, hydroxyl, C1~C 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylamino, (CH2) x’ (OCH2-CH2) y’ OCH3 (each x' is an independent integer between 0 and 20, and each y' is an independent integer between 0 and 50), Z-(CH2) n -SO2-QR 3 , C2~C 18 (Hetero)aryl groups, amides, amines, carbamates, carboxylic acids, carboxylic acid esters, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazones, azides, aldehydes, thiols and their protected forms are independently selected from the group, each W 1 This is independently a water-soluble portion, L 1 , L 2 and L 3 Each is an independently selected linker portion, and each E is independently selected from the group consisting of a chromophore, a functional group portion, a substrate, and a bonding partner, and each R 7 H, hydroxyl, C1-C 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C18 (hetero)aryloxy, C2~C 18 (hetero)arylamino, C2~C 12 Carboxylic acids, C2-C 12 Carboxylic acid esters and C1-C 12 Independently selected from the group consisting of alkoxys, R 1 , R 2 , R 3 or R 4 At least one of them includes a water-soluble portion, Each M 1 R is further substituted as needed. 4 and / or arylene substituted with trifluoromethyl, further substituted as needed, R 4 and / or heteroarylenes substituted with trifluoromethyl, further substituted as needed, R 4 Independently selected from the group consisting of 9,10-dihydrophenanthrene substituted with and / or trifluoromethyl, and binaphthyl substituted as needed, each M 2 R is further substituted as needed. 4 and / or arylene substituted with trifluoromethyl, further substituted as needed, R 4 and / or heteroarylenes substituted with trifluoromethyl, further substituted as needed, R 4 Independently selected from the group consisting of 9,10-dihydrophenanthrene substituted with and / or trifluoromethyl, and binaphthyl substituted as needed, M 2 M 1 It has a different structure, M 2 and M 1The linkers L are evenly or randomly distributed along the polymer backbone, and each linker L as needed is an aryl or heteroaryl group evenly or randomly distributed along the polymer backbone, and is substituted by one or more pendant chains terminated with functional groups selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols and their protected groups, for conjugation to another substrate, acceptor dye, molecule or binding partner, G 1 and G 2 [where a, c, d, and e are independently selected from the group consisting of unmodified and modified polymer ends, which are conjugated to E as needed, and a, c, d, and e define the mol% of each unit in the structure which can be repeated evenly or randomly along the polymer backbone, where a is 10 to 100% (mol%), c is >0 to 90% (mol%), each d is 0 to 90% (mol%), each e is 0 to 25% (mol%), each b is independently 0 or 1, each f is independently an integer from 0 to 50, m is an integer from 1 to about 10,000, each n is independently an integer from 1 to 20, s is 1 or 2, and t is 0, 1, 2, or 3].

[0108] In the UV-absorbing polymer dye according to formula (I), each X can be independently selected from C and Si. Each Y is a bond, CR 1 R 2 , CHR 1 , CHR 2 SiHR 2 SiHR 1 and SiR 1 R 2 They can be selected independently, and if Y is a bond, then X is directly bonded to both rings. 1This includes polyethylene glycol (PEG), alkylammonium salts, alkyloxyammonium salts, oligoetherammonium salts, alkyl sulfonates, alkoxysulfonates, sulfonamide oligoethers, and -Z-(CH2) n -SO2-QR 3 It can be selected independently of R. In some embodiments, Y is a bond and R is a bond. 1 and R 2 These are, independently, -Z-(CH2) n -SO2-QR 3 Each R 2 H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG group, alkylammonium salt, alkyloxyammonium salt, oligoetherammonium salt, alkyl sulfonate, alkoxysulfonate, oligoethersulfonate, sulfonamide oligoether, and -Z-(CH2) n -SO2-QR 3 Each R can be selected independently. 3 These include H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino and PEG groups (e.g., -PEG-R 5 Alternatively, it can be selected independently from -PEG-OMe). Each Z can be selected independently from C, O, and N. Each Q is bound, NH, NR 4 , C1~C 12 Alkylene and CH2 can be selected independently. Each R 4 These include chromophores (e.g., acceptor dyes), halogens, hydroxyls, and C1-C2 compounds. 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18(hetero)arylamino, (CH2) x’ (OCH2-CH2) y’ OCH3 (each x' is an independent integer between 0 and 20, and each y' is an independent integer between 0 and 50), -Z-(CH2) n -SO2-QR 3 and C2~C 18 (Hetero)aryl groups can be selected independently. Each modification unit M 1 and M 2 This can be independently selected from arylene or heteroarylene, which allows for modification of the polymer's band cap. 1 R is further substituted as needed. 4 and / or arylene substituted with trifluoromethyl, further substituted as needed, R 4 and / or heteroarylenes substituted with trifluoromethyl, further substituted as needed, R 4 9,10-dihydrophenanthrene substituted with trifluoromethyl and / or binaphthyl, which may be selected independently as needed. For example, each M 1 This is 1 to 4 (for example, 1, 2, 3 or 4) R 4 Alternatively, it may have a trifluoromethyl substituent. Each M 2 R is further substituted as needed. 4 and / or arylene substituted with trifluoromethyl, further substituted as needed, R 4 and / or heteroarylenes substituted with trifluoromethyl, further substituted as needed, R 4 9,10-dihydrophenanthrene substituted with trifluoromethyl and / or binaphthyl substituted as needed can be independently selected, M 2 M 1 It has a different structure. For example, each M 2 This is 1 to 4 (for example, 1, 2, 3 or 4) R 4Alternatively, it may have a trifluoromethyl substituent. Each linker L may be an aryl or heteroaryl group evenly or randomly distributed along the polymer backbone and is substituted with one or more pendant chains terminated with functional groups selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols and their protected groups, for conjugation to another substrate, acceptor dye, molecule or binding partner.

[0109] In some examples, variable element G 1 and G 2 Each of these may be independently selected from hydrogen, halogens, alkynes, halogen-substituted aryls, silyls, diazonium salts, triflates, acetyloxys, azides, sulfonates, phosphates, boronic acid-substituted aryls, boronic acid ester-substituted aryls, boronic acid esters, boronic acids, optionally substituted aryls, optionally substituted heteroaryls, optionally substituted dihydrophenanthrene (DHP), or optionally substituted fluorenes, wherein the optionally substituted aryls, heteroaryls, fluorenes, or DHPs may be substituted by one or more pendant chains terminated with functional groups selected from, for example, amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxylsuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and their protected groups, for conjugation to a substrate or binding partner.

[0110] Variables a, c, d, and e define the mol% of each unit in the structure, and each of these structures can be repeated evenly or randomly, where a is 10 to 100% (mol%), c is >0 to 90% (mol%), each d is 0 to 90% (mol%), and each e is 0 to 25% (mol%). Each b can independently be 0 or 1. The variable m can be an integer from 1 to about 10,000 (e.g., at least 2 or less than 10,000, but greater than, less than, or equal to 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, 500, 1,000, 5,000, or 7,500). Each n can be an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) independently. The UV-absorbing polymer can be water-soluble.

[0111] In the polymers described herein, the units defined by a, c, d, and e may exist in any order within the polymer backchain, such as the order shown in the structure of formula (I), or in a different order. The units may exist in an even or random arrangement within the polymer backchain.

[0112] The term "polymer dye," as used herein, may refer to a UV-absorbing polymer dye, a UV-absorbing polymer dye conjugate, a UV-absorbing polymer tandem dye, a UV-absorbing polymer tandem dye conjugate, or a quenched UV-absorbing polymer dye. Examples of polymer dyes include R 1 or R 2 In this, for example, one or more acceptor dye moieties may be attached to the main chain via a linker L or to the monomer, thereby allowing monitoring of the luminescence of the acceptor dye attached to the main chain via energy transfer. In some embodiments, R 1 or R 2 At least one of them is -Z-(CH2) n -SO2-N (chromophore)-R 3 ,-Z-(CH2)n -SO2-N(L 2 -Color-developing group)-R 3 , [ka] That is the case.

[0113] The chromophore can be an acceptor dye that enables excitation of the polymer backbone and allows monitoring of the luminescence of the acceptor dye bound to the backbone.

[0114] Acceptor dyes useful for tandem polymer dyes may include, for example, cyanine dyes, xanthene dyes, coumarin dyes, thiadin dyes, acridine dyes, FITC, CY3B, Cy55, Alexa488, Alexa750, Texas red, Cy3B, Cy3.5, Cy5, Cy7, Cy55, Alexa750, 800CW, Biotium CF 555, diethylcoumarin, DY705 (Dyomics), DY431, DY485XL, DY500XL, DY610, DY640, DY654, DY682, DY700, DY701, DY704, DY730, DY731, DY732, DY734, DY752, DY778, DY782, DY800, and DY831. The acceptor dye may also be a pendant acceptor dye.

[0115] For example, acceptor dyes useful for tandem polymer dyes include, for example, Dyomics DY704, FITC, CY3B, Cy55, Alexa488, Texas red, Cy5, Cy7, Alexa750, and 800CW. The tandem dye may be a UV polymer according to the disclosure comprising one or more, two or more, three or more, 1 to 30, 2 to 20, or 2.5 to 10 acceptor dye moieties.

[0116] In some embodiments, the acceptor dye moiety may be, for example, Dyomics DY704 or derived therefrom. [ka]

[0117] In some embodiments, UV polymer dyes are, for example, R 1 or R 2 In this, for example, a quenched UV polymer can be made which includes one or more quenchable moieties bonded to the main chain or monomer via a linker L. In some embodiments, at least one R 2 is -Z-(CH2) n -SO2-N (quenching moiety)-R 3 In some embodiments, the acceptor dye can be a quenching portion. For example, the quenching portion may be selected from, for example, DABCYL, DABSYL, Black Hole Quencher 1 (BHQ1), BHQ-0, Deep Dark Quencher I, DDQI, EDQ, QSY7, QSY9, QSY35, TAMRA (carboxytetramethylrhodamine), Dabcyl Q, Dabcyl plus, Anaspec490Q, Dyomics425Q, and Dyomics505Q. Non-limiting examples of the quenching portion are, for example, [ka] It can include...

[0118] In some embodiments, quenched UV polymer dyes according to the Disclosure are provided, comprising 1 to 30, 2 to 20, or 2.5 to 10 quenched moieties. In some embodiments, the quenched moiety is a dabcyl moiety. In some embodiments, the quenched UV polymer dye comprises 2.5 to 10 dabcyl moieties (poly-Dabcyl UV polymer). As provided herein, tandem dyes or quenched polymer dyes may be prepared by reacting an active ester, such as an NHS ester of an acceptor moiety, e.g., an NHS ester of a quenched moiety, with the UV polymer dye of the Disclosure. Such acceptor dye NHS esters are commercially available, e.g., DY-705NHS ester from Dyomics, or Dabcyl SE (Dabcyl succinimidyl ester) from Abcam.

[0119] A buffer composition for dyeing is provided, comprising a quenched UV polymer dye according to the Disclosure, comprising at least one quenched moiety, optionally comprising 1 to 30, 2 to 20, or 2.5 to 10 quenched moieties. The quenched moieties may be selected from any suitable quenched moieties. Non-limiting examples may include DABCYL, DABSYL, BHQ1, BHQ0, DDQI, EDQ, QSY7, QSY9, QSY35, TAMRA, Dabcyl Q, Dabcyl plus, 490Q, 425Q, and 505Q.

[0120] The polymer is, Formula II: [ka] It can have the following structure.

[0121] The polymer is given by formula III: [ka] It can have the following structure. Each f can be an integer between 0 and 50 independently. Each R 5 H, C1~C 12 Alkyl, C2~C 12Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylaminos and C1-C 12 They can be independently selected from the group consisting of alkoxys.

[0122] The polymer is, Formula IV: [ka] [In the formula, each f can be an integer between 0 and 50, 10 and 20, or 11 and 18, independently.] It can have the following structure.

[0123] The polymer is, formula V: [ka] The copolymer can have the following structure. The variables g and h together can be 10 to 100% (mol%). Each f can independently be an integer from 0 to 50. Each R 5 H, C1~C 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylaminos and C1-C 12 They can be independently selected from the group consisting of alkoxys.

[0124] The polymer is, Formula VI: [ka] [In the formula, each f can be an integer between 0 and 50 independently.] It can have a structure. Each R 5 H, C1~C 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylaminos and C1-C 12 They can be independently selected from the group consisting of alkoxys.

[0125] The polymer is, formula VII: [ka] The copolymer can have the following structure. The variables g and h together can be 10 to 100% (mol%). Each f can independently be an integer from 0 to 50.

[0126] The polymer is, Formula VIII: [ka] It can have the following structure. Each f can be an integer between 0 and 50, independently.

[0127] In the formulas described herein, each f (i.e., the multiplicity of the PEG group) can be an integer that is 0 to 50 or less than 50, such as 5 to 40, 3 to 30, 5 to 20, 10 to 25, 10 to 20, 11 to 18, but greater than or equal to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, or 45.

[0128] In the formulas described herein, the modifying unit M 1 This may allow for modification of the polymer's band gap. Each M 1 R is further substituted as needed. 4 and / or arylene substituted with trifluoromethyl, and further substituted as needed, R 4 Each M can be independently selected from heteroarylenes substituted with trifluoromethyl. 1 Each M can be a halide, MeO-PEG-CH2 and / or an arylene (e.g., phenylene) substituted with MeO-PEG, which may be further substituted as needed. 1 This can be independently selected from arylenes substituted with halides (e.g., fluorine) and / or trifluoromethyl, which are further substituted as needed, and heteroarylenes substituted with halides (e.g., fluorine) and / or trifluoromethyl, which are further substituted as needed. 1 Each M can independently be a halide-substituted arylene having 1 to 4 halide substituents. 1 Each M can independently be a fluorine-substituted arylene having 1 to 4 fluorine substituents. 1 This can independently be a halide-substituted phenylene having 1 to 4 halide substituents, and the phenylene may be further substituted as needed. 1 This can independently be a fluorine-substituted phenylene having 1 to 4 fluorine substituents, and the phenylene may be further substituted as needed. Each M 1 This can be independently a halide-substituted phenylene having two or three halide substituents, or a fluorine-substituted phenylene having two or three fluorine substituents.

[0129] Each M 1 This can be phenylene substituted with a dihalide. Each M1 This can be independently selected from phenylene in which the 1st and 4th positions are substituted with the polymer main chain and dihalide-substituted with halides at positions 2 and 3, 2 and 5, or 2 and 6; phenylene in which the 1st and 4th positions are substituted with the polymer main chain and trihalide-substituted with halides at positions 2, 3 and 5; phenylene in which the 1st and 3rd positions are substituted with the polymer main chain and dihalide-substituted with halides at positions 2 and 4, 2 and 5, 4 and 5, or 4 and 6; and phenylene in which the 1st and 3rd positions are substituted with the polymer main chain and trihalide-substituted with halides at positions 4, 5 and 6, 2, 4 and 5, or 2, 4 and 6. 1 This can be independently selected from phenylene in which the 1st and 4th positions are substituted with the main chain of the polymer and which is dihalide-substituted with a halide at the 2nd and 3rd positions, the 2nd and 5th positions or the 2nd and 6th positions, and phenylene in which the 1st and 3rd positions are substituted with the main chain of the polymer and which is dihalide-substituted with a halide at the 2nd and 4th positions, the 2nd and 5th positions, the 4th and 5th positions or the 4th and 6th positions.

[0130] Each M 1 This can be phenylene substituted with a difluoropolymer. Each M 1This can be independently selected from phenylene in which the 1st and 4th positions are substituted with the polymer main chain and the 2nd and 3rd, 2nd and 5th, or 2nd and 6th positions are dihalo (e.g., difluoro) substituted with fluorine; phenylene in which the 1st and 4th positions are substituted with the polymer main chain and the 2nd, 3rd, and 5th positions are trihalo (e.g., trifluoro) substituted with fluorine; phenylene in which the 1st and 3rd positions are substituted with the polymer main chain and the 2nd and 4th, 2nd and 5th, 4th and 5th, or 4th and 6th positions are dihalo (e.g., difluoro) substituted with fluorine; and phenylene in which the 1st and 3rd positions are substituted with the polymer main chain and the 4th, 5th, and 6th, 2nd, 4th, and 5th, or 2nd, 4th, and 6th positions are trihalo (e.g., trifluoro) substituted with fluorine. 1 This can be independently selected from phenylene in which the 1st and 4th positions are substituted with the polymer main chain and which is difluorosubstituted with fluorine at the 2nd and 3rd positions, the 2nd and 5th positions, or the 2nd and 6th positions, and phenylene in which the 1st and 3rd positions are substituted with the polymer main chain and which is difluorosubstituted with fluorine at the 2nd and 4th positions, the 2nd and 5th positions, the 4th and 5th positions, or the 4th and 6th positions.

[0131] Some implementation methods, each R 4 is F, Cl, -CF3, -OCH3, -CN, -CH3, -O(CH2CH2O) f OCH3 and -CO2H may be selected independently.

[0132] Each M 1 teeth, [ka] [ka] [ka] It can be selected independently of the others.

[0133] Each M 1 teeth, [ka] It can be selected independently of the others.

[0134] Each M 1 teeth, [ka] It can be selected independently of the others.

[0135] Each M 1 This can be phenylene in which the 1st and 4th positions are substituted in the main chain of the polymer, and which is 2,5-difluorosubstituted. 1 teeth, [ka] It can be done this way.

[0136] Each M 1 This can be replaced with binaphthyl as needed. Each M 1 teeth, [ka] It can be done this way.

[0137] In the formulas described herein, the modifying unit M 2 This may allow for modification of the polymer's band gap.

[0138] M 2 M 1 It can have a different structure.

[0139] Each M 2 teeth, [ka] [ka] [ka] It can be selected independently of the others.

[0140] Each M 2 teeth, [ka] It can be selected independently of the others.

[0141] Each M 2 teeth, [ka] It can be selected independently of the others.

[0142] Each M 2 R is further substituted as needed. 4 and / or arylene substituted with trifluoromethyl, and further substituted as needed, R 4 Each M can be independently selected from heteroarylenes substituted with trifluoromethyl. 2 Each M can be a halide, MeO-PEG-CH2 and / or an arylene (e.g., phenylene) substituted with MeO-PEG, which may be further substituted as needed. 2Each M can be independently selected from arylenes substituted with fluorine and / or trifluoromethyl, which are further substituted as needed, and heteroarylenes substituted with fluorine and / or trifluoromethyl, which are further substituted as needed. 2 Each M can independently be a halogen-substituted arylene having 1 to 4 halide substituents. 2 Each M can independently be a fluorine-substituted arylene having 1 to 4 fluorine substituents. 2 This can independently be a halide-substituted phenylene having 1 to 4 halide substituents, and the phenylene may be further substituted as needed. 2 This can independently be a fluorine-substituted phenylene having 1 to 4 fluorine substituents, and the phenylene may be further substituted as needed. Each M 2 This can be independently a halide-substituted phenylene having two or three halide substituents, or a fluorine-substituted phenylene having two or three fluorine substituents.

[0143] Each M 2 teeth, [ka] It can be selected independently of the others.

[0144] Each M 2 This can be phenylene substituted with a tri-halide. Each M 2 This can be independently selected from phenylene in which the 1st and 4th positions are substituted with the polymer main chain and phenylene in which the 2nd, 3rd, and 5th positions are trihalide-substituted with halides, and phenylene in which the 1st and 3rd positions are substituted with the polymer main chain and phenylene in which the 4th, 5th, and 6th positions, 2nd, 4th, and 5th positions, or 2nd, 4th, and 6th positions are trihalide-substituted with halides. 2This can be phenylene in which the 1st and 3rd positions are substituted in the main chain of the polymer, and which is 4,5,6-trihalide substituted.

[0145] Each M 2 This can be phenylene substituted with a trifluoropolymer. Each M 2 This can be independently selected from phenylene in which the 1st and 4th positions are substituted with the polymer main chain and phenylene trifluorosubstituted with fluorine at the 2nd, 3rd, and 5th positions, and phenylene in which the 1st and 3rd positions are substituted with the polymer main chain and phenylene trifluorosubstituted with fluorine at the 4th, 5th, and 6th positions, 2nd, 4th, and 5th positions, or 2nd, 4th, and 6th positions. 2 This is a phenylene in which the 1st and 3rd positions are substituted in the main chain of the polymer, and can be a 4,5,6-trifluorosubstituted phenylene. 2 teeth, [ka] It can be done this way.

[0146] Each M 2 This can be replaced with binaphthyl as needed. Each M 2 teeth, [ka] It can be done this way.

[0147] Modification unit M 1 and M 2 These can be arranged uniformly or randomly along the polymer chain. For example, Figure 11 shows an exemplary scheme relating to a UV-absorbing polymer according to this disclosure, with two different modification units M 1 and M 2The repeating DHP units are randomly distributed in alternating positions. The number of segments represents the average Mn of individual polymer molecules with different chain lengths.

[0148] Each M 1 and M 2 Each of them is, [ka] It can be selected independently from M 1 and M 2 They are different.

[0149] In some embodiments, this disclosure relates to formula XIV: [ka] [In the formula, each R 2 , R 3 , G 1 , G 2 L, Q, X, Y, Z, a, b, c, e, n and m are independently as described herein, and each R 4’ R 4 Selected independently from, and at least one R 4’ It is not H, but each R 4’’ R 4 Selected independently from, and at least one R 4’’ It's R, not H. 9 [where f is a C1-C8 alkyl group, each f is an integer independently between 0 and 50 or between 10 and 20, each o is an integer independently selected from 1, 2, 3 or 4, and each p is an integer independently selected from 1, 2, 3 or 4] The invention provides UV-absorbing polymers according to formula XIV. In some examples, UV-absorbing polymers according to formula XIV have near-ultraviolet excitation spectra and / or absorption maxima in the range of 300 nm to 400 nm or 350 nm to 400 nm.

[0150] In the formulas described herein, each linker L may be an aryl or heteroaryl group evenly or randomly distributed along the polymer backbone and is substituted by one or more pendant chains terminated with functional groups selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols and their protected groups, which can be conjugated to another substrate, acceptor dye, molecule or binding partner. Each L is, [ka] [ka] Each R can be selected independently. 6 H, OH, SH, NHCOO-t-butyl, (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) fOCH3, N-maleimide, halogen, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 (hetero)aryl, C1~C 12 (hetero)arylamino, optionally substituted benzyl, halogen, hydroxyl, C1-C 12 Alkoxy or (OCH2CH2) f Each can be selected independently from OCH3. Each f can be an integer between 0 and 50, 10 and 20, or 11 and 18, independently. Each n can be an integer between 1 and 20, independently.

[0151] UV-absorbing polymers are G 1 and G 2 This may include the capping units represented in the formulas herein. 1 and G 2 These can be independently unmodified polymer ends and modified polymer ends. For example, G 1 and G 2Each can be independently selected from hydrogen, halogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, boronic acid-substituted aryl, boronic acid ester-substituted aryl, boronic acid ester, boronic acid, optionally substituted dihydrophenanthrene (DHP), and optionally substituted fluorene. The optionally substituted aryl, heteroaryl, fluorene, or DHP may be substituted by one or more pendant chains terminated with functional groups selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxylsuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, and their protected groups for conjugation to a substrate or binding partner. In some examples, at least one capping unit G 1 or G 2 It is conjugated to the substrate or binding partner. Capping unit G 1 and G 2 Each of these can be independently selected from dihydrophenanthrene (DHP) which is optionally substituted, fluorene which is optionally substituted, aryl which is substituted with one or more pendant chains terminated with a functional group, and heteroaryl which is substituted with one or more pendant chains terminated with a functional group. In some examples, the capping unit G 1 and G 2 Each of them is, [ka] [In the formula, each R 6 H, OH, SH, NHCOO-t-butyl, (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 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 (hetero)aryl, C1~C 12 (hetero)arylamino, optionally substituted benzyl, halogen, hydroxyl, C1-C 12 Alkoxy or (OCH2CH2) f [Can be selected independently from OCH3] Each can be independently selected from the others. Each f can be an integer between 0 and 50 or between 11 and 18. Each n can be an integer between 1 and 20 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

[0152] Variables a, c, d, and e define the mol% of each unit in the structure, each of which can be repeated equally or randomly, where a is 10–100% (mol%), c is >0–90% (mol%), each d is 0–90% (mol%), and each e is 0–25% (mol%). Variable a is in mol%, and can be 10–100%, 25–75%, 35–65%, 45–55%, or higher than or equal to 10%, 15, 20, 25, 30, 35, 40, 42, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 60, 65, 70, 75, 80, 85, 90, or 95%. The variable c is in mol%, and is >0-90%, 5-80%, 10-40%, 15-35%, 20-30%, or less than or equal to 90%, but can be greater than or equal to 1%, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85%. Variable d is in mol% and is between 0 and 90%, 5 and 80%, 10 and 40%, 15 and 35%, 20 and 30%, or less than or equal to 90%, but greater than or equal to 0%, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85%. The variable e is in mol%, and can be 0-25%, 0-20%, 0-10%, or less than or equal to 25%, but greater than or equal to 0%, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, or 24%.

[0153] The polymer is, formula IX: [ka] It can have the following structure.

[0154] The variable f can independently be an integer between 0 and 50. The units in the polymer structure represented by formula IX may exist in any preferred order, such as the same or different order, within the polymer backbone, as shown in formula IX. For example, the units in the polymer structure represented by formula IX are given by formula X: [ka] They can exist in the order shown.

[0155] For example, the units in the polymer structure represented by formula IX or X are given by formula XI: [ka] They can exist in the order shown.

[0156] In formulas X and XI, the variables m, p, and n specify the mol% of each unit in the structure. The variable m may be the same as that described herein for formulas I-IX or XIV. In the formulas described herein, the base M 1 and M 2 The UV-absorbing polymer can have any preferred molar ratio of each other. For example, M 1 Original M 2 The molar ratio to the base is 0.5:1 to 1.5:1, 0.7:1 to 1.3:1, 0.9:1 to 1.1:1, approximately 1:1, or less than or equal to 1.5:1, but may be greater than or equal to 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, or 1.4:1.

[0157] UV-absorbing polymers are available in the ranges of 300nm-400nm, 320nm-380nm, 330nm-380nm, 335nm-380nm, 340nm-380nm, 350nm-380nm, 350nm-375nm, 340nm-360nm, 345nm-356nm, or shorter than or equal to 380nm, but also 320nm, 322nm, 324nm, 326nm. It may have absorption maxima longer than or equal to 328, 330, 332, 334, 336, 338, 340, 342, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376 or 378 nm. The polymer may have an emission maximum of approximately 380 nm or longer, or within the range of approximately 380 nm to approximately 1000 nm, approximately 380 nm to approximately 800 nm, 380 nm to 430 nm, 406 nm to 415 nm, or shorter than or equal to 430 nm, but longer than or equal to 380 nm, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 418, 420, 422, 424, 426, or 428 nm. In some examples, the emission maximum may be longer than 1000 nm.

[0158] UV-absorbing polymer dyes can have any suitable molecular weight (MW), which can be expressed, for example, in g / mol or kilodaltons (kDa). In some cases, the MW of a UV-absorbing polymer dye may be expressed as an average molecular weight. In some examples, UV polymer dyes can have average molecular weights in the range of 1,000 to 500,000, such as 2,000 to 400,000, 5,000 to 300,000, 10,000 to 200,000, 25,000 to 175,000, 30,000 to 150,000, 40,000 to 150,000, or even as high as 50,000 to 100,000. UV-absorbing polymer dyes can have average molecular weights of 20-150 kDa, 30-130 kDa, 40-120 kDa, 50-100 kDa, or 60-70 kDa.

[0159] The monomers for preparing the UV-absorbing polymers of this disclosure may include dihydrophenanthrene (DHP)-based monomers, such as 9,10-phenanthrenedione-based monomers and / or fluorene-based mp monomers. For example, the monomers of this disclosure are [ka] It may contain a halogen atom, boronic acid ester or boronic acid, silyl, diazonium salt, triflate, acetyloxy, sulfonate or phosphate, which can independently or together undergo Pd or nickel salt-catalyzed polymerization. Variable element (variable) R 1 , R 2 X, Y, Z, n, R 3 , f and R 5 This is as described herein.

[0160] In some embodiments, the monomers of the present invention also include crosslinked monomers. For example, the crosslinked monomers of this disclosure are [ka] It can include...

[0161] In various embodiments of the present invention, the polymer further comprises a binding partner linked to the polymer. In some embodiments, the binding partner may be an antibody. The “binding partner” in this disclosure may be any molecule or complex of molecules that can specifically bind to a target analyte. Examples of binding partners in this disclosure include proteins, small organic molecules, carbohydrates (including polysaccharides), oligonucleotides, polynucleotides, lipids, affinity ligands, antibodies, antibody fragments, aptamers, and the like. In some embodiments, the binding partner is an antibody or a fragment thereof. Specific binding in the context of this disclosure refers to a binding reaction that determines the presence of a target analyte in the presence of a heterogeneous population. Thus, under specified assay conditions, the identified binding partner preferentially binds to a specific protein or isoform of a specific protein and does not bind in significant amounts to other proteins or other isoforms present in the sample.

[0162] When the binding partner is an antibody, they may be a monoclonal antibody or a polyclonal antibody. The term antibody, as used herein, refers to immunoglobulin molecules and the immunologically active portion of immunoglobulin (Ig) molecules. Such antibodies may include polyclonal antibodies, monoclonal antibodies, single-specific polyclonal antibodies, antibody mimetic compounds, chimeric compounds, single chains, Fab, Fab' and F(ab')2 fragments, Fv, and Fab expression libraries.

[0163] In general, the UV-absorbing polymers of this disclosure can be conjugated to a bonding partner using techniques known to those skilled in the art, or using methods known in the art in combination with the methods described herein. [ka]

[0164] For example, the preparation of polymer NHS ester can be carried out as follows: Take 5 mg of polymer in a clean vial and dissolve it in 1 mL of dry CH3CN. Add 15 mg of TSTU and stir for a further 2 minutes. Add 100 μL of DIPEA and continue stirring overnight using a cap sealed with Parafilm. After this, remove the organic solvent from the reaction mixture by distillation. Dissolve the crude NHS in approximately 750 μL of 1 × BBS buffer (pH 8.8) by rapid vortexing and transfer this to a Zebra column 40K MWCO. Centrifuge the sample at 2200 RPM for 2 minutes and use the polymer NHS immediately.

[0165] Conjugation of polymer NHS with CD4 can be carried out as follows: Take polymer NHS in centrifugated 1×BBS (approximately 800 μL), add 0.6 mg of CD4, and mix with 100 μL of 0.5 M borate buffer (pH 9.0). Quickly vortex for 30 seconds and mix in Coultermix for 3-4 hours.

[0166] The purification of conjugates using a Histrap HP column can be carried out as follows: Method 1: After the crude reaction, purify the conjugate using a Histrap HP column. Load the sample with 1×PBS buffer and collect the unbound fraction. This can be done using 20 CV of buffer. After this, change the buffer and wash the bound fraction containing both the conjugate and the free antibody. This can be done using 1×PBS containing 0.25 M imidazole and running 10 CV. Method 2: Histrap SP Sepharose FF column. Equilibrate the column and load the sample with 20 mM citrate buffer (pH 3.5) and collect the unbound fraction. This can be done using 20 CV of buffer. After this, change the buffer and elute the bound fraction containing both the conjugate and the free antibody. This can be done using 20 mM Tris buffer (pH 8.5) and running 20 CV. Method 3: Load the crude conjugate into a tangential flow filtration system equipped with a 300K MWCO membrane. The conjugate can be washed with 1×PBS until the filtrate no longer shows absorption at 405 nm. After this, concentrate the compound.

[0167] The purification of the conjugate using an SEC column can be carried out as follows: Load the crude conjugate containing the free antibody onto a size exclusion column using 1×PBS. After confirming the absorption spectrum, pool the tube and concentrate it in an Amicon Ultra-15 with a 30 kDa MWCO centrifugal concentrator. Method for detecting analytes in a sample

[0168] This disclosure provides a method for detecting an analyte in a sample, comprising the step of contacting a sample suspected of containing an analyte with a binding partner conjugated to a UV-absorbing polymer of this disclosure (including, but not limited to, UV-absorbing polymer-tandem polymers) (e.g., UV-absorbing polymers shown in formulas I-XI or XIV, e.g., any one of formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI and / or XIV according to this disclosure, and their polymer tandem dyes). The binding partner can interact with the analyte. If an analyte is present, the binding partner and the analyte can form a polymer dye conjugate complex. The binding partner may be bound to a substrate as needed. The binding partner may be a protein, peptide, affinity ligand, antibody, antibody fragment, sugar, lipid, nucleic acid or aptamer. A light source capable of exciting the polymer is applied to the sample, and light emitted from the conjugated polymer complex is detected. In a typical assay, the UV-absorbing polymers of this disclosure are excitable by light having wavelengths of 320nm–380nm, 340nm–360nm, 345nm–356nm, or shorter than or equal to 380nm, but longer than or equal to 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, or 378nm. Synchrotron radiation typically has wavelengths of 380nm–430nm, 406nm–415nm, or shorter than or equal to 430nm, but longer than or equal to 380nm, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 418, 420, 422, 424, 426, or 428nm.

[0169] A method for detecting an analyte in a sample is provided, comprising the steps of: adding at least one polymer dye conjugate to a composition according to the present disclosure to form a polymer dye conjugate composition; contacting a biological sample suspected to contain an analyte with the polymer dye conjugate composition to form a fluorescent polymer dye conjugate complex with the analyte; applying a light source capable of exciting at least one fluorescent polymer dye conjugate complex to the sample; and detecting light emitted from the fluorescent polymer dye conjugate complex.

[0170] In some embodiments, the light from the light source has a wavelength between approximately 340 nm and approximately 450 nm. In some embodiments, the synchrotron radiation has a wavelength between approximately 380 nm and approximately 1000 nm, or between 380 nm and 800 nm. The step of detecting the light includes a step of analyzing it by flow cytometry to obtain a first flow cytometry plot, the first flow cytometry plot, when compared to a second flow cytometry plot obtained by contacting a biological sample with a composition that does not contain nonionic surfactants and does not contain UV-absorbing polymer dyes or quenched UV polymer dyes, exhibits one or more of the group consisting of reduced nonspecific interactions of polymer dye conjugates; and reduced aggregation of polymer dye conjugates.

[0171] The biological sample in the methods of this disclosure may be, for example, blood, bone marrow, spleen cells, lymphocytes, bone marrow aspirate (or any cells obtained from bone marrow), urine (wash fluid), serum, saliva, cerebrospinal fluid, urine, amniotic fluid, interstitial fluid, feces, mucus, or tissue (e.g., tumor sample, deagglutinated tissue, deagglutinated solid tumor). In some embodiments, the sample is a blood sample. In some embodiments, the blood sample is whole blood. Whole blood can be obtained from a subject using standard clinical procedures. In some embodiments, the sample is a subset of one or more cells from whole blood (e.g., erythrocytes, leukocytes, lymphocytes, phagocytes, monocytes, macrophages, granulocytes, basophils, neutrophils, eosinophils, platelets, or any cells containing one or more detectable markers). Examples of lymphocyte cells include T cells, B cells, or NK cells. In some embodiments, the sample may be derived from a cell culture.

[0172] The subjects may be humans (e.g., patients suffering from a disease) or commercially important mammals (e.g., including monkeys, cattle, or horses). Samples may also be obtained from household pets, including, for example, dogs or cats. In some embodiments, the subjects are experimental animals used as animal models of diseases or for drug screening, such as mice, rats, rabbits, or guinea pigs.

[0173] "Analyte," as used herein, refers to a substance, such as a molecule, whose abundance / concentration is determined by certain analytical procedures. For example, in this disclosure, the analyte may be a protein, peptide, nucleic acid, lipid, carbohydrate, or small molecule.

[0174] The 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, the target analytes are proteins, such as structural microfilaments, microtubules 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. The proteins can be activatable proteins, or proteins differentially expressed or activated in diseased or abnormal cells, including, but not limited to, transcription factors, DNA and / or RNA-binding proteins and modified proteins, nuclear import and export receptors, and apoptotic or survival regulators. The analytes can be proteins expressed on the cell surface.

[0175] Assay systems that utilize binding partners and fluorescent labeling for the quantification of bound molecules are well known. Examples of such systems include flow cytometers, scanning cytometers, imaging cytometers, fluorescence microscopes, and confocal fluorescence microscopes.

[0176] In some embodiments, the method may be configured for flow cytometry. Fluorescence can be detected using flow cytometry. Several devices suitable for this use are available and known to those skilled in the art. Examples include BCI Navios, Gallios, Aquios, and CytoFLEX flow cytometers.

[0177] In other embodiments, the method may be configured as an immunoassay. Examples of immunoassays useful in this disclosure include fluoroluminescence assays (FLAs). The assay may also be performed using a protein array.

[0178] When the binding partner is an antibody, antibody or multi-antibody sandwich assays can also be used. A sandwich assay refers to the use of a series of recognition events that construct layers of various binding partners and reporting elements to signal the presence of a specific analyte. Examples of sandwich assays are disclosed in U.S. Patent No. 4,486,530 and the references cited therein.

[0179] In some embodiments, the method may include a step of providing additional binding partners (e.g., more than one binding partner) to simultaneously detect further targets for analysis. Buffer composition for staining

[0180] Polymer dyes (PDs) are hydrophobic and have a large apparent molecular weight, which causes them to aggregate in aqueous buffers. Therefore, when polymer dyes are conjugated to antibodies, the resulting conjugates are also highly likely to interact with each other and / or with other polymer dye conjugates present in the same sample. When more than one polymer dye conjugate is used to stain the same sample, nonspecific interactions between polymer dyes usually occur, which can lead to data deficiency and potentially inaccurate data analysis. Prior art competitive staining buffer compositions are commercially available. However, these staining buffers are somewhat dye-specific and show reduced effectiveness in inhibiting dye-to-dye interactions across different dye classes in multicolor panels. A universal staining buffer capable of suppressing dye-to-dye interactions caused by all types of polymer dye conjugates is desirable.

[0181] To develop a universal staining buffer suitable for use with multicolor panels of polymer dye conjugates, various detergents, PEGs, amino acids, DNA, peptides, proteins, polymers (such as violet polymers and ultraviolet polymers), and ureas were tested individually or in combination.

[0182] This disclosure provides a staining buffer composition capable of reducing, substantially reducing, or eliminating polymer-polymer interactions in a multicolor panel across a whole class of dyes. For example, a universal staining buffer solution suitable for use in multicolor panels has been developed, including various polymer dye conjugates from various commercial suppliers. The multicolor panel may contain one or more, or two or more, different types of polymer dye conjugates.

[0183] In some embodiments, the polymer dye conjugates in the multicolor panel may be fluorescent dye conjugates that can be excited by, for example, ultraviolet (e.g., 351nm, 355nm, 375nm, 334-364nm, 351-356nm), violet (e.g., 405nm, 407nm, 414nm, 395-425nm), blue (e.g., 436nm, 458nm), blue-green (e.g., 488nm), green (e.g., 514nm, 532nm, 541nm, 552nm), yellow-green (e.g., 561nm, 563nm), yellow (e.g., 568nm), red (e.g., 627-640nm, 633nm, 637nm, 640nm, 647nm) and / or near-infrared lasers (e.g., in the range of 673nm, 750nm, 780nm or 660-800nm).

[0184] This disclosure provides a staining buffer composition comprising a UV polymer dye or quenched UV polymer dye and a nonionic surfactant for reducing or inhibiting nonspecific interactions between polymer dye conjugates. A composition is provided for use with at least one fluorescent polymer dye conjugated to a binding partner for use in staining biological samples, comprising at least one UV-absorbing polymer dye or quenched UV-absorbing dye; a nonionic surfactant; and optionally a biological buffer. In some embodiments, the UV polymer dye or quenched UV polymer dye may be the dyes of this disclosure. The composition reduces the nonspecific binding of at least one fluorescent polymer dye conjugate compared to the at least one fluorescent polymer dye conjugate in the absence of the composition.

[0185] The staining buffer composition may be added to a multicolor panel of polymer dye conjugates before staining cells, and can effectively reduce or inhibit nonspecific interactions of polymer dye conjugates, for example, in flow cytometry (FCA) analysis of biological samples. The staining buffer composition was found to substantially reduce nonspecific polymer dye conjugate interactions in a multicolor dye conjugate panel. This was demonstrated in FCA of treated whole blood samples compared to the same sample without UV-absorbing polymer dyes, quenched UV polymer dyes, or nonionic surfactants.

[0186] The ready-to-use staining buffer compositions of this disclosure are universal solutions that function with all types of polymer dye conjugates across all polymer dye classes, including, for example, violet excitable polymer dye conjugates, ultraviolet excitable polymer dye conjugates, blue excitable polymer dye conjugates, and red excitable polymer dye conjugates. The staining buffer compositions have been found to substantially reduce or completely eliminate nonspecific interactions that may occur between cell staining with multiple polymer dye conjugates. UV-absorbing polymer dyes

[0187] The buffer compositions for staining according to this disclosure may comprise one or more UV-absorbing polymer dyes, one or more UV-absorbing tandem polymer dyes, and / or one or more quenched UV-absorbing polymer dyes. In some embodiments, the UV-absorbing polymer dyes, UV-absorbing tandem polymer dyes, or quenched UV-absorbing polymer dyes for use in the buffer compositions for staining may be DHP-based dyes, fluorene-based dyes, binaphthyl-based dyes, carbazole-based dyes, oxepin-based dyes (e.g., fluorenooxepine-based dyes), or combinations thereof. The UV polymer dyes, UV tandem polymer dyes, or quenched UV polymer dyes for use in the buffer compositions for staining may have one or more water-soluble moieties. The UV-absorbing polymer dyes, UV-absorbing tandem polymer dyes, or quenched UV-absorbing polymer dyes may be conjugated to a binding partner. The UV-absorbing polymer dye, UV-absorbing tandem polymer dye, or quenched UV-absorbing polymer dye may be a water-soluble UV-absorbing polymer dye. The UV-absorbing polymer dye, UV-absorbing tandem polymer dye, or quenched UV-absorbing polymer dye may be those provided for in this disclosure.

[0188] Ultraviolet (UV) is the electromagnetic spectrum region from approximately 10 nm to approximately 400 nm. In some examples, this disclosure provides UV-absorbing polymer dyes having a near-ultraviolet excitation spectrum and / or a near-UV absorption maximum. In some examples, this disclosure provides water-soluble UV-absorbing polymer dyes having a near-ultraviolet excitation spectrum and / or a near-UV absorption maximum. Near ultraviolet (UV) is the electromagnetic spectrum region from approximately 300 nm to approximately 400 nm, such as 350 nm to 400 nm. The term “near-ultraviolet excitation spectrum” can refer to the absorption spectrum of a UV-absorbing polymer dye having a full width at half maximum (FWHM) that defines a wavelength range within the near-UV region of the electromagnetic spectrum.

[0189] UV polymer dyes for use in dyeing buffer compositions may have a structure according to any of formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI and / or XIV. In some examples, the UV polymer dye may be a UV-absorbing polymer tandem dye ("UV polymer tandem dye"), for example, a quenched UV-absorbing polymer dye ("quenched UV polymer dye") according to any of formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI and / or XIV. In some embodiments, the UV-absorbing polymer dye does not contain a binding partner. In some embodiments, the quenched UV polymer dye does not contain a binding partner.

[0190] In some embodiments, UV polymer dyes or quenched UV polymer dyes for use in staining buffers may include UV polymer dyes known in the art, such as those taught in US9,719,998;US10,228,375;US11,119,107;US10,605,813;US2019 / 0194467A1 or WO2022 / 013198, each of which is incorporated herein by reference in whole.

[0191] In some embodiments, the staining buffer according to this disclosure comprises a UV polymer dye or a quenched UV polymer dye, including a binding partner. In some embodiments, the UV polymer dye or quenched UV polymer dye for use in a staining buffer does not include a binding partner.

[0192] The quenched polymer may contain polymer dyes according to this disclosure comprising one or more or more quenched moieties, for example, 1 to 50, 2 to 25, or 5 to 8 quenched moieties. In some embodiments, the quenched polymer exhibits a quantum yield (QY) of 0.1Φ or less, or 0.06Φ or less, or 0.056Φ or less, 0.05Φ or less, 0.02Φ or less, or 0.015Φ or less.

[0193] In some embodiments, the UV-absorbing dye may have an average molecular weight in the range of about 5 to about 150 kDa, about 10 to about 150 kDa, about 20 to about 150 kDa, about 40 to about 120 kDa, about 50 to about 100 kDa, or about 60 to about 70 kDa.

[0194] The staining buffer composition may contain 0.01 to 10 mg / mL, 0.02 to 5 mg / mL, 0.05 to 2 mg / mL, 0.1 to 1 mg / mL, 0.2 to 0.8 mg / mL, or about 0.5 mg / mL of the UV polymer dye or quenched UV polymer according to this disclosure. The amount of UV polymer dye or quenched UV polymer per test may be about 1 to about 50 ug / test, about 2 to about 30 ug / test, or about 5 to about 20 ug / test.

[0195] Nonionic surfactants

[0196] The composition may contain one or more nonionic surfactants. A sufficient amount of nonionic surfactant may be included to prevent aggregation of the polymer dye conjugate. Non-limiting examples of nonionic surfactants include poloxamer surfactants such as poloxamer 188 (e.g., PLURONIC® F-68; PF-68), polysorbate nonionic surfactants such as TWEEN® 20 and TWEEN® 80, and ether-linked nonionic surfactants such as polyoxyethylene glycol alkyl ether (BRIJ®), polyoxyethylene glycol octylphenol ether (TRITON®), or polyoxyethylene nonylphenyl ether (IGEPAL) surfactants. In some embodiments, the surfactant is a poloxamer nonionic surfactant.

[0197] The term "poloxamer nonionic surfactant" or "poloxamer" refers to polyethylene oxide-polypropylene oxide-polyethylene oxide (PEG-PPG-PEG) nonionic triblock copolymer. Poloxamer nonionic surfactants are known by trademark names such as PLURONIC® (BASF) nonionic surfactants, Kolliphor® (BASF) nonionic surfactants, and Synperonic® (CRODA) nonionic surfactants. Non-limiting examples of PLURONIC® surfactants include, for example, PLURONIC® F68, F77, F87, F98, F108, F123, F127, P103, P104, P105, P123, PE 3100, PE4300, PE6100, PE6200, PE6400, PE6800, PE8100, PE9400, PE10100, PE10400, and PE10500 (BASF Corporation). Poloxamer surfactants include nonionic triblock copolymers such as polyoxyethylene oxide-polyoxypropylene oxide-polyoxyethylene oxide (PEO-PPO-PEO), characterized by a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) sandwiched between two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)).

[0198] The dyeing buffer composition may contain a nonionic surfactant, which is a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer. An example nonionic triblock copolymer has the structure shown by formula XII. [ka] [In the formula, each a is an independent integer in the range of 2 to 130, and b is an integer in the range of 15 to 67.] This may include: In some embodiments, a is in the range of 50 to 100 and b is in the range of 20 to 40. In some embodiments, a is in the range of 70 to 90 and b is in the range of 25 to 30. The nonionic surfactant may be poloxamer 188.

[0199] Non-limiting examples of poloxamers may include, for example, poloxamer 188, also known as Pluronic F-68 or KOLLIPHOR® P188, having a=80 and b=27. Other poloxamers include poloxamer 338, also known as Synperonic® PE / F108, poloxamer 407, also known as Synperonic® PE / F127, and poloxamer 331, also known as Synperonic® PE / L101.

[0200] Since the length of the polymer block can be customized, there are many different poloxamers with slightly different properties. Poloxamer copolymers are generally named using the letter "P" (in the case of poloxamers) followed by a three-digit number, where the first two digits × 100 indicate the approximate molecular mass of the polyoxypropylene core, and the last digit × 10 indicates the percentage of polyoxyethylene content (e.g., P407 = 4,000 g / mol of polyoxypropylene molecular mass and a poloxamer with 70% polyoxyethylene content). In the case of the trademark names Pluronic and Synperonic poloxamers, the coding of these copolymers begins with a letter that defines its physical form at room temperature (L = liquid, P = paste, F = flake (solid)), followed by a two- or three-digit number. In the numerical representation, multiplying the first digit (or second digit in the case of a three-digit number) by 300 indicates the approximate molecular weight of the hydrophobic chain, and multiplying the last digit by 10 indicates the percentage of polyoxyethylene content (for example, F-68 indicates that the molecular weight of polyoxypropylene is 1,800 g / mol and the polyoxyethylene content is 80%).

[0201] The term "PLURONIC® F68," "Pluronic F-68," or "PF-68," also known as Poloxamer 188, refers to a poly(ethylene glycol)-block-poly(propylene glycol)-block poly(ethylene glycol) copolymer having an average molecular weight (average Mn) of 8350-8400. The term "PLURONIC® F127," also known as Poloxamer 407, refers to a triblock copolymer consisting of a central hydrophobic block of polypropylene glycol sandwiched between two hydrophilic blocks of polyethylene glycol (PEG). The approximate length of the two PEG blocks is 101 repeating units, while the approximate length of the propylene glycol block is 56 repeating units. This is also known as Croda® Synperonic PE / F127 with an average weight of 12,600 g / mol. The term "PLURONIC® F108" refers to poly(ethylene glycol)-block-poly(propylene glycol)-block poly(ethylene glycol) with an average Mn of approximately 14,600. The term "PLURONIC® P103" refers to poly(ethylene glycol)-block-poly(propylene glycol)-block poly(ethylene glycol) with an average Mw of approximately 4,950. The term "PLURONIC® P104" refers to poly(ethylene glycol)-block-poly(propylene glycol)-block poly(ethylene glycol) with an average Mw of approximately 5,900. The term "PLURONIC® P123" refers to poly(ethylene glycol)-block-poly(propylene glycol)-block poly(ethylene glycol) with an average Mn of approximately 5,800.

[0202] Exemplary poloxamer surfactants include, but are not limited to, Pluronic F-68. PF-68 is a nonionic triblock copolymer, such as poloxamer 188, which is a polyoxyethylene oxide-polyoxypropylene oxide-polyoxyethylene oxide (PEO-PPO-PEO). The concentration of the surfactant used can be determined empirically (i.e., titrated so that conjugate precipitation does not occur). In some embodiments, the staining buffer composition may contain a nonionic surfactant, such as a poloxamer surfactant. The nonionic surfactant may be Pluronic F-68. In some examples, the nonionic surfactant can be used alone (i.e., without UV dyes or UV-quenched dyes in the buffer) to reduce or block nonspecific interactions. Nonionic surfactants may be present in a composition, for example, a working concentrated staining buffer composition (1×), in the range of 0.1% to 20%, 0.1% to 15%, 0.2% to 9%, 0.5% to 8%, or 1% to 7% (wt / vol). Nonionic surfactants may be present in a final concentration (wt / vol) of about 0.1% to 2%, 0.5% to 1.5%, or about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or any value in between. Nonionic surfactants may be present in the concentrated dyeing composition (10×) in amounts ranging from, for example, 1-90%, 1-80%, 1-70%, 2-60%, 3-50%, 4-40%, or 5-25% (wt / vol). Nonionic surfactants may also be present in the concentrated dyeing composition (10×) in amounts ranging from, for example, 0.1-70%, 0.2-60%, 0.3-50%, 0.4-40%, or 0.5-25% (wt / vol). biological buffer

[0203] The term "biological buffer" refers to a physiologically compatible aqueous solution containing one or more biological buffers that maintains a pH within the biological range of pH 6–8, 6.5–8, or 7–8 in a cell-free system. The aqueous solution may include water for injection, milli-Q water, or other forms of highly purified water. The aqueous solution may include physiological saline or alcohol. A biological buffer may contain water and one or more biological buffers. A biological buffer may include PBS, Hanks' solution, Ringer's solution, or physiological saline buffer.

[0204] In certain embodiments, the biological buffer is, in particular among the buffers, N-(2-acetamide)-aminoethanesulfonic acid (ACES), acetate, N-(2-acetamide)-iminodiacetic acid (ADA), 2-aminoethanesulfonic acid (AES), ammonia, 2-amino-2-methyl-1-propanol (AMP), 2-amino-2-methyl-1,3-propanediol (AMPD), and N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfone Acid (AMPSO), N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), bicarbonate, N,N'-bis-(2-hydroxyethyl)-glycine, [bis-(2-hydroxyethyl)-imino]-tris-(hydroxymethylmethane) (BIS-Tris), 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BIS-Tris-propane), boric acid, dimethylarsinic acid, 3-(cyclohexylamino)-propanesulfonic acid (CAP S), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO), carbonate, cyclohexylaminoethanesulfonic acid (CHES), citrate, 3-[N-bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), formate, glycine, glycylglycine, N-(2-hydroxyethyl)-piperazine-N'-ethanesulfonic acid (HEPES), lactate, N-(2-hydroxyethyl)-piperazine-N'-3-propanesulfonic acid Piperazine acid (HEPPS, EPPS), N-(2-hydroxyethyl)-piperazine-N'-2-hydroxypropanesulfonic acid (HEPPSO), imidazole, malate, maleate, 2-(N-morpholino)-ethanesulfonic acid (MES), 3-(N-morpholino)-propanesulfonic acid (MOPS), 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO), phosphate, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) (POPSO), pyridine, polyvinylpyrrolidone (PVP), succinate, 3-{[tris(hydroxymethyl)-methyl]-amino}-propanesulfonic acid (TAPS), 3-[N-tris(hydroxymethyl)-methylamino]-2-hydroxypropanesulfonic acid (TAPSO), 2-aminoethanesulfonic acid, AES (taurine), trehalose, triethanolamine (TEA), 2-[tris(hydroxymethyl)-methylamino]-ethanesulfonic acid (TES), N-[tris(hydroxymethyl)-methylamino] It may contain one or more of the following: glycine (tricine), tris(hydroxymethyl)-aminomethane (Tris), glyceraldehyde, mannose, glucosamine, mannoheptulose, sorbose-6-phosphate, trehalose-6-phosphate, iodoacetate, sodium citrate, sodium acetate, sodium phosphate, sodium tartrate, sodium succinate, sodium maleate, magnesium acetate, magnesium citrate, potassium phosphate, magnesium phosphate, ammonium acetate, ammonium citrate, and ammonium phosphate. Typical buffers include salts of organic acid salts such as citric acid, ascorbic acid, gluconic acid, carbonate, tartaric acid, succinic acid, acetic acid, or phthalic acid; it may also contain Tris(tromethamine) hydrochloride or phosphate. Common examples of biological buffers include phosphate-buffered saline (PBS), N-2-hydroxyethylpiperazine-N'-2-hydroxypropanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), 2-([2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)ethanesulfonic acid (TES), 3-[N-tris(hydroxy-methyl)ethylamino]-2-hydroxyethyl]-1-piperazinepropanesulfonic acid (EPPS), tris[hydroxymethyl]-aminomethane (THAM), 1,This may include 4-piperazinediethanesulfonic acid (PIPES) and tris[hydroxymethyl]methylaminomethane (TRIS) buffers. Conventional biological buffers have a pK within the physiological range and can function most effectively within this range. Biological buffers may be aqueous solutions with concentrations of, for example, 10–100 mM or 5–25 mM.

[0205] The biological buffer may be PBS. The term "PBS" refers to phosphate-buffered saline, which is an aqueous buffer that may contain sodium chloride, disodium hydrogen phosphate, potassium chloride, and / or potassium dihydrogen phosphate. For example, PBS may contain milli-Q water or deionized water and 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4. The pH may be approximately pH 7.0 to 7.4. PBS may or may not be stored with an azide such as sodium azide. PBS may be an isotonic solution. The buffer may be a PBA buffer. The PBA buffer may contain PBS, BSA, and sodium azide. The PBA buffer may contain 1 × PBS, approximately 2 mg / mL of BSA, and approximately 0.1% (wt / vol) of sodium azide. Additional components

[0206] The compositions of this disclosure can be used, for example, as a staining buffer composition in flow cytometry sample analysis, and thus may include additional components, including, but not limited to, one or more of any suitable carriers, stabilizers, salts, chelating agents (e.g., EDTA), colorants, or preservatives. The compositions may also include one or more additional surfactants (e.g., ionic surfactants and zwitterionic surfactants). Ionic surfactants may also be anionic surfactants. The staining buffer composition may include formulation agents such as suspending agents, solubilizers, stabilizers, and / or dispersants. For example, the staining buffer composition may contain a carrier such as water, or a solvent such as DMSO, DMF, and acetonitrile as a solubilizer. The compositions may also contain pH adjusters, and typically the buffer is a salt prepared from an organic acid or base.

[0207] The staining buffer composition may contain a protein stabilizer. The term "protein stabilizer" refers to a protein that works to reduce nonspecific binding, for example, to reduce cell-cell interactions, or to help prevent nonspecific binding between an antibody and a non-target molecule. The compositions according to this disclosure may contain a protein stabilizer. The protein stabilizer may be selected from one or more of the group consisting of serum albumin, e.g., bovine serum albumin (BSA), casein, or gelatin. The protein stabilizer may be BSA. The protein stabilizer may be present in the compositions according to this disclosure at concentrations of 0.1–10 mg / mL, 0.5–5 mg / mL, 1–3 mg / mL, or about 2 mg / mL.

[0208] The staining buffer composition may contain any suitable preservative. The preservative may be an antioxidant, biocide, or antimicrobial agent. The preservative may be an inorganic salt. For example, the preservative may be sodium azide, 2-chloroacetamide, 2-methylisothiazolinone, salicylic acid, ProClin®, Kathon® CG, 5-chloro-2-methyl-4-isothiazolin-3-one, or 2-methyl-4-isothiazolin-3-one. The preservative may be present in the composition of this disclosure at a concentration of 0.01-0.5%, 0.05-0.3%, or about 0.1% (wt / vol).

[0209] The staining buffer compositions of this disclosure may contain additional surfactants. Suitable additional surfactants that can be used as needed according to the methods described herein include zwitterionic surfactants such as alkylbetaines, alkylamidebetaines, amidazolinium betaines, sulfobetaines (INCI sultaines), and betaines such as phosphobetaine. Examples of suitable zwitterionic surfactants include those with the general formula R 1’ [CO-X(CH2)] j ] g -N + (R 2’ )(R 3’ )-(CH2) f -[CH(OH)CH2] h -Y - [In the formula, R 1’ C 8~18 Alkyl, saturated C 10~16 Alkyl or saturated C 12~14 Saturated or unsaturated C such as alkyl groups 6~22 It is alkyl, and X is NH, NR 4’ And R 4’ C 1~4 Alkyl, O or S, j is an integer from 1 to 10 such as 2 to 5 and 3, g is 0 or 1, R 2’ and R 3’ Each of them is independent of C 1~4Alkyl, hydroxy optionally substituted with a hydroxyethyl group or methyl group, f is an integer from 1 to 4 such as 1, 2, or 3, h is 0 or 1, and Y is COO, SO3, OPO(OR 5’ )O or P(O)(OR 5’ )O and R 5’ is H or C 1~4 It contains surfactants that are alkyl.

[0210] Examples of suitable zwitterionic surfactants include formula: R 1’ -N + (CH3)2-CH2COO - ; R 1’ -CO-NH(CH2)3-N + (CH3)2-CH2COO - ; R 1’ -N + (CH3)2-CH2CH(OH)CH2SO3 - and R 1’ -CO-NH-(CH2)3-N + (CH3)2-CH2CH(OH)CH2SO3 - It contains alkyl betaines such as those found in [this product].

[0211] Examples of suitable betaines and sulfobetaines are listed below (as indicated in accordance with INCI): almondamidopropyl betaine, apricotamidopropyl betaine, avocadoamidopropyl betaine, babassuamidopropyl betaine, behenamidopropyl betaine, behenyl betaine, canolamidopropyl betaine. Capryl / Capramidopropyl Betaine, Carnitine, Cetyl Betaine, Cocamidoethyl Betaine, Cocamidopropyl Betaine, Cocamidopropyl Hydroxysultaine, Cocobetaine, Cocohydroxysultaine, Coco / Oleamidopropyl Betaine, Cocosultaine, Decyl Betaine, Dihydroxyethyl Oleyl Glycinate, Dihydroxyethyl Soy Glycinate, Dihydroxyethyl Stearyl Glycinate, Dihydroxyethyl Fat Glycinate, PG-Betaine Dimethicone Propylene Glycol, Dorcamidopropyl Hydroxysultaine, Hydrogenated Fat Betaine, Isostearamidopropyl Betaine, Lauramidopropyl Betaine, Lauryl Betaine, Lauryl Hydroxysultaine, Lauryl Saltaine, Milk Amidopropyl Betaine, Milk Amidopropyl Betaine These include betaine, myristamidopropyl betaine, myristyl betaine, oleamidopropyl betaine, oleamidopropyl hydroxysultaine, oleyl betaine, olibamidopropyl betaine, palmamidopropyl betaine, palmitamidopropyl betaine, palmitoyl carnitine, palm kernel amidopropyl betaine, polytetrafluoroethylene acetoxypropyl betaine, ricinolamidopropyl betaine, sesamidopropyl betaine, soyamidopropyl betaine, stearamidopropyl betaine, stearyl betaine, tallow amidopropyl betaine, tallow amidopropyl hydroxysultaine, tallow betaine, tallow dihydroxyethyl betaine, undecylenamidopropyl betaine, and wheat germ amidopropyl betaine.

[0212] For example, coconut dimethyl betaine is commercially available from Seppic under the trademark name AMONYL265®, and lauryl betaine is commercially available from Sigma-Aldrich under the trademark name EMPIGEN BB®. A further example of betaine is lauryl-imino-dipropionate, commercially available from Rhodia under the trademark name MIRATAINE H2C-HA®. The presence of a zwitterionic surfactant as needed in the staining buffer composition may reduce nonspecific binding in the biological sample, for example, to monocytes or other blood components. The zwitterionic surfactant may be present in the composition at concentrations of 0-0.5% or 0.01-0.3% as needed. composition

[0213] Staining buffer compositions are provided to reduce polymer-polymer interactions between polymer dye conjugates in biological samples and to reduce precipitation of dye conjugates. Staining buffer compositions are also provided to reduce polymer-polymer interactions between polymer dye conjugates in a multicolor panel containing two or more polymer dye conjugates.

[0214] The staining buffer composition can be used with one, more, or more fluorescent polymer dye conjugates. The staining buffer composition of this disclosure can substantially reduce nonspecific binding between multiple fluorescent polymer dye conjugates.

[0215] The compositions according to this disclosure may be used with a mixture of dye conjugates containing one or more, two or more, or three or more polymer dye conjugates before, simultaneously with, or after addition to a biological sample, in order to reduce, substantially reduce, and / or prevent nonspecific bonding, such as polymer-polymer interactions, between polymer dye conjugates. The mixture of dye conjugates may contain one or more, two or more, or three or more polymer dye conjugates.

[0216] A buffer composition for dyeing is provided, comprising a UV polymer dye or a quenched UV polymer dye and a nonionic surfactant. The UV polymer dye or quenched UV polymer dye may be as described in this disclosure. The buffer composition for dyeing may contain, in a biological buffer, the UV polymer dye or quenched UV polymer dye according to this disclosure and a nonionic surfactant.

[0217] The staining buffer composition can supply 5-20 ug / test of UV polymer dye and 0.1-2% (wt / vol) / test of nonionic surfactant in a biological buffer.

[0218] The UV polymer dye may be any suitable UV polymer dye or a quenched UV polymer. The UV polymer dye may be as described in this disclosure. In some embodiments, the UV polymer dye does not include a binding partner. In some embodiments, the UV polymer dye includes a binding partner. In some embodiments, the binding partner is neither an antibody nor a fragment thereof. The UV polymer may be a tandem UV polymer comprising one or more acceptor dyes. The UV polymer may be a quenched UV polymer comprising one or more quenching moieties. The quenched UV polymer dye may or may not include a binding partner. The nonionic surfactant may be a poloxamer nonionic surfactant. The poloxamer may be Pluronic F-68. Optionally, the composition further comprises a protein stabilizer. Optionally, the composition comprises a preservative. Optionally, the composition comprises a zwitterionic surfactant. The biological buffer may be a PBS buffer. The protein stabilizer may be BSA. The preservative may be NaN3. The composition can reduce, substantially reduce, or eliminate polymer-polymer interactions in a multicolor panel.

[0219] Typical buffer compositions for staining are shown in Table 1A.

[0220] [Table 1A]

[0221] The dyeing buffer compositions of this disclosure can reduce, substantially reduce, or eliminate polymer-polymer interactions in multicolor panels.

[0222] The term "multicolor panel" refers to a conjugate of one or more, two or more, or three or more fluorescent polymer dyes, and, as appropriate, a conjugate of fluorescein, coumarin, cyanine, rhodamine dyes, such as FITC, PE, ECD, PC5, PC5.5, PC7, APC, AA700, AA750, PBE, Alexa Fluor® 488 (AF488), AF532, AF647, AF700, AF750, Atlantis Bioscience CF® 350 dye, CF® 405S, CF® 405, CF® 405L, CF® 430, CF® 440, CF® 450, CF® 488A, CF® 514, and AAT Bioquest. This refers to a mixture of dye conjugates that may contain one or more, two or more, or three or more fluorescent dye conjugates, such as iFluor(trademark) 488, iFluor(trademark) 350, iFluor(trademark) 405, mFluor(trademark) Blue 570, mFluor(trademark) Blue 580, mFluor(trademark) Blue 590, mFluor(trademark) Blue 620, mFluor(trademark) Blue 630, mFluor(trademark) Blue 660, ThermoFisher Scientific NovaFluor Blue 510, NovaFluor Blue 530, NovaFluor Blue 555, NovaFluor Blue 585, NovaFluor Blue 610 / 30S, NovaFluor Blue 660 / 40S, NovaFluor Blue 660 / 120S, BioLegend(registered trademark) Kiravia Blue 520(trademark), and KrO dye conjugates.

[0223] The term "fluorescent dye" refers to a dye containing a photoexcitable fluorophore that can re-emit light upon photoexcitation. The term "fluorophore" refers to a fluorescent chemical compound that can re-emit light upon photoexcitation. Fluorophores can typically contain several mixed aromatic groups or planar and cyclic molecules having several pi bonds. The term "fluorescent dye" encompasses both fluorescent polymer dyes and fluorescent nonpolymer dyes, including fluorescent monomers and other conventional fluorescent dyes. Fluorescent polymer dyes may be any suitable fluorescent polymer dye.

[0224] The compositions according to this disclosure can be used with any polymer dye conjugate. The polymer dye conjugate may be a tandem polymer dye conjugate. The polymer dye conjugate may include any previously disclosed or commercially available fluorescent polymer dyes. For example, the polymer dyes are published in PCT application WO2017 / 180998; US application 2021 / 0047476; US application 2020 / 0190253; US application 2020 / 0048469; US application 2020 / 0147615; US application 2021 / 0108083; US application 2019 / 0194467; US application 2018 / 0364245; US Application No. 2018 / 0224460; U.S. Patent No. 11,034,840; U.S. Patent No. 11,119,107; U.S. Patent No. 10,962,546; U.S. Patent No. 10,920,082; U.S. Patent No. 10,001,475; U.S. Patent No. 10,107,818; U.S. Patent No. 10,228,375; U.S. Patent No. 10,844,228; U.S. Patent No. 10,605,813; These may be any dyes disclosed in U.S. Patent No. 10,604,657; U.S. Patent No. 10,545,137B2; U.S. Patent No. 10,533,092; U.S. Patent No. 10,472,521; U.S. Patent No. 10,240,000; U.S. Patent No. 9,971,998; U.S. Patent No. 9,758,625; U.S. Patent No. 9,719,998; U.S. Patent No. 7,214,489; U.S. Patent No. 9,012,643; U.S. Patent No. 8,623,332; U.S. Patent No. 8,431,416; U.S. Patent No. 8,354,239; U.S. Patent No. 8,575,303; U.S. Patent No. 8,969,509; and any dyes disclosed in WO2022 / 013198, each of which is incorporated by reference as if they were entirely described herein. The polymer dye may have the structure of any water-soluble fluorescent polymer dye disclosed in published U.S. Patent Application No. 2020 / 0190253A1, which is incorporated by reference as if the whole were fully described herein.The polymer dye conjugate may have the structure of any water-soluble fluorescent polymer dye disclosed in published U.S. Patent Application No. 2019 / 0144601, which is incorporated by reference as if the whole were fully described herein.

[0225] The polymer dye conjugate can be any commercially available polymer dye that can be excited by ultraviolet (e.g., 351nm, 355nm, 375nm, 334-364nm, 351-356nm), violet (e.g., 405nm, 407nm, 414nm, 395-425nm), blue (e.g., 436nm, 458nm), blue-green (e.g., 488nm), green (e.g., 514nm, 532nm, 541nm, 552nm), yellow-green (e.g., 561nm, 563nm), yellow (e.g., 568nm), red (e.g., 627-640nm, 633nm, 637nm, 640nm, 647nm) and / or near-infrared lasers (e.g., in the range of 673nm, 750nm, 780nm or 660-800nm). The polymer dye may include a polymer dye that is excitable by a violet laser. The polymer dye or polymer dye conjugate may include a polymer dye that is excitable by a violet laser with a wavelength of approximately 395 nm to approximately 425 nm, for example, 405 nm, 407 nm, or 414 nm. The polymer dye or polymer dye conjugate may include a polymer dye that is excitable by a violet laser (405 nm).

[0226] In some embodiments, the polymer dye conjugate may include SuperNova polymer dyes (SN) (Beckman Coulter, Inc.). SuperNova polymer dyes are a new generation of polymer dyes useful for flow cytometry applications. The polymer dye or polymer dye conjugate may include SNv428, SNv605, or SNv786. SNv428 has unique photophysical properties that, when conjugated to an antibody or other binder, result in a fairly bright conjugate. For example, SNv428 is a polymer dye that is optimally excited by a violet laser (e.g., 405 nm) with an excitation maximum at 414 nm and an emission peak at 428 nm, and can be detected using a 450 / 50 bandpass filter or equivalent. SNv428 is one of the brightest dyes that can be excited by a violet laser and is therefore particularly suitable for evaluating faintly expressed markers. SuperNova polymer dyes conjugated with antibodies can include dye conjugates of anti-CD19 antibody-SNv428, anti-CD22 antibody-SNv428, anti-CD25 antibody-SNv428, and anti-CD38 antibody-SNv428 antibody-polymer. SNv605 and SNv786 (Beckman Coulter, Inc.) are tandem polymer dyes derived from core SNv428. Both share the same absorbance characteristics, with maximum excitation at 414 nm. Using SNv605 and SNv786, which have emission peaks at 605 nm and 786 nm respectively, they are optimally detected using 610 / 20 nm and 780 / 60 nm bandpass filters on a flow cytometer. SNv605 and SNv786 may be conjugated with, for example, anti-CD19 antibody, anti-CD22 antibody, anti-CD25 antibody, and anti-CD38 antibody.

[0227] Polymer dye conjugates may contain polymer dyes that are excitable by ultraviolet ("UV") lasers. Polymer dyes or polymer dye conjugates may contain polymer dyes that are excitable by UV lasers with wavelengths of 320 nm to 380 nm, 340 nm to 360 nm, 345 nm to 356 nm, or shorter than or equal to 380 nm but longer than or equal to 320 nm. Polymer dyes or polymer dye conjugates may contain UV-excitable polymer dyes. UV-excitable polymer dyes or polymer dye conjugates can typically emit light at wavelengths of 380 nm to 1000 nm, 380 nm to 800 nm, 380 nm to 430 nm, 406 nm to 415 nm, or shorter than or equal to 430 nm but longer than or equal to 380 nm.

[0228] Polymer dye conjugates include Brilliant Violet421(trademark) (excitation maximum 405nm, emission maximum 421nm, 450 / 50 filter), Brilliant Violet510(trademark) (excitation maximum 405nm, emission maximum 510nm, 510 / 50 filter), Brilliant Violet570(trademark) (excitation maximum 405nm, emission maximum 570nm, 585 / 42 filter), Brilliant Violet605(trademark) (excitation maximum 405nm, emission maximum 603nm, 610 / 20 filter), Brilliant Violet650(trademark) (excitation maximum 405nm, emission maximum 645nm, 660 / 20 filter), Brilliant Violet711(trademark) (excitation maximum 405nm, emission maximum 711nm, 710 / 50 filter), and Brilliant Violet This may include Brilliant Violet® dyes (BioLegend® / Sirigen Group Ltd) such as Violet750® (excitation maximum 405nm, emission maximum 750nm, 780 / 60 filter) and Brilliant Violet785® (excitation maximum 405nm, emission maximum 785nm, 780 / 60 filter). Polymer dyes or polymer dye conjugates may include Spark Violet® 538 (BioLegend, Inc) (excitation maximum 405nm, emission maximum 538nm).

[0229] The polymer dye conjugate may include a Super Bright polymer dye (Invitrogen, ThermoFisher Scientific). The Super Bright dye may be excited by a violet laser (405 nm). The Super Bright dye may be Super Bright 436 (excitation maximum 414 nm, emission maximum 436 nm, 450 / 50 bandpass filter), Super Bright 600 (emission maximum 600 nm, 610 / 20 bandpass filter), Super Bright 645 (emission maximum 645 nm, 660 / 20 bandpass filter), or Super Bright 702 (emission maximum 702 nm, 710 / 50 bandpass filter).

[0230] The polymer dye conjugate may contain BD Horizon Brilliant® Violet ("BV") polymer dyes (Becton, Dickinson and Co., BD Life Sciences). The polymer dyes may also contain BD Horizon Brilliant® BV421 (450 / 40 or 431 / 28 filter), BV480 (525 / 40 filter), BV510 (525 / 40 filter), BV605 (610 / 20 filter), BV650 (660 / 20 filter), BV711 (710 / 50 filter), and BV786 (786 / 60 filter) polymer dyes. method

[0231] A method is provided for reducing or eliminating nonspecific binding, such as polymer-polymer interactions, between at least one polymer dye conjugate or at least two polymer dye conjugates in a biological sample, such as a blood sample, comprising the step of contacting at least one dye conjugate with at least one UV polymer dye and a nonionic surfactant before, during, or after contact between the polymer dye conjugate and the biological sample, thereby resulting in a reduction of nonspecific binding, such as a reduction in polymer-polymer interactions between at least one or at least two polymer dye conjugates in the biological sample.

[0232] In some embodiments, the Disclosure provides a method for reducing or eliminating nonspecific binding, such as polymer-polymer interactions, between at least one or at least two polymer dye conjugates in a biological sample, comprising the step of contacting the at least one polymer dye conjugate with a UV-absorbing polymer dye and a nonionic surfactant before, during, or after contacting the dye conjugate with a biological sample, resulting in a reduction of nonspecific binding between the polymer dye conjugates in the sample. In some embodiments, the Disclosure provides a method for reducing or eliminating polymer-polymer interactions between at least one or at least two polymer dye conjugates in a multicolor panel, comprising the step of contacting the at least one or at least two polymer dye conjugates with at least one UV-absorbing polymer dye and / or a nonionic surfactant before, during, or after contacting the polymer dye conjugate with a blood sample, resulting in a reduction of nonspecific binding between the at least one or at least two polymer dye conjugates in a biological sample. The compositions and methods of the Disclosure reduce or eliminate nonspecific binding of polymer dye conjugates in a blood sample. [Examples]

[0233] Various embodiments of this disclosure can be better understood by referring to the following examples presented by illustration. This disclosure is not limited to the examples shown herein. (Example 1) Initial screening

[0234] The initial focus was on identifying one or more modification units that, when reacted with monomers such as DHP monomers, could shift the absorption maximum of the polymer dye to near 355 nm, thereby minimizing absorption at 405 nm. A general reaction scheme for polymer preparation is shown in Scheme 1.

[0235] Scheme 1 [ka]

[0236] Initial screening of monomers and modification units was performed using test polymerization reactions.

[0237] Modified units that produced polymers with good absorption at 355 nm and minimal excitation at 405 nm were selected for large-scale polymerization.

[0238] Test polymerization reactions were performed using DHP monomers and one or two modification units (in a ratio of 1:0.5:0.5). Table 1B shows the evaluation of DHP monomer units (f=11~40). Both absorbance and emission maxima were monitored. Item 1 in Table 1B shows an absorption maximum at 348 nm and an emission maximum at 408 nm. Furthermore, the absorption spectrum was relatively sharp, and therefore, crosstalk at 405 nm was minimal (crosstalk was 2.1). The polymer exhibited excellent photophysical properties with respect to brightness, low crosstalk, and high conjugation yield. Test polymerization of DHP monomers using one and two modification units was performed as shown in items 1-16 of Table 1B. Based on these results, it was decided to proceed with DHP monomers together with difluoro and trifluoro-based modification units as components of the target polymer (shown in item 1). Other preferred target polymers include the polymerization products of Table 1B, items 2, 3, 4, 5, 6, 7, 12, or 15. The variable "f" in Table 1B is an integer between 11 and 40. The variable "p" in Table 1B is 18.

[0239] [Table 1B-1] [Table 1B-2] [Table 1B-3] [Table 1B-4] (Example 2) Cap polymer synthesis

[0240] Method 1: Both the modified units and DHP monomers were placed in a round-bottom flask in a (DMF-water) mixture 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. Subsequently, a capping agent containing appropriate functional groups (selected from G1) was added to the reaction mixture, and after 3 hours, a second capping agent (selected from G2) was added. After the reaction, the crude reaction mixture was removed by distillation and passed through a gel filtration column to remove organic low molecular weight and low MW oligomers. The crude polymer was then passed through a tangential flow filtration system.

[0241] Method 2: Both the modified units and DHP monomers (1:1) were taken into a round-bottom flask and dissolved in a THF-water (4:1) mixture containing 10 equivalents of K2CO3 and 3% Pd(PPh3)4. The reaction mixture was attached to a Schlenkline and degassed by three freeze-pump-thaw cycles, then heated to 80°C under nitrogen with vigorous stirring for 18 hours. Subsequently, a capping agent containing appropriate functional groups (selected from G1) was added to the reaction mixture via a cannula under nitrogen overpressure, and after 3 hours, a second capping agent (selected from G2) was added. After the reaction, the crude reaction mixture was removed by distillation and passed through a gel filtration column to remove organic low molecular weight and low MW oligomers. The crude polymer was then passed through a tangential flow filtration system.

[0242] The polymer was further characterized using NMR (polymer quality), GPC (Mw, Mn, and PDI), and spectroscopy (molar extinction coefficient, quantum yield, brightness), as well as capping efficiency.

[0243] The polymer has the following structure X (where "f" is an integer from 0 to 50, or from 11 to 40, G 1 and G 2 (As described herein): [ka] It possessed.

[0244] Figure 1 illustrates a comparison of the signal-to-noise ratio at a 405 nm channel for three different lots (B, C, and D) of a CD4 antibody conjugated with a UV-absorbing polymer, compared to a BUV395-CD4 conjugate (A) (Becton Dickinson Biosciences). Laser excitation was performed at 355 nm.

[0245] Figure 1 shows that the conjugated polymer exhibited a brightness of over 2× in the 405nm channel compared to BUV395CD4 conjugate A, as measured by a 355nm laser flow cytometer. Table 2 shows the absorbances of the BUV395-CD4 conjugate and polymer CD4 conjugate at 375nm and 355nm. Subsequently, this polymer CD4 conjugate was analyzed by a 375nm laser flow cytometer, and it exhibited a brightness of approximately 5.5× at 405nm compared to the BUV395CD4 conjugate. As shown in Table 2, the polymer CD4 conjugate exhibited approximately 75% of the absorbance at 375nm compared to 355nm, while the BUV395CD4 conjugate showed only about a 5% difference in absorbance at 375nm compared to 355nm, thus justifying the above results. Therefore, the UV-absorbing polymer CD4 conjugate of the present invention exhibited superior performance in a 375 nm laser system compared to the comparative BUV395CD4 conjugate. This may be advantageous for polymers in both 355 nm and 375 nm laser flow systems.

[0246] [Table 2] (Example 3) Polymer tandem dyes

[0247] In this example, the polymer tandem dye having an acceptor dye was prepared according to the general method shown in Example 2, and the polymer had the structure shown below (where "f" is an integer from 0 to 50, G 1 and G 2 The compound (as described herein) was then conjugated to a CD4 antibody. [ka]

[0248] Polymers from Example 2 were used to form polymer tandem dyes. The reaction scheme for the synthesis is shown in Scheme 2.

[0249] Scheme 2. Synthesis of polymer tandem dyes. [ka]

[0250] Procedure for synthesizing NHBoc UV polymer: Under a nitrogen atmosphere, the polymer solution was transferred to a 10 mL reaction flask containing cesium carbonate (100 equivalents). The tert-butyl-3-iodopropyl-carbamate solution was diluted from the storage solution (10 mg / mL in anhydrous DMF) and 10 equivalents were added to the polymer mixture. The sealed reaction flask was heated to 50°C and the reaction was continued for 1 hour with stirring at 500 rpm. The reaction mixture was cooled to room temperature and the DMF was evaporated in a rotary evaporator under high vacuum. The crude reaction mixture was diluted with chloroform (25 mL) and washed with 15% w / v brine solution (25 mL). The organic layer was collected in a 250 mL conical flask, an additional chloroform (12 mL) was added, and the mixture was washed three times with 30% w / v brine solution (10 mL). The organic fraction was dried by adding 20 g of anhydrous sodium sulfate, then filtered through Whatman Paper 2 and placed in a 150 mL flat-bottom flask. The filtered sodium sulfate was washed twice with chloroform (15 mL) to recover the remaining polymer dye, which was filtered and placed in the same flat-bottom flask. The chloroform was evaporated in a rotary evaporator at 45 °C and 150–200 rpm. The residual DMF was removed under a high vacuum pump at 50 °C for 30–40 minutes. The dried polymer was washed with diethyl ether (2 × 2 mL) and sonicated for 2 minutes to remove unreacted tert-butyl-3-iodopropyl-carbamate. After drying the polymer under high vacuum for 5 minutes, the polymer yield was calculated relative to the initial polymer amount. The dried polymer product was characterized using 1H NMR. The proton signal at 1.4 ppm indicates the presence of the NH-Boc moiety in the polymer.

[0251] Procedure for synthesizing NH2 UV polymer: 50 mg of NHBoc polymer prepared as described above was added to a 20 mL round-bottom flask and dissolved by vortexing in 1 mL of methanol and 1 mL of water for 5 minutes, followed by sonication for 5 minutes. 12 M HCl (2 mL) was added to the resulting solution, and the mixture was allowed to react at room temperature for 2 hours. The reaction mixture was then transferred to a small beaker, the pH was adjusted to 9-10 using a 15% w / v K2CO3 solution, and the mixture was stirred for a further 15 minutes. The polymer was extracted using 25 mL of chloroform in a 100 mL separation funnel, and the organic layer was collected in a conical flask. Brine solution (15% w / v) was added to the aqueous layer, and the remaining polymer was recovered using additional chloroform. The extraction process was monitored using a UV lamp.

[0252] The organic layer was dried over approximately 40 g of anhydrous sodium sulfate, filtered through Whatman filter paper 2, and placed in a 250 mL flat-bottom flask. The polymer remaining from the filtered sodium sulfate was recovered using additional chloroform washings (2 × 20 mL). The combined chloroform layer was evaporated in a rotary evaporator at approximately 40°C. After complete evaporation of the solvent, the solid was dissolved again in chloroform (10 mL), centrifuged at 3000 rpm for 5 minutes, and salt impurities were removed in a 15 mL Falcon tube. The supernatant was decanted in a 20 mL vial, concentrated in a rotary evaporator, and dried under high vacuum. The yield of the deprotected amine-functionalized polymer was calculated relative to the amount of protected polymer.

[0253] To form polymer tandem dyes, 10 mg of polymer was weighed into a glass vial and dissolved in 200 μL of anhydrous DMSO. To ensure complete dissolution of the polymer, a combination of vortexing, sonication, and incubation in a 50°C water bath for approximately 10–15 minutes was applied. 200 μL of acetonitrile and 20 μL of diisopropylethylamine were then added. A 10 mg / mL (w / v) solution of acceptor dye NHS ester (e.g., an acceptor dye with emission centered at approximately 700–800 nm) was prepared in anhydrous DMSO, and 8 equivalents of the dye were added to the polymer solution. The mixture was stirred at room temperature for 2 hours and protected from light, yielding a product containing an average of 2–3 dyes per polymer chain. Products containing 1–6 dyes per polymer chain can be prepared by adjusting the amount of acceptor dye used in the reaction. The polymer tandem dyes were conjugated with CD4 antibody.

[0254] Absorption and emission measurements were performed on the purified polymer CD4 conjugate. The acceptor-to-donor ratio was calculated to be 1.7. Fluorescence resonance energy transfer (FRET) was investigated by exciting the polymer at 355 nm, resulting in emission from the acceptor dye with FRET accompanied by >90% quenching of the donor emission. When this polymer was excited at 405 nm, almost no fluorescence was observed from either the polymer or the acceptor dye. This suggests that the current backbone can be modified using standardization techniques, and efficient energy transfer can be achieved by finding a suitable acceptor dye.

[0255] Figure 2 illustrates the signal-to-noise ratio in the S / N 740 / 40 nm channel (using laser excitation at 355) of a comparative CD4 conjugate of BD Horizon BUV737 compared to the UV polymer-Dy704 tandem-CD4 conjugate polymer tandem dye of the present invention formed in this embodiment.

[0256] The terms and expressions used are for illustrative purposes only, not restrictive, and are not intended to exclude any equivalent or part thereof of the features shown and described, although it is recognized that various modifications are possible within the scope of the embodiments of this disclosure. Therefore, although this disclosure is specifically disclosed by specific embodiments and features as needed, it should be understood that modifications and variations of the concepts disclosed herein are usable by those skilled in the art, and that such modifications and variations are considered to be within the scope of the embodiments of this disclosure. (Example 4) Procedure for surface staining with co-immobilized buffer in sample preparations for flow cytometry

[0257] In this procedure, to avoid any possible nonspecific interactions that may occur between dye conjugates over time, the staining buffer according to this disclosure is added to the test tube before the dye conjugates are added. Immobilization is a step that allows the leukocyte preparation to be stored for several hours without degradation after staining with a fluorescent antibody. In the preparation of biological samples for flow cytometry, a lysis solution may be used to lyse erythrocytes.

[0258] 1. Immediately prepare the "fixation and dissolution" mixture by adding 25 μL of undiluted IOTest 3 10× Fixative Solution (AO7800, Beckman Coulter, Inc.) to 1 mL of VersaLyse® dissolution solution (AO9777, Beckman Coulter, Inc.). Prepare a sufficient amount of the "fixation and dissolution" mixture (1 μL of mixture per tube) depending on the number of biological test samples to be dissolved. 2. Add 10 μL or 20 μL of the staining buffer according to this disclosure to each test tube. 3. Add an appropriate amount of dye conjugate. Gently vortex the tube. 4. Add 100 μL of the test sample to each tube. Gently vortex the tubes. 5. Protect from light and incubate at room temperature (18-25°C) for 15-20 minutes. Next, perform erythrocyte lysis: 6. Immediately add 1 ml of the prepared "fixation and dissolution" mixture and vortex for 1 second. 7. Protect from light and incubate at room temperature for 10 minutes. 8. Centrifuge at 150 × g for 5 minutes at room temperature. 9. Remove the supernatant by suction. 10. Resuspend the cell pellet using 3 mL of PBS. 11. Centrifuge at 150 × g for 5 minutes at room temperature. 12. Remove the supernatant by suction. 13. Resuspend the cell pellet using 0.5 mL of PBS and 0.1% formaldehyde (0.1% formaldehyde in PBS can be obtained by diluting 12.5 μL of IOTest 3 Fixative Solution (see catalog for PN) in 1 mL of PBS at its 10x concentration).

[0259] These preparations may be kept at a temperature of 2–8°C for 24 hours and protected from light before analysis by flow cytometry. (Example 5) Selection of components for staining buffer compositions to avoid nonspecific interactions with cells

[0260] We developed a staining buffer composition for use with a multicolor panel containing one or more polymer dye conjugates for use in staining biological samples during flow cytometry. The primary objective was to select compositional components that avoid nonspecific interactions with cells in the biological sample.

[0261] Human whole blood was treated with various concentrations of UV-absorbing polymers according to this disclosure, with or without 1% PF-68. Red blood cells were lysed, and white blood cells were washed twice, and the samples were subjected to a CytoFlex LX flow cytometer. FCA dot plots are shown in Figure 3. The top panel shows only blood without additives; the four middle panels show the addition of UV-absorbing polymers according to this disclosure at 2.5 ug / test, 5 ug / test, 10 ug / test, and 20 ug / test (from left to right); and the four bottom panels show the addition of UV-absorbing polymers according to this disclosure at 2.5 ug, 5 ug, 10 ug, and 20 ug (from left to right), together with 1% PF-68. The upper right panel shows the addition of 10 ug of quenched polymer 3. The MFI values ​​of monocytes indicate that UV polymers containing or not containing 1% PF-68 (2.5 ug to 20 ug / test) did not show strong nonspecific binding to cells compared to control cells (blood-only panel).

[0262] An exemplary staining buffer composition provides PF-68 at 0.1-2% / test of the UV polymer dye according to this disclosure (5-20 ug / test) in PBA buffer (PBS / BSA / NaN3).

[0263] Table 4 shows, for example, typical compositions containing polymer dye conjugates to be added to a multicolor panel for staining biological samples before FCA analysis. One exemplary staining buffer composition contains 0.5 mg / mL of UV polymer, 7% PF-68, 2 mg / mL of BSA, and 0.02% NaN3 in PBS buffer. The UV polymer may be any UV polymer according to this disclosure. The UV polymer may be a tandem UV polymer containing one or more acceptor dyes. The UV polymer may be a quenched UV polymer containing one or more quenching moieties.

[0264] We developed a method for preparing a buffer composition for dyeing.

[0265] The preservation solutions were prepared as follows: UV polymer: 1.3 mg of polymer was weighed, and then 130 uL of DMSO was added and the polymer was dissolved by vortexing to prepare a 10 mg / mL UV polymer preservation solution. PF-68: A commercially available 10% PF-68 solution was used as is. BSA: 20 mg of BSA was weighed, and then 1 mL of buffer-PBS was added to prepare a 20 mg / mL preservation solution. NaN3: 10 mg of NaN3 was weighed, and then 1 mL of buffer-PBS was added to prepare a 1% preservation solution.

[0266] The staining buffer composition was formulated using the preservation solutions shown in Table 3.

[0267] [Table 3]

[0268] For example, before staining cells, the staining buffer compositions listed in Table 4 can be added to the polymer dye conjugate. For example, 20 μL of the staining buffer composition, followed by the polymer dye conjugate, may be added to the test tube before adding the biological sample. (Example 6) Performance of a dyeing buffer composition containing two different UV polymer dye conjugates

[0269] Human whole blood was stained with two polymer dye conjugates according to this disclosure: CD20-UV excitable polymer dye (UVEPD) and SN uv408-CD4 (Beckman Coulter Life Sciences), in the presence of various concentrations of UV polymer, with and without additives. FCA dot plots of cells stained with the two polymer dye conjugates and various additives are shown in Figure 4. The upper left panel shows cells stained without additives, the upper center panel shows cells stained with 1% PF-68, the upper right panel shows cells stained with the UV polymer according to this disclosure (10 ug / test), the lower left panel shows cells stained with 5 ug of UV polymer and 1% PF-68, the lower center panel shows cells stained with 10 ug / test of UV polymer and 1% PF-68, and the lower right panel shows cells stained with 20 ug of UV polymer and 1% PF-68. In this example, cells stained in the presence of a combination of UV polymer (5–20 μg / test) + 1% PF-68 (the three lower panels) showed less spillover than the control, including samples without buffer (upper left panel), 1% PF-68 (upper center panel), and UV polymer alone (upper right panel). The values ​​in each panel represent the MFI values. (Example 7) Performance of a dyeing buffer composition containing two different purple polymer dye conjugates

[0270] Human whole blood was stained and lysed with CD20-SN v428 (Beckman Coulter Life Sciences) and CD4-BV650 (BD Biosciences) polymer dye conjugates, with or without additives and / or UV polymers. FCA dot plots are shown in Figure 5. The upper left panel shows stained cells in PBS without additives, the upper center panel shows cells stained with 1% PF-68, the upper right panel shows cells stained with the UV polymer (10 ug / test) according to this disclosure, the lower left panel shows cells stained with 5 ug / test UV polymer and 1% PF-68, the lower center panel shows cells stained with 10 ug / test UV polymer and 1% PF-68, and the lower right panel shows cells stained with 20 ug / test UV polymer and 1% PF-68.

[0271] In this example, cells stained in the presence of a combination of UV polymer (5–20 μg / test) + 1% PF-68 (the three lower panels) showed better separation than the control, including samples without buffer (upper left), with 1% PF-68 (upper center), and with UV polymer alone (upper right). The values ​​in each panel represent the MFI values. (Example 8) Performance of dyeing buffer compositions containing UV polymers and nonionic surfactants at various concentrations

[0272] In this example, the effects of various concentrations of nonionic surfactants (0.1-1% PF-68) were investigated using test staining buffers. Human whole blood was stained with CD20-SN v428 (Beckman Coulter Life Sciences) and CD4-BV650 (BD Biosciences). The FCA dot plots of the stained cells are shown in Figure 6. The upper right panel shows cells stained with UV polymer (10 ug), the middle central panel shows cells stained in PBS containing 1% PF-68, the upper left panel shows samples without buffer, the lower left panel shows cells stained with UV polymer and 1% PF-68, the lower central panel shows cells stained with UV polymer and 0.5% PF-68, and the lower right panel shows cells stained with UV polymer and 0.1% PF-68. In this example, cells stained in the presence of UV polymer combined with PF-68 at various concentrations (0.1–1%) (lower left, lower center, and lower right panels) showed better separation than the control, including samples without buffer (upper left panel), 1% PF-68 (upper center panel), and UV polymer alone (10 ug / test; upper right panel). The values ​​in each panel represent the MFI values. (Example 9) Flow cytometry performance of a buffer composition for dyeing UV polymers and quenched UV polymers in a mixture of two different polymer dye conjugates.

[0273] Three different quenched UV polymers (quenched polymers 1-3) according to this disclosure were prepared from a UV-absorbing polymer and a dabcyl dye quenching moiety. The ratio of quenching agent to polymer (D / P) was determined to be 2.5, 5, or 10 for the three quenched UV polymers. The intensity of the emission spectra of the quenched polymers after excitation at 355 nm is shown in Figure 7. Emission spectra from 365 to 600 nm are shown. The quantum yields (QY) at 405 nm for quenched polymer 1 (D / P=2.5), quenched polymer 2 (D / P=5.0), and quenched polymer 3 (D / P=10) are 0.072, 0.030, and 0.003, respectively. The quantum yield of the non-quenched polymer is 0.739.

[0274] In mixtures of whole blood cells with two commercially available polymer dye conjugates, CD20-SN v428 (Beckman Coulter Life Sciences) and CD4-BV650 (BD Biosciences), three quenched polymers (quenched polymer 1, quenched polymer 2, and quenched polymer 3) having D / P = 2.5, 5, and 10 were used as additives to a staining buffer containing 1% PF-68 at concentrations of 5 ug, 10 ug, and 20 ug per test, respectively. Figure 8 shows FCA dot plots of stained and lysed cells in a mixture with CD20-SN v428- and CD4-BV650, containing no additives (lower left panel), a control with 1% PF-68 (upper left panel), a comparative 10 ug of UV polymer dye containing 1% PF-68 (center left panel), and quenched polymers 1, 2, and 3 containing 1% PF-68 in 5 ug, 10 ug, or 20 ug / test (panels in the second, third, and rightmost columns). Cells stained in the presence of a test staining buffer composition containing a combination of UV polymer (10 μg / test) + 1% PF-68 (center left panel), or cells stained in the presence of quenched polymers 1, 2, and 3 containing 1% PF-68 in 5 ug, 10 ug, or 20 ug / test (the three rightmost columns, upper, middle, and lower panels, respectively), each showed better separation than the control without additives (lower left panel). The values ​​in each panel represent the MFI (Multi-Factor Indication) values. (Example 10) Flow cytometry performance of a nonionic surfactant alone in a mixture of two different polymer dye conjugates

[0275] Nonionic surfactants were found to be desirable additives for reducing nonspecific interactions of polymer dye conjugates in staining buffer compositions containing UV-absorbing polymers or quenched UV polymers. The effect of various concentrations of nonionic surfactants alone on FCA of stained and lysed cells was evaluated using mixtures of two different polymer dye conjugates. Figure 9 shows FCA dot plots of cells stained and lysed with a mixture of CD4-BV650 (BD Biosciences) and CD19-SNv428 (Beckman Coulter Life Sciences) in the following cases: no buffer (left panel), with 0.1% PF-68 (second panel from the left), with 0.5% PF-68 (second panel from the right), and with 1% PF-68 (wt / vol) (right panel). The presence of increased concentrations of PF-68 (0.1–1% wt / vol) is associated with reduced nonspecific interactions in the mixture, as evidenced by improved separation compared to the case without PF-68. (Example 11) Flow cytometry performance of a staining buffer containing a zwitterionic surfactant as needed in a mixture of two different polymer dye conjugates.

[0276] In some cases, it may be desirable to add a zwitterionic surfactant to the composition according to this disclosure. Figure 10 shows evaluations of cells stained with no buffer (top panel), a staining buffer (composition of UV polymer + PF-68), and various concentrations (0, 0.03%, 0.05%, and 0.07%) of Empigen (center panel: left, second from the left, second from the right, and right, respectively). The presence of various concentrations of Empigen in the staining buffer does not affect the performance of the staining buffer. Bottom panel: The MFI values ​​of monocytes show that the addition of various concentrations of Empigen to the staining buffer (bottom panel: left to right) reduces nonspecific binding to cells compared to the control sample (bottom left panel). Exemplary Embodiments

[0277] The following exemplary embodiments are presented, and their numbering should not be interpreted as representing a level of importance.

[0278] Embodiment 1a provides a UV-absorbing polymer having the structure of formula I: [ka] [In the formula, Each X is independently selected from the group consisting of C and Si. Each Y is a bond, CR 1 R 2 , CHR 1 , CHR 2 SiHR 2 SiHR 1 and SiR 1 R 2 Independently selected from the group consisting of, if Y is a bond, then X is directly bonded to both rings. Each R 1 This includes water-soluble moieties, alkyl groups, alkenes, alkynes, cycloalkyl groups, haloalkyl groups, (hetero)aryloxy groups, (hetero)arylamino groups, aryl groups, heteroaryl groups, polyethylene glycol (PEG) groups, carboxylic acids, alkylammonium salts, alkyloxyammonium salts, oligoetherammonium salts, alkyl sulfonates, alkoxysulfonates, sulfonamide oligoethers, sulfonamides, sulfinamides, phosphoamides, phosphineamides, [ka] Independently selected from the group consisting of, Each R 2This includes the water-soluble portion, the linker portion, H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, heteroaryl, (hetero)arylamino, PEG group, sulfonamide-PEG, phosphoramide-PEG, alkylammonium salt, alkyloxyammonium salt, oligoetherammonium salt, alkyl sulfonate, alkoxysulfonate, oligoethersulfonate, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamide, phosphineamide, [ka] Independently selected from the group consisting of, Each R 3 This is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, water-soluble moiety, and PEG group. Each Z corresponds to CH2, CHR 4 , O, NH and NR 4 Independently selected from the group consisting of, Each Q is a combination of NH and NR. 4 , C1~C 12 Alkylene, CHR 4 And independently selected from the group consisting of CH2, Each R 4 H, PEG group, water-soluble portion, linker portion, chromophore, linked chromophore, functional group, linked functional group, substrate, linked substrate, binding partner, linked binding partner, quenching portion, L 2 -E, halogen, hydroxyl, C1~C 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylamino, (CH2)x’ (OCH2-CH2) y’ OCH3 (each x' is an independent integer between 0 and 20, and each y' is an independent integer between 0 and 50), Z-(CH2) n -SO2-QR 3 , C2~C 18 Independently selected from the group consisting of (hetero)aryl groups, amides, amines, carbamates, carboxylic acids, carboxylic acid esters, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazones, azides, aldehydes, thiols, and their protected forms, Each W 1 This is independently a water-soluble portion, L 1 , L 2 and L 3 These are linker parts that are selected independently. Each E is independently selected from the group consisting of a chromophore, a functional group moiety, a substrate, and a binding partner. Each R 7 H, hydroxyl, C1-C 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylamino, C2~C 12 Carboxylic acids and C2-C 12 Independently selected from the group consisting of carboxylic acid esters, R 1 , R 2 , R 3 or R 4 At least one of them includes a water-soluble portion, Each M 1 R is further substituted as needed. 4 and / or arylene substituted with trifluoromethyl, further substituted as needed, R 4and / or heteroarylenes substituted with trifluoromethyl, further substituted as needed, R 4 Independently selected from the group consisting of 9,10-dihydrophenanthrene substituted with and / or trifluoromethyl, and binaphthyl substituted as needed, Each M 2 R is further substituted as needed. 4 and / or arylene substituted with trifluoromethyl, further substituted as needed, R 4 and / or heteroarylenes substituted with trifluoromethyl, further substituted as needed, R 4 Independently selected from the group consisting of 9,10-dihydrophenanthrene substituted with and / or trifluoromethyl, and binaphthyl substituted as needed, M 2 M 1 It has a different structure, M 2 and M 1 These are distributed evenly or randomly along the polymer backbone. Each linker L, as needed, is an aryl or heteroaryl group evenly or randomly distributed along the polymer backbone and is substituted by one or more pendant chains terminated with functional groups selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols and their protected groups, for conjugation to another substrate, acceptor dye, molecule or binding partner. G 1 and G 2 These are independently selected from the group consisting of unmodified polymer ends and modified polymer ends, which are conjugated to E as needed. a, c, d, and e define the mol% of each unit in the structure, which may be repeated evenly or randomly along the polymer backbone, where a is 10-100% (mol%), c is >0-90% (mol%), each d is 0-90% (mol%), and each e is 0-25% (mol%). Each b is independently either 0 or 1. Each f is an independent integer between 0 and 50. m is an integer between 1 and approximately 10,000. Each n is an independent integer between 1 and 20. s is either 1 or 2, t is 0, 1, 2, or 3.

[0279] Embodiment 1b provides the polymer of Embodiment 1a having the structure of Formula I: [ka] [In the formula, Each X is selected independently from C and Si. Each Y is a bond, CR 1 R 2 and SiR 1 R 2 If selected independently, and Y is a bond, then X is directly bonded to both rings. Each R 1 This includes polyethylene glycol (PEG), alkylammonium salts, alkyloxyammonium salts, oligoetherammonium salts, alkyl sulfonates, alkoxysulfonates, sulfonamide oligoethers, and -Z-(CH2) n -SO2-QR 3 Selected independently from, Each R 2H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG group, alkylammonium salt, alkyloxyammonium salt, oligoetherammonium salt, alkyl sulfonate, alkoxysulfonate, oligoethersulfonate, sulfonamide oligoether, and -Z-(CH2) n -SO2-QR 3 Selected independently from, Each R 3 The group is independently selected from H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino and PEG groups. Each Z corresponds to CH2, CHR 4 , O, NH and NR 4 Selected independently from, Each Q is a combination of NH and NR. 4 , C1~C 12 Alkylene, CHR 4 and selected independently from CH2, Each R 4 Chromophores, linked chromophores, halogens, hydroxyls, C1-C 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylamino, (CH2) x’ (OCH2-CH2) y’ OCH3 (each x' is an independent integer between 0 and 20, and each y' is an independent integer between 0 and 50), -Z-(CH2) n -SO2-QR 3 and C2~C 18 (Independently selected from heteroaryl groups, Each M 1 R is further substituted as needed. 4and / or arylene substituted with trifluoromethyl, further substituted as needed, R 4 and / or heteroarylenes substituted with trifluoromethyl, further substituted as needed, R 4 Independently selected from 9,10-dihydrophenanthrene substituted with trifluoromethyl and / or binaphthyl substituted as needed, Each M 2 R is further substituted as needed. 4 and / or arylene substituted with trifluoromethyl, further substituted as needed, R 4 and / or heteroarylenes substituted with trifluoromethyl, further substituted as needed, R 4 Independently selected from 9,10-dihydrophenanthrene substituted with trifluoromethyl and / or binaphthyl substituted as needed, M 2 M 1 It has a different structure, Each linker L is an aryl or heteroaryl group evenly or randomly distributed along the polymer backbone and is substituted by one or more pendant chains terminated with functional groups selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols and their protected groups, for conjugation to another substrate, acceptor dye, molecule or binding partner. G 1 and G 2These are independently selected from hydrogen, halogens, alkynes, halogen-substituted aryls, silyls, diazonium salts, triflates, acetyloxys, azides, sulfonates, phosphates, boronic acid-substituted aryls, boronic acid ester-substituted aryls, boronic acid esters, boronic acids, optionally substituted aryls, optionally substituted heteroaryls, optionally substituted dihydrophenanthrene (DHP), or optionally substituted fluorenes, and optionally substituted aryls, heteroaryls, fluorenes, or DHPs may be substituted or conjugated to a substrate or binding partner by one or more pendant chains terminated with functional groups selected from, for example, amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxylsuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and their protected groups. a, c, d, and e define the mol% of each unit in the structure, which may be repeated evenly or randomly along the polymer backbone, where a is 10-100% (mol%), c is >0-90% (mol%), each d is 0-90% (mol%), and each e is 0-25% (mol%). Each b is independently either 0 or 1. m is an integer between 1 and approximately 10,000. Each n is an independent integer between 1 and 20.

[0280] Embodiment 2a is given by formula II: [ka] The present invention provides one polymer from either Embodiment 1a or Ib having the structure shown.

[0281] Embodiment 3 is given by formula III: [ka] [In the formula, each f is an integer between 0 and 50 independently, and each R 5 H, C1~C 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylaminos and C1-C 12 [Independently selected from the group consisting of alkoxys] The present invention provides one polymer having the structure described in Embodiments 1a to 2b.

[0282] Embodiment 4 is given by formula IV: [ka] [In the formula, each f is an independent integer between 0 and 50.] The present invention provides one polymer having the structure of any one of embodiments 1a to 3.

[0283] Embodiment 5 is given by formula V: [ka] [In the formula, g and h together are 10 to 100% (mol%), each f is an integer between 0 and 50, and each R 5 H, C1~C 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylaminos and C1-C 12 [Independently selected from the group consisting of alkoxys] The present invention provides one polymer having the structure of any one of embodiments 1a to 4.

[0284] Embodiment 6 is given by formula VI: [ka] [In the formula, each f is an integer between 0 and 50 independently, and each R 5 H, C1~C 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylaminos and C1-C 12 [Independently selected from the group consisting of alkoxys] The present invention provides one polymer having the structure of any one of embodiments 1a to 5.

[0285] Embodiment 7 is given by formula VII: [ka] [In the formula, g and h together represent 10 to 100% (mol%), and each f is an integer between 0 and 50.] The present invention provides one polymer having the structure of any one of embodiments 1a to 6.

[0286] Embodiment 8 is given by formula VIII: [ka] [In the formula, each f is an independent integer between 0 and 50.] The present invention provides one polymer having the structure of any one of embodiments 1a to 7.

[0287] Embodiment 9a is given by formula XIV: [ka] [In the formula, R 2 , R 3 , G 1 , G 2 Each of L, Q, X, Y, Z, a, b, c, e, n, and m is independently as described herein. Each R 4’ These are F, Cl, -CH3, -CF3 and -(OCH2CH2) f Ure 9 Selected independently from each R 4’’ These are F, Cl, -CH3, -CF3 and -(OCH2CH2) f Ure 9 Selected independently from, R 9 [where f is a C1-C8 alkyl group, each f is an integer independently between 0 and 50 or between 10 and 20, each o is an integer independently selected from 1, 2, 3 or 4, and each p is an integer independently selected from 1, 2, 3 or 4] The present invention provides one polymer from any of embodiments 1a to 8, which includes a structure based on the given formula.

[0288] Embodiment 9b is each M 1 However, independently, they are arylenes substituted with fluorine having 1 to 4 fluorine substituents, or each M 1 The present invention provides one polymer from any of Embodiments 1a to 9a, which is independently and optionally further substituted with a halide, MeO-PEG-CH2 and / or an arylene (e.g., phenylene) substituted with MeO-PEG.

[0289] Embodiment 10 is each M 1 However, the present invention provides one polymer from any of embodiments 1a to 9b, which is a fluorine-substituted phenylene having 1 to 4 fluorine substituents, and in which the phenylene is further substituted as needed.

[0290] Embodiment 11 is each M 1However, the present invention provides one polymer of any of Embodiments 1a to 10, which is a fluorine-substituted phenylene having two or three fluorine substituents.

[0291] Embodiment 12 is each M 1 However, the present invention provides one polymer from any of embodiments 1a to 11, which is a difluorosubstituted phenylene.

[0292] Embodiment 13 is each M 1 but, The phenylene is substituted at positions 1 and 4 in the polymer's main chain, and is difluorosubstituted with fluorine at positions 2 and 3, 2 and 5, or 2 and 6. The phenylene is substituted at positions 1 and 4 in the polymer's main chain, and at positions 2, 3 and 5, it is trifluorosubstituted with fluorine. Phenylene in which the 1st and 3rd positions are substituted in the polymer main chain, and which is difluorosubstituted with fluorine at the 2nd and 4th positions, 2nd and 5th positions, 4th and 5th positions, or 4th and 6th positions, and The phenylene is substituted at positions 1 and 3 in the polymer's main chain, and is trifluorosubstituted with fluorine at positions 4, 5 and 6, 2, 4 and 5, or 2, 4 and 6. The present invention provides one polymer from any of embodiments 1a to 12, which can be independently selected from the others.

[0293] Embodiment 14 is each M 1 but, Phenylene in which the 1st and 4th positions are substituted in the polymer main chain, and phenylene in which the 2nd and 3rd positions, 2nd and 5th positions or 2nd and 6th positions are difluorosubstituted with fluorine, and The phenylene is substituted at positions 1 and 3 in the polymer's main chain, and is difluorosubstituted with fluorine at positions 2 and 4, 2 and 5, 4 and 5, or 4 and 6. The present invention provides one polymer from any of embodiments 1a to 13, which can be independently selected from the others.

[0294] Embodiment 15 is each M 1 but, [ka] [In the formula, each f is an integer between 0 and 50, 10 and 20, or 11 and 18, independently.] The present invention provides one polymer from any of embodiments 1a to 14, which is independently selected from the others.

[0295] Embodiment 16 is each M 1 but, [ka] The present invention provides one polymer from any of embodiments 1a to 15.

[0296] Embodiment 17 is each M 1 The present invention provides one polymer from any of Embodiments 1a to 16, which is a phenylene in which the 1st and 4th positions are substituted in the main chain of the polymer, and is 2,5-difluorosubstituted.

[0297] Embodiment 18 is each M 2 However, independently, they are fluorine-substituted arylenes having 1 to 4 fluorine substituents, or each M 2 The present invention provides any one polymer from Embodiments 1a to 17, which is independently and optionally further substituted with a halide, MeO-PEG-CH2 and / or an arylene (e.g., phenylene) substituted with MeO-PEG.

[0298] Embodiment 19 is each M 2 However, independently, there is a polymer of any one of embodiments 1a to 18, which is a fluorine-substituted phenylene having 1 to 4 fluorine substituents, and the phenylene is further substituted as needed.

[0299] Embodiment 20 is each M 2 However, the present invention provides one polymer from any of Embodiments 1a to 19, which is a fluorine-substituted phenylene having two or three fluorine substituents.

[0300] Embodiment 21 is each M 2 The present invention provides one polymer of any of embodiments 1a to 20, which is a trifluorosubstituted phenylene.

[0301] Embodiment 22 is each M 2 but, The phenylene is substituted at positions 1 and 4 in the polymer's main chain, and is trifluorosubstituted with fluorine at positions 2, 3 and 5, and The phenylene is substituted at positions 1 and 3 in the polymer's main chain, and is trifluorosubstituted with fluorine at positions 4, 5 and 6, 2, 4 and 5, or 2, 4 and 6. The present invention provides one polymer from any of embodiments 1a to 21, which can be independently selected from the others.

[0302] Embodiment 23 is each M 2 but, [ka] [Each f is an integer between 0 and 50, 10 and 20, or 11 and 18, and M 2 M 1 [Different from] The present invention provides one polymer from any of embodiments 1a to 22.

[0303] Embodiment 24 is each M 2 The present invention provides one polymer from any of Embodiments 1a to 23, wherein the phenylene is substituted at the 1st and 3rd positions in the main chain of the polymer, and is 4,5,6-trifluorosubstituted.

[0304] Embodiment 25 is such that each L is [Chemical formula] [wherein, each R 6 is H, OH, SH, NHCOO-t-butyl, (CH2) n [[ID=1�]]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 alkene, C2-C 12 alkyne, C3-C 12 cycloalkyl, C1-C 12 haloalkyl, C1-C 12 (hetero)aryl, C1-C 12 (hetero)arylamino, and one or more halogens, hydroxyls, C_{1}-C 12 alkoxy or (OCH2CH2) f OCH3, and is independently selected from benzyl optionally substituted by each f is independently an integer from 0 to 50, Each n is independently an integer from 1 to 20 Provided is any one polymer of Embodiments 1a to 24, independently selected therefrom.

[0305] Embodiment 26 is G 1 and G 2 wherein G and G are each independently selected from dihydrophenanthrene (DHP) optionally substituted, fluorene optionally substituted, aryl substituted by one or more pendant chains terminated with a functional group, and heteroaryl substituted by one or more pendant chains terminated with a functional group, and provides any one polymer of Embodiments 1a to 25.

[0306] Embodiment 27 is G 1 and G 2 wherein G and G are [Chemical formula] [Chemical formula] [wherein each R 6 is H, OH, SH, NHCOO-t-butyl, (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 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 (hetero)aryl, C1~C 12 (Hetero)arylamino, and one or more halogens, hydroxyl, C1-C 12 Alkoxy or (OCH2CH2) f Each benzyl is independently selected from those which are substituted as needed by OCH3. Each f is an independent integer between 0 and 50. Each n is an independent integer between 1 and 20. The present invention provides one polymer from any of embodiments 1a to 26, which can be independently selected from the others.

[0307] Embodiment 28 is given by formula IX: [ka] [In the formula, f is an integer between 0 and 50, independently.] The present invention provides one polymer having the structure of any one of embodiments 1a to 27.

[0308] Embodiment 29 is M 1 Original M 2 The present invention provides one polymer from any of Embodiments 1a to 28, wherein the molar ratio to the group is 0.5:1 to 1.5:1.

[0309] Embodiment 30 is M 1 Original M 2 The present invention provides one polymer from any of the embodiments 1a to 29, wherein the molar ratio to the group is 0.7:1 to 1.3:1.

[0310] Embodiment 31 is M 1 Original M 2 The present invention provides one polymer from any of the embodiments 1a to 30, wherein the molar ratio to the group is 0.9:1 to 1.1:1.

[0311] Embodiment 32 is M 1 Original M 2 The present invention provides one polymer from any of embodiments 1a to 31, wherein the molar ratio to the group is approximately 1:1.

[0312] Embodiment 33 provides one of the polymers from Embodiments 1a to 32, wherein b is 0.

[0313] Embodiment 34 provides one of the polymers from Embodiments 1a to 33, wherein a is 25% to 75%.

[0314] Embodiment 35 provides one of the polymers from Embodiments 1a to 34, wherein a is present in an amount of 35% to 65%.

[0315] Embodiment 36 provides one of the polymers from Embodiments 1a to 35, wherein a is 45% to 55%.

[0316] Embodiment 37 provides one of the polymers from Embodiments 1a to 36, wherein c is 5% to 80%.

[0317] Embodiment 38 provides one of the polymers from Embodiments 1a to 37, wherein c is 10% to 40%.

[0318] Embodiment 39 provides one of the polymers from Embodiments 1a to 38, wherein c is 15% to 35%.

[0319] Embodiment 40 provides one of the polymers from Embodiments 1a to 39, wherein c is 20% to 30%.

[0320] Embodiment 41 provides one of the polymers from Embodiments 1a to 40, wherein d is 0%.

[0321] Embodiment 42 provides one of the polymers from Embodiments 1a to 41, wherein d is 5% to 80%.

[0322] Embodiment 43 provides one of the polymers from Embodiments 1a to 42, wherein d is 10% to 40%.

[0323] Embodiment 44 provides one of the polymers from Embodiments 1a to 43, wherein d is 15% to 35%.

[0324] Embodiment 45 provides one of the polymers from Embodiments 1a to 44, wherein d is 20-30%.

[0325] Embodiment 46 provides one of the polymers from Embodiments 1a to 45, wherein e is 0%.

[0326] Embodiment 47 provides one of the polymers from Embodiments 1a to 46, wherein e is 0% to 20%.

[0327] Embodiment 48a has at least one R 2 However, -Z-(CH2) n -SO2-N (chromophore)-R 3 The present invention provides one polymer from any of embodiments 1a to 47.

[0328] Embodiment 49 provides one of the polymers from Embodiments 1a to 48, having an absorption maximum in the range of 320 nm to 380 nm.

[0329] Embodiment 50 provides one of the polymers from Embodiments 1a to 49, having an absorption maximum in the range of 340 nm to 360 nm.

[0330] Embodiment 51 provides one of the polymers from Embodiments 1a to 50 having an absorption maximum at 345 nm to 356 nm.

[0331] Embodiment 52 provides one of the polymers from Embodiments 1a to 51 having an emission maximum in the range of 380 nm to 430 nm.

[0332] Embodiment 53 provides one of the polymers from Embodiments 1a to 52 having an emission maximum in the range of 406 nm to 415 nm.

[0333] Embodiment 54 provides one of the polymers from Embodiments 1a to 53, further comprising a binding partner linked to the polymer.

[0334] Embodiment 55 provides the polymer of Embodiment 54, wherein the binding partner is an antibody.

[0335] Embodiment 56a is a method for detecting an analyte in a sample, Samples suspected of containing the analyte should be treated with Formula I: [ka] [In the formula, Each X is independently selected from the group consisting of C and Si. Each Y is a bond, CR 1 R 2 , CHR 1 , CHR 2 and SiR 1 R 2 Independently selected from the group consisting of, if Y is a bond, then X is directly bonded to both rings. Each R 1This includes water-soluble portion, linker portion, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, (hetero)aryloxy, (hetero)arylamino, aryl, heteroaryl, polyethylene glycol (PEG) group, carboxylic acid, alkylammonium salt, alkyloxyammonium salt, oligoetherammonium salt, alkyl sulfonate, alkoxysulfonate, sulfonamide oligoether, sulfonamide, sulfinamide, phosphoamide, phosphineamide, [ka] Independently selected from the group consisting of, each R 2 This includes the water-soluble portion, the linker portion, H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, heteroaryl, (hetero)arylamino, PEG group, sulfonamide-PEG, phosphoramide-PEG, alkylammonium salt, alkyloxyammonium salt, oligoetherammonium salt, alkyl sulfonate, alkoxysulfonate, oligoethersulfonate, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamide, phosphineamide, [ka] Independently selected from the group consisting of, Each R 3 This is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, water-soluble moiety, and PEG group. Each Z corresponds to CH2, CHR 4 , O, NH and NR 4 Independently selected from the group consisting of, Each Q is a combination of NH and NR. 4 , C1~C 12 Alkylene, CHR 4 And independently selected from the group consisting of CH2, Each R 4 is H, a PEG group, a water-solubilizing moiety, a linker moiety, a chromophore, a linked chromophore, a functional group, a linked functional group, a substrate, a linked substrate, a binding partner, a linked binding partner, a quenching moiety, L 2 -E, a halogen, a hydroxyl group, C1-C 12 alkyl, C2-C 12 alkene, C2-C 12 alkyne, C3-C 12 cycloalkyl, C1-C 12 haloalkyl, C1-C 12 alkoxy, C2-C 18 (hetero)aryloxy, C2-C 18 (hetero)arylamino, (CH2) x’ (OCH2-CH2) y’ OCH3 (each x' is independently an integer from 0 to 20, and each y' is independently an integer from 0 to 50), Z-(CH2) n -SO2-Q-R 3 、C2-C 18 (hetero)aryl group, amide, amine, carbamate, carboxylic acid, carboxylic acid ester, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazone, azide, aldehyde, thiol and their protected groups, and is independently selected from the group consisting of Each W 1 is independently a water-solubilizing moiety, L 1 、L 2 and L 3 are each independently selected linker moieties, Each E is independently selected from the group consisting of a chromophore, a functional group moiety, a substrate and a binding partner, Each R 7 is H, a hydroxyl group, C1-C 12 alkyl, C2-C 12 alkene, C2-C 12 alkyne, C3-C 12 cycloalkyl, C1-C 12 haloalkyl, C1-C 12 alkoxy, C2-C 18 (hetero)aryloxy, C2-C18 (hetero)arylamino, C2~C 12 Carboxylic acids and C2-C 12 Independently selected from the group consisting of carboxylic acid esters, R 1 , R 2 , R 3 or R 4 At least one of them includes a water-soluble portion, Each M 1 R is further substituted as needed. 4 and / or arylene substituted with trifluoromethyl, further substituted as needed, R 4 and / or heteroarylenes substituted with trifluoromethyl, further substituted as needed, R 4 Independently selected from the group consisting of 9,10-dihydrophenanthrene substituted with and / or trifluoromethyl, and binaphthyl substituted as needed, Each M 2 R is further substituted as needed. 4 and / or arylene substituted with trifluoromethyl, further substituted as needed, R 4 and / or heteroarylenes substituted with trifluoromethyl, further substituted as needed, R 4 Independently selected from the group consisting of 9,10-dihydrophenanthrene substituted with and / or trifluoromethyl, and binaphthyl substituted as needed, M 2 M 1 It has a different structure, M 2 and M 1 These are distributed evenly or randomly along the polymer backbone. Each linker L, as needed, is an aryl or heteroaryl group evenly or randomly distributed along the polymer backbone and is substituted by one or more pendant chains terminated with functional groups selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols and their protected groups, for conjugation to another substrate, acceptor dye, molecule or binding partner. G 1 and G 2 These are independently selected from the group consisting of unmodified polymer ends and modified polymer ends, which are conjugated to E as needed. a, c, d, and e define the mol% of each unit in the structure, which may be repeated evenly or randomly along the polymer backbone, where a is 10-100% (mol%), c is >0-90% (mol%), each d is 0-90% (mol%), and each e is 0-25% (mol%). Each b is independently either 0 or 1. Each f is an independent integer between 0 and 50. m is an integer between 1 and approximately 10,000. Each n is an independent integer between 1 and 20. s is either 1 or 2, t is 0, 1, 2, or 3. The step of bringing a polymer containing the structure into contact with a binding partner that is conjugated to it. This provides a method that includes [something].

[0336] Embodiment 56b is a polymer of formula I [In the formula, Each X is selected independently from C and Si. Each Y is a bond, CR 1 R 2 and SiR 1 R 2 If selected independently, and Y is a bond, then X is directly bonded to both rings. Each R 1 This includes polyethylene glycol (PEG), alkylammonium salts, alkyloxyammonium salts, oligoetherammonium salts, alkyl sulfonates, alkoxysulfonates, sulfonamide oligoethers, and -Z-(CH2) n -SO2-QR 3 Selected independently from, Each R 2 H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG group, alkylammonium salt, alkyloxyammonium salt, oligoetherammonium salt, alkyl sulfonate, alkoxysulfonate, oligoethersulfonate, sulfonamide oligoether, and -Z-(CH2) n -SO2-QR 3 Selected independently from, Each R 3 The group is independently selected from H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino and PEG groups. Each Z corresponds to CH2, CHR 4 , O, NH and NR 4 Selected independently from, Each Q is a combination of NH and NR. 4 , C1~C 12 Selected independently from alkylenes and CH2, Each R 4 These are chromophores, halogens, hydroxyls, and C1-C 12 Alkyl, C2~C 12 Alkenes, C2~C 12 Alkyne, C3~C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1~C 12 Alkoxy, C2~C 18 (hetero)aryloxy, C2~C 18 (hetero)arylamino, (CH2) x’ (OCH2-CH2) y’OCH3 (each x' is an independent integer between 0 and 20, and each y' is an independent integer between 0 and 50), -Z-(CH2) n -SO2-QR 3 and C2~C 18 (Independently selected from heteroaryl groups, Each M 1 R is further substituted as needed. 4 and / or arylene substituted with trifluoromethyl, further substituted as needed, R 4 and / or heteroarylenes substituted with trifluoromethyl, further substituted as needed, R 4 Independently selected from 9,10-dihydrophenanthrene substituted with trifluoromethyl and / or binaphthyl substituted as needed, Each M 2 R is further substituted as needed. 4 and / or arylene substituted with trifluoromethyl, further substituted as needed, R 4 and / or heteroarylenes substituted with trifluoromethyl, further substituted as needed, R 4 Independently selected from 9,10-dihydrophenanthrene substituted with trifluoromethyl and / or binaphthyl substituted as needed, M 2 M 1 It has a different structure, Each linker L is an aryl or heteroaryl group evenly or randomly distributed along the polymer backbone and is substituted by one or more pendant chains terminated with functional groups selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols and their protected groups, for conjugation to another substrate, acceptor dye, molecule or binding partner. G 1 and G 2Each is independently selected from the group consisting of hydrogen, halogens, alkynes, halogen-substituted aryls, silyls, diazonium salts, triflates, acetyloxys, azides, sulfonates, phosphates, boronic acid-substituted aryls, boronic acid ester-substituted aryls, boronic acid esters, boronic acids, optionally substituted aryls, optionally substituted heteroaryls, optionally substituted dihydrophenanthrene (DHP), and optionally substituted fluorenes, wherein the substituted aryls, heteroaryls, fluorenes, or DHPs are substituted by one or more pendant chains terminated with functional groups selected from, for example, amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxylsuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and their protected groups, for conjugation to a substrate or binding partner. a, c, d, and e define the mol% of each unit in the structure, which may be repeated evenly or randomly along the polymer backbone, where a is 10-100% (mol%), c is >0-90% (mol%), each d is 0-90% (mol%), and each e is 0-25% (mol%). Each b is independently either 0 or 1. m is an integer between 1 and approximately 10,000. Each n is an independent integer between 1 and 20. It has a structure, The binder can interact with the analyte or target-binding biomolecule. The method of embodiment 56a is provided.

[0337] Embodiment 57 provides the method of Embodiment 56a or 56b, wherein the binding partner is a protein, peptide, affinity ligand, antibody, antibody fragment, sugar, lipid, nucleic acid, or aptamer.

[0338] Embodiment 58 provides one of the methods from Embodiments 56a to 57, wherein the binding partner is an antibody.

[0339] Embodiment 59 provides one of the methods from Embodiments 56a to 58, configured for flow cytometry.

[0340] Embodiment 60 provides one of the methods from Embodiments 56a to 59, wherein the bonding partner is bonded to the substrate.

[0341] Embodiment 61 provides one of the methods described in Embodiments 56a to 60, wherein the analyte is a protein expressed on the cell surface.

[0342] Embodiment 62 provides one of the methods from Embodiments 56a to 61, configured as an immunoassay.

[0343] Embodiment 63 provides one of the methods from Embodiments 56a to 62, further including the step of providing additional binding partners to simultaneously detect further targets for analysis.

[0344] Embodiment 64 is a bond, and R 1 and R 2 However, each operates independently, -Z-(CH2) n -SO2-QR 3 The present invention provides one polymer from any of embodiments 1a to 55 or one method from any of embodiments 56a to 63.

[0345] Embodiment 65 provides any one polymer from Embodiments 1a to 55 or any one method from Embodiments 56a to 63, wherein each f is independently an integer between 5 and 30, and each n is independently an integer between 2 and 10.

[0346] Embodiment 66 provides any one polymer from Embodiments 1a to 55 or any one method from Embodiments 56a to 63, wherein each f is independently an integer between 10 and 25, and each n is independently an integer between 3 and 5.

[0347] Embodiment 67 provides a polymer according to any one of Embodiments 1a to 55, 64, or 65, wherein the acceptor dye is a quenchable portion.

[0348] Embodiment 68 provides one of the polymers from Embodiments 1a-55 or 64-67, which does not include a binding partner.

[0349] Embodiment 69 provides a polymer or method of any one or any combination of Embodiments 1a to 68, configured as necessary so that all enumerated elements or options are available for use or selection.

[0350] Embodiment 70 provides a composition for use with at least one fluorescent polymer dye conjugated to a binding partner for use in staining biological samples, comprising at least one UV-absorbing polymer dye or quenched UV polymer dye (optionally, the UV-absorbing polymer dye or quenched polymer dye comprises any of formulas I, II, III, IV, V, VI, VII, VIII, IX, X, XI, X and / or XIV, or any structure according to any one of Embodiments 1a to 55), a nonionic surfactant; and a biological buffer, which reduces the nonspecific binding of at least one fluorescent polymer dye conjugate compared to the at least one fluorescent polymer dye conjugate in the absence of the composition.

[0351] Embodiment 71 provides the composition of Embodiment 70, wherein the quenched UV polymer dye comprises a UV-absorbing polymer dye having at least one quenchable portion, and optionally 1 to 30, 2 to 20, or 2.5 to 10 quenchable portions.

[0352] Embodiment 72 provides the composition of Embodiment 70 or 71, wherein the quenching portion is selected from the group consisting of DABCYL, DABSYL, BHQ1, BHQ0, DDQI, EDQ, QSY7, QSY9, QSY35, TAMRA, Dabcyl Q, Dabcyl plus, 490Q, 425Q, and 505Q.

[0353] Embodiment 73 provides one composition from any of Embodiments 70 to 72, wherein the nonionic surfactant is a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer.

[0354] Embodiment 74 is a nonionic surfactant of formula (XII) [ka] [In the formula, each a is independently in the range of 2 to 130, and each b is in the range of 15 to 67.] The present invention provides one composition from any of embodiments 70 to 73, which includes a structure based on the above.

[0355] Embodiment 75 provides one of the compositions of Embodiments 70 to 74, wherein the composition further comprises additional additives selected from the group consisting of protein stabilizers, preservatives, and further surfactants, and optionally the further surfactant is a zwitterionic surfactant or an ionic surfactant.

[0356] Embodiment 76 provides one of the compositions from Embodiments 70 to 75, comprising a plurality of fluorescent polymer dye conjugates, which substantially reduces nonspecific binding between the plurality of fluorescent polymer dye conjugates. The present invention provides, for example, the following items: (Item 1) UV-absorbing polymer dyes containing the structure of formula I: [ka] [In the formula, Each X is independently selected from the group consisting of C and Si. Each Y is a bond, CR 1 R 2 , CHR 1 , CHR 2 SiHR 2 SiHR 1 and SiR 1 R 2 Independently selected from the group consisting of, if Y is a bond, then X is directly bonded to both rings. Each R 1 This includes water-soluble portion, linker portion, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, (hetero)aryloxy, (hetero)arylamino, aryl, heteroaryl, polyethylene glycol (PEG) group, carboxylic acid, alkylammonium salt, alkyloxyammonium salt, oligoetherammonium salt, alkyl sulfonate, alkoxysulfonate, sulfonamide oligoether, sulfonamide, sulfinamide, phosphoamide, phosphineamide,

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Claims

1. UV-absorbing polymer dyes containing the structure of formula I: 【Chemistry 83】 [In the formula, Each X is C, Each Y is CR １ R ２ _CHR １ and CHR ２ Independently selected from the group consisting of, Each R １ This includes a water-soluble portion, polyethylene glycol (PEG) group, carboxylic acid, sulfonamide oligoether, sulfonamide, sulfinamide, phosphoamide, phosphineamide, 【Chemical 84】 【Chemical 85】 Independently selected from the group consisting of, wherein, if necessary, at least one R １ is -Z-(CH ２ ) ｎ -SO ２ -N(Chromophore)-R ３ , -Z-(CH ２ ) ｎ -SO ２ -N(Linked chromophore)-R ３ , -Z-(CH ２ ) ｎ -SO ２ -N(Quenching moiety)-R ３ or -Z-(CH ２ ) ｎ -SO ２ -N(Linked quenching moiety)-R ３ ; Each R ２ This includes the water-soluble portion, the linker portion, H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, heteroaryl, (hetero)arylamino, PEG group, sulfonamide-PEG, phosphoramide-PEG, oligoether sulfonate, sulfonamide oligoether, sulfonamide, sulfinamide, phosphoamide, phosphineamide, 【Chemical 86】 【Transformation 87】 Independently selected from the group consisting of, Each R ３ This is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, water-soluble moiety, chromophore, and PEG group. Each Z is CH ２ _CHR ４ , O, NR ４ and independently selected from the group consisting of NH, Each Q is a combination of NH and NR. ４ , C １ ~C １２ Alkylene, CHR ４ and CH ２ Independently selected from the group consisting of, Each R ４ H, PEG group, water-soluble portion, linker portion, chromophore, linked chromophore, functional group, linked functional group, substrate, linked substrate, binding partner, linked binding partner, quenching portion, L ２ -E, halogen, hydroxyl, C １ ~C １２ Alkyl, C ２ ~C １２ Alken, C ２ ~C １２ Alkin, 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, (CH ２ ) ｘ’ (OCH ２ -CH ２ ) ｙ’ OR ９ (In the formula, each R ９ C １ ~C ８ (It is an alkyl group, where x' is an integer from 0 to 20 independently, and each y' is an integer from 0 to 50 independently), Z-(CH ２ ) ｎ -SO ２ -Q-R ３ , C ２ ~C １８ A group independently selected from the group consisting of (hetero)aryl groups, amides, amines, carbamates, carboxylic acids, carboxylic acid esters, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazones, azides, aldehydes, thiols, and their protected forms, Each W １ This is independently a water-soluble portion, L, L １ , L ２ and L ３ These are linker parts that are selected independently. Each E is independently selected from the group consisting of a chromophore, a functional group moiety, a substrate, and a binding partner. Each R ７ is independently selected from the group consisting of H, hydroxyl, C １ to C １２ alkyl, C ２ to C １２ alkene, C ２ to C １２ alkyne, C ３ to C １２ cycloalkyl, C １ to C １２ haloalkyl, C １ to C １２ alkoxy, C ２ to C １８ (hetero)aryloxy, C ２ to C １８ (hetero)arylamino, C ２ to C １２ carboxylic acid, C ２ to C １２ carboxylic acid ester and -OC １ to C １２ and hydroxy, R １ , R ２ , R ３ or R ４ At least one of them includes a water-soluble portion, Each M １ is a modifying unit independently selected from the group consisting of phenylene further substituted, if necessary, with R ４ and / or trifluoromethyl, heteroarylene further substituted, if necessary, with R ４ and / or trifluoromethyl, and binaphthyl optionally substituted Each M as needed ２ R is further substituted as needed. ４ and / or arylene substituted with trifluoromethyl, further substituted as needed, R ４ and / or heteroarylenes substituted with trifluoromethyl, further substituted as needed, R ４ A modification unit independently selected from the group consisting of 9,10-dihydrophenanthrene substituted with and / or trifluoromethyl, and binaphthyl substituted as needed, M ２ M １ It has a different structure from M ２ and M １ These are distributed evenly or randomly along the polymer backbone. Each linker L, as needed, is an independent linker component. G １ and G ２ These are independently selected from the group consisting of unmodified polymer ends and modified polymer ends, and are conjugated to E as needed. a, c, d, and e define the mol% of each unit in the structure, which may be repeated evenly or randomly along the polymer main chain, where a is 10 to 100% (mol%), c is >0 to 90% (mol%), each d is 0 to 90% (mol%), and each e is 0 to 25% (mol%). Each b is independently either 0 or 1. Each f is an independent integer between 0 and 50. m is an integer between 1 and approximately 10,000. Each n is an independent integer between 1 and 20. s is either 1 or 2. t is 0, 1, 2, or 3. The aforementioned UV-absorbing polymer dye exhibits an absorption maximum in the range of 300 nm to 400 nm. [Optionally, the polymer further comprises a chromophore covalently bonded to it near its energy acceptor, and optionally, the UV-absorbing polymer dye is a water-soluble UV-absorbing polymer dye.]

2. G １ and G ２ The UV-absorbing polymer dye according to claim 1, wherein each is independently selected from the group consisting of hydrogen, halogens, alkynes, halogen-substituted aryls, silyls, diazonium salts, triflates, acetyloxys, azides, sulfonates, phosphates, boronic acid-substituted aryls, boronic acid ester-substituted aryls, boronic acid esters, boronic acids, optionally substituted aryls, optionally substituted heteroaryls, optionally substituted dihydrophenanthrene (DHP), and optionally substituted fluorene, wherein the substituted aryl, heteroaryl, fluorene, or DHP is optionally substituted by one or more pendant chains terminated with functional groups optionally selected from the group consisting of amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxylsuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and their protected groups, and optionally conjugated to E.

3. Each linker L, as needed, is independently selected from the group consisting of aryl or heteroaryl groups evenly or randomly distributed along the polymer main chain; it is substituted with one or more pendant chains terminated with functional groups selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols and their protected groups, and is conjugated to E as needed. If necessary, each linker L as needed, 【Chemical 88】 【Chemical 89】 [In the formula, Each R ６ 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 ) 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 Alken, C ２ ~C １２ Alkin, C ３ ~C １２ Cycloalkyl, C １ ~C １２ Haloalkyl, C １ ~C １２ (hetero)aryl, C １ ~C １２ (Hetero)arylamino, optionally substituted benzyl, halogen, hydroxyl, C １ ~C １２ Alkoxy, (OCH ２ CH ２ ) ｆ OCH ３ , [Chemical 90] 【Chemistry 91】 [Selected independently from the group consisting of] A UV-absorbing polymer dye according to claim 1, independently selected from the group consisting of the following.

4. The polymer is defined as formula II or formula III: 【Chemistry 92】 or 【Chemistry 93】 [In the formula, each f is an integer between 0 and 50, and each R ５ H, C １ ~C １２ Alkyl, C ２ ~C １２ Alken, C ２ ~C １２ Alkin, 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)arylaminos and C １ ~C １２ [Independently selected from the group consisting of alkoxys] A UV-absorbing polymer dye according to any one of claims 1 to 3, having the structure of [the specified structure].

5. If necessary, the polymer may be of formula V: 【Chemical 94】 [In the formula, g and h together are between 10 and 100% (mol%), each f is an integer between 0 and 50, and each R ５ H, C １ ~C １２ Alkyl, C ２ ~C １２ Alken, C ２ ~C １２ Alkin, 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)arylaminos and C １ ~C １２ [Independently selected from the group consisting of alkoxys] A UV-absorbing copolymer dye comprising the polymer according to any one of claims 1 to 3, which is a copolymer having the structure of [the polymer].

6. The aforementioned polymer is defined by formula IX or formula XIV: 【Chemistry 97】 or 【Chemistry 96】 [In the formula, Each R ４’ F, Cl, -CH ３ , -CF ３ and - (OCH ２ CH ２ ) ｆ OR ９ Selected independently from, Each R ４’’ F, Cl, -CH ３ , -CF ３ and - (OCH ２ CH ２ ) ｆ OR ９ Selected independently from, Each R ９ C １ ~C ８ It is alkyl, Each f is an integer independent of 0 to 50, 11 to 40, or 10 to 20. Each o is an integer independently selected from 1, 2, 3, or 4. Each p is an integer independently selected from 1, 2, 3, or 4. A UV-absorbing polymer dye according to any one of claims 1 to 3, comprising the structure of [the specified structure].

7. Each M １ but, 【Chem.98】 Fluorine-substituted arylenes having 1 to 4 fluorine substituents, and halides, MeO-PEG-CH3, which may be further substituted as needed. ２ and / or arylene substituted with MeO-PEG, Independently selected from the group consisting of, Each f is an integer between 0 and 50, 10 and 20, or 11 and 18, Each M as needed ２ However, independently, fluorine-substituted arylenes having 1 to 4 fluorine substituents, and halides, MeO-PEG-CH3, which may be further substituted as needed. ２ and / or arylene substituted with MeO-PEG, and further, if necessary, M １ and M ２ Different, A UV-absorbing polymer dye according to any one of claims 1 to 3.

8. Each M ２ but, Halide, MeO-PEG-CH, which may be further substituted as needed. ２ and / or arylenes substituted with MeO-PEG, fluorine-substituted arylenes having 1 to 4 fluorine substituents, 【Chem.99】 Independently selected from the group consisting of, Each f is an independent integer between 0 and 50, and M is used as needed. １ and M ２ They differ, and furthermore, each M １ but, 【Chemistry 101】 Independently selected from the group consisting of, A UV-absorbing polymer dye according to any one of claims 1 to 3.

9. M １ Original M ２ A UV-absorbing polymer dye according to any one of claims 1 to 3, wherein the molar ratio to the group is 0.5:1 to 1.5:

1.

10. The polymer has an absorption maximum in the range of 320 nm to 400 nm, 320 nm to 380 nm, or 350 nm to 400 nm, and A UV-absorbing polymer dye according to any one of claims 1 to 3, having an emission maximum in the range of 380 nm to 1000 nm, 380 nm to 800 nm, or 380 nm to 430 nm.

11. A UV-absorbing polymer according to any one of claims 1 to 3, comprising a binding partner covalently bonded to the polymer, wherein the binding partner is a protein, peptide, affinity ligand, antibody, antibody fragment, sugar, lipid, nucleic acid, or aptamer, and optionally the polymer is covalently bonded to a chromophore near its energy acceptor.

12. A method for detecting the target of analysis in a sample, The steps include: contacting a sample suspected of containing the analyte with the UV-absorbing polymer described in claim 11 to form a fluorescent polymer dye conjugate complex with the analyte; The steps include applying a light source capable of exciting at least one of the fluorescent polymer dye conjugate complexes to the sample, The steps include detecting light emitted from the fluorescent polymer dye conjugate complex and A method comprising, optionally, the method being configured for flow cytometry or the method being configured as an immunoassay.

13. A composition for use in staining biological samples, for use with at least one fluorescent polymer dye conjugated to a binding partner, At least one UV-absorbing polymer dye, UV-absorbing tandem polymer dye, or quenched UV polymer dye, wherein the UV-absorbing polymer dye, UV-absorbing tandem polymer dye, or quenched polymer dye has a structure according to formula I: 【Chemistry 83】 [In the formula, Each X is C, Each Y is bonded, CR １ R ２ _CHR １ and CHR ２ Independently selected from the group consisting of, if Y is a bond, then X is directly bonded to both rings. Each R １ This includes a water-soluble portion, polyethylene glycol (PEG) group, carboxylic acid, sulfonamide oligoether, sulfonamide, sulfinamide, phosphoamide, phosphineamide, 【Chemical 84】 【Chemical 85】 Independently selected from the group consisting of, Each R ２ This includes the water-soluble portion, the linker portion, H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, heteroaryl, (hetero)arylamino, PEG group, sulfonamide-PEG, phosphoramide-PEG, oligoether sulfonate, sulfonamide oligoether, sulfonamide, sulfinamide, phosphoamide, phosphineamide, 【Chemical 86】 【Transformation 87】 Independently selected from the group consisting of, Each R ３ This is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, water-soluble moiety, chromophore, and PEG group. Each Z is CH ２ _CHR ４ , O, NR ４ and independently selected from the group consisting of NH, Each Q is a combination of NH and NR. ４ , C １ ~C １２ Alkylene, CHR ４ and CH ２ Independently selected from the group consisting of, Each R ４ H, PEG group, water-soluble portion, linker portion, chromophore, linked chromophore, functional group, linked functional group, substrate, linked substrate, binding partner, linked binding partner, quenching portion, L ２ -E, halogen, hydroxyl, C １ ~C １２ Alkyl, C ２ ~C １２ Alken, C ２ ~C １２ Alkin, 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, (CH ２ ) ｘ’ (OCH ２ -CH ２ ) ｙ’ OR ９ (In the formula, each R ９ C １ ~C ８ (It is an alkyl group, where x' is an integer from 0 to 20 independently, and each y' is an integer from 0 to 50 independently), Z-(CH ２ ) ｎ -SO ２ -Q-R ３ , C ２ ~C １８ A group independently selected from the group consisting of (hetero)aryl groups, amides, amines, carbamates, carboxylic acids, carboxylic acid esters, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazones, azides, aldehydes, thiols, and their protected forms, Each W １ This is independently a water-soluble portion, L, L １ , L ２ and L ３ These are linker parts that are selected independently. Each E is independently selected from the group consisting of a chromophore, a functional group moiety, a substrate, and a binding partner. Each R ７ H, hydroxyl, C １ ~C １２ Alkyl, C ２ ~C １２ Alken, C ２ ~C １２ Alkin, 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, C ２ ~C １２ Carboxylic acid, C ２ ~C １２ Carboxylic acid esters and -OC １ ~C １２ Independently selected from the group consisting of hydroxyl, R １ , R ２ , R ３ or R ４ At least one of them includes a water-soluble portion, Each M １ R is independently and further substituted as needed. ４ and / or phenylene substituted with trifluoromethyl, further substituted as needed, R ４ A modification unit selected from the group consisting of heteroarylenes substituted with trifluoromethyl and / or binaphthyl as needed, Each M as needed ２ R is independently and further substituted as needed. ４ and / or arylene substituted with trifluoromethyl, further substituted as needed, R ４ and / or heteroarylenes substituted with trifluoromethyl, further substituted as needed, R ４ A modification unit selected from the group consisting of 9,10-dihydrophenanthrene substituted with and / or trifluoromethyl, and binaphthyl substituted as needed, M ２ M １ It has a different structure from M ２ and M １ These are distributed evenly or randomly along the polymer backbone. Each linker L, as needed, is an independent linker component. G １ and G ２ These are independently selected from the group consisting of unmodified polymer ends and modified polymer ends, and are conjugated to E as needed. a, c, d, and e define the mol% of each unit in the structure, which may be repeated evenly or randomly along the polymer main chain, where a is 10 to 100% (mol%), c is >0 to 90% (mol%), each d is 0 to 90% (mol%), and each e is 0 to 25% (mol%). Each b is independently either 0 or 1. Each f is an independent integer between 0 and 50. m is an integer between 1 and approximately 10,000. Each n is an independent integer between 1 and 20. s is either 1 or 2. t is 0, 1, 2, or 3. The aforementioned UV-absorbing polymer dye exhibits an absorption maximum in the range of 300 nm to 400 nm. [Optionally, further comprising a chromophore covalently bonded to the polymer near its energy receptor.] A UV-absorbing polymer dye, a UV-absorbing tandem polymer dye, or a quenched UV polymer dye, including at least one UV-absorbing polymer dye, Nonionic surfactants, and biological buffer Includes, Compared to the at least one fluorescent polymer dye conjugate in the absence of the composition, the nonspecific binding of the at least one fluorescent polymer dye conjugate is reduced. composition.

14. The quenched UV polymer dye comprises a UV-absorbing polymer dye having at least one quenchable portion, and optionally 1 to 30, 2 to 20, or 2.5 to 10 quenchable portions. The composition according to claim 13, wherein, if necessary, the quenching portion is selected from the group consisting of DABCYL, DABSYL, BHQ1, BHQ0, DDQI, EDQ, QSY7, QSY9, QSY35, TAMRA, DABCYL Q, DABCYL plus, 490Q, 425Q, and 505Q.

15. The nonionic surfactant is a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer. If necessary, the nonionic surfactant may be derived from formula XII: 【Chemistry 107】 The composition according to claim 13, comprising a structure in which [each a is independently in the range of 2 to 130, and b is in the range of 15 to 67].

16. It further contains additional additives selected from the group consisting of protein stabilizers, preservatives, and further surfactants, The composition according to any one of claims 13 to 15, wherein, if necessary, the further surfactant is selected from the group consisting of zwitterionic surfactants and ionic surfactants.

17. The composition according to any one of claims 13 to 15, comprising a plurality of fluorescent polymer dye conjugates, wherein the nonspecific binding between the plurality of fluorescent polymer dye conjugates is substantially reduced.

18. A method for detecting the target of analysis in a sample, A step of adding at least one polymer dye or tandem polymer dye, which is conjugated to a binding partner, to the composition according to any one of claims 13 to 15 to form a polymer dye conjugate composition, A step of contacting a biological sample suspected of containing the analyte with the polymer dye conjugate or tandem polymer dye conjugate composition to form a fluorescent polymer dye conjugate complex with the analyte, The steps include applying a light source capable of exciting the at least one fluorescent polymer dye conjugate complex to the sample, The steps include detecting light emitted from the fluorescent polymer dye conjugate complex and Includes, and as needed, The aforementioned biological sample is selected from the group consisting of blood, bone marrow, spleen cells, lymphocytes, bone marrow aspirate, urine, serum, saliva, cerebrospinal fluid, urine, amniotic fluid, interstitial fluid, feces, mucus, or tissue, and if necessary, A method comprising adding two or more polymer dye conjugates to the composition.

19. The aforementioned biological sample is a blood sample, and if necessary, a whole blood sample, and further if necessary, one or more types of cells from whole blood, and if necessary, The method according to claim 18, wherein the one or more cells of whole blood are selected from the group consisting of any cells including red blood cells, white blood cells, lymphocytes, phagocytes, monocytes, macrophages, granulocytes, basophils, neutrophils, eosinophils, platelets, and one or more detectable markers.

20. The step of detecting the light further includes the step of analyzing it by flow cytometry to obtain a first flow cytometry plot, wherein the first flow cytometry plot is When the biological sample is compared with a second flow cytometry plot obtained by the step of contacting it with a composition that does not contain the nonionic surfactant and does not contain the UV-absorbing polymer dye, the UV-absorbing tandem polymer dye, or the quenched UV polymer dye, Reduction of nonspecific interactions of polymer dye conjugates, and Reduction of aggregation of polymer dye conjugates This indicates one or more of the groups consisting of The method according to claim 18.

21. A kit comprising the composition according to any one of claims 13 to 15, the kit comprising a container containing the composition, and optionally the at least one fluorescent polymer dye conjugate.

22. A kit comprising a UV-absorbing polymer dye according to any one of claims 1 to 3, wherein the UV-absorbing polymer dye is contained in at least one container.

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