Additives to reduce nonspecific interactions between fluorescent polymer conjugates and cells in biological samples.

Abstract

The present disclosure relates to methods and compositions for reducing or eliminating nonspecific binding of at least one dye conjugate to cells in a biological sample, wherein the dye conjugate is contacted with at least one zwitterionic or anionic surfactant before, during, or after contacting the dye conjugate with a blood sample, resulting in a substantial reduction in nonspecific binding of the dye conjugate to cells in the biological sample.

Description

[Technical Field]

[0001] This application was filed on November 12, 2021, as a PCT international patent application and claims priority to U.S. Provisional Application No. 63 / 113,703, filed on November 13, 2020, which is incorporated herein by reference in its entirety. [Background technology]

[0002] background Polymer dye conjugates are bright and offer excellent performance for use in monochromatic or multichromatic flow cytometry assays. Generally, polymer dye conjugates exhibit high brightness due to their unique and complex structure. However, this same unique and complex structure can also present some significant limitations. This disclosure addresses these limitations. [Overview of the project] [Means for solving the problem]

[0003] summary Due to their nature, polymer dye conjugates can nonspecifically bind to cells in biological samples, such as monocytes and granulocytes in peripheral blood samples. Nonspecific binding can lead to misinterpretations and false positive conclusions. For example, if a polymer dye conjugate comes into contact with blood during the analysis of cell markers, the conjugate may nonspecifically bind to cells, such as monocytes and / or granulocytes, thereby producing a signal that can be misinterpreted as a positive population or highlighting populations other than the desired ones.

[0004] This disclosure provides solutions to these problems and other problems associated with the use of polymer dye conjugates. In some embodiments, this disclosure provides compositions for reducing or removing nonspecific binding of dye conjugates to cells in a biological sample, comprising the dye conjugates described herein and surfactants.

[0005] In some embodiments, the Disclosure provides a method for reducing or removing the nonspecific binding of at least one dye conjugate to cells in a biological sample, comprising the steps of contacting at least one dye conjugate with at least one zwitterionic surfactant before, during, or after contacting the dye conjugate with the biological sample, wherein the contact results in a reduction of the nonspecific binding of at least one dye conjugate in the sample. In some embodiments, the Disclosure provides a method for reducing or removing the nonspecific binding of at least one to cells in a blood sample, comprising the steps of contacting at least one dye conjugate with at least one anionic surfactant before, during, or after contacting the dye conjugate with the blood sample, wherein the contact results in a reduction of the nonspecific binding of at least one dye conjugate in the blood sample. The compositions and methods of the Disclosure reduce or remove the nonspecific binding of polymer dye conjugates or nonpolymer dye conjugates to monocytes and / or granulocytes in a blood sample.

[0006] A method for reducing or removing non-specific binding of at least one dye conjugate in a biological sample, such as a blood sample, comprising contacting at least one dye conjugate with at least one zwitterionic or anionic surfactant before, during, or after contacting the polymer dye conjugate with the biological sample, wherein contacting results in a reduction of non-specific binding of at least one polymer dye conjugate in the biological sample.

[0007] In some embodiments, the biological sample may be a blood sample. In some embodiments, the cells may be white blood cells, and the reduction of non-specific binding may include a reduction of non-specific binding to white blood cells in the blood sample. In some embodiments, the white blood cells are selected from the group consisting of monocytes and granulocytes.

[0008] In some embodiments, the method includes adding the surfactant to the polymer dye conjugate before contacting the polymer dye conjugate with a biological sample, such as a peripheral blood sample.

[0009] In some embodiments, the method includes adding the surfactant to the blood sample before contacting it with the polymer dye conjugate.

[0010] The surfactant has the formula: R 1’ [CO-X(CH2) j g -[N + (R 2’ )(R 3’ )] k -(CH2) f -[CH(OH)CH2] h -Y - (wherein R 1’ is saturated or unsaturated C 5~24 alkyl; X is NH, NR 4 ’, where R 4’ is C 1~4 ​It is alkyl, O, or S; j is an integer from 1 to 10; g is either 0 or 1; R 2’ and R 3’ C 1~4 It is alkyl; k is either 0 or 1; Hydroxyl is substituted as needed with methyl, ethyl, hydroxymethyl, or hydroxyethyl; f is an integer between 0 and 4; h is either 0 or 1; Y is COO, SO3, OPO(OR 5’ )O, or P(O)(OR 5’ )O, and here, R 5’ is H or C 1~4 (It is alkyl.) It may be a compound of the same, and when k=0, the surfactant may be in an acidic form or its sodium or potassium salt.

[0011] In some embodiments, the surfactant is given by 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 is either saturated or unsaturated. 5~24 It is alkyl; X is NH or NR 4’ And here, R 4’ C 1~4 It is alkyl, O, or S; j is an integer from 1 to 10; g is either 0 or 1; R 2’ and R 3’ C 1~4 It is alkyl; Hydroxyl is substituted as needed with methyl, ethyl, hydroxymethyl, or hydroxyethyl; f is an integer from 1 to 4; h is either 0 or 1; Y is COO, SO3, OPO(OR 5’ )O or P(O)(OR 5’ )O, and here, R 5’ is H or C 1~4 (It is an alkyl residue.) It may also be a zwitterionic surfactant compound.

[0012] Zwitterionic surfactants are defined by the 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 - ;or R 1’ -CO-NH-(CH2)3-N + (CH3)2-CH2CH(OH)CH2SO3 - It may also be a compound of the following.

[0013] In some embodiments, the zwitterionic surfactant is 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. (glycinate), dihydroxyethyl stearyl glycinate, dihydroxyethyl tallow glycinate, dimethicone propyl PG-betaine, dorucamidopropyl hydroxysultaine, hydrogenated tallow betaine, isostearamidopropyl betaine, lauramidopropyl betaine, lauryl betaine, lauryl hydroxysultaine, lauryl sultaine, milk amidopropyl (milkThe group may be selected from amidopropyl betaine, milkamidopropyl betaine, myristamidopropyl betaine, myristyl betaine, oleamidopropyl betaine, oleamidopropyl hydroxysultaine, oleyl betaine, oliamidopropyl betaine, palmamidopropyl betaine, palmitamidopropyl betaine, palmitoyl carnitine, palm kernelamidopropyl betaine, polytetrafluoroethylene acetoxypropyl betaine, ricinolamidopropyl betaine, sesamidopropyl betaine, soyamidopropyl betaine, stearamidopropyl betaine, stearyl betaine, tallowamidopropyl betaine, tallowamidopropyl hydroxysultaine, tallow betaine, tallow dihydroxyethyl betaine, undecylenamidopropyl betaine, and wheat germamidopropyl betaine. In some embodiments, the surfactant is lauryl betaine.

[0014] In some embodiments, the surfactant is given by formula: R 1’ [CO-X(CH2)] j ] g -(CH2) f -[CH(OH)CH2] h -Y - (In the formula, R 1’ C is either saturated or unsaturated. 5~24 It is alkyl; X is NH, NR 4 ' and here, R 4’ C 1~4 It is alkyl, O, or S; j is an integer from 1 to 10; g is either 0 or 1; R 2’ and R 3’ C 1~4 It is alkyl; Hydroxyl is substituted as needed with methyl, ethyl, hydroxymethyl, or hydroxyethyl; f is an integer between 0 and 4; h is either 0 or 1; Y is COO, SO3, OPO(OR 5’ )O or P(O)(OR 5’ )O, and here, R 5’ is H or C 1~4 It may be an anionic surfactant compound (which is alkyl), and the anionic surfactant may be in an acidic form or in the form of its sodium or potassium salt. In some embodiments, f=0. In some embodiments, f=1. In some embodiments, f=3. In some embodiments, f=4. In some embodiments, Y is COO or SO3. In some embodiments, R 2’ and R 3’ It is methyl.

[0015] Anionic surfactants are, R 1’ -CO-N(CH3)-CH2-COO - ;or R 1 '-CO-N(CH3)-CH2-SO3-(in the formula, R 1’ C is either saturated or unsaturated. 5~24 Compounds (which are alkyl) and their sodium or potassium salts. In some embodiments, R 1’ is saturated or unsaturated C 7~19 Alkyl, or C 11~17 Alkyl is also acceptable.

[0016] In some embodiments, the anionic surfactant may be selected from the group consisting of N-lauroyl sarcosine, sodium lauroyl sarcosinate, sodium palmitoyl sarcosinate, sodium stearoyl sarcosinate, sodium N-methyl-N-(1-oxotetradecyl)-glycine salt, sodium caproyl sarcosinate, sodium capryloyl sarcosinate, sodium N-methyl-N-(1-oxo-9-octadecen-1-yl)-glycine, sodium salt, sodium oleoyl sarcosinate, and sodium linoleoyl sarcosinate. In some embodiments, the anionic surfactant is N-lauroyl sarcosine.

[0017] In some embodiments, the polymer dye conjugate is derived from formula III: [ka] (In the formula, Each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers; Each required M is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, bandgap-modified monomers, optionally substituted ethylenes, and ethynylenes; Each required L is a linker part; each G 1 and G 2 These are selected independently from unmodified and modified polymer ends; a, c, and d each define the mol% of each unit, which may be repeated uniformly or randomly, with each a being from 10 to 100% mol%, each c being from 0 to 90% mol%, and each d being from 0 to 25% mol%; Each b is independently either 0 or 1; (Each m is an integer between 1 and approximately 10,000) It includes a binding partner conjugated to a polymer dye having the structure.

[0018] In some embodiments, A comprises a DHP moiety. In some embodiments, A comprises a fluorene moiety. In some embodiments, A comprises both a DHP and a fluorene moiety.

[0019] In some embodiments, the polymeric dye conjugate has the formula I:

Chemical formula

Chemical formula

Chemical formula

[0020] The binding partner may be a molecule or a molecular complex that can specifically bind to the target analyte. The binding partner may be a protein, affinity ligand, antibody, or antibody fragment. In some embodiments, the binding partner may be selected from the group consisting of monoclonal antibodies, polyclonal antibodies, immunoglobulins, immunoactive moieties of immunoglobulins, single-chain antibodies, Fab fragments, Fab' fragments, and F(ab')2 fragments and scFv fragments.

[0021] A composition comprising a polymer dye conjugate; an aqueous buffer; and a zwitterionic or anionic surfactant is provided. The composition may contain the zwitterionic or anionic surfactant at a concentration below the critical micelle concentration (CMC). In some embodiments, the surfactant may be at a concentration of 0.05 to 0.25% (w / v), 0.06 to 0.20% (w / v), or 0.08 to 0.16% (w / v). The aqueous buffer may contain additional additives selected from the group consisting of protein stabilizers, preservatives, and additional surfactants. Depending on the composition, after exposure to a blood sample and flow cytometry analysis, a reduction in the nonspecific binding of the polymer dye conjugate to leukocytes in the sample may be shown. The reduction in nonspecific binding may be compared to the same composition without the zwitterionic or anionic surfactant. The leukocytes may be selected from the group consisting of monocytes and granulocytes. [Brief explanation of the drawing]

[0022] [Figure 1A] Figure 1A shows plots of fluorescence intensity as a function of wavelength for fluorene (FF), dihydrophenanthrene (DD), and fluorene-DHP(DF) polymer dyes.

[0023] [Figure 1B]Figure 1B shows graphs of the absorption spectra of fluorene (Fl-Fl) polymer and dihydrophenanthrene (DHP-DHP) polymer. The DHP-DHP polymer (black curve) shows lambda max (λmax) at 390 and 410 nm, while the Fl-Fl polymer (gray curve) shows lambda max (λmax) at approximately 400 nm.

[0024] [Figure 2] Figure 2 shows flow cytometry point plots of unstained blood cells (top panel); blood cells stained with polymer dyes and without surfactants (bottom left panel); and blood cells stained with compositions containing polymer dyes and surfactants (bottom right). Blood samples were stained with the antibody-free fluorescent polymer dye SN v605 and analyzed by flow cytometry. The surfactant-free polymer dye showed nonspecific binding to monocytes / granulocytes (bottom left). The polymer dye containing EMPIGEN BB® showed substantial reduction in nonspecific binding to monocytes / granulocytes (bottom right).

[0025] [Figure 3] Figure 3 shows dot plots for blood cells without polymer dye conjugates (top panel), blood cells stained with SN 605-CD20 conjugates and containing surfactants (bottom left panel), and blood cells stained with SN 605-CD20 conjugates and without surfactants (bottom right panel). The percentage of nonspecifically bound granulocytes decreased with the use of surfactants (see the "P2" gate in the dot plots). Furthermore, the percentage of positive cells was similar in both cases, indicating no change in the functional aspects of the conjugates (see the "P1" gate in the dot plots).

[0026] [Figure 4]Figure 4 shows a bar graph of median fluorescence intensity (MdFI) values ​​of monocytes in the presence and absence of surfactants for two lots of polymer dye conjugate (SN v605-CD20) compared to unstained monocytes (autofluorescent). In the presence of surfactants, nonspecific interactions on monocytes were substantially reduced for both lot 1 and lot 2 of the SN605 CD20 conjugate.

[0027] [Figure 5] Figure 5 shows bar graphs of MdFI values ​​of granulocytes in the presence and absence of surfactants for two lots of polymer dye conjugates (SN v605-CD20) compared with unstained granulocytes (autofluorescent). In the presence of surfactants, nonspecific interactions with granulocytes were reduced for both lot 1 and lot 2 of the SN605 CD20 conjugate.

[0028] [Figure 6] Figure 6 shows dot plots of blood cells without polymer dye conjugate (top left), blood cells stained with SN v786-CD103 conjugate and containing Empigen BB® surfactant (bottom left), and blood cells stained with SN v786-CD103 conjugate but without surfactant (top right). The dot plots compare one of the polymers described in the claims with BV786-CD103, a tandem fluorescent dye (bottom right) (available from Becton Dickinson). In the presence of the surfactant, the percentage of nonspecifically bound granulocytes and monocytes was reduced (see gates "P1" and "P2" in the dot plots, respectively).

[0029] [Figure 7]Figure 7 shows bar graphs of MdFI values ​​of monocytes in the presence and absence of surfactants for two lots of polymer dye conjugates (SN v786-CD103) compared to unstained monocytes (autofluorescent). In the presence of surfactants, nonspecific binding of polymer conjugates to monocytes was substantially reduced for both lots of polymer dye conjugates.

[0030] [Figure 8] Figure 8 shows bar graphs of MdFI values ​​of granulocytes in the presence and absence of surfactants for two lots of polymer conjugates, compared to unstained granulocytes (autofluorescent). In the presence of surfactants, nonspecific binding of polymer conjugates to granulocytes was substantially reduced for both lots of polymer dye conjugates.

[0031] [Figure 9] Figure 9 shows dot plots of blood cells stained with SN v605-CD20 conjugates containing a surfactant and SN v605-CD20 conjugates without a surfactant (top panel). The lower left panel is stained with the nonionic surfactant Tween®-20, and the lower right panel is stained with the nonionic surfactant Pluronic F-68. The percentage of nonspecifically bound monocytes did not decrease with the use of the nonionic surfactants Tween®-20 and Pluronic F-68 (see the "nonspecific monocyte" gate in the dot plots).

[0032] [Figure 10]Figure 10 shows point plots of blood cells without dye conjugate (upper left panel); blood cells stained with SN v605-CD20 conjugate and containing BSA (upper right panel); blood cells stained with SN v605-CD20 conjugate and containing oxidized BSA (lower left panel); and blood cells stained with SN v605-CD20 conjugate and containing BSA-Cy5-ox (lower right panel). The percentage of nonspecifically bound monocytes and granulocytes was not substantially reduced even with the use of protein blockers (see the "P1" gate in the point plots).

[0033] [Figure 11-1] Figure 11 shows three graphs illustrating the effect of surfactant concentration on negative monocytes (MFIs) in unstained and stained donor 1 (D1) and donor 2 (D2) blood samples, respectively, regarding the specificity of SN v428 CD19 (Figure 11, top panel), SN v428 CD22 (Figure 11, bottom panel), and SN v428 CD25 (Figure 11, middle panel). SN conjugates showed lower nonspecific monocyte interactions in the presence of 0.06 to 0.20% Empigen BB® compared to the absence of surfactants.

[0034] [Figure 11-2] Figure 11 (continued) shows three graphs illustrating the effect of surfactant concentration on negative monocytes (MFI) in unstained and stained donor 1 (D1) and donor 2 (D2) blood samples for SN v428 CD19 (Figure 11, top panel), SN v428 CD22 (Figure 11, bottom panel), and SN v428 CD25 (Figure 11, middle panel), respectively. The data are shown as negative monocyte MFI% without Empigen BB® sample. Samples stained with BD polymer dye conjugates showed a lower percentage of nonspecific monocyte interactions in the presence of 0.06 to 0.20% Empigen BB® compared to the absence of surfactant.

[0035] [Figure 12]Figure 12 (9 graphs) shows the effect of surfactant concentration on negative granulocytes (top three panels), positive lymphocytes (middle panel), and positive lymphocyte percentage without Empigen BB® sample (bottom three panels). Unstained and stained blood samples from donor 1 (D1) and donor 2 (D2) are shown. For CD19 BD, CD25 BD, and CD22 BD polymer dye conjugates, negative interactions with granulocytes were slightly lower in the presence of 0.06 to 0.20% surfactant compared to the absence of surfactant (top three panels). For CD19 BD, CD25 BD, and CD22 BD polymer dye conjugates, positive lymphocyte data were similar or slightly higher in the presence of surfactant compared to the absence of surfactant (middle three and bottom three panels).

[0036] [Figure 13] Figure 13 shows point plots of SS / FL9 staining patterns for blood samples from Donor 1 (upper panel) and Donor 2 (lower panel) at 0.5 μg / test of SN v428 CD19 lot number D19-094 polymer dye conjugate, as well as CD19 BV-421 conjugate (Becton Dickinson) at its commercially available dose, for surfactant-free, 0.06%, 0.12%, and 0.2% Empigen BB® surfactant-containing samples, and for the commercially available CD19 BV-421 conjugate (Becton Dickinson).

[0037] [Figure 14] Figure 14 shows point plots of SS / FL9 staining patterns for blood samples from Donor 1 (upper panel) and Donor 2 (lower panel) with SN v428 CD25 lot number D19-107 polymer dye conjugate at 0.5 μg / test, with surfactant-free, 0.06%, 0.12%, and 0.2% surfactant content, as well as with the commercially available CD25 BV-421 conjugate (Becton Dickinson).

[0038] [Figure 15]Figure 15 shows point plots of SN v428 CD22 lot number D19-109 polymer dye conjugate at 0.5 μg / test for donor 1 (upper panel) and donor 2 (lower panel) blood samples, with surfactant-free, 0.06%, 0.12%, and 0.2% surfactant content, as well as for the CD22 BV-421 conjugate (Becton Dickinson) at its commercially available dose.

[0039] [Figure 16] Figure 16 shows a point plot containing the percentage of dead cells with up to 0.2% surfactant. CD19-SNv428D19-094, without EMPIGEN BB® (negative control) and containing 0.06%, 0.12%, and 0.2% EMPIGEN BB®, was tested on whole blood samples from donors 1 and 4 stained with 7-ADD to evaluate the percentage of dead cells under each condition. Whole blood samples stored for more than 24 hours were added as a positive control for 7-AAD staining (left panel, 12% dead cells). The percentage of dead cells did not substantially increase with the presence of up to 0.2% EMPIGEN BB® compared to the surfactant-free sample.

[0040] [Figure 17A] Figure 17A shows a point plot of peripheral blood samples without monochromatic conjugates, clearly demonstrating that no clusters are observed at the CD20+ gate.

[0041] [Figure 17B]Figure 17B shows a positive control plot of peripheral blood samples in a buffering composition containing BSA, sodium azide, Pluronic® F-68 (PF-68), and Empigen BB® as additives, in the presence of a CD20-SN v605 monochromatic conjugate. Compared to the negative control plot (Figure 17C), the population percentages at the "monocyte nonspecific binding" and "granulocyte nonspecific binding" gates are significantly reduced, demonstrating the effectiveness of Empigen BB® in eliminating or reducing nonspecific binding to monocytes and granulocytes.

[0042] [Figure 17C] Figure 17C shows a negative control point plot of peripheral blood samples in a buffer composition containing only BSA, PF-68, and sodium azide as additives, in the presence of a CD20-SN v605 monochromatic conjugate.

[0043] [Figure 17D] Figure 17D shows a test point plot of peripheral blood samples in a buffering composition containing BSA, sodium azide, PF-68, and NLS (0.16% w / v) as additives, in the presence of a CD20-SN v605 monochromatic conjugate.

[0044] [Figure 17E] Figure 17E shows a test point plot of peripheral blood samples in a buffering composition containing BSA, sodium azide, PF-68, and NLS (0.08% w / v) as additives, in the presence of a CD20-SN v605 monochromatic conjugate. [Modes for carrying out the invention]

[0045] Detailed explanation of this disclosure Herein, specific embodiments of the disclosed subject matter will be referenced in detail, examples of which will be partially illustrated in the accompanying drawings and embodiments. The disclosed subject matter will be described together with the enumerated claims, but it will be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter. General disclosure

[0046] This disclosure relates to compositions and methods for detecting analytes in a sample, using compositions comprising at least one surfactant and at least one polymer dye conjugated to a binding partner (e.g., an antibody), such as a fluorescent polymer dye conjugated to a binding partner. More specifically, this disclosure relates to a method for reducing or removing nonspecific binding of at least one polymer dye conjugate in a biological sample, such as a blood sample, comprising the steps of contacting at least one polymer dye conjugate with at least one zwitterionic or anionic surfactant before, during, or after contacting the polymer dye conjugate with the biological sample, such as a blood sample, wherein the contact results in a reduction of nonspecific binding of the at least one polymer dye conjugate to cells, such as leukocytes in a blood sample. The surfactant may be added to the blood sample before contact. The surfactant may be added to the polymer dye conjugate before contact with the biological sample. definition

[0047] Abbreviations used herein have their conventional meanings within the fields of chemistry and biology.

[0048] The singular forms "a," "an," and "the" are intended to include the plural forms unless the context clearly indicates a different meaning.

[0049] The terms "and / or" refer to and encompass any and all possible combinations of one or more of the related enumeration items. Unless otherwise specified, the term "room temperature" refers to a temperature between 18 and 27°C.

[0050] Unless otherwise specified, the term "percent" or "%" refers to weight percentage.

[0051] All patents, patent applications, and publications referenced herein are incorporated in their entirety by reference.

[0052] The term "analyte" refers to molecules, compounds, or other components in a sample. Analytes may include, but are not limited to, peptides, proteins, polynucleotides, organic molecules, sugars and other carbohydrates, and lipids.

[0053] The term "binding partner" refers to a molecule that can specifically bind to the analyte. A binding partner may be any of several different types of molecules, including antibodies or their antigen-binding fragments, or other proteins, peptides, polysaccharides, lipids, nucleic acids or nucleic acid analogs, such as oligonucleotides, aptamers, or PNA (peptide nucleic acids).

[0054] The term "CD" refers to Cluster of Differentiation.

[0055] In flow cytometry, the term "correction" refers to a mathematical process that corrects fluorescence overflow (spectral overlap in multi-parameter flow cytometry data). For example, correction may be performed by removing the signal of any given fluorescent dye from all detectors except those specifically designed to measure that dye. Because fluorescent dyes can have a wide range of spectra, they can overlap, leading to undesirable confusion during data analysis.

[0056] The term “labeled binding partner” refers to the binding partner conjugated to the dye. The term “reaction solution” refers to the solution containing the labeled binding partner. In some embodiments, in addition to the labeled binding partner, the reaction solution may further contain stabilizers, salts, buffers, surfactants, and / or other reagents.

[0057] The term “linker” or “linkage” refers to a bonding portion that connects two groups and has a skeleton of 100 or fewer atoms in length. A linker or linkage may be a covalent bond connecting two groups, or a chain of atoms between 1 and 100 in length, for example, a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, or more carbon atoms, and the linker may be linear, branched, cyclic, or single-atom. In some embodiments, a linker is a branched linker referring to a bonding portion connecting three or more groups. In certain particular cases, one, two, three, four, or five, or more carbon atoms in the linker skeleton may be optionally substituted with sulfur, nitrogen, or oxygen heteroatoms. In some embodiments, the linker skeleton may be a linking functional group, such as an ether, thioether, amino, amide, sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester, or imine. The bonds between the skeletal atoms may be saturated or unsaturated, and in some cases, there may be no more than one, two, or three unsaturated bonds in the linker skeleton. The linker may be, for example, one or more substituents with alkyl, aryl, or alkenyl groups. The linker may be, but is not limited to, polyethylene glycol, ether, thioether, tertiary amine, or alkyl, which may be linear or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, or 1,1-dimethylethyl (t-butyl). The linker skeleton may be a cyclic group, such as an aryl, heterocycle, or cycloalkyl group, and two or more atoms of the cyclic group, such as two, three, or four atoms, may be included in the skeleton. The linker may be cleavable or incleavable.

[0058] The linker portion may be bonded to “A” as taught in U.S. Publication Application 2020 / 0190253A1, which is incorporated herein by reference in its entirety, or it may be bonded to “L” as taught in U.S. Publication Application 2019 / 0144601, which is incorporated herein by reference in its entirety. The linker portion may contain a sulfonamide, disulfonamide, serenomide, sulfinamide, sultam, disulfinamide, amide, selenamide, phosphonamide, phosphinamide, phosphoamide, or secondary amine.

[0059] As described therein, each is related to the linker part, so the term "sulfonamide" refers to the -S(O)2NR- part; the term "disulfonamide" refers to the -S(O)2NRS(O)2- part; the term "selenonamide" refers to the -Se(O)2NR- part; the term "sulfinamide" refers to the -S(O)NR- part; the term "disulfinamide" refers to the -S(O)NRS(O)- part; the term "selenamide" refers to the, The term "-Se(O)NR-" refers to the -NR-PR(O)NR- moiety; the term "phosphonamide" refers to the -NR-PR(O)NR- moiety; the term "phosphineamide" refers to the -PR(O)NR- moiety; the term "phosphonamide date" refers to the -O-PR(O)NR- moiety; the term "sultam" refers to a cyclic sulfonamide (where, for example, the R group is bonded to a sulfur atom via an alkylene moiety); for each term, the R group is independently H, alkyl, haloalkyl, or aryl.

[0060] The term “terminus,” as used herein, refers to the end of a conjugated polymer chain that may contain a functional group that provides bioconjugation. In some cases, such a functional group is referred to as a terminal linker. The terminus may be an aryl or heteroaryl substituted with one or more pendant chains ending with a functional group selected from, for example, hydrogen, halogens, alkynes, optionally substituted aryls, optionally substituted heteroaryls, 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 tetrahydropyrene (THP), optionally substituted fluorene, optionally substituted dihydrophenanthrene (DHP); amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkynes, aldehydes, thiols, and protecting groups thereof for conjugation to a substrate or binder.

[0061] The term "MdFI" or "MDFI" refers to the median fluorescence intensity.

[0062] The term "recruitment %" refers to the number of gated cells in a suitable population.

[0063] The term "multiplex" as used herein refers to an assay or other analytical method that allows multiple analytes to be assayed simultaneously.

[0064] The term "PEG" refers to polyethylene glycol or poly(ethylene glycol). The number after "PEG" indicates the average molecular weight, Mw indicates the weight-average molecular weight, and Mn indicates the number-average molecular weight.

[0065] The term "PBS" refers to phosphate-buffered saline, which is an aqueous buffer that may contain sodium chloride, disodium hydrogen phosphate, potassium chloride, and 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 azides, such as sodium azide. PBS is an isotonic solution.

[0066] The acronym "SSC" refers to lateral scattering.

[0067] The acronym "WBC" refers to white blood cells.

[0068] A “dye” is a portion that provides a detectable signal and may be directly or indirectly bound to or incorporated into a binding partner. Dyes used in this disclosure may be colored, fluorescent, or luminescent and are typically detected by a detector in a flow cytometer, e.g., a PMT or APD. Fluorescent dyes may be monomers or polymers. Fluorescent dyes may be fluorescent polymer dyes. Polymer dyes are particularly useful for the analysis of chemical and biological targets. They are highly responsive optical reporters and efficient light absorbers because they contain multiple chromophores. Fluorescent polymer dyes suitable for use in this disclosure are described herein, for example, in US 2019 / 0144601 and US 2020 / 0190253. Examples of polymer dyes, but not limited to, include conjugate polymers having repeating units of chromophores, aggregates of conjugate molecules, luminescent dyes bound to saturated polymers via side chains, semiconductor quantum dots, and dendritic structures. Polymers and monomer dyes disclosed in U.S. Patents No. 7,214,489, No. 8,354,239, and No. 8,575,303 may also be used in the present invention.

[0069] As used herein, the term "ammonium" refers to formula NHR3 + This refers to a cation having the following characteristics, where each R group is independently hydrogen, or a substituted or unsubstituted alkyl, aryl, aralkyl, or alkoxy group. Preferably, each R group is hydrogen.

[0070] As used herein, “oligoether” is understood to mean an oligomer containing a structural repeating unit having an ether functional group. As used herein, “oligomer” is understood to mean a molecule containing one or more identifiable structural repeating units of the same or different formulas.

[0071] The terms "sulfonate functional group" or "sulfonate," as used herein, refer to both the free sulfonate anion (-S(=O)2O-) and its salts. Thus, the term sulfonate encompasses sulfonates, such as sodium sulfonate, lithium sulfonate, potassium sulfonate, and ammonium sulfonate.

[0072] As used herein, the term "sulfonamide" refers to the group of the formula -SO2NR-, where R is hydrogen, alkyl, or aryl.

[0073] As used herein, the term "alkyl" refers to a straight-chain or branched saturated aliphatic radical having the indicated number of carbon atoms. For example, C1-C6 alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, and the like. Other alkyl groups include, but are not limited to, heptyl, octyl, nonyl, decyl, and the like. Alkyl may contain any number of, for example, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6, and 5-6 carbons. An alkyl group is typically monovalent, but may be divalent, for example, when two alkyl groups are joined together.

[0074] As used herein, the term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic, fused bicyclic, or bridged polycyclic ring assembly containing 3 to 12 ring atoms, or as a monocyclic ring with the indicated number of atoms, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Bicyclic and polycyclic rings include, for example, norbornane, decahydronaphthalene, and adamantane. For example, C 3~8 Cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and norbornane.

[0075] The term "haloalkyl," as used herein, refers to an alkyl group as defined above, in which some or all of the hydrogen atoms are substituted with halogen atoms. The halogen (halo) preferably represents chloro or fluoro, but may also represent bromo or iodine. Examples of haloalkyls include trifluoromethyl, fluoromethyl, and 1,2,3,4,5-pentafluorophenyl. The term "perfluoro" defines a compound or radical in which at least two available hydrogens are substituted with fluorine. For example, perfluorophenyl refers to 1,2,3,4,5-pentafluorophenyl, perfluoromethane refers to 1,1,1-trifluoromethyl, and perfluoromethoxy refers to 1,1,1-trifluoromethoxy.

[0076] As used herein, the term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0077] As used herein, the term "alkoxy" refers to an alkyl group having an oxygen atom bonded to the alkyl group, as defined above. Examples of alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, butoxy, 2-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, and hexoxy. The alkoxy group may be further substituted with various substituents described within this specification. For example, the alkoxy group may be substituted with a halogen to form a "halo-alkoxy" group.

[0078] As used herein, the term "alkene" refers to a linear or branched hydrocarbon having at least one double bond. Examples of alkene groups, but not limited to, include vinyl, propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexadienyl. Alkene groups are typically monovalent, but may be divalent, for example, if the alkenyl group combines two parts.

[0079] As used herein, the term "alkyne" refers to a linear or branched hydrocarbon having at least one triple bond. Examples of alkynyl groups, but not limited to, include acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiinyl, 1-pentynyl, 2-pentynyl, isopentinyl, 1,3-pentadinyl, 1,4-pentadinyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadinyl, 1,4-hexadinyl, 1,5-hexadinyl, 2,4-hexadinyl, or 1,3,5-hexadinyl. Alkynyl groups are typically monovalent, but may be divalent, for example, if the alkynyl group combines two parts.

[0080] The term "aryl," as used herein, refers to a monocyclic, fused bicyclic, tricyclic, or more cyclic aromatic ring assembly containing 6 to 16 ring carbon atoms. For example, the aryl may be phenyl, benzyl, or naphthyl, and preferably phenyl. "Arylene" means a divalent radical derived from an aryl group. The aryl group may be alkyl, alkoxy, aryl, hydroxy, halogen, cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy, and oxy-C2~C3-alkylene; all of these may be further substituted as needed, for example, as defined above; or mono-, di-, or tri-substituted by one, two, or three radicals selected from 1- or 2-naphthyl; or 1- or 2-phenantrenyl. An alkylenedioxy is a divalent substituent bonded to two adjacent carbon atoms of phenyl, such as methylenedioxy or ethylenedioxy. Oxy-C2~C3-alkylenes are also divalent substituents bonded to two adjacent carbon atoms of phenyl, such as oxyethylene or oxypropylene. An example of an oxy-C2~C3-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

[0081] Preferred aryls are naphthyl, phenyl, or phenyl mono- or disubstituted with alkoxy, phenyl, halogen, alkyl, or trifluoromethyl, and in particular phenyl, or phenyl mono- or disubstituted with alkoxy, halogen, or trifluoromethyl, and especially phenyl.

[0082] The term "aryloxy," as used herein, refers to an O-aryl group, where aryl is as defined above. The aryloxy group may be unsubstituted or substituted with one or two preferred substituents. The term "phenoxy" refers to an aryloxy group in which the aryl portion is a phenyl ring. The term "heteroaryloxy," as used herein, refers to an -O-heteroaryl group, where heteroaryl is as defined below. The term "(hetero)aryloxy" is used to indicate that the portion is an aryloxy or heteroaryloxy group.

[0083] When used herein, the terms "polyethylene 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. 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, less than 15, e.g., 3 to 15, or 10 to 15. It should be understood that the PEG polymer groups may be of any convenient length, but may include a variety of terminal groups and / or further substituents, including alkyl, aryl, hydroxyl, amino, acyl, carboxylic acid, carboxylate ester, acyloxy, and amide terminals and / or substituents.

[0084] The term "heteroaryl," as used herein, refers to a monocyclic, fused bicyclic, or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, where 1 to 4 of the ring atoms are heteroatoms such as N, O, or S. Examples of heteroaryls include pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any other radical, particularly mono- or disubstituted, substituted by alkyl, nitro, or halogen, for example. Pyridyl represents 2-, 3-, or 4-pyridyl, and preferably 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, and most preferably 4-thiazolyl. Triazolyl preferably represents 1-, 2-, or 5-(1,2,4-triazolyl). Tetrazolyl preferably represents 5-tetrazolyl.

[0085] Preferably, the heteroaryl is any of pyridyl, indolyl, quinolinyl, pyrrolyl, thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl, benzothienyl, oxazolyl, indazolyl, or a substituted, particularly mono- or disubstituted, radical.

[0086] Similarly, substituents for aryl and heteroaryl groups are diverse, ranging from zero to the total number of open valences in 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(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2)=NR', -S(O)R', -S(O) The formulas are selected from 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-C5)alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4)alkyl, and (unsubstituted aryl)oxy-(C1-C4)alkyl.

[0087] Two of the substituents on the atoms adjacent to the aryl or heteroaryl ring are of the formula -TC(O)-(CH2) q -U- substituents may be substituted as needed, where T and U are independently -NH-, -O-, -CH2-, or single bonds, and q is an integer from 0 to 2. Alternatively, two of the substituents of atoms adjacent to the aryl or heteroaryl ring may be -A-(CH2) r -B- may be replaced as needed, 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 of the novel ring thus formed may be replaced as needed with a double bond. Alternatively, two of the substituents of atoms adjacent to 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.

[0088] 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.

[0089] The term "amine," as used herein, refers to an alkyl group having one or more amino groups, as defined herein. The amino groups may be primary, secondary, or tertiary. The alkylamine may be further substituted with a hydroxyl group. Amines useful in the present invention include, but are not limited to, ethylamine, propylamine, isopropylamine, ethylenediamine, and ethanolamine. The amino groups may be bonded to the alkylamine at a bonding site with the rest of the compound, at the omega position of the alkyl group, or bonded together with at least two carbon atoms of the alkyl group. Those skilled in the art will understand that other alkylamines may be useful in the present invention.

[0090] As used herein, the term "carbamate" refers to a functional group having the structure -NR"CO2R', where R' and R'' are independently selected from hydrogen, (C1-C8) alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4) alkyl, and (unsubstituted aryl)oxy-(C1-C4) alkyl. Examples of carbamates include t-Boc, Fmoc, benzyloxycarbonyl, 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, V-(2-pivaloylamino)-1,1-dimethylethyl carbamate, and NpSSPeoc.

[0091] 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.

[0092] As used herein, the term “activated ester” refers to a carboxyl activating group used in peptide chemistry to facilitate the easy 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.

[0093] The terms "hydrazine" and "hydrazide" refer to compounds containing a single-bonded nitrogen atom, one of which is a primary amine functional group.

[0094] As used herein, the term "aldehyde" refers to a chemical compound having a -CHO group.

[0095] As used herein, the term "thiol" 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 carbon-containing atomic group.

[0096] The term "silyl" as used herein refers to Si(R z ) refers to 3, and in the formula, each R z These are, independently, alkylaryl atoms or other carbon-containing groups.

[0097] When used herein, the term "diazonium salt" refers to R-N2 + X - This refers to a group of an organic compound having the structure shown in the formula, where R may be any organic residue (e.g., alkyl or aryl), and X may be an inorganic or organic anion (e.g., halogen).

[0098] The term "triflate" refers to a group with the formula CF3SO3, also known as trifluoromethanesulfonate.

[0099] As used herein, the term "boronic acid" refers to the structure -B(OH)2. Those skilled in the art will recognize that boronic acids may exist as boronate esters at various stages in the synthesis of quenchers. Boronic acid means including such esters. The terms "boronic acid ester" or "boronate ester" as used herein refer to -B(Z 1 )(Z 2 This refers to a chemical compound containing the ) part, where Z1 and Z 2 In all cases, the atom that forms the part together and is bonded to boron is an oxygen atom. The boronic acid ester portion may be a 5-membered ring. The boronic acid ester portion may be a 6-membered ring. The boronic acid ester portion may be a mixture of a 5-membered ring and a 6-membered ring.

[0100] Values ​​expressed in range format should be interpreted in a flexible manner to include not only the numerical values ​​explicitly listed as limits to the range, but also all 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 to include not only approximately 0.1% to approximately 5%, but also the individual values ​​within the indicated range (e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%). The phrase "approximately X to Y" has the same meaning as "approximately X to approximately Y" unless otherwise specified. Similarly, the phrase "approximately X, Y, or approximately Z" has the same meaning as "approximately X, approximately Y, or approximately Z" unless otherwise specified.

[0101] As used herein, “dye conjugate” refers to a binding partner that is conjugated to a nonpolymer or polymer dye.

[0102] As used herein, "SN" refers to the "Super Nova" dye commercially available from Beckman Coulter, Inc.

[0103] As used herein, "EMPIGEN BB®" refers to a zwitterionic surfactant (CAS number 66455-29-6) containing N,N-dimethyl-N-dodecylglycine betaine at a betaine concentration of approximately 30% in aqueous solution.

[0104] When used herein, the term "approximately" may allow a degree of variation in a value or range, for example, within 10%, 5%, or 1% of the limit of a given value or range.

[0105] As used herein, “specific binding” refers to the binding of an antibody or other binding partner (e.g., in polymer conjugate dyes) to an epitope of the cell or target analyte targeted by the antibody or binding partner.

[0106] As used herein, “non-specific binding” refers to the binding of an antibody or other binding partner (e.g., in polymer conjugate dyes) to a cell or sample component that does not contain the epitope targeted by the antibody or other binding partner. For example, non-specific binding occurs when an antibody binds to a cell that does not have an epitope specific to that antibody.

[0107] As used herein, “reducing” or “removing” nonspecific binding of polymer dye conjugates may mean that the mean fluorescence intensity (MFI) of “negative” cells (e.g., negative granulocytes, monocytes, and lymphocyte populations) is reduced by at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least 99%, or more; about 50% to about 95%, about 50% to about 75%, about 60% to about 80%, or about 65% to about 90%) compared to when no surfactant is used. In other words, the percentage reduction in background staining of at least one of the monocytes, granulocytes, and lymphocytes is at least about 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least 99%, or more; about 50% to about 95%, about 50% to about 75%, about 60% to about 80%, or about 65% to about 90%) compared to when no surfactant is used.

[0108] As used herein, the terms "substantially" or "substantially consisting of" refer to a majority or a large portion, 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 more.

[0109] As used herein, the terms "substantially free of" or "substantially not containing" refer to less than about 1%, less than 0.5%, less than 0.1%, less than 0.05%, less than 0.001%, or less than about 0.0005% or less, about 0%, below the limit of quantification, below the limit of detection, or 0%.

[0110] In this document, the terms "a", "an", or "the" are used to include one or more than one, unless the context clearly indicates otherwise. The term "or" is used to mean a non-exclusive "or" unless otherwise indicated. Further, it should be understood that the grammar or terminology used herein, and in particular those not specifically defined, are for illustrative purposes only and are not limiting. Any use of section headings is intended to facilitate reading of this document and should not be construed as limiting. Further, information related to a section heading may be present within or outside that particular section. Further, all publications, patents, and patent documents referred to in this document are hereby incorporated by reference in their entirety as if individually incorporated by reference. In case of any conflict in the use between this document and the documents incorporated by reference in this way, the use in the incorporated reference should be considered as supplementing that of this document; for irresolvable conflicts, the use in this document prevails.

[0111] In certain embodiments, a dye composition is provided that includes at least one polymeric dye conjugate and at least one suitable zwitterionic surfactant.

[0112] In certain embodiments, a dye composition is provided that includes at least one polymeric dye conjugate and at least one suitable anionic surfactant.

[0113] In some embodiments, a method for reducing or eliminating non-specific binding of at least one polymeric dye conjugate to cells in a biological sample, such as a blood sample, comprising contacting the at least one polymeric dye conjugate with at least one zwitterionic and / or anionic surfactant before, during, and / or after contacting the at least one polymeric dye conjugate with the biological sample is provided. The steps may be performed in any order without departing from the principles of the invention, except where the temporal or operational order is explicitly recited. Further, certain steps may be performed simultaneously, unless explicitly recited in the language of the claims to be performed separately. For example, the step of performing X recited in the claims and the step of performing Y recited in the claims may be performed simultaneously in one operation, and the process thus obtained will fall within the literal scope of the process recited in the claims. Thus, in some cases, the at least one polymeric dye conjugate may be contacted with the at least one zwitterionic or anionic surfactant before contacting the at least one polymeric dye conjugate with the blood sample. In some cases, the at least one polymeric dye conjugate may be contacted with the at least one zwitterionic or anionic surfactant simultaneously with contacting the at least one polymeric dye conjugate with the blood sample. Surfactant

[0114] Various types of surfactants were investigated to reduce or inhibit nonspecific interactions between polymer dye conjugates and biological samples.

[0115] Suitable surfactants may be zwitterionic surfactants or certain anionic surfactants. Examples of suitable surfactants include general formulas.

[0116] R 1’ [CO-X(CH2)] j ] g -[N + (R 2’ )(R 3’ )] k -(CH2) f -[CH(OH)CH2] h -Y - (In the formula, R 1’ C is either saturated or unsaturated. 5~24 Alkyl, for example, C 6~22 , C 5~21 , C 7~19 , C 11~17 , or C 8~18 Alkyl, saturated C 10~16 Alkyl or saturated C 12~14 It is alkyl; X is NH, NR 4 ' and here, R 4’ C 1~4 It is alkyl, O, or S; j is an integer from 1 to 10, e.g., 2 to 5 and 3; g is 0 or 1, R 2’ and R 3’ Each of them is independent of C 1~4 Alkyl, e.g., ethyl or methyl; hydroxy, optionally substituted with a hydroxyethyl group or methyl; k is 0 or 1; f is an integer from 0 to 4, e.g., 0, 1, 2, 3, or 4; h is 0 or 1; Y is COO, SO3, OPO(OR 5’ )O, or P(O)(OR 5’ )O, and here, R 5’ is H or C 1~4There is a surfactant (which is alkyl), and when k=0, the surfactant may be in an acidic form or its sodium or potassium salt.

[0117] The surfactant may be present in the buffering agent or other suitable aqueous composition according to this disclosure at concentrations ranging from about 0.05% to about 0.25%, about 0.06% to about 0.2%, or about 0.08% to about 0.16% (w / v).

[0118] Suitable zwitterionic surfactants that may be used according to the methods described herein include betaine zwitterionic surfactants, such as alkylbetaine, alkylamidebetaine, amidazolinium betaine, sulfobetaine (INCI sultaine), and phosphobetaine.

[0119] Suitable zwitterionic surfactants include alkyl betaines, for example, 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 - There are some of these.

[0120] Examples of suitable betaines and sulfobetaines are as follows (as indicated by 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 soybean glycinate, dihydroxyethyl stearyl glycinate, dihydroxyethyl tallow glycinate, dimethicone propyl PG-betaine, dourcamidopropyl hydroxysultaine, hydrogenated tallow betaine, isostearamidopropyl betaine These include betaine, lauramidopropyl betaine, lauryl betaine, lauryl hydroxysultaine, lauryl sultaine, milk amidopropyl betaine, milk amidopropyl betaine, myristamidopropyl betaine, myristyl betaine, oleamidopropyl betaine, oleamidopropyl hydroxysultaine, oleyl betaine, olive amidopropyl betaine, palm amidopropyl betaine, palmitamidopropyl betaine, palmitoyl carnitine, palm kernel amidopropyl betaine, polytetrafluoroethylene acetoxypropyl betaine, ricinol amidopropyl betaine, sesamidopropyl betaine, soy amidopropyl betaine, stearamidopropyl betaine, stearyl betaine, tallow amidopropyl betaine, tallow amidopropyl hydroxysultaine, tallow betaine, tallow dihydroxyethyl betaine, undecylenamidopropyl betaine, and wheat germ amidopropyl betaine.

[0121] A suitable betaine zwitterionic surfactant is N-(alkylC 10~16 )-N,N-dimethylglycine betaine, N-(alkyl C 12~14The compounds may be n-tetradecyl-N,N-dimethylglycine betaine, N,N-dimethyl-N-dodecylglycine betaine, lauryldimethylbetaine (also known as laurylbetaine), myristylsulfobetaine, or n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate. Laurylbetaine is commercially available as EMPIGEN BB® (Huntsman Corporation) and has a CMC of 1.6-2.1 mM (20-25°C). Myristylsulfobetaine (n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, also known as DMMA) is available under the trademark name ZWITTERGENT® 3-14 (Merck KGaA, Darmstadt, Germany) and has a CMC of 100-400 μM. n-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (also known as 3-N,N-dimethylpalmitylammonio)propanesulfonate, DMPA) is available under the trademark name ZWITTERGENT® 3-16 and has a CMC of 10-60 μM. For example, coconut dimethylbetaine is marketed by Seppic under the trademark name AMONYL265®; lauryl betaine is marketed by Sigma-Aldrich under the trademark name EMPIGEN BB®. A further example of betaine is lauryl-imino-dipropionate, marketed by Rhodia under the trademark name MIRATAINE H2C-HA®.

[0122] The zwitterionic surfactant may be present in the buffer or other suitable aqueous composition according to this disclosure in an amount ranging from about 0.06% to about 0.2% or from about 0.08% to about 0.16%.

[0123] Examples of suitable anionic surfactants include sarcosinate surfactants in acidic or neutral forms. For example, a suitable anionic surfactant may be a sarcosinate surfactant in neutral form. The sarcosinate surfactant may be an alkanoyl sarcosinate surfactant.

[0124] Examples of suitable anionic surfactants include those with the general formula R 1’ [CO-X(CH2)] j ] g -(CH2) f -[CH(OH)CH2] h -Y - (In the formula, R 1’ C is either saturated or unsaturated. 5~24 Alkyl, for example, C 8~18 Alkyl, saturated C 10~16 Alkyl or saturated C 12~14 It is alkyl; X is NH, NR 4 ' and here, R 4’ C 1~4 It is alkyl, O, or S; j is an integer from 1 to 10, e.g., 2 to 5 and 3; g is 0 or 1; f is an integer from 0 to 4, e.g., 0, 1, 2, 3, or 4; h is 0 or 1; Y is COO, SO3, OPO(OR 5’ )O, or P(O)(OR 5’ )O, and here, R 5’ is H or C 1~4 There are surfactants (which are alkyl), and anionic surfactants may be in acidic form or in the form of their sodium or potassium salts.

[0125] Suitable anionic surfactants have a structure CH3(CH2) a CH2 (CH2CH = CH) b CH2(CH2) c The formula may include CH2(C=O)N(CH3)CH2CO2X (where a=1 to 8; b=0 to 2; c=0 to 6; and X=H, Na, or K).

[0126] An example of an alkanoyl sarcosinate is formula: R 1’ -CO-N(CH3)-CH2-COO - and R 1’ -CO-N(CH3)-CH2-SO3 - (Here, R1’ is saturated or unsaturated C 5~24 Alkyl, C 7~19 Alkyl, or C 11~17 (They may be alkyl groups), and for example, those of the sodium or potassium salts.

[0127] Examples of suitable alkanoyl sarcosinates and their acidic or salt forms include N-lauroyl sarcosine, sodium lauroyl sarcosinate, sodium palmitoyl sarcosinate, sodium stearoyl sarcosinate, sodium N-methyl-N-(1-oxotetradecyl)-glycine sodium salt, sodium caproyl sarcosinate, sodium capryloyl sarcosinate, N-methyl-N-(1-oxo-9-octadecen-1-yl)-glycine, sodium salt, sodium oleoyl sarcosinate, and sodium linoleoyl sarcosinate.

[0128] Anionic surfactants may be present in buffers or other suitable aqueous compositions according to this disclosure in amounts ranging from about 0.06% to about 0.2% or from about 0.08% to about 0.16%.

[0129] The composition can be used in flow cytometry and thus may contain one or more additional components including, but not limited to, any suitable carrier, stabilizer, buffer, salt, chelating agent (e.g., EDTA), or preservative. The composition may also contain one or more additional surfactants in addition to the zwitterionic surfactant and / or anionic surfactant described herein. Non-limiting examples of one or more additional surfactants include polysorbates, such as TWEEN® 20 (polyoxyethylene sorbitan monolaurate) and TWEEN® 80 (polyoxyethylene sorbitan monooleate). The carrier may be an aqueous solution, such as water, saline, alcohol, or a physiologically compatible buffer, such as Hank's solution, Ringer's solution, or physiological saline buffer. The carrier may contain formulation agents, such as suspending agents, stabilizers, and / or dispersing agents. The composition may also contain a buffer or pH adjuster, typically the buffer is a salt prepared from an organic acid or base. Representative buffers include salts of organic acids, such as citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; tris tromethamine hydrochloride, or phosphate buffers.

[0130] The composition may contain a protein stabilizer selected from the group consisting of bovine serum albumin (BSA or "fraction V"), casein, and gelatin. The protein stabilizer may be BSA, a commercially available bovine serum albumin protein derived from cows. The protein stabilizer may be present in a buffer or other suitable aqueous composition according to the disclosure at about 0.1 - 5 mg / mL, about 0.5 - 3 mg / mL, or about 2 mg / mL.

[0131] The stabilizer may be gelatin or protein, usually derived from collagen obtained from parts of animal bodies. It is brittle when dry and rubbery when wet. After hydrolysis, it may also be called hydrolyzed collagen, collagen hydrolysate, gelatin hydrolysate, hydrolyzed gelatin, and collagen peptide. Several types of gelatin are commercially available, including gelatin type A, gelatin type B, Prionex® highly purified gelatin type A, and cold-water fish gelatin.

[0132] The composition may also contain any suitable preservative. The preservative may be an antioxidant, biocide, or antibacterial agent. The preservative may be an inorganic salt. The preservative may be sodium azide. The preservative may be present in a concentration range of about 0.01 to about 1%, about 0.05 to about 0.5%, or about 0.1%. Polymer dyes

[0133] In another embodiment, the composition may be used in conjunction with a polymer dye. The polymer dye may be a fluorescent polymer dye or a fluorescent polymer tandem dye. Polymer dyes are particularly useful for the analysis of chemical and biological target analytes. They are highly responsive optical reporters and efficient light absorbers because they contain multiple chromophores. The polymer dye conjugate may contain any previously disclosed fluorescent polymer dye or fluorescent polymer tandem dye.

[0134] For example, polymer dyes or tandem polymer dyes may be any dyes disclosed in published PCT applications WO2017 / 180998; U.S. applications 2021 / 0047476; U.S. applications 2020 / 0190253; U.S. applications 2020 / 0147615; U.S. applications 2021 / 0108083; U.S. applications 2018 / 0224460; U.S. patents 11,034,840; 10,228,375; 10,545,137B2; 10,533,092; U.S. patents 7,214,489; 8,354,239; and 8,575,303, each of which is incorporated by reference as if it were entirely described herein. A polymer dye conjugate may have the structure of any water-soluble fluorescent polymer dye disclosed in U.S. Application No. 2020 / 0190253A1, which is incorporated by reference as if it were described in its entirety herein. A polymer dye conjugate may have the structure of any water-soluble fluorescent polymer dye disclosed in U.S. Application No. 2019 / 0144601, which is incorporated by reference as if it were described in its entirety herein.

[0135] The polymer dye or polymer dye conjugate may be any commercially available polymer dye, or a polymer dye conjugated to a binding partner. The polymer dye or polymer dye conjugate may contain a polymer dye that is excitable by a violet laser. The polymer dye or polymer dye conjugate may contain a polymer dye that is excitable by a violet laser, for example at 405 nm. The polymer dye or polymer dye conjugate may contain a violet laser (405 nm) excitable polymer dye.

[0136] In some embodiments, the polymer dye or polymer dye conjugate may contain SuperNova® dyes (Beckman Coulter, Inc.). SuperNova® polymers are a new generation of polymer dyes useful for flow cytometry applications. The polymer dye or polymer dye conjugate may contain SuperNova® v428, SuperNova® v605, or SuperNova® v786 (Beckman Coulter, Inc.). SuperNova® v428 has unique photophysical properties that result in extremely bright conjugates when conjugated to antibodies or other binding partners. For example, SuperNova® v428 (SN v428) (Beckman Coulter, Inc.) is a polymer dye that is optimally excited by a violet laser (e.g., 405 nm), has a maximum excitation wavelength of 414 nm, an emission peak of 428 nm, and can be detected using a 450 / 50 bandpass filter or equivalent.

[0137] SuperNova® v428 is one of the brightest dyes that can be excited by a violet laser, making it particularly suitable for evaluating dimly expressed markers. Possible SuperNova® conjugate antibodies include anti-CD19 antibody-SuperNova® v428, anti-CD22 antibody-SuperNova® v428, anti-CD25 antibody-SuperNova® v428, and anti-CD38 antibody-SuperNova® v428 antibody-polymer dye conjugates.

[0138] SuperNova® v605 and SuperNova® v786 (Beckman Coulter, Inc.) are tandem polymer dyes derived from the core of the SuperNova® v428 polymer dye. Both share the same absorbance characteristics and a maximum excitation wavelength of 414 nm. Since the emission peaks of SuperNova® v605 and SuperNova® v786 are 605 nm and 786 nm, respectively, they are best detected using 610 / 20 and 780 / 60 nm bandpass filters on a flow cytometer. SuperNova® v605 and SuperNova® v786 may be conjugated with, for example, anti-CD19 antibodies, anti-CD22 antibodies, anti-CD25 antibodies, and anti-CD38 antibodies.

[0139] Polymer dyes or 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 less than or equal to 380 nm but greater 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 may typically emit light at wavelengths of 380 nm to 430 nm, 406 nm to 415 nm, or less than or equal to 430 nm but greater than or equal to 380 nm.

[0140] Polymer dyes or polymer dye conjugates include Brilliant Violet® dyes (BioLegend® / Sirigen Group Ltd.), such as Brilliant Violet 421® (maximum excitation wavelength 405 nm, maximum emission wavelength 421 nm, 450 / 50 filter), Brilliant Violet 510® (maximum excitation wavelength 405 nm, maximum emission wavelength 510 nm, 510 / 50 filter), Brilliant Violet 570® (maximum excitation wavelength 405 nm, maximum emission wavelength 570 nm, 585 / 42 filter), Brilliant Violet 605® (maximum excitation wavelength 405 nm, maximum emission wavelength 603 nm, 610 / 20 filter), Brilliant Violet 650® (maximum excitation wavelength 405 nm, maximum emission wavelength 645 nm, 660 / 20 filter), and Brilliant Violet The polymer dye or polymer dye conjugate may contain Spark Violet 538 (BioLegend, Inc.) (maximum excitation wavelength 405 nm, maximum emission wavelength 711 nm, 710 / 50 filter), Brilliant Violet 750 (trademark) (maximum excitation wavelength 405 nm, maximum emission wavelength 750 nm, 780 / 60 filter), or Brilliant Violet 785 (trademark) (maximum excitation wavelength 405 nm, maximum emission wavelength 785 nm, 780 / 60 filter). The polymer dye or polymer dye conjugate may contain Spark Violet 538 (BioLegend, Inc.) (maximum excitation wavelength 405 nm, maximum emission wavelength 538 nm).

[0141] The polymer dye or polymer dye conjugate may contain a Super Bright dye (Invitrogen, ThermoFisher Scientific). The Super Bright dye can be excited by a violet laser (405 nm). The Super Bright dye may be Super Bright 436 (maximum excitation wavelength 414 nm, maximum emission wavelength 436 nm, 450 / 50 bandpass filter), Super Bright 600 (maximum emission wavelength 600 nm, 610 / 20 bandpass filter), Super Bright 645 (maximum emission wavelength 645 nm, 660 / 20 bandpass filter), or Super Bright 702 (maximum emission wavelength 702 nm, 710 / 50 bandpass filter).

[0142] The polymer dye or polymer dye conjugate may contain BD Horizon Brilliant® Violet polymer dye (Becton, Dickinson and Co., BD Life Sciences). The polymer dye may also be 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), or BV786 (786 / 60 filter).

[0143] Polymer dyes may be synthetically prepared by polymerization of monomers, thereby forming a highly conjugate fluorescent skeleton. Capping may be performed on the polymer by activation using appropriate functional groups, thereby obtaining a polymer that can be conjugated to a binding partner. Alternatively, the polymer may be activated and conjugated by attaching appropriate functional groups to the polymer skeleton. The activated polymer can be conjugated to a binding partner. Any suitable binding partner may be purified, for example, using an antibody, followed by purification using, for example, a standard procedure. The functional groups may be selected from the group consisting of amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and their protecting groups for conjugation to substrates or binding partners.

[0144] Polymer dye conjugates may include, but are not limited to, fluorescent polymers having monomer subunits including dihydrophenanthrene (DHP), fluorene, and combinations thereof. In some embodiments, the polymer dye conjugate is formula III: [ka] It may also contain polymer dyes having the structure shown.

[0145] Each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers. Each A may be substituted with a functional group that will be conjugated with a bonding partner.

[0146] Each required M is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, bandgap-modified monomers, optionally substituted ethylenes, and ethynylenes, and is distributed uniformly or randomly along the polymer backbone. Each M is, [ka] [ka] [ka] (wherein each M may be substituted and terminated with a functional group selected from amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, amides, sulfonamides, ethers, thioethers, thiocarbamates, hydroxyls, iodoacetyls, hydrazides, hydrazinos, ketones, phosphines, epoxides, ureas, thioureas, thioesters, imines, disulfides, and their protecting groups for conjugation to another substrate, acceptor dye, molecule, or binder.) Each R 5 These are 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)aryl group, C2~C 18 (hetero)aryloxy, C2~C 18 (Hetero)arylamino, carboxylic acid, carboxylate ester, (CH2) x’ (OCH2-CH2) y’ OCH3 and (CH2) x’ (OCH2-CH2) y’ Independently selected from the group consisting of OCF3, each x' is an integer between 0 and 20, and each y' is an integer between 0 and 50; Each R 1 These are independently ammonium alkyl salts, ammonium alkyl oxy salts, ammonium oligo ether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligo ether salts, sulfonamide oligo ethers, or partly: [ka] and; Each R 2 These are independently H, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG group, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, or partial [ka] and; Each R 3 The group is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and PEG groups; Each Z is independently selected from the group consisting of C, O, and N; Each Q is a combination of NH and NR. 4 Independently selected from the group consisting of , and CH2; (Each subscript n is an integer between 0 and 20, independently.) You may also choose independently from the group consisting of these elements.

[0147] The linker is represented by formula III as L. Each required linker L may be an aryl or heteroaryl group uniformly or randomly distributed along the polymer backbone, and may be substituted with one or more pendant chains ending with a functional group selected from the group consisting of amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and their protecting groups for conjugation to a substrate or binding partner.

[0148] The polymer composites disclosed herein are each G1 and G 2 This also includes the terminus represented by formula III. The terminus may be modified or not. The terminus may be independently selected from the group consisting of aryl or heteroaryl substituted with one or more pendant chains ending with a functional group selected from hydrogen, halogens, alkynes, optionally substituted aryls, optionally substituted heteroaryls, 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 dihydrophenanthrene (DHP), optionally substituted fluorene;amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and protecting groups which may be conjugated to a substrate or binding partner.

[0149] In the structure of Equation III, a, c, and d each define the mol% of each unit, which may be repeated uniformly or randomly, with each a being from 10 to 100% mol%, each c being from 0 to 90% mol%, and each d being from 0 to 25% mol%; each b being independently 0 or 1; and each m being an integer from 1 to approximately 10,000.

[0150] In some embodiments, the polymer dye conjugate is of formula I: [ka] (In the formula: Each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers; L 1 , L 2 , and L 3 This is the linker part; W is the water-soluble part; Each E is an independently selected chromophore, sensual part, or bonding partner; Each B is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, bandgap-modified monomers, optionally substituted ethylenes, and ethynylenes; G 1 and G 2 These are selected independently from unmodified and modified polymer ends; The subscripts n and m are independently integers in the range of 1 to 10,000. The subscript p is an integer in the range of 0 to 10,000. The sum of the subscripts n, m, and p is in the range of 2 to 10,000; The subscript q is 1, 2, 3, or 4; The subscript r is 1, 2, 3, or 4; The subscript s is 0, 1, 2, or 3; The subscript t is either 1 or 2. The sum of the subscripts r and s is in the range of 1 to 4; A and B are distributed randomly or non-randomly in the conjugate polymer. It may have a structure. L 1 This may be a sulfonamide, sulfonamide, sultam, disulfinamide, amide, phosphonamide, phosphoamidate, phosphineamide, or secondary amine. Or, L 1 This may be a sulfonamide, amide, phosphonamide, or secondary amine. The subscript q can be equal to the sum of the subscripts r and s, where the subscript r may be 1 or 2, and if the subscript r is 1, the subscript s is 0 or 1, and if the subscript r is 2, the subscript s is 0. Each L 3 This could be a covalent bond.

[0151] The conjugate polymer is given by formula II: [ka] (In the formula: L 1a This is the linker part; R 1 (Selected from the group consisting of H and amine protecting groups) It may have a structure based on the following. Various linkers L as described herein 1a and L 2 This can be used for the synthesis of polymers according to formulas I and II. For example: L 1a Covalent bond, C 1~8 Alkylenes, 2- to 8-membered heteroalkylenes (e.g., divalent alkoxylinkers), C 3~8 Cycloalkylene, C 6~10 Arirene, 5- to 12-membered heteroarirene, 5- to 12-membered heterocycline, -NHC(O)L a -, -C(O)NHL a -, -C(O)L a - and combinations thereof may be selected from the group; L 2 Covalent bond, C 1~8 Alkylenes, 2- to 8-membered heteroalkylenes (e.g., divalent alkoxylinkers), C 3~8 Cycloalkylene, C 6~10 Arylene, 5- to 12-membered heteroarylene, 5- to 12-membered heterocyclylene, -L b NHC(O)-, -L b C(O)NH-, -L b C(O)-, -C(O)NHL b -, -C(O)L b - and combinations thereof may be selected from the group; L a and L b C 1~8 Alkylenes and 2- to 8-membered heteroalkylenes may be independently selected; R 1 This may be selected from the group consisting of H and amine protecting groups. A polymer according to formula II is provided (wherein: L 1a Covalent bond, C 1~8 Alkylene, 2- to 8-membered heteroalkylene, -NHC(O)L a -, -C(O)NHL a -, and -C(O)L a - Selected from the group consisting of, L 2 Covalent bond, C 1~8 Alkylenes; 2 to 8-membered heteroalkylenes, -L b NHC(O)-, -L b C(O)NH-, -L b C(O)-, -C(O)NHL b -, and -C(O)L b -Selected from the group consisting of; L a and L b C 1~8 Independently selected from the group consisting of alkylenes and 2- to 8-membered heteroalkylenes; R 1 (Selected from the group consisting of H and amine protecting groups). W may contain one or more ethylene glycol monomers. Alternatively, W may contain poly(ethylene glycol). L 3 is the first L 1 Part (or the first L) 1a The first joining point to the part; the second L 1 Part (or second L) 1a The trivalent arylalkyl moiety may have a second bonding site to the (part) and a third bonding site to the A monomer.

[0152] For example, this disclosure is based on formula VI: [ka] (In the formula, L 1a This is defined as above; L 2 This is defined as above; W is as defined above; L 3a Covalent bond, C 1~8 Alkylene, 2- to 8-membered heteroalkylene, -NHC(O)L a -, -C(O)NHL a -, and -C(O)L a -Selected from the group consisting of; L a C 1~8 Selected from the group consisting of alkylenes and 2- to 8-membered heteroalkylenes; the wavy line indicates the bond point to the monomer.) The present invention provides a conjugate polymer in which two or more chromophores are bonded together, as shown. Each A monomer in a polymer having the structure of formula I, II, or III may be the same monomer. Each A monomer in a polymer having the structure of formula I, II, or III may be a different monomer. A may be a fluorescent monomer. A may be a 9,10-phenanthrenedion monomer (e.g., a dihydrophenanthrene (DHP) monomer), a fluorene monomer, or a fluoreneoxepin monomer.

[0153] In polymers having the structure of formula I, II, or III, monomer A is a DHP-based monomer, for example: [ka] (In the formula: Each X is independently either C or Si; Each Y is independent of CR 1 R 2 or SiR 1 R 2 and; Each R 1 These are independently ammonium alkyl salts, ammonium alkyl oxy salts, ammonium oligo ether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligo ether salts, sulfonamide oligo ethers, or partial [ka] and; Each R 2 These are independently H, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG group, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, or partial [ka] and; Each R 3 The group is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and PEG groups; Each Z is independently selected from the group consisting of C, O, and N; Each Q is a combination of NH and NR. 4 Independently selected from the group consisting of , and CH2; (Each subscript n is an integer between 0 and 20, independently.) That's fine.

[0154] R 1 The structure is as follows: [ka] (In the formula, Q is NH, and each R 3 (Z is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and PEG groups; each Z is independently selected from the group consisting of C, O, and N.) It may have.

[0155] DHP monomers have the following structure: [ka] (In the formula: Each subscript character f is an integer between 0 and 50, independently of the others; Each subscript n is an integer between 0 and 20, independently of the others; Each R 2 These are independently H, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG group, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, or partial [ka] and; Each R 5 These are H, C1~C independently. 20 Alkyl, C2~C 20 Alkenyl, C2~C 20 Alkinyl, C3~C 20 Cycloalkyl, C1-C 20 Haloalkyl, C1~C 20 Alkoxy, C2~C 26 Aryloxy, C2~C 26 Heteroaryloxy, C2~C 26 Arylamino, or C2-C 26 It is a heteroarylamino; Each Z is independently selected from the group consisting of C, O, and N. It may have.

[0156] DHP monomer has the following structure: [ka] (In the formula: Each subscript character f is an integer between 0 and 50, independently of the others; Each subscript n is an integer between 0 and 20, independently of the others; Each R 2These are independently H, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG group, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, or partial [ka] and; Each R 5 These are H, C1~C independently. 20 Alkyl, C2~C 20 Alkenyl, C2~C 20 Alkinyl, C3~C 20 Cycloalkyl, C1-C 20 Haloalkyl, C1~C 20 Alkoxy, C2~C 26 Aryloxy, C2~C 26 Heteroaryloxy, C2~C 26 Arylamino, or C2-C 26 It is a heteroarylamino; Each Z is independently selected from the group consisting of C, O, and N. It may have.

[0157] In polymers having the structure of formula I, II, or III, monomer A is a fluorene monomer, for example: [ka] (In the formula, X, Z, R 1 , R 2 , R 5 (The subscripts n and f are as defined herein.) That's fine.

[0158] R 1 base and R 2Groups, for example, ammonium alkyl salts, ammonium alkyl oxy salts, ammonium oligo ether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligo ether salts, sulfonamide oligo ethers, or structures: [ka] The portion having can provide solubility in water / buffer. In some embodiments, for example, the polymer is soluble at levels above 10 mg / mL, above 15 mg / mL, above 20 mg / mL, above 25 mg / mL, above 30 mg / mL, above 35 mg / mL, above 40 mg / mL, above 45 mg / mL, above 50 mg / mL, above 60 mg / mL, above 70 mg / mL, above 80 mg / mL, above 90 mg / mL, or above 100 mg / mL.

[0159] Monomer A is also a crosslinked monomer. For example, the crosslinked monomer of the present invention is: [ka] (In the formula, X, Y, R 2 , and R 5 (As defined above) There is.

[0160] In polymers having the structure of formula I, II, or III, monomer A is an oxepin monomer (e.g., a fluoreneoxepin monomer), e.g.: [ka] (In the formula, X, R 1 , and R 2 (This is as defined herein.) That's fine. Tandem polymer dyes

[0161] The polymer may have acceptor dyes bonded to its backbone, thereby providing monitoring of the luminescence of the acceptor dyes bonded to the backbone via energy transfer. Useful acceptor dyes in tandem polymer dyes include, for example, FITC, CY3B, Cy55, Alexa 488, Texas red, Cy5, Cy7, Alexa 750, and 800CW. For example, the acceptor dye is linker L: [ka] They may be bonded to the polymer via a mediated linkage. As described in U.S. Publication No. 2020 / 0190253, which is incorporated herein by reference in its entirety, the acceptor dye can also be directly bonded to monomer A as group E in the structure shown in Figure I or II above. SuperNova tandem dyes SuperNova v605 and SuperNova v786 (Beckman Coulter, Inc.) are tandem polymer dyes derived from core SuperNova v428. Both SuperNova v605 and SuperNova v786 share the same absorbance characteristics, with a maximum excitation wavelength of 414 nm. Since their emission peaks are 605 nm and 786 nm, respectively, they are best detected using 610 / 20 and 780 / 60 nm bandpass filters on a flow cytometer. Conjugate dyes

[0162] Polymer dyes may be conjugated to binding partners of different specificities, such as target analyte-specific antibodies, to synthesize binding partner-dye conjugates, such as CD19-SN v428, CD20-SN v605, etc.

[0163] Polymer dyes and polymer dye conjugates may be formulated using an aqueous buffer. Any suitable aqueous buffer, such as an isotonic aqueous buffer, such as PBS buffer, may be used. Additives may be used as aqueous buffers. For example, aqueous buffers may include BSA, sodium azide, a nonionic surfactant, such as PF-68, and a zwitterionic surfactant, such as Empigen BB®, or an anionic surfactant, such as NLS, as described herein. BSA helps stabilize the conjugate, sodium azide prevents any microbial contamination, and surfactants, such as Empigen BB®, significantly reduce or eliminate nonspecific binding to monocytes and granulocytes. BSA may be present in the range of 0-3 mg / mL, 0.5-2.5 mg / mL, or about 2 mg / mL. Sodium azide may be present in the range of 0-0.05%, 0.05-0.03%, or about 0.01% (w / v). Integration Partners

[0164] As used herein, “binding partner” refers to any molecule or molecular complex that can specifically bind to a target analyte. A binding partner may be, for example, a protein (e.g., an antibody or antigen-binding antibody fragment), a small organic molecule, a carbohydrate (e.g., a polysaccharide), an oligonucleotide, a polynucleotide, a lipid, an affinity ligand, an aptamer, and the like. In some embodiments, the binding partner is an antibody or a fragment thereof. Specific binding in the context of the present invention refers to a binding reaction that determines the presence of a target analyte in the presence of a heterogeneous population. Therefore, under certain assay conditions, a designated 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.

[0165] In some cases, the antibodies are intravenous immunoglobulin (IVIG) and / or antibodies from IVIG (e.g., concentrated, purified, or affinity-purified). IVIG is a blood product containing IgG (immunoglobulin G) pooled from the plasma of many (e.g., sometimes more than 1,000 to 60,000) normal, healthy blood donors (e.g., in some cases, without any other proteins). IVIG is commercially available. Embodiments of IVIG are described, for example, in U.S. Patent Applications Publications 2010 / 0150942; 2004 / 0101909; 2013 / 0177574; 2013 / 0108619; and 2013 / 0011388, which are incorporated herein by reference.

[0166] When the binding partner is an antibody, they may be monoclonal or polyclonal antibodies. The term “antibody,” as used herein, refers to, for example, immunoglobulin molecules and the immunologically active portion of an immunoglobulin (Ig) molecule that specifically bind to an antigen in a target analyte. Such antibodies include, but are not limited to, polyclonal, monoclonal, single-specific polyclonal antibodies, antibody mimes, chimeric, single-stranded, Fab, Fab' and F(ab')2 fragments, Fv, and Fab expression libraries. In some cases, the antibody is a monoclonal antibody of a defined subclass (e.g., IgG1, IgG2, IgG3, or IgG4, IgA, IgD, IgE, IgG2a, IgG2b, IgG3, and IgM). When a combination of antibodies is used, the antibodies may be from the same subclass or from different subclasses. For example, the antibody may be an IgG1 antibody. In some embodiments, the monoclonal antibody is humanized. The antibody fragment may include molecules such as Fab, scFv, F(ab')2, and Fab' molecules. Antibody derivatives include antibodies or fragments thereof that have been added or substituted, such as chimeric antibodies. The antibody may be of human or animal origin, from hybridomas, via recombinant methods, or by any other method known in the art.

[0167] Other binding partners besides antibody fragments or derivatives specific to the antibody or target analyte may also be used in this system and method. For example, the binding partner may be a nucleic acid or nucleic acid analog, such as an oligonucleotide or PNA probe. In one embodiment, an aptamer can be used as a specific binding partner. An aptamer is a single-stranded DNA or RNA (ssDNA or ssRNA) molecule that can bind with high affinity and specificity to pre-selected targets, including proteins and peptides. Other binding partners that can bind to the target analyte to form receptor-ligand, enzyme-substrate, enzyme-inhibitor, and enzyme-cofactor pairs may also be used. Specific examples of such binding partner pairs include carbohydrates and lectins, biotin and avidin or streptavidin, folic acid and folate-binding proteins, vitamin B12 and intrinsic factor, protein A and immunoglobulins, and protein G and immunoglobulins. Binding partners that form covalent bonds with the target analyte are also included. Conjugate

[0168] The polymer dye conjugate may include any known polymer dye conjugated to a binding partner using techniques known to those skilled in the art. In some embodiments, the polymer dye may be conjugated to a binding partner to form the polymer dye conjugate using a method of directly modifying a core polymer described in U.S. Publication Application No. 2020 / 0190253, which is incorporated herein by reference in its entirety.

[0169] In some cases, the polymer dye may be conjugated to a binding partner using the method described in U.S. Publication Application No. 2019 / 0144601, which is incorporated herein by reference in whole, to form a polymer dye conjugate. The method can be illustrated as follows: [ka]

[0170] SuperNova v428 (SN v428 (Beckman Coulter)) is a bright polymer dye that can be activated by amine activation in the case of a tandem dye, followed by maleimide activation in the case of a tandem conjugate. The rigidity of the polymer dye structure helps to reduce rotational energy, resulting in brighter luminescence. SuperNova v428 is one of the brightest dyes that can be excited by a violet laser, and is therefore particularly suitable for evaluating dimly expressed markers. Possible SuperNova conjugate antibodies include anti-CD19 antibody-SuperNova v428, anti-CD22 antibody-SuperNova v428, anti-CD25 antibody-SuperNova v428, and anti-CD38 antibody-SuperNova v428 antibody-polymer dye conjugate. target analyte

[0171] This disclosure also relates to a method for detecting a target analyte in a sample, wherein the target analyte contains a target antigen and may be a substance, such as a molecule, whose abundance / concentration is determined by several analytical steps. The present invention is designed to detect the presence, and in some cases the amount thereof, of a specific target analyte. The term “target analyte” refers to a target molecule containing a target antigen to be detected in a biological sample, such as peptides, proteins, polynucleotides, organic molecules, sugars and other carbohydrates, lipids, and small molecules. A key aspect of this disclosure is that the target analyte is contained in a liquid sample and is accessible or becomes accessible at some point to bind to the target analyte-specific binding partner of the present invention. The target analyte may be found in biological samples, such as blood samples, cell line development samples, tissue culture samples, etc.

[0172] The target analyte may 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. The target analyte may also be a protein, such as structural microfilaments, microtubules, and intermediate filament proteins, organelle-specific markers, proteasomes, transmembrane proteins, surface receptors, pore proteins, protein / peptide translocases, protein folding chaperones, signaling scaffolds, ion channels, etc. The protein may be, but is not limited to, an activatable protein or a protein differentially expressed or activated in diseased or abnormal cells, and may include transcription factors, DNA and / or RNA binding and modification proteins, nuclear uptake and transport receptors, apoptosis or survival regulators, etc.

[0173] The target analyte may be present on the cell surface or accessible therefrom. Examples of useful analytes, but not limited to, include: 1) specific cell surface macromolecules and antigens (including hormones, protein complexes, and molecules recognized by cell receptors), and 2) cellular proteins, DNA, or RNA in permeable cells, including those containing abnormal DNA or RNA sequences or specific messenger RNA in abnormal amounts. Detection of these analytes may be particularly useful in situations where they are present in and / or identify rare cells, for example, in the early stages of various cancers.

[0174] In some cases, the target analyte may be CD2, CD3, CD4, CD8, CD10, CD11c, CD14, CD15, CD16, CD19, CD20, CD22, CD25, CD27, CD38, CD45, CD45RA, CD56, CD62L, CD64, CD95, CD103, HLA-DR, IFN-γ, TNF-α, or ZAP-70, or any other target analyte for the purpose. Biological samples

[0175] Non-limiting examples of biological samples include blood, serum, plasma, urine, semen, breast milk, sputum, mucus, buccal swab specimens, vaginal swab specimens, rectal swab specimens, aspirates, needle biopsies, for example, tissue sections obtained by surgical or dissection, plasma, serum, cerebrospinal fluid, lymph, exocrine secretions from the skin, respiratory, intestinal, and urogenital tract, tears, saliva, tumors, organs, and samples of in vitro cell culture components (but not limited to these, including conditioned media obtained from cell proliferation in cell culture media, putative virus-infected cells, recombinant cells, and cellular components).

[0176] The sample in the methods of this disclosure may be, for example, blood. The blood sample may be whole blood. Whole blood can be obtained from a subject using standard clinical procedures. The sample may be a subset of one or more cells from whole blood (e.g., erythrocytes, leukocytes, lymphocytes (e.g., T cells, B cells, or NK cells), phagocytes, monocytes, macrophages, granulocytes, basophils, neutrophils, eosinophils, platelets, or any cell having one or more detectable markers). The sample may be from a cell culture. The sample may contain the target analyte naturally or may be prepared entirely or partially by synthetic means. subject

[0177] The subjects may be humans (e.g., patients with or suspected of having a disease), commercially important food chain mammals, such as cattle, castrated cattle, pigs, goats, sheep, birds, fish, or horses. Samples may also be obtained from domestic pets or companion animals, such as dogs, cats, rabbits, birds, or ferrets. The subjects may be laboratory animals used as animal models of disease or for drug screening, such as monkeys, mice, rats, rabbits, or guinea pigs. The subjects may be exotic animals, such as zoo animals or wild animals, such as elephants, antelopes, zebras, bison, giraffes, lions, tigers, leopards, orangutans, gorillas, whales, dolphins, sharks, or reptiles. reaction vessel

[0178] The reaction vessels disclosed herein may be any vessel in which a reaction between the binding partner or its polymer dye conjugate and the target analyte can occur. For example, the reaction vessel may be a tube, a plate, a well of a microtiter plate, a chamber, and a slide. In preferred embodiments, the reaction vessel has a lid or cap so that the binding reaction can occur in a closed environment. substrate

[0179] The reaction vessel comprises one or more substrates. The substrate may be any suitable surface, including, but is not limited to, plastics, nitrocellulose, cellulose acetate, quartz, and glass. Non-limiting examples of plastics may include polystyrene, polypropylene, cycloolefins, and polycarbonates. In some embodiments, the substrate is a membrane. The substrate may be the inner surface of the reaction vessel body, e.g., a plastic tube or a well in a microtiter plate. The substrate may also be beads. In some embodiments, at least one of the substrates (e.g., membranes) that accepts a labeled binding partner is bound to the inner surface of the reaction vessel body. In some embodiments, the membrane substrate is a sheet or roll, which facilitates the deposition and drying of the solution. In some embodiments, the membrane may be cut to separate individual dried reaction spots. In some embodiments, the cut membrane is simply dropped into the reaction vessel. In some preferred embodiments, the cut membrane is bound to the surface of the reaction vessel so that the spots do not escape from the vessel when the liquid is pipetted in or out of the reaction vessel. liquid sample

[0180] The reaction vessel is configured to receive a liquid sample. The liquid sample used in this invention typically includes a target analyte obtained mostly as an aqueous medium or dispersed therein.

[0181] The sample may be, for example, a biological sample, such as blood, bone marrow, spleen cells, lymphocytes, bone marrow aspirate (or any cells obtained from bone marrow), urine (lavage fluid), serum, plasma, saliva, cerebrospinal fluid, lymph, urine, amniotic fluid, interstitial fluid, feces, mucus, breast milk, semen, buccal swab, nasopharyngeal swab, vaginal swab, rectal swab, aspirate, needle biopsy, for example, a section of tissue obtained by surgery or dissection, or a tissue sample (e.g., tumor sample, non-aggregated tissue, non-aggregated solid tumor). The sample may also be a blood sample. The blood sample may be a whole blood sample. Whole blood can be obtained from the subject using standard clinical procedures. The sample may be a subset of one or more cells from whole blood (e.g., red blood cells, white blood cells, lymphocytes (e.g., T cells, B cells, or NK cells), phagocytes, monocytes, macrophages, granulocytes, basophils, neutrophils, eosinophils, platelets, or any cells having one or more detectable markers). The sample may also be from cell cultures, in vitro cell culture components (including, but not limited to, conditioned media obtained from cell proliferation in cell culture media, putative virus-infected cells, recombinant cells, and cellular components).

[0182] The sample may be any raw material of the biological material and may contain proteins, carbohydrates, and / or polynucleotides that can be obtained directly or indirectly from living organisms. Examples of samples may include cells, tissues, or bodily fluids, as well as sediments left by organisms containing viruses, mycoplasmas, and fossils. The sample may contain a target analyte. The target analyte may be naturally occurring in the biological sample or may be prepared entirely or partially by synthetic means. Labeled binding partners

[0183] Dyes can be conjugated to their binding partners by various binding chemistry between the binding partner and the reactive pair present in the label. Reactive pairs include, but are not limited to, maleimide / thiol, succimidyl ester (NHS ester) / amine, azide chemistry, carboxy / EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) / amine, amine / sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate) / thiol, and amine / BMPH (N-[ ~ Possible conjugates include [maleimidepropionic acid]hydrazide (TFA) / thiol. Methods for conjugation are well known in the art. Commercial kits for conjugation are readily available, for example, from Innova Biosciences (Cambridge, UK), Novus Biologicals (Littleton, Colo.), and Thermo Fisher Scientific (Waltham, Mass.).

[0184] Either dry or liquid polymer dye conjugates can be used in this method and composition. Dry polymer dye conjugates may be prepared using any technique known in the art, such as those described in US2019 / 0242882, which is incorporated herein by reference.

[0185] The polymer dye conjugate may be used in the compositions of this disclosure, or it may be used directly to stain blood and analyze it with a flow cytometer. Assay system

[0186] Assay systems for quantifying binding molecules using binding partners and fluorescent labeling are well known. Examples of such systems include flow cytometers, scanning cytometers, imaging cytometers, fluorescence microscopes, and confocal fluorescence microscopes.

[0187] Flow cytometry is used to detect fluorescence. 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.

[0188] The assay may be an immunoassay. Examples of immunoassays useful in the present invention, but not limited to these, include fluorescence assays (FLA). The assay may also be performed using a protein array.

[0189] If the binding partner is an antibody, an antibody or multi-antibody sandwich assay may also be used. A sandwich assay is one in which a series of recognition events are used to construct layers of various binding partners and report the elements that indicate the presence of a particular analyte. Examples of sandwich assays are disclosed in U.S. Patent No. 4,486,530 and the references listed therein.

[0190] A light source is applied to a sample in which the polymer can be excited, and the light emitted from the conjugate polymer complex is detected. In a typical assay, the fluorescent polymer dye conjugate used in the present invention can be excited with light having a wavelength between approximately 395 nm and approximately 415 nm. The emitted light is typically between approximately 415 nm and approximately 475 nm. Alternatively, the excitation light may have a wavelength between approximately 340 nm and approximately 370 nm, and the emitted light is between approximately 390 nm and approximately 420 nm. Applicable

[0191] The compositions according to this disclosure may comprise monochromatic, i.e., single polymer dye conjugates, such as a single SN polymer dye conjugate. For example, a biological sample may be stained using an SN conjugate to monitor or identify a specific cell population depending on the antibody conjugated to the polymer dye.

[0192] In some embodiments, the compositions according to this disclosure may comprise monochromatic polymer dye conjugates and conventional nonpolymer dye conjugates. For example, SN conjugates may be used together with nonpolymer dye conjugates, such as CD4-FITC, CD7-PE, CD25-ECD, CD56-PC5.5, in a panel for identifying cell subpopulations in human whole blood samples by flow cytometry.

[0193] In some embodiments, one or more compositions according to the Disclosure may be brought into contact with a biological sample, such as a blood sample. For example, the biological sample may be stained with a composition comprising multiple SN conjugates to monitor or identify a specific cell population in response to antibodies conjugated with polymer dyes. In some embodiments, two or more, three or more, or four compositions according to the Disclosure may be brought into contact with a biological sample. In some embodiments, a composition comprising multiple polymer dye conjugate compositions may further comprise non-polymer dye conjugates, such as CD4-FITC, CD7-PE, CD25-ECD, CD56-PC5.5, etc., in a panel for identifying cell subpopulations in a human whole blood sample by flow cytometry. [Examples]

[0194] The present invention can be better understood by referring to the following examples provided for illustrative purposes. The present invention is not limited to the examples given herein. (Example 1) Preparation of DHP polymer composites [ka]

[0195] Method 1: In a round-bottom flask, dibromo DHP monomer and diboronic acid DHP monomer (1:1) as described in WO2017 / 180998 were placed 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 evaporated and passed through a gel filtration column to remove small organic molecules and low MW oligomers. The crude polymer was then passed through a tangential flow filtration system with a 100K MWCO membrane. The filtrate was washed with 20% ethanol until absorption decreased.

[0196] Method 2: Alternatively, polymerization may be carried out by the autopolymerization of the bromoboronic acid ester of the DHP molecule. In a round-bottom flask, the DHP bromoboronic acid ester was placed in a (DMF-water) mixture and purged with nitrogen for 10 minutes. Under nitrogen, approximately 10 equivalents of CsF and 5% Pd(OAc)2 were mixed and heated 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 evaporated and passed through a gel filtration column to remove small organic molecules and low MW oligomers. The crude polymer was then passed through a tangential flow filtration system equipped with a 100K MWCO membrane. The filtrate was washed with 20% ethanol until the absorption decreased.

[0197] Method 3: Both dibromodihydrophenanthrene and diboronic acid dihydrophenanthrene monomer (1:1) were placed in 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 placed in a Schlenkle line and degassed with three freeze-pump-thaw cycles, then heated to 80°C for 18 hours under nitrogen with vigorous stirring. Subsequently, a capping agent containing appropriate functional groups (selected from G1) was added to the reaction mixture via a cannula under excess nitrogen pressure, and after 3 hours, a second capping agent (selected from G2) was added. After the reaction, the crude reaction mixture was evaporated and passed through a gel filtration column to remove small organic molecules and low MW oligomers. The crude polymer was then passed through a tangential flow filtration system with a 100K MWCO membrane. The filtrate was washed with 20% ethanol until absorption decreased.

[0198] Method 4: Alternatively, polymerization may be carried out by the autopolymerization of the bromoboronic acid ester of the dihydrophenanthrene molecule. Dihydrophenanthrene bromoboronic acid ester was placed in 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 placed in a Schlenkle line and degassed by three freeze-pump-thaw cycles, and then heated to 80°C for 18 hours under nitrogen with vigorous stirring. Subsequently, a capping agent containing appropriate functional groups (selected from G1) was added to the reaction mixture via a cannula under excess nitrogen pressure, and after 3 hours, a second capping agent (selected from G2) was added. After the reaction, the crude reaction mixture was evaporated and passed through a gel filtration column to remove small organic molecules and low MW oligomers. The crude polymer was then passed through a tangential flow filtration system equipped with a 100K MWCO membrane. The filtrate was washed with 20% ethanol until absorption decreased. (Example 2) Preparation of fluorene-DHP copolymer complex [ka]

[0199] Method 1: In a round-bottom flask, both dibromo DHP and diboronic fluorene monomer (1:1) were placed 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 evaporated and passed through a gel filtration column to remove small organic molecules and low MW oligomers. The crude polymer was then passed through a tangential flow filtration system with a 100K MWCO membrane. The filtrate was washed with 20% ethanol until absorption decreased.

[0200] Method 2: In a round-bottom flask, both dibromofluorene and diboronic acid DHP monomer (1:1) were placed 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 evaporated and passed through a gel filtration column to remove small organic molecules and low MW oligomers. The crude polymer was then passed through a tangential flow filtration system with a 100K MWCO membrane. The filtrate was washed with 20% ethanol until absorption decreased.

[0201] Method 3: Both dibromodihydrophenanthrene and diboronic fluorene monomer (1:1) were placed in 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 placed in a Schlenkle line and degassed with three freeze-pump-thaw cycles, then heated to 80°C for 18 hours under nitrogen with vigorous stirring. Subsequently, a capping agent containing appropriate functional groups (selected from G1) was added to the reaction mixture via a cannula under excess nitrogen pressure, and after 3 hours, a second capping agent (selected from G2) was added. After the reaction, the crude reaction mixture was evaporated and passed through a gel filtration column to remove small organic molecules and low MW oligomers. The crude polymer was then passed through a tangential flow filtration system with a 100K MWCO membrane. The filtrate was washed with 20% ethanol until absorption decreased.

[0202] Method 4: Dibromofluorene and dihydrophenanthrene diboronic acid monomer (1:1) were placed in 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 placed in a Schlenkle line and degassed by three freeze-pump-thaw cycles, then heated to 80°C for 18 hours under nitrogen with vigorous stirring. Subsequently, a capping agent containing appropriate functional groups (selected from G1) was added to the reaction mixture via a cannula under excess nitrogen pressure, and after 3 hours, a second capping agent (selected from G2) was added. After the reaction, the crude reaction mixture was evaporated and passed through a gel filtration column to remove small organic molecules and low MW oligomers. The crude polymer was then passed through a tangential flow filtration system with a 100K MWCO membrane. The filtrate was washed with 20% ethanol until absorption decreased. (Example 3) Comparison of fluorescence emission spectra

[0203] The fluorescence emission spectra of fluorene (Fl-Fl), dihydrophenanthrene (DHP-DHP), and fluorene-DHP (DHP-Fl) polymers were compared. After excitation at 405 nm, the DHP-containing polymer showed a significant difference in its maximum fluorescence wavelength at 426–428 nm, while the fluorene-based polymer showed a maximum wavelength of 421 nm, as shown in Figure 1A. (Example 4) Comparison of absorption spectra

[0204] The absorption spectra of both fluorene (Fl-Fl) polymer and dihydrophenanthrene (DHP-DHP) polymer were measured. The DHP-DHP polymer (black curve) shows lambda max (λmax) at 390 and 410 nm, as shown in Figure 1B, while the Fl-Fl polymer (gray curve) shows lambda max (λmax) at approximately 400 nm. Samples were measured under different concentrations. (Example 5) Polymer dye properties

[0205] The polymer dyes of this disclosure have been found to possess certain physical and chemical properties, such as absorption, fluorescence, brightness, molecular weight, polydispersity, and the ratio of dye to protein when conjugated to an antibody. Preferred ranges for these parameters are shown in Table 1A.

[0206] [Table 1A]

[0207] The excitation and emission spectra of the tandem polymer were measured. Excitation was performed at the polymer's maximum wavelength (405 nm), and emission was observed from various acceptor dyes bound to the backbone. (Example 6) Experiments using non-conjugate dyes

[0208] Blood samples were stained with the non-conjugate polymer dye SN v605 containing Empigen BB® (antibody-free) and the non-conjugate polymer dye SN v605 without Empigen BB® (antibody-free), and analyzed by flow cytometry. As shown in the lower right of Figure 2, the presence of EMPIGEN BB® showed a significant reduction in the nonspecific interaction of the fluorescent polymer conjugated to its binding partner with leukocytes in the blood. In Figure 2, blood samples were stained using the antibody-free fluorescent polymer dye SN v605 and analyzed by flow cytometry. In the lower left of Figure 2, it is clear that the polymer dye without Empigen BB® binds nonspecifically to monocytes / granulocytes. While we do not wish to be bound to any particular theory, it is likely that the polymer is adsorbed to the cell surface of monocytes and granulocytes. When EMPIGEN BB® is added, the cell surface is blocked by EMPIGEN BB® molecules, and the nonspecific binding of the polymer to monocytes and granulocytes is substantially reduced.

[0209] When EMPIGEN BB® is added to the polymer, the dot plot appears the same as that of the unstained sample in the upper panel of Figure 2, which contains no polymer. The diffusion of granulocyte populations corresponds to the unstained tube, suggesting that when EMPIGEN BB® is added to the polymer dye, the nonspecific interaction between the polymer dye and monocytes and granulocytes is dramatically reduced. (Example 7) Experiments using the conjugate dye SN 605-CD20

[0210] EMPIGEN BB® was formulated with the conjugates described herein (e.g., SN605-CD20, SN786-CD103, and SN428 conjugates), bovine serum albumin (BSA; 2 mg / mL), sodium azide (0.1%), and pluronic F-68 (polyethylene oxide-polypropylene oxide-polyethylene oxide nonionic triblock copolymer) to a dose of 0.12% per 10 μl of conjugate.

[0211] CD20 is a B-lineage cell marker that is expressed during pre-B lymphocyte development, persists in B lymphocyte expression, and is lost simultaneously with plasma cell differentiation. CD20 is not expressed on other leukocyte populations, including monocytes, granulocytes, and NK cells. Figure 3 shows the performance of the SN 605-CD20 conjugate without EMPIGEN BB® and the SN 605-CD20 conjugate in the presence of EMPIGEN BB®.

[0212] The percentage of nonspecifically bound granulocytes decreased with the use of EMPIGEN BB® (see the "P2" gate in the point plot). Furthermore, the functional aspects of the conjugate remained unchanged because the percentage of positive cases was similar in both instances (see the "P1" gate in the point plot).

[0213] To confirm the effect of EMPIGEN BB®, two lots of SN 605-CD20 conjugate were tested in and without EMPIGEN BB®. The mean fluorescence intensity (MFI) was compared to the autofluorescence of monocytes from unstained samples (median fluorescence intensity of the negative population (MdFI)). The results shown in Figure 4 demonstrate that in the presence of the surfactant, nonspecific interactions on monocytes were reduced to 75% and 67% for lot 1 and lot 2 of the SN605 CD20 conjugate, respectively. A similar effect (reduction in nonspecific binding) was observed in granulocytes, but the percentage reduction was not as pronounced (13.7% and 17.7% for lot 1 and lot 2, respectively, Figure 5). (Example 8) Experiment using the conjugate dye SN 786-CD103

[0214] Since the CD103 conjugate is not normally expressed in normal whole blood, the test is performed using the cell line (MOLT16). Because CD103 is not expressed in normal blood, a positive signal should not be present. However, due to nonspecific interactions, the SN786 CD103 conjugate tends to bind to whole blood as well. Adding EMPIGEN BB® to this formulation helps to contain these nonspecific interactions, as shown in Figure 6.

[0215] Two lots of the SN 786-CD103 conjugate were tested in and without EMPIGEN BB® to compare the autofluorescence (MdFI in the negative population) of monocytes and granulocytes with that of unstained samples. As shown in Figure 7, the signal from nonspecific binding of the conjugate to monocytes was reduced by 149.3% and 202.1% for lot 1 and lot 2 of the SUPERNOVA® conjugate, respectively. The same effect (reduction in nonspecific binding) was observed in the granulocyte population, decreasing by 150.8% and 253% for lot 1 and lot 2, respectively, as shown in Figure 8. (Example 9) Effect of zwitterionic surfactants on reducing monocyte background using SN 428 conjugates.

[0216] The efficiency of the zwitterionic surfactant EMPIGEN BB® in reducing nonspecific binding to monocytes was evaluated. Other populations, namely lymphocytes and granulocytes, were also studied to assess the no-effect relationship of the cleanser on MFI and cell percentage.

[0217] The experimental conditions were generally as follows: • CD19-SN 428 (Lot D19-094, Polymer Lot RDS-042919 (82.7kD), 1 dose (0.5μg / test)). CD22-SN 428 (Lot D19-109, Polymer Lot WX-20190624 (86.4kD), 1 dose (0.5μg / test)). CD25-SN 428 (Lot D19-107, Polymer Lot RDS-062419 (72.8kD), 1 dose (0.5μg / test)). • Becton Dickinson CD19, CD22, and CD25-BV 421, commercially available dose 1x. Three doses of EMPIGEN BB®—0.06%, 0.12%, and 0.2%—were prepared as conjugate final formulations. • 10 μL of the sample and 100 μL of whole blood were added. • Two donors were tested, and the cells were dissolved using VersaLyse (a lysate used to dissolve red blood cells, Beckman Coulter, Inc.) + Fix, with one wash. • Navios flow cytometry data acquisition at FL9.

[0218] Prepare the required number of tubes (x), where "number of tubes x" depended on the performance being tested. Add the calculated volume (at the required dose) of conjugate antibody to each tube. Add whole blood (100 μL) to each tube. Vortex the tubes slowly for 15 seconds and incubate at room temperature (18-25°C) for 15-20 minutes, protected from light. Add a mixture of VersaLyse and IOTest3 fixative (Versalyse Ref. A09777 2 mL + IOTest3 fixative Ref. A07800 10 × 50 μl) to the tubes. Vortex the tubes immediately for 1 second and incubate at room temperature (18-25°C) for 10 minutes, protected from light. Centrifuge the tubes at 300 g at room temperature for 5 minutes, remove the supernatant by aspirate, and resuspend the cell pellet using PBS 1 × 3 mL. The tube was centrifuged again at room temperature (18-25°C) for 5 minutes with a 300g sample, the supernatant was removed by aspirate, and the cell pellet was resuspended using PBS 1× or PBS 1× 0.5 ml of 0.1% formaldehyde (which can be obtained by diluting PBS 1× 1 ml + IOTest3 fixative 10× 12.5 μl).

[0219] In cytometry, correction involves mathematically adjusting for signal overlap between channels in the emission spectra of different fluorescent dyes. Therefore, this correction factor was used to exclude signal outflow to other undesirable channels. Manual correction was performed to evaluate conjugate performance.

[0220] Table 1B shows raw data obtained from two donors using CD19, CD22, and CD25 SUPERNOVA® v428 conjugates, data normalized to the condition without EMPIGEN®, and the final percentage reduction in monocyte background staining. The data indicate that in the presence of Empigen BB®, nonspecific background monocyte binding was substantially reduced by between 52 and 73% compared to the condition without EMPIGEN®, where monocyte background was at its maximum.

[0221] [Table 1B]

[0222] Table 2 shows data demonstrating the effect of Empigen BB® on reducing granulocyte background. Granulocyte background reduction was observed in the range of 4 to 21% compared to the condition without EMPIGEN®, where granulocyte background was at its maximum.

[0223] [Table 2]

[0224] Table 3 shows the effect of EMPIGEN® on the positive lymphocyte population: The presence of EMPIGEN® did not induce a significant change in positive signaling to lymphocytes compared to the condition without EMPIGEN®.

[0225] [Table 3]

[0226] The data from Tables 1-3 is summarized in Figures 11-15.

[0227] Table 4 describes additional experiments and shows the percentage reduction in background monocytes and granulocytes.

[0228] [Table 4] (Example 10) Effect of EMPIGEN BB® on sample cell integrity

[0229] EMPIGEN BB® is a surfactant and may therefore cause cell membrane permeabilization and cell death. Studies described herein have concluded that the concentrations of EMPIGEN BB® used with polymer dye conjugates do not induce permeabilization or death of whole blood cells and do not affect the performance of the conjugates.

[0230] Micelle concentration studies were conducted in the samples and during the staining period to ensure that the critical micelle concentration (CMC) was not exceeded. See Table 5, which shows the evaluation of CMC in the conjugate formulation and during the staining period. CMC studies were performed in the conjugate formulation and during the staining period in 100 μL of whole blood. Experiments were conducted to evaluate the effects of EMPIGEN BB® on whole blood cell integrity and on peripheral blood mononuclear cells (PMBCs).

[0231] [Table 5]

[0232] The percentage of dead cells in whole blood samples containing 7-AAD was evaluated. 7-AAD is a DNA marker that stains positively when the cell membrane becomes permeable. Whole blood samples of CD19-SNv428 D19-094 containing 0.06%, 0.12%, and 0.2% EMPIGEN BB® (negative control), as well as EMPIGEN BB®-free samples, were tested from four donors with 7-ADD, and the percentage of dead cells under each condition was evaluated. The goal of the experiment was to determine whether the percentage of dead cells increased with increasing EMPIGEN BB® concentration.

[0233] Two whole blood samples stored for more than 24 hours were added as positive controls for 7-AAD staining.

[0234] Protocol for 7-AAD staining: • 100 μl whole blood + 10 μl CD19-SNv428+ / -Empigen, incubated for 20 minutes. Dissolve using Versalyse, wash once, • Resuspend in 500 μl of PBS in 1x. Add 20 μl of 7-AAD (Ref B88526) and incubate for 15-20 minutes. • Navios flow cytometer acquisition: FL4 for 7-AAD, FL9 for CD19-SNv428

[0235] The data is shown in Figure 16.

[0236] Finally, EMPIGEN BB® demonstrated its effectiveness in reducing nonspecific interactions of the polymer dye conjugates described herein (SN 605-CD20, SN786-CD103, SN428-CD25, SN428-CD19, and SN428-CD22). When tested against five specificities of the conjugates, EMPIGEN BB® efficiently reduced nonspecific background binding to monocyte and granulocyte populations. This efficiency of EMPIGEN BB® was donor-independent. A clear difference was observed in the reduction of monocyte nonspecific extraction when the conjugates were compared with BV786-CD103 and BV 421. In addition to its performance, the presence of EMPIGEN BB® and the polymer dye conjugates did not induce whole blood cell membrane permeabilization or whole blood cell death at concentrations of at least 0.2% in the composition. (Example 11) Effects of nonionic surfactants

[0237] The nonionic surfactants Tween®-20, tergitol, NP-40, and Pluronic F-68 (PF-68) were additional detergents / surfactants tested to remove the nonspecific binding of the conjugates described herein to monocytes. Figure 9 shows the inefficiency of Tween®-20 and PF-68 in avoiding the nonspecific binding of the conjugates to monocytes. (Example 12) The effects of protein blockers

[0238] The problem of nonspecific interactions with monocytes was also observed with conventional tandem dyes (e.g., PC5, PC5.5, PC7, AA700, available from Beckman Coulter, Inc.). To overcome this problem, we hypothesized that known protein blockers, BSA-ox (oxidized BSA) and BSA-Cy5-ox (oxidized Cy5-BSA), could inhibit nonspecific binding. However, BSA, BSA-ox, and BSA-Cy5-ox were all found to be inefficient in controlling nonspecific interactions between polymer dye conjugates and granulocytes / monocytes. See Figure 10. (Example 13) The effect of anionic surfactants on nonspecific binding.

[0239] The anionic surfactant N-lauryl sarcosine (NLS) was found to be effective in reducing nonspecific staining of monocytes and granulocytes at concentrations of 0.16% and 0.08% (w / v). [ka]

[0240] NLS is an anionic surfactant with a CMC of 14.57 mM (30°C). NLS sodium was evaluated to determine the effective concentration for inhibiting the nonspecific binding of polymer dye conjugate (SN v605-CD20) to monocytes and granulocytes.

[0241] In this example, NLS at different concentrations (0.16%, 0.08%, 0.04%, and 0.02% w / v) was formulated using SN v605CD20 conjugates in the presence of BSA and sodium azide, and peripheral blood samples were stained. Flow cytometry was performed after sample staining.

[0242] Figures 17A-E show point plots of blood samples in the absence and presence of different concentrations of conjugates, including Empigen, Empigen, and NLS. The point plot of peripheral blood samples without monochromatic conjugates is shown in Figure 17A, clearly demonstrating the absence of a population at the CD20+ gate.

[0243] Figure 17B shows a positive control plot of peripheral blood samples in the presence of a CD20-SN v605 monochromatic conjugate in a buffering composition containing BSA, sodium azide, and the zwitterionic surfactant Empigen BB® as additives. Compared to the negative control plot (Figure 17C), the percentages of the "monocyte nonspecific binding" (0.64%) and "granulocyte nonspecific binding" (0.68%) gates were significantly reduced, demonstrating the effectiveness of Empigen BB® in inhibiting nonspecific binding to monocytes and granulocytes.

[0244] Figure 17C shows negative control point plots of peripheral blood samples in the presence of CD20-SN v605 monochromatic conjugate in a buffering composition containing only BSA and sodium azide as additives. Without surfactants, granulocyte nonspecific staining was 1.20%, and monocyte nonspecific staining was 1.63%.

[0245] Figure 17D shows test point plots of peripheral blood samples in the presence of CD20-SN v605 monochromatic conjugate in a buffering composition containing BSA, sodium azide, and NLS (0.16% w / v) as additives. Nonspecific staining of granulocytes was substantially reduced to 0.60% and nonspecific staining of monocytes was substantially reduced to 0.39% compared to the negative control (Figure 17C).

[0246] Figure 17E shows test point plots of peripheral blood samples in the presence of CD20-SN v605 monochromatic conjugate in a buffering composition containing BSA, sodium azide, and NLS (0.08% w / v) as additives. Compared to the negative control (Figure 17C), granulocyte nonspecific staining decreased to 0.76%, and monocyte nonspecific staining decreased to 0.93%.

[0247] The point plot in Figure 17B (Empigen BB®) shows that the population percentages at the gates for "monocyte nonspecific binding" and "granulocyte nonspecific binding" are very similar when compared with Figures 17D and 17E (NLS), indicating that the efficacy of NLS equivalent to Empigen in blocking nonspecific binding to cells is 0.16% and 0.08%, respectively.

[0248] In CD20-SN v605, the effective concentration of NLS for reducing or removing nonspecific staining of monocytes and granulocytes was found to be 0.16% to 0.08% w / v. Therefore, NLS anionic surfactants in this concentration range were demonstrated to be effective in reducing nonspecific binding in monochromatic fluorescent polymer dye conjugate compositions. In one embodiment, for example, the following items are provided. (Item 1) A method for reducing or removing nonspecific binding of at least one polymer dye conjugate in a biological sample, A step of contacting the polymer dye conjugate with at least one zwitterionic or anionic surfactant before, during, or after contacting the polymer dye conjugate with a blood sample, wherein the contact results in a reduction of the nonspecific binding of the at least one polymer dye conjugate to cells in the biological sample. A method that includes this. (Item 2) The method according to item 1, wherein the reduction in nonspecific binding includes a reduction in nonspecific binding to leukocytes in the biological sample; and optionally, the biological sample is a blood sample. (Item 3) The method according to item 2, wherein the leukocytes are selected from the group consisting of monocytes and granulocytes. (Item 4) The method according to any one of items 1 to 3, comprising contacting the surfactant with the biological sample before contacting the polymer dye conjugate with the biological sample. (Item 5) The method according to any one of items 1 to 3, comprising contacting the polymer dye conjugate with the surfactant before contacting the polymer dye conjugate with the biological sample. (Item 6) The aforementioned surfactant is of the formula: R1’ [CO-X(CH 2 ) j ] g -[N + (R 2’ )(R 3’ )] k -(CH 2 ) f -[CH(OH)CH 2 ] h -Y - (In the formula, R 1’ C is either saturated or unsaturated. 5~24 It is alkyl; X is NH, NR 4 ' and here, R 4’ C 1~4 It is alkyl, O, or S; j is an integer from 1 to 10; g is either 0 or 1; R 2’ and R 3’ C 1~4 It is alkyl; k is either 0 or 1; Hydroxyl is substituted as needed with methyl, ethyl, hydroxymethyl, or hydroxyethyl; f is an integer between 0 and 4; h is either 0 or 1; Y is COO, SO 3 , OPO(OR 5’ )O, or P(O)(OR 5’ )O, and here, R 5’ is H or C 1~4 (It is alkyl.) The method described in item 1, which is a compound of the above. (Item 7) The aforementioned surfactant is of the formula: R1’ [CO-X(CH 2 ) j ] g -N + (R 2’ )(R 3’ )-(CH 2 ) f -[CH(OH)CH 2 ] h -Y - (In the formula, R 1’ C is either saturated or unsaturated. 5~24 It is alkyl; X is NH or NR 4’ And here, R 4’ C 1~4 It is alkyl, O, or S; j is an integer from 1 to 10; g is either 0 or 1; R 2’ and R 3’ C 1~4 It is alkyl; Hydroxyl is substituted as needed with methyl, ethyl, hydroxymethyl, or hydroxyethyl; f is an integer from 1 to 4; h is either 0 or 1; Y is COO, SO 3 , OPO(OR 5’ )O, or P(O)(OR 5’ )O, and here, R 5’ is H or C 1~4 (It is an alkyl residue.) The method according to any one of items 1 to 6, wherein the zwitterionic surfactant compound is... (Item 8) The aforementioned zwitterionic surfactant is given by formula: R 1’ -N + (CH 3 ) 2 -CH 2 COO - ; R 1’ -CO-NH(CH 2 ) 3 -N + (CH 3 ) 2 -CH 2 COO - ; R 1’ -N + (CH 3 ) 2 -CH 2 CH(OH)CH 2 SO 3 - ;or R 1’ -CO-NH-(ch 2 ) 3 -N + (CH 3 ) 2 -CH 2 CH(OH)CH 2 SO 3 - The compound described in item 7. (Item 9) The surfactants mentioned above include 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, and cocohydroxysultaine. Sultaine, Coco / Oleamidopropyl Betaine, Cocosultaine, Decyl Betaine, Dihydroxyethyl Oleyl Glycinate, Dihydroxyethyl Soybean Glycinate, Dihydroxyethyl Stearyl Glycinate, Dihydroxyethyl Fat Glycinate, Dimethicone Propyl PG-Betaine, Dorcamidopropyl Hydroxysultaine, Hydrogenated Fat Betaine, Isostearamidopropyl Betaine, Lauramidopropyl Betaine, Lauryl Betaine, Lauryl The method according to any one of items 1 to 8, selected from the group consisting of hydroxysultaine, laurylsultaine, milkamidopropyl betaine, milkamidopropyl betaine, myristamidopropyl betaine, myristyl betaine, oleamidopropyl betaine, oleamidopropyl hydroxysultaine, oleyl betaine, oliamidopropyl betaine, palmamidopropyl betaine, palmitamidopropyl betaine, palmitoylcarnitine, palm kernelamidopropyl betaine, polytetrafluoroethyleneacetoxypropyl betaine, ricinolamidopropyl betaine, sesamidopropyl betaine, soybeanamidopropyl betaine, stearamidopropyl betaine, stearyl betaine, tallowamidopropyl betaine, tallowamidopropyl hydroxysultaine, tallow betaine, tallow dihydroxyethyl betaine, undecylenamidopropyl betaine, and wheat germamidopropyl betaine. (Item 10) The method according to any one of items 1 to 9, wherein the surfactant is lauryl betaine. (Item 11) The aforementioned surfactant is of the formula: R 1’ [CO-X(CH 2 ) j ] g -(CH 2 ) f -[CH(OH)CH 2 ] h -Y - (In the formula, R 1’ C is either saturated or unsaturated. 5~24 It is alkyl; X is NH, NR 4 ' and here, R 4’ C 1~4 It is alkyl, O, or S; j is an integer from 1 to 10; g is either 0 or 1; Hydroxyl is substituted as needed with methyl, ethyl, hydroxymethyl, or hydroxyethyl; f is an integer between 0 and 4; h is either 0 or 1; Y is COO, SO 3 , OPO(OR 5’ )O or P(O)(OR 5’ )O, and here, R 5’ is H or C 1~4 (It is alkyl.) The method according to any one of items 1 to 6, wherein the anionic surfactant compound is an anionic surfactant, and the anionic surfactant may be in an acidic form or in the form of a sodium or potassium salt thereof. (Item 12) The aforementioned anionic surfactant is, R 1’ -CO-N(CH 3 )-CH 2 -COO - ;or R 1’ -CO-N(CH 3 )-CH 2 -SO 3 - (In the formula, R 1’ C is either saturated or unsaturated. 5~24 (It is alkyl.) The method according to item 11, wherein the compound is a compound thereof, and the anionic surfactant is in an acidic form or its sodium or potassium salt. (Item 13) The method according to item 11 or 12, wherein the anionic surfactant is selected from the group consisting of N-lauroyl sarcosine, sodium lauroyl sarcosinate, sodium palmitoyl sarcosinate, sodium stearoyl sarcosinate, sodium N-methyl-N-(1-oxotetradecyl)-glycine salt, sodium caproyl sarcosinate, sodium capryloyl sarcosinate, sodium N-methyl-N-(1-oxo-9-octadecen-1-yl)-glycine, sodium salt, sodium oleoyl sarcosinate, and sodium linoleoyl sarcosinate. (Item 14) The method according to item 13, wherein the anionic surfactant is N-lauroyl sarcosine or a salt thereof. (Item 15) The polymer dye conjugate is, Formula III:

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Claims

1. A method for reducing or removing the nonspecific binding of at least one fluorescent polymer dye conjugate to leukocytes in a biological sample, wherein the method is Before contacting the fluorescent polymer dye conjugate with the biological sample, the steps include: contacting at least one fluorescent polymer dye conjugate with at least one zwitterionic or anionic surfactant; A step of contacting the biological sample with the surfactant before contacting the fluorescent polymer dye conjugate with the biological sample, wherein the contact step results in a reduction of the nonspecific binding of the at least one fluorescent polymer dye conjugate to the leukocytes in the biological sample. Includes, The fluorescent polymer dye conjugate is of formula III: 【Chemistry 31】 (In the formula, Each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers; Each M is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, bandgap-modified monomers, ethylene, substituted ethylene, and ethynylene, and is distributed uniformly or randomly along the polymer backbone; Each L is a linker portion, or a linker portion conjugated to a substrate or binding partner; Each G1 and G2 is independently selected from the unmodified polymer ends and the modified polymer ends conjugated to the substrate or binding partner; a, c, and d each define the mol% of each unit, which may be repeated uniformly or randomly, with each a being mol% from 10 to 100%, each c being mol% from 0 to 90%, and each d being mol% from 0 to 25%; Each b is independently either 0 or 1; (Each m is an integer between 1 and approximately 10,000.) A method comprising a conjugated binding partner to a fluorescent polymer dye having the structure of [structure name].

2. The method according to claim 1, wherein the biological sample is a blood sample.

3. The method according to claim 1, wherein the leukocytes are selected from the group consisting of monocytes and granulocytes.

4. The aforementioned zwitterionic or anionic surfactant is of the formula: R １’ [CO-X(CH ２ ) ｊ ] ｇ -[N ＋ (R ２’ )(R ３’ )] ｋ -(CH ２ ) ｆ -[CH(OH)CH ２ ] ｈ -Y － (In the formula, R １’ is saturated or unsaturated C ５～２４ It is alkyl; X is NH or NR ４ ' and here, R ４’ C １～４ It is alkyl, O, or S; j is an integer between 1 and 10; g is either 0 or 1; R ２’ and R ３’ C is independent １～４ It is alkyl; k is either 0 or 1; Hydroxyl is either unsubstituted or substituted with methyl, ethyl, hydroxymethyl, or hydroxyethyl; f is an integer between 0 and 4; h is either 0 or 1; Y stands for COO, SO ３ , OPO (OR ５’ )O, or P(O)(OR ５’ ) O, and here, R ５’ is H or C １～４ (It is alkyl.) The method according to claim 1, wherein the compound is such that, when k=0, the surfactant is in an acidic form or in the form of a salt thereof.

5. The aforementioned surfactant is given by formula: R １’ [CO-X(CH ２ ) ｊ ] ｇ -N ＋ (R ２’ )(R ３’ )-(CH ２ ) ｆ -[CH(OH)CH ２ ] ｈ -Y － (In the formula, R １’ is saturated or unsaturated C ５～２４ It is alkyl; X is NH or NR ４’ And here, R ４’ C １～４ It is alkyl, O, or S; j is an integer between 1 and 10; g is either 0 or 1; R ２’ and R ３’ C is independent １～４ It is alkyl; Hydroxyl is either unsubstituted or substituted with methyl, ethyl, hydroxymethyl, or hydroxyethyl; f is an integer from 1 to 4; h is either 0 or 1; Y stands for COO, SO ３ , OPO (OR ５’ )O, or P(O)(OR ５’ ) O, and here, R ５’ is H or C １～４ (It is an alkyl residue.) The method according to any one of claims 1 to 4, wherein the zwitterionic surfactant compound is...

6. The aforementioned zwitterionic surfactant is given by formula: R １’ -N ＋ (CH ３ ) ２ -CH ２ COO － ; R １’ -CO-NH(CH) ２ ) ３ -N ＋ (CH) ３ ) ２ -CH ２ COO － ; R １’ -N ＋ (CH ３ ) ２ -CH ２ CH(OH)CH ２ SO ３ － ;or R １’ -CO-NH-(CH ２ ) ３ -N ＋ (CH) ３ ) ２ -CH ２ CH(OH)CH ２ SO ３ － The method according to claim 5, wherein the compound is [the compound].

7. The aforementioned zwitterionic surfactants include 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, and cocohyalopropyl betaine. Droxysultaine, Coco / Oleamidopropyl Betaine, Cocosultaine, Decyl Betaine, Dihydroxyethyl Oleyl Glycinate, Dihydroxyethyl Soybean Glycinate, Dihydroxyethyl Stearyl Glycinate, Dihydroxyethyl Fat Glycinate, Dimethicone Propyl PG-Betaine, Dorcamidopropyl Hydroxysultaine, Hydrogenated Fat Betaine, Isostearamidopropyl Betaine, Lauramidopropyl Betaine, Lauryl Betaine, Lauryl The method according to any one of claims 1 to 4, selected from the group consisting of lylhydroxysultaine, laurylsultaine, milkamidopropyl betaine, milkamidopropyl betaine, myristamidopropyl betaine, myristyl betaine, oleamidopropyl betaine, oleamidopropyl hydroxysultaine, oleyl betaine, oliveamidopropyl betaine, palmamidopropyl betaine, palmitamidopropyl betaine, palmitoylcarnitine, palm kernelamidopropyl betaine, polytetrafluoroethyleneacetoxypropyl betaine, ricinolamidopropyl betaine, sesamidopropyl betaine, soybeanamidopropyl betaine, stearamidopropyl betaine, stearyl betaine, tallowamidopropyl betaine, tallowamidopropyl hydroxysultaine, tallow betaine, tallow dihydroxyethyl betaine, undecylenamidopropyl betaine, and wheat germamidopropyl betaine.

8. The aforementioned surfactant is given by formula: R １’ [CO-X(CH ２ ) ｊ ] ｇ -(CH ２ ) ｆ -[CH(OH)CH ２ ] ｈ -Y － (In the formula, R １’ is saturated or unsaturated C ５～２４ It is alkyl; X is NH or NR ４ ', where R ４’ is C １～４ alkyl, O, or S; j is an integer between 1 and 10; g is either 0 or 1; Hydroxyl is either unsubstituted or substituted with methyl, ethyl, hydroxymethyl, or hydroxyethyl; f is an integer between 0 and 4; h is either 0 or 1; Y stands for COO, SO ３ , OPO (OR ５’ ) O or P (O) ( OR ５’ ) O, and here, R ５’ is H or C １～４ (It is alkyl.) The method according to any one of claims 1 to 4, wherein the anionic surfactant compound is in the form of an acidic form or a salt thereof.

9. The aforementioned anionic surfactant is, R １’ -CO-N(CH ３ ) - CH ２ - COO － ;or R １’ -CO-N(CH ３ )-CH ２ -SO ３ - (In the formula, R １’ is saturated or unsaturated C ５～２４ (It is alkyl.) The method according to claim 8, wherein the compound is a compound thereof, and the anionic surfactant is in an acidic form or its sodium or potassium salt.

10. The method according to claim 8, wherein the anionic surfactant is selected from the group consisting of N-lauroyl sarcosine or its salts, sodium lauroyl sarcosinate, sodium palmitoyl sarcosinate, sodium stearoyl sarcosinate, sodium N-methyl-N-(1-oxotetradecyl)-glycine salt, sodium caproyl sarcosinate, sodium capryloyl sarcosinate, sodium N-methyl-N-(1-oxo-9-octadecen-1-yl)-glycine, sodium salt, sodium oleoyl sarcosinate, and sodium linoleoyl sarcosinate.

11. The method according to any one of claims 1 to 4, wherein A comprises dihydrophenanthrene (DHP portion), fluorene portion, or DHP portion and fluorene portion.

12. The aforementioned at least one polymer dye conjugate is of formula I: 【Chemistry 32】 (In the formula, Each X is independently either C or Si; Each Y is independent of CR １ R ２ or SiR １ R ２ And; Each R １ These are independently ammonium alkyl salts, ammonium alkyl oxy salts, ammonium oligo ether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligo ether salts, sulfonamide oligo ethers, or partly: 【Transformation 33】 And; Each R ２ These are independently H, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG group, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, or partial 【Transformation 34】 And; Each R ３ The group is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and PEG groups; Each R ４ is, L ２ -E is; Each E is independently a chromophore, a sensual part, or a bonding partner; Each Z is independently selected from the group consisting of C, O, and N; Each Q is a combination of NH and NR. ４ , and CH ２ Independently selected from the group consisting of; Each subscript n is an integer between 0 and 20, independently of the others; Each M is a unit that can change the polymer band gap and is distributed uniformly or randomly along the polymer backbone; L is a linker, or a linker conjugated to a substrate or binding partner; Each L ２ It is a linker; G １ and G ２ Each G is independently selected from the group consisting of hydrogen, halogens, alkynes, aryls, substituted aryls, heteroaryls, substituted heteroaryls, 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, dihydrophenanthrene (DHP), substituted DHP, fluorene, substituted fluorene; amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and aryls or heteroaryls substituted with one or more pendant chains ending with functional groups selected from their protecting groups, and each G １ and G ２ They are either independently unconjugated or conjugated to a substrate or binding partner; a, c, and d each define the mol% of each unit in the structure, which may be repeated uniformly or randomly, where a is 10 to 100% mol%, c is 0 to 90% mol%, and each d is 0 to 25% mol%; Each b is independently either 0 or 1; m is an integer from 1 to approximately 10,000; Each n is an independent integer between 1 and 20. The method according to any one of claims 1 to 4, comprising a binding partner conjugated to a polymer having the structure of the following.

13. The method according to claim 12, wherein each L is an aryl or heteroaryl group substituted with one or more pendant chains that are independently and uniformly or randomly distributed along the polymer main chain and terminated with a functional group selected from the group consisting of amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and protecting groups thereof that are conjugated to a binding partner.

14. The method according to any one of claims 1 to 4, wherein the binding partner is a molecule or molecular complex that can specifically bind to a target analyte.

15. The method according to any one of claims 1 to 4, wherein the binding partner is a protein, affinity ligand, antibody, or antibody fragment, and the antibody or antibody fragment is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, an immunoglobulin, an immunoactive moiety of an immunoglobulin, a single-chain antibody, a Fab fragment, a Fab' fragment, and an F(ab')2 fragment and an scFv fragment.

16. Fluorescent polymer dyes conjugated to binding partners; Aqueous buffering agents; and Zwitterionic or anionic surfactants Includes, The polymer dye conjugate is of formula III: 【Chemistry 31】 (In the formula, Each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers; Each M is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, bandgap-modified monomers, ethylene, substituted ethylene, and ethynylene, and is distributed uniformly or randomly along the polymer backbone; Each L is a linker portion, or a linker portion conjugated to a substrate or binding partner; Each G1 and G2 is independently selected from the unmodified polymer ends and the modified polymer ends conjugated to the substrate or binding partner; a, c, and d each define the mol% of each unit, which may be repeated uniformly or randomly, with each a being mol% from 10 to 100%, each c being mol% from 0 to 90%, and each d being mol% from 0 to 25%; Each b is independently either 0 or 1; (Each m is an integer between 1 and approximately 10,000.) A composition comprising a binding partner conjugated to a fluorescent polymer dye having the structure of [structure name].

17. The composition according to claim 16, further comprising a nonpolymer dye conjugate.

18. The concentration of the zwitterionic or anionic surfactant is below the critical micelle concentration (CMC), and / or The composition according to claim 16, wherein the concentration of the surfactant is 0.05 to 0.25% (w / v), 0.06 to 0.20% (w / v), or 0.08 to 0.16% (w / v).

19. The composition according to claim 16, wherein the aqueous buffer comprises one or more additional additives selected from the group consisting of protein stabilizers, preservatives, and additional surfactants.

20. The composition according to any one of claims 16 to 19, further comprising a biological sample, wherein the composition exhibits a reduction in the nonspecific binding of the fluorescent polymer dye conjugate to leukocytes in the sample compared to a composition comprising the fluorescent polymer dye conjugate without the zwitterionic or anionic surfactant, and the leukocytes are selected from the group consisting of monocytes and granulocytes.

21. The aforementioned zwitterionic or anionic surfactant is of the formula: R １’ [CO-X(CH ２ ) ｊ ｇ -[N ＋ (R ２’ )(R ３’ )] ｋ -(CH ２ ) ｆ -[CH(OH)CH ２ ｈ -Y － (In the formula, R １’ is saturated or unsaturated C ５～２４ It is alkyl; X is NH or NR ４ ' and here, R ４’ C １～４ It is alkyl, O, or S; j is an integer between 1 and 10; g is either 0 or 1; R ２’ and R ３’ C is independent １～４ It is alkyl; k is either 0 or 1; Hydroxyl is either unsubstituted or substituted with methyl, ethyl, hydroxymethyl, or hydroxyethyl; f is an integer between 0 and 4; h is either 0 or 1; Y stands for COO, SO ３ , OPO (OR ５’ )O, or P(O)(OR ５’ ) O, and here, R ５’ is H or C １～４ (It is alkyl.) The composition according to any one of claims 16 to 19, wherein the surfactant is a compound of the same compound, and when k=0, the surfactant is in the form of an acidic form or a salt thereof.

22. The fluorescent polymer dye conjugate is of formula I: 【Transformation 36】 (In the formula, Each X is independently either C or Si; Each Y is independent of CR １ R ２ or SiR １ R ２ And; Each R １ These are independently ammonium alkyl salts, ammonium alkyl oxy salts, ammonium oligo ether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligo ether salts, sulfonamide oligo ethers, or partly: 【Chemistry 37】 And; Each R ２ These are independently H, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, PEG group, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, or part of: 【Transformation 38】 And; Each R ３ The group is independently selected from the group consisting of H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, and PEG groups; Each R ４ is, L ２ -E is; Each E is independently a chromophore, a sensual part, or a bonding partner; Each Z is independently selected from the group consisting of C, O, and N; Each Q is a combination of NH and NR. ４ , and CH ２ Independently selected from the group consisting of; Each subscript n is an integer between 0 and 20, independently of the others; Each M is a unit that can change the polymer band gap and is distributed uniformly or randomly along the polymer backbone; L is a linker, or a linker portion conjugated to a substrate or binding partner; Each L ２ It is a linker; G １ and G ２ Each G is independently selected from the group consisting of hydrogen, halogens, alkynes, aryls, substituted aryls, heteroaryls, substituted heteroaryls, 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, dihydrophenanthrene (DHP), substituted DHP, fluorene, substituted fluorene; amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and aryls or heteroaryls substituted with one or more pendant chains ending with functional groups selected from their protecting groups, and each G １ and G ２ They are either independently unconjugated or conjugated to a substrate or binding partner; a, c, and d each define the mol% of each unit in the structure, which may be repeated uniformly or randomly, where a is 10 to 100% mol%, c is 0 to 90% mol%, and each d is 0 to 25% mol%; Each b is independently either 0 or 1; m is an integer from 1 to approximately 10,000; Each n is an independent integer between 1 and 20. The composition according to any one of claims 16 to 19, comprising a binding partner conjugated to a polymer dye having the structure of the aforementioned.

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