Compositions and methods for preventing non-specific interactions between polymer dye-antibody conjugates

Abstract

To provide compositions and methods for preventing non-specific interactions between polymer dye-antibody conjugates.SOLUTION: The disclosure relates to compositions comprising a non-fluorescent component of a polymer dye such as a monomeric component of a polymer dye, a photo-bleached polymer dye, and / or a polymer dye comprising a quenching moiety, for reducing non-specific interactions of polymer dye conjugates, for example, in flow cytometric analysis of a biological sample. Methods for using such compositions, and kits comprising such compositions are also provided. Because of their nature, polymer dye conjugates can interact with one another, leading to non-specific binding. The disclosure provides compositions for decreasing or preventing non-specific interactions between polymer dye conjugates.SELECTED DRAWING: None

Description

[Technical field]

[0001] CITED RELATED APPLICATIONS This application was filed as a PCT international patent application on February 04, 2022, and claims the benefit of and priority to U.S. Provisional Application No. 63 / 146,498, filed February 05, 2021, which is incorporated by reference in its entirety. [Background technology]

[0002] background Polymer-dye conjugates are bright and provide excellent performance that can be utilized in multicolor flow cytometry assays. In general, polymer-dye conjugates exhibit high brightness due to their unique complex structure. However, such same unique complex structure also brings some serious limitations.

[0003] Polymer dyes are hydrophobic and have large apparent molecular weights, which makes them prone to aggregation in aqueous buffers.Therefore, when polymer dyes are conjugated to antibodies, the resulting conjugates may also tend to interact with each other and / or with other polymer dye conjugates present in the same sample.When more than one polymer dye conjugate is used to stain the same sample, non-specific interactions between the polymer dyes may occur, which may result in under-compensation of data.

[0004] A dedicated staining buffer that can eliminate non-specific interactions between polymer-dye conjugates is highly desirable. Summary of the Invention [Means for solving the problem]

[0005] Abstract Polymer-dye conjugates, by their nature, can interact with one another, resulting in non-specific binding. The present disclosure provides compositions that reduce or prevent non-specific interactions between polymer-dye conjugates.

[0006] The present disclosure provides a composition that can reduce non-specific interactions between dye conjugates, comprising one or more non-fluorescent components of polymer dyes and a buffer.The composition may optionally further comprise a non-ionic surfactant.The composition may optionally further comprise a protein stabilizer.The composition may optionally further comprise a preservative.

[0007] The present disclosure provides a staining buffer composition comprising one or more non-fluorescent components of a polymer dye, a non-ionic surfactant and a buffer, which can reduce non-specific binding, such as polymer-polymer interactions, between one or more fluorescent polymer dye conjugates.

[0008] The non-fluorescent component of the polymeric dye may be one or more of a monomeric component of a polymeric dye, a polymeric dye that includes a quenching moiety, such as a photobleached polymeric dye, and a quenched polymeric dye.

[0009] The present disclosure provides a composition that includes monomeric components of a polymeric dye in a buffer solution.

[0010] The present disclosure provides a composition comprising a photobleached dye in a buffer.

[0011] The present disclosure provides a composition comprising a quenched polymeric dye in a buffer solution.

[0012] The present disclosure provides a composition including monomer components of a polymeric dye and a quenched polymeric dye in a buffer solution.

[0013] The present disclosure provides a composition that includes monomer components of a polymeric dye and a photobleached dye in a buffer solution.

[0014] The compositions of the present disclosure may further comprise a protein stabilizer, a non-ionic surfactant, and / or a preservative, as desired. For example, a working concentration composition (1×) of the present disclosure may further comprise a protein stabilizer at 0.1-10 mg / mL, 0.5-5 mg / mL, 1-3 mg / mL, or about 2 mg / mL.

[0015] The deep staining buffer composition (10x) of the present disclosure may further comprise a protein stabilizer at 1-100 mg / mL, 2-50 mg / mL, 5-40 mg / mL, 10-30 mg / mL, or about 20 mg / mL. In some embodiments, the protein stabilizer is present at 0.1-100 mg / mL, 0.2-50 mg / mL, 1-20 mg / mL, or 1-10 mg / mL.

[0016] Working concentration compositions (1×) of the present disclosure may contain 0.01-10%, 0.01-4%, 0.01-2%, 0.01-0.4% or about 0.02% (weight / volume) of a non-ionic surfactant.

[0017] The deep dye buffer composition (10x) of the present disclosure may contain 0.1-40%, 0.5-20%, 1-10% or about 5% (weight / volume) of a non-ionic surfactant.

[0018] The compositions of the present disclosure may further comprise a preservative at 0.01-0.5%, 0.03-0.3%, or 0.05-0.2% or about 0.1% (weight / volume) as required.

[0019] For example, the disclosure provides a working concentration staining buffer composition (1×) containing 0.3-1.5 mg / mL, 0.5-1.2 mg / mL, or about 1 mg / mL of quenched polymeric dye in a biological buffer, such as PBS buffer, containing 0.01-10% or 0.1-4% (weight / volume) non-ionic surfactant and, optionally, 0.1-10 mg / mL or 0.2-5 mg / mL of a protein stabilizer.

[0020] The present disclosure provides a deep staining buffer composition (10X) comprising 3-15 mg / mL, 5-12 mg / mL or about 10 mg / mL of a quenched polymeric dye in a biological buffer, such as PBS buffer, containing 0.1-40% or 1-10% (weight / volume) of a non-ionic surfactant and optionally 1-100 mg / mL or 2-50 mg / mL of a protein stabilizer. This composition was found to significantly reduce non-specific polymeric dye conjugate interactions in a multi-color dye conjugate panel. This was demonstrated in FCA flow cytometry analysis of stained and lysed blood samples when compared to the same samples without the quenched polymeric dye.

[0021] The present disclosure provides a working concentration staining buffer composition (1×) comprising 20-40 mg / mL or about 30 mg / mL of monomeric components of a polymeric dye; 0.2-0.8 mg / mL or 0.3-0.7 mg / mL of a quenched polymeric dye; and 0.01-10% or 0.1-4% of a non-ionic surfactant in a biological buffer. The working concentration composition (1×) may further comprise 0.1-10 mg / mL, 0.5-5 mg / mL of a protein stabilizer.

[0022] The present disclosure provides a concentrated staining buffer composition (10x) comprising 200-400 mg / mL or about 300 mg / mL of monomeric components of a polymeric dye, 2-8 mg / mL or 3-7 mg / mL of a quenched polymeric dye, and 0.1-40% or 1-20% of a non-ionic surfactant in a biological buffer. The concentrated composition (10x) may further comprise 1-100 mg / mL or 5-50 mg / mL of a protein stabilizer.

[0023] This composition was found to significantly reduce non-specific polymer-dye conjugate interactions in a multicolor dye conjugate panel, as demonstrated in flow cytometric analysis (FCA) of stained and lysed whole blood samples when compared to the same samples without the monomer component and without the quenched polymer dye.

[0024] The present disclosure provides a working concentration staining buffer composition (1×) comprising 20-40 mg / mL or about 30 mg / mL of monomeric components of polymeric dye; 0.2-0.8 mg / mL, 0.3-0.7 mg / mL or about 0.5 mg / mL of photobleached polymeric dye; and 0.01-10% or 0.1-4% of a non-ionic surfactant in a biological buffer. The working concentration composition (1×) may further comprise 0.1-10 mg / mL or 0.5-5 mg / mL of a protein stabilizer.

[0025] The present disclosure provides a concentrated staining buffer composition (10x) comprising 200-400 mg / mL or about 300 mg / mL of monomeric components of a polymeric dye, 2-8 mg / mL, 3-7 mg / mL or about 5 mg / mL of photobleached polymeric dye, and 0.1-40% or 1-20% of a non-ionic surfactant in a biological buffer. The concentrated composition (10x) may further comprise 1-100 mg / mL or 2-50 mg / mL of a protein stabilizer.

[0026] This composition was found to substantially reduce non-specific polymer-dye conjugate interactions in a multicolor dye conjugate panel, as demonstrated in FCA analysis of stained and lysed whole blood samples when compared to the same samples without the monomer component and without the photobleached polymer dye.

[0027] A composition for use with at least one fluorescent polymer dye conjugated to a binding partner for use in staining a biological sample is provided, the composition comprising a non-ionic surfactant; and a biological buffer, the composition reducing non-specific binding of the at least one fluorescent polymer dye conjugate when compared to the at least one fluorescent polymer dye conjugate in the absence of the composition. The non-ionic surfactant may be a poloxamer.

[0028] A method for detecting an analyte in a sample is provided, comprising the steps of: adding at least one or at least two polymer dye conjugates to a staining buffer composition according to the present disclosure to form a polymer dye conjugate composition; contacting the polymer dye conjugate composition with a biological sample suspected of containing the analyte to form a fluorescent polymer dye conjugate complex with the analyte; applying a light source to the sample that can excite the at least one or at least two fluorescent polymer dye conjugate complexes; and detecting light emitted from the fluorescent polymer dye conjugate complexes. The step of detecting light can include analyzing by flow cytometry to obtain a first flow cytometry plot, and the first flow cytometry plot shows one or more of the group consisting of reduced non-specific interactions of the polymer dye conjugate; and reduced aggregation of the polymer dye conjugate when compared with a second flow cytometry plot obtained by contacting the biological sample with a composition that does not include a non-ionic surfactant and does not include a non-fluorescent component of the first polymer dye.

[0029] The biological sample can be blood, bone marrow, spleen cells, lymphocytes, bone marrow aspirate, urine, serum, saliva, cerebrospinal fluid, urine, amniotic fluid, interstitial fluid, stool, mucus, a tissue sample or a cell culture sample. The biological sample may be a whole blood sample.

[0030] The light from the light source may have a wavelength within the range between about 340 nm and about 800 nm, or within the range between about 340 nm and about 450 nm.

[0031] The present disclosure provides a kit comprising a dyeing buffer composition according to the present disclosure, comprising separate containers comprising one or more non-fluorescent components of a first polymer dye; and at least one fluorescent polymer dye conjugate. The dyeing buffer composition may comprise a non-ionic surfactant in the same container comprising one or more non-fluorescent components. The dyeing buffer composition may comprise two or more non-fluorescent components of a first polymer dye and optionally a non-ionic surfactant in one container; and at least one fluorescent polymer dye conjugate in another container. [Brief description of the drawings]

[0032] [Figure 1] FIG. 1 shows a comparison of FCA dot plots of test staining buffers using a mixture of three SuperNova (SNv) dye-antibody conjugates, CD56-SNv428, CD20-SNv605, and CD4-SNv786, containing stained lysed whole blood, with or without pre-addition of test staining buffer. Gating is on lymphocytes (LY). As shown in the top three panels, staining anomalies can occur in the absence of test staining buffers, and data can appear under-corrected. As shown in the three lower panels, a substantial reduction in non-specific polymer-dye conjugate interactions occurs in the presence of test staining buffers containing photobleached polymer and monomeric components of the polymer dye.

[0033] [Figure 2A]Figure 2A shows an FCA dot plot of a lysed whole blood sample stained with a mixture of three SuperNova violet polymer-dye conjugates, including CD56-SNv428+CD19-SNv605+CD4-SNv786, without staining buffer components. The positive and negative cell populations in this dot plot are not aligned and appear tilted, indicating non-specific polymer-dye interactions.

[0034] [Figure 2B] Figure 2B shows an FCA dot plot of a lysed whole blood sample stained with a mixture of three SuperNova violet polymer dye conjugates, including CD56-SNv428+CD19-SNv605+CD4-SNv786, with a commercial comparative staining buffer. Some reduction in nonspecific interactions is evident, as compared to Figure 2A.

[0035] [Figure 2C] Figure 2C shows an FCA dot plot of a lysed whole blood sample stained with a mixture of three SuperNova violet polymer-dye conjugates including CD56-SNv428+CD19-SNv605+CD4-SNv786 in a biological buffer containing a poloxamer non-ionic surfactant, and a test staining buffer according to the present disclosure containing monomer A and photobleached dye 428. The test staining buffer composition shows reduced spillover compared to Figure 2A and some reduction in non-specific polymer-polymer interactions compared to the prior art comparison buffer in Figure 2B.

[0036] [Diagram 3]Figure 3 shows the graph of the fluorescence profile of wavelength vs. AFU of quenched polymers: polymer 1 Dabcyl (QY 0.01), polymer 2 Dabcyl (QY 0.005), polymer 2 DY425Q (QY 0.01), polymer 3 Dabcyl (QY 0.005), polymer 3 Dabcyl plus (QY 0.015) and polymer 3 DY425Q (QY 0.009). The inset of Figure 3 shows the graph of a representative parent fluorescent polymer (polymer 3 QY 0.54) before conjugation to a quenching moiety and after conjugation to a quenching moiety to obtain a quenched polymer that shows a substantial decrease in fluorescence QY when excited by a 405 nm laser. QY refers to quantum yield.

[0037] [Figure 4] Figure 4 shows FCA dot plots (top row, left to right) of stained and lysed whole blood samples after staining with a mixture of two polymer dye conjugates, CD4-UV-excitable polymer dye (CD4-UVEPD) and CD20-violet-excitable polymer dye (CD20-VEPD) (both Beckman Coulter Life Sciences, top left panel), and a mixture of two polymer dye conjugates, CD4-BUV395 (BD Biosciences) and CD20-VEPD (Beckman Coulter Life Sciences, top right panel), when gated on lymphocytes without the use of a quenched polymer staining buffer additive. When the two different dye conjugates are mixed without a quenched polymer, the dot plots show nonspecific interactions and associated spillover. Addition of a quenched polymer according to the present disclosure (quenched polymer 2-DYQ425) during staining with CD4-UVEPD and CD20-VEPD (lower left panel) or BUV395-CD4 and CD20-VEPD (lower right panel) reduced nonspecific interactions and spillover.

[0038] [Diagram 5]Figure 5 shows FCA dot plots (top row, left to right) of stained and lysed whole blood samples after staining with a mixture of two polymer dye conjugates, CD4-UV-excitable polymer dye (CD4-UVEPD) and CD20-violet-excitable polymer dye (CD20-VEPD) (both Beckman Coulter Life Sciences, top left panel), and a mixture of two polymer dye conjugates, CD4-BUV395 (BD Biosciences) and CD20-VEPD (Beckman Coulter Life Sciences, top right panel), when gated on lymphocytes without the use of a quenched polymer staining buffer additive. When the two different dye conjugates are mixed without a quenched polymer, the dot plots show nonspecific interactions and associated spillover. Addition of a quenched polymer according to the present disclosure (quenched polymer 2-Dabcyl) during staining with CD4-UVEPD and CD20-VEPD (lower left panel) or BUV395-CD4 and CD20-VEPD (lower right panel) reduced nonspecific interactions and spillover.

[0039] [Figure 6] Figure 6 shows a two-dimensional FCA dot plot of the SuperNova polymer-dye conjugate CD56-SNv428 / CD4-SNv786 in a sample prepared with a composition containing an ineffectively photobleached dye (left panel), resulting in undesired spillover of the conjugate. In contrast, the right panel shows the effect of a composition according to the present disclosure containing a correctly photobleached dye with a QY≦0.056 and <47 AFU at 10ug / mL when excited with a 405nm laser. Spillover of the conjugate is significantly reduced.

[0040] [Figure 7]Figure 7 shows two-dimensional FCA dot plots of two SuperNova polymer-dye conjugates CD3-SNv428 / CD19-SNv605, individually preformulated with or without Monomer A + Poly-Dabcyl additive, prior to addition into a biological sample. In the absence of additive (left panel), nonspecific interactions are evident. In the presence of Monomer A + Poly-Dabcyl additive (right panel), nonspecific interactions between the polymer-dye conjugates are substantially reduced.

[0041] [Figure 8] Figure 8 shows FCA dot plots of lysed cells stained with a mixture of dye conjugates CD19-SNv428 (Beckman Coulter Life Sciences) and CD4-BV650 (BD Biosciences) in blood samples without quenched polymer (upper left), with 1% PF-68 (upper right), with 10ug quenched polymer 2-Dabcyl (lower left), and with 10ug quenched polymer 2-Dabcyl and 1% PF-68 non-ionic surfactant (lower right). Test sample compositions with 10ug quenched polymer 2-Dabcyl (MFI 2172) and 1% PF-68 (MFI 2400) showed reduced non-specific binding compared to the control sample without buffer (MFI 7804). A test sample composition containing 10 ug of quenched polymer 2-Dabcyl and 1% PF-68 (MFI 1327) showed improved reduction in MFI, improved reduction in non-specific binding, and improved reduction in polymer-polymer interactions compared to the control sample.

[0042] [Figure 9]Figure 9 shows FCA dot plots of cells lysed and stained with a mixture of CD4-BV650 (BD Biosciences) and CD19-SNv428 (Beckman Coulter Life Sciences) in no buffer (top panel), with 0.1% non-ionic detergent (lower left panel), with 0.5% non-ionic detergent (lower middle panel), and with 1% non-ionic detergent (weight / volume) (lower right panel). The presence of increasing concentrations of non-ionic detergent (0.1-1% weight / volume) was accompanied by a reduction in spillover and non-specific interactions in the mixture, as evidenced by improved separation compared to when no non-ionic detergent was present.

[0043] [Figure 10] Figure 10 shows a graph of the emission spectra in the range of 415-700 nm and the quantum yields of two non-fluorescent polymeric dyes useful for reducing non-specific interactions and spillover in FCA assays in a staining buffer composition. The structures of DHP-pyrrole polymer (QY0.043) and DHP-nitrocapped polymer (QY0.092) are also shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] explanation Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings and examples. While the disclosed subject matter is described in conjunction with the enumerated claims, it will be understood that the example subject matter is not intended to limit the claims to the disclosed subject matter.

[0045] Provided herein are compositions and methods that reduce non-specific interactions between polymer-dye conjugates, for example in multicolor panels that include a multiplicity of dye conjugates. The compositions and methods are useful, for example, in flow cytometry, to reduce polymer-polymer interactions that can lead to increased spillover into other channels.

[0046] The present disclosure generally relates to a method of detecting an analyte in a sample using a staining buffer designed to reduce non-specific interactions between fluorescent polymer-dye conjugates, a composition comprising such a polymer, and a composition comprising a fluorescent polymer conjugated to a binding partner (e.g., a polymer dye conjugated to an antibody, referred to herein as a "polymer-dye conjugate"). For example, a composition according to the present disclosure comprises a liquid staining buffer comprising at least one or at least two non-fluorescent components of a polymer dye, a buffer, and optionally a non-ionic surfactant. The non-fluorescent components of the polymer dye may be one or more of a monomeric component of the polymer dye, a photobleached polymer dye, and a polymer dye comprising a quenching moiety.

[0047] For example, the compositions described herein may include a monomer component of a polymer dye and a photobleached polymer dye; a monomer component of a polymer dye and a polymer dye comprising a quenching moiety; or a photobleached polymer dye and a non-ionic surfactant. The compositions according to the present disclosure can reduce non-specific polymer-polymer interactions between various polymer-dye conjugates in a multicolor panel.

[0048] Polymer Dyes The staining buffer composition of the present disclosure may include one or more non-fluorescent components of a polymeric dye. The non-fluorescent component may be a non-fluorescent polymeric dye. The non-fluorescent component of the polymeric dye may be a polymeric dye that includes a quenching moiety. The non-fluorescent component of the polymeric dye may be a photobleached polymeric dye. The non-fluorescent component of the polymeric dye may be a monomeric component ("monomer") of the polymeric dye. In some embodiments, the non-fluorescent component of the polymeric dye does not include a binding partner. In some embodiments, the polymeric dye also includes a polymeric tandem dye. In some embodiments, the polymeric dye also includes a polymeric dye that is conjugated to a functional moiety.

[0049] The composition of the present disclosure can be used with one or more fluorescent polymer dye conjugates.In some embodiments, the polymer dye also comprises a polymer dye conjugated to a binding partner.In some embodiments, the polymer dye also comprises a polymer dye conjugated to a functional moiety. Fluorescent polymer dyes are particularly useful in the analysis of chemical and biological target analytes: they are highly responsive optical reporters and, due to the multiple chromophores they contain, are efficient light absorbers.

[0050] The polymer dye can include any previously disclosed or commercially available fluorescent polymer dye. For example, the polymer dye can be any of those described in PCT Publication No. WO2022 / 013198; PCT Publication No. WO2017 / 180998; U.S. Application No. 2021 / 0047476; U.S. Application No. 2020 / 0190253; U.S. Application No. 2020 / 0048469; U.S. Application No. 2020 / 0147615; U.S. Application No. 2021 / 0108083; U.S. Application No. 2019 / No. 0194467; U.S. Application No. 2018 / 0364245; U.S. Application No. 2018 / 0224460; U.S. Patent No. 11,034,840; U.S. Patent No. 11,119,107; U.S. Patent No. 10,962,546; U.S. Patent No. 10,920,082; U.S. Patent No. 10,001,475; U.S. Patent No. 10,107,818; U.S. Patent No. 10,228,37 No. 5; U.S. Pat. No. 10,844,228; U.S. Pat. No. 10,604,657; U.S. Pat. No. 10,545,137(B2); U.S. Pat. No. 10,533,092; U.S. Pat. No. 10,472,521; U.S. Pat. No. 10,240,000; U.S. Pat. No. 9,758,625; U.S. Pat. No. 9,719,998; U.S. Pat. No. 7,214,489; U.S. Pat. No. 9,012,643; U.S. Pat. No. 8,623,332; U.S. Pat. No. 8,431,416; U.S. Pat. No. 8,354,239; U.S. Pat. No. 8,575,303; U.S. Pat. No. 8,969,509, each of which is incorporated by reference as if fully set forth in their entirety herein. The polymer dye can have the structure of any water-soluble fluorescent polymer dye disclosed in U.S. Patent Publication No. 2020 / 0190253(A1), the entirety of which is incorporated by reference as if fully set forth herein. The polymer dye conjugate can have the structure of any water-soluble fluorescent polymer dye disclosed in U.S. Patent Publication No. 2019 / 0144601, the entirety of which is incorporated by reference as if fully set forth herein.

[0051] The polymer dye can be any commercially available polymer dye. In some embodiments, the polymer dye can be excitable by, for example, ultraviolet (e.g., 351 nm, 355 nm, 375 nm, 334-364 nm, 351-356 nm), violet (e.g., 405 nm, 407 nm, 414 nm, 395-425 nm), blue (e.g., 436 nm, 458 nm), blue-green (e.g., 488 nm), green (e.g., 514 nm, 532 nm, 541 nm, 552 nm), yellow-green (e.g., 561 nm, 563 nm), yellow (e.g., 568 nm), red (e.g., 627-640 nm, 633 nm, 637 nm, 640 nm, 647 nm) and / or near-infrared lasers (e.g., in the range of 673 nm, 750 nm, 780 nm, or 660-800 nm). The polymer dye may comprise a polymer dye excitable by a violet laser. The polymer dye or polymer dye conjugate may comprise a polymer dye excitable by a violet laser at a wavelength of about 395 nm to about 425 nm, for example, 405 nm, 407 nm or 414 nm. The polymer dye or polymer dye conjugate may comprise a polymer dye excitable by a violet laser (405 nm). In some embodiments, the polymer dye may be a non-fluorescent polymer dye.

[0052] In some embodiments, the polymer dye or polymer dye conjugate may comprise SuperNova polymer dye (SN) (Beckman Coulter, Inc.). SuperNova polymer dye is a new generation polymer dye useful for flow cytometry applications. The polymer dye or polymer dye conjugate may comprise SNv428, SNv605 or SNv786. SNv428 has unique photophysical properties that result in a fairly bright conjugate when conjugated to an antibody or other binding partner. For example, SNv428 is a polymer dye that is optimally excited by a violet laser (e.g., 405 nm) with an excitation maximum of 414 nm, an emission peak of 428 nm, and can be detected using a 450 / 50 bandpass filter or equivalent.

[0053] SNv428 is one of the brightest dyes that can be excited by violet laser, and is therefore particularly suitable for the evaluation of faintly expressed markers. SuperNova polymer dyes conjugated with antibodies can include the following antibody-polymer dye conjugates: anti-CD19 antibody-SNv428, anti-CD22 antibody-SNv428, anti-CD25 antibody-SNv428 and anti-CD38 antibody-SNv428.

[0054] SNv605 and SNv786 (Beckman Coulter, Inc.) are tandem polymer dyes derived from the core SNv428. Both share the same absorbance characteristics, with an excitation maximum at 414 nm. SNv605 and SNv786 have emission peaks at 605 nm and 786 nm, respectively, so they are best detected using 610 / 20 and 780 / 60 nm bandpass filters on a flow cytometer. SNv605 and SNv786 can be conjugated with binding partners, such as, for example, anti-CD19, anti-CD22, anti-CD25 or anti-CD38 antibodies.

[0055] The polymer dye may comprise a polymer dye excitable by an ultraviolet ("UV") laser. The polymer dye or polymer dye conjugate may comprise a polymer dye excitable by a UV laser at a wavelength 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 longer than or equal to 320 nm. The polymer dye or polymer dye conjugate may comprise a UV-excitable polymer dye. The UV-excitable polymer dye or polymer dye conjugate can typically emit at a wavelength of 380 nm to 430 nm, 406 nm to 415 nm, or less than or equal to 430 nm, but longer than or equal to 380 nm.

[0056] The polymer dyes are Brilliant Violet 421™ (excitation maximum 405 nm, emission maximum 421 nm, 450 / 50 filter), Brilliant Violet 510™ (excitation maximum 405 nm, emission maximum 510 nm, 510 / 50 filter), Brilliant Violet 570™ (excitation maximum 405 nm, emission maximum 570 nm, 585 / 42 filter), Brilliant Violet 605™ (excitation maximum 405 nm, emission maximum 603 nm, 610 / 20 filter), Brilliant Violet 650™ (excitation maximum 405 nm, emission maximum 645 nm, 660 / 20 filter), Brilliant Violet 711™ (excitation maximum 405 nm, emission maximum 711 nm, 710 / 50 filter), Brilliant Violet 721™ (excitation maximum 405 nm, emission maximum 721 nm, 710 / 50 filter), Brilliant Violet 711™ (excitation maximum 405 nm, emission maximum 721 nm, 710 / 50 filter), Brilliant Violet 72 ... These dyes may include Brilliant Violet™ dyes (BioLegend® / Sirigen Group Ltd.), such as Brilliant Violet 750™ (excitation maximum 405 nm, emission maximum 750 nm, 780 / 60 filter), and Brilliant Violet 785™ (excitation maximum 405 nm, emission maximum 785 nm, 780 / 60 filter).

[0057] The polymeric dye or polymeric dye conjugate may include BD Horizon Brilliant™ Violet ("BV") polymeric dye (Becton, Dickinson and Co., BD Life Sciences). The polymeric dye may 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), BV786 (786 / 60 filter).

[0058] The polymer dyes may be synthetically prepared by polymerization of monomers, resulting in the formation of highly conjugated fluorescent backbones. Capping may be performed on the polymer by activation using appropriate functional groups, resulting in a polymer that can be conjugated to a binding partner. Alternatively, the polymer may be activated to conjugate or bind an acceptor dye by attaching an appropriate functional group from the polymer backbone. The activated polymer can be conjugated, for example, to a binding partner, an acceptor dye, or a quenching moiety. Any suitable binding partner, for example, an antibody, may be used, and then purified, for example, by using standard procedures. The functional group 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 protected groups for conjugation to substrates or binding partners.

[0059] The polymeric dye may include fluorescent polymeric dyes having aryl and / or heteroaryl monomeric subunits, including, but not limited to, dihydrophenanthrene (DHP), fluorene, and combinations thereof. In some embodiments, the polymeric dye has the structure of Formula I: [ka] and During the ceremony, each monomer A is independently an aromatic comonomer or a heteroaromatic comonomer; each optional M is an aromatic or heteroaromatic comonomer; each optional L is a linker; G 1 and G 2 are modified or unmodified polymer ends, respectively; Each a is a mol% of 10 to 100%, each c is a mol% of 0 to 90%, each d is a mol% of 0 to 25%; each b is independently 0 or 1; and each m is an integer of 1 to about 10,000.

[0060] As used throughout this disclosure, "a," "b," "c," and "d" define the mol % of each unit, each of which may be repeated evenly or randomly.

[0061] Each monomer A may be substituted with a water-solubilizing group and / or an optional functional group that may be conjugated, for example, to an acceptor dye, a binding partner, or a quenching moiety. Each monomer A in a polymer having the structure of formula I may be the same monomer. Each monomer A in a polymer dye having the structure of formula I may be a different monomer. Monomer A may be, for example, a 9,10-phenanthrene dione-based monomer (e.g., a dihydrophenanthrene (DHP)-based monomer), a fluorene-based monomer, or a fluorenooxepin-based monomer.

[0062] Monomer A may, for example, have the structure of formula (II): [ka] The monomer may be a DHP-based monomer, a fluorene-based monomer, or a carbazole monomer having the formula:

[0063] As used herein, each "X" can independently be C, N, or Si.

[0064] As used herein, "Y" is CH, CR 1 R 2 , SiR 1 R 2 or a bond. When Y is a bond, X is directly attached to both rings.

[0065] As used throughout this disclosure, “R 1 " each independently represents a water solubilizing moiety, alkene, alkyne, cycloalkyl, haloalkyl, (hetero)aryloxy, (hetero)arylamino, PEG, carboxylic acid, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamide [ka] It is.

[0066] As used herein, "R 2 " each independently represents a water solubilizing moiety, a linker moiety, H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, (hetero)aryloxy, (hetero)arylamino, sulfonamido-PEG, phosphoramido-PEG, ammonium alkyl salt, ammonium alkyloxy salt, ammonium oligoether salt, sulfonate alkyl salt, sulfonate alkoxy salt, sulfonate oligoether salt, sulfonamide oligoether, sulfonamide, sulfinamide, phosphonamidate, phosphinamide [ka] It is.

[0067] As used herein, "R 3 Each "R" may independently be a water-solubilizing moiety. 3 " can each independently be H, alkyl, alkene, alkyne, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, or a PEG group.

[0068] As used herein, "R 4 " is each independently H, an alkyl, a PEG, a water-solubilizing moiety, a linker moiety, a chromophore, a carboxylamine, an amine, a carbamate, a carboxylic acid, a carboxylic ester, a maleimide, an activated ester, N-hydroxysuccinimidyl, a hydrazine, a hydrazide, a hydrazone, an azide, an alkyne, an aldehyde, or a thiol, or a protected group thereof.

[0069] As used herein, "R 7 " are H, hydroxyl, C1-C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, C3-C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 Alkoxy, C2-C 18 (Hetero)aryloxy, C2-C 18 (Hetero)arylamino, C2-C 12 Carboxylic acids, C2-C 12 Carboxylic acid ester or C1-C 12 It is an alkoxy.

[0070] As used herein, each "Q" is independently a bond, NR 4 Or -CH2.

[0071] As used herein, each "Z" is independently CH, O, or NR 4 It is.

[0072] As used herein, in some cases, R 1 , R 2 , R 3 or R 4 At least one of the comprises a water-solubilizing portion.

[0073] As used herein, each f is independently an integer of 0 to 50, 1 to 50, 2 to 40, or 5 to 20, and each n is independently an integer of 1 to 20.

[0074] The DHP-based monomer A may, for example, have the structure of formula (III): [ka] may have the following structure:

[0075] DHP monomers can be, for example, those having the structure of formula (IV): [ka] may have the following structure:

[0076] Monomer A in a polymer having the structure of formula I can be, for example, a structure of formula (Va) or (Vb) where X is C or N, respectively: [ka] The monomer may be a fluorene-based monomer or a carbazole-based monomer having the formula:

[0077] Monomer A may also be a cross-linking monomer. For example, the cross-linking monomer may have the structure of formula (VIa), (VIb) or (VIc): [ka] may have the following structure:

[0078] Monomer A in the polymer having the structure of formula I can be, for example, an oxepin-based monomer having the structure of formula (VIIa), (VIIb) or (VIIc) (e.g., a fluorenoxepin-based monomer): [ka] may be also possible.

[0079] Monomer A in the polymer having the structure of formula I may be a binaphthinyl monomer, as described in WO2022 / 013198, the entirety of which is incorporated herein by reference. The binaphthinyl-based monomer is represented by formula (VIId): [ka] may have the following structure:

[0080] Each optional M in a polymer having the structure of Formula I may be a polymeric modifying unit evenly or randomly distributed along the polymer chain and may include one or more optionally substituted R 1 , R 2 , R 3 or R 4 It may be optionally substituted with a group.

[0081] Each optional M may be an optionally substituted ethylene or ethynylene. Each M may be an optionally substituted ethylene moiety, i.e., a carbon-carbon double bond having the formula -CR=CR-, and each R is independently H, alkyl, alkenyl, alkynyl, cycloalkyl, haloalkyl, alkoxy, (hetero)aryloxy, aryl, (hetero)arylamino, a PEG group, an ammonium alkyl salt, an ammonium alkyloxy salt, an ammonium oligoether salt, a sulfonate alkyl salt, a sulfonate alkoxy salt, a sulfonate oligoether salt, a sulfonamide oligoether or moiety. [ka] Each M may be an ethynylene moiety, i.e., a carbon-carbon triple bond having the formula -C≡C-.

[0082] Each optional M may be evenly or randomly distributed along the polymer backbone. Each optional M may be a band gap modifying monomer. Each optional M may independently be any of the following: [ka] [ka] [ka] wherein M can each be substituted and terminated with a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, amide, sulfonamide, ether, thioether, thiocarbamate, hydroxyl, iodoacetyl, hydrazide, hydrazino, ketone, phosphine, epoxide, urea, thiourea, thioester, imine, disulfide, and protected groups thereof for conjugation to another substrate, acceptor dye, molecule, or binding partner.

[0083] As used herein, "R 5 " are independently H, C1~C 20 Alkyl, C2-C 20 Alkenyl, C2-C 20 Alkynyl, C3-C 20 Cycloalkyl, C1-C 20 Haloalkyl, C1-C 20 Alkoxy, C2-C 26 Aryloxy, C2-C 26 Heteroaryloxy, C2-C 26Arylamino or C2-C 26 It is heteroarylamino.

[0084] Each optional L in a polymer having the structure of formula I is a linker. Each L may be an aryl or heteroaryl group, evenly or randomly distributed along the polymer backbone. L may be an aryl or heteroaryl group, evenly or randomly distributed along the polymer backbone, optionally substituted with one or more pendant chains terminated with a functional group selected from the group consisting of amines, carbamates, carboxylic acids, carboxylates, maleimides, activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azides, alkynes, aldehydes, thiols, and protected groups thereof for conjugation to a substrate or binding partner. Each optional L is independently: [ka] [ka] may be also possible.

[0085] As used herein, R 6 are each independently H, OH, SH, NHCOO-t-butyl, (CH2) n COOH, (CH2) n COOCH3, (CH2) n NH2, (CH2) n NH-(CH2) n -CH3, (CH2) n NHCOOH, (CH2) n NHCO-(CH2) n -CO-(CH2) n -CH3, (CH2) n NHCOO-(CH2) n -CH3, (CH2) n NHCOOC(CH3)3, (CH2) n NHCO(C3~C 12) cycloalkyl, (CH2) n NHCO(CH2CH2O) f , (CH2) n NHCO(CH2) n COOH, (CH2) n NHCO(CH2) n COO(CH2) n CH3, (CH2) n (OCH2CH2) f OCH3, N-maleimide, halogen, C2~C 12 Alkenes, C2-C 12 Alkynes, C3-C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 (Hetero)aryl, C1-C 12 (Hetero)arylamino, benzyl (one or more halogens, hydroxyl, C1-C 12 Alkoxy or (OCH2CH2) f (optionally replaced by OCH3) [ka] It is.

[0086] G 1 and G 2Each of G may independently be hydrogen, halogen, alkyne, optionally substituted aryl, optionally substituted heteroaryl, halogen-substituted aryl, silyl, diazonium salt, triflate, acetyloxy, azide, sulfonate, phosphate, boronate-substituted aryl, boronate ester-substituted aryl, boronate ester, boronic acid, optionally substituted dihydrophenanthrene (DHP), optionally substituted tetrahydropyrene (THP), optionally substituted fluorene, or aryl or heteroaryl (substituted with one or more pendant chains terminated with a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol), and protected groups thereof, which may be conjugated to a substrate or binding partner. 1 and G 2 are each independently [ka] [ka] may be also possible.

[0087] Capping units may be conjugated to the polymer backbones of the present invention by mechanisms known in the art and as taught in U.S. Application Publication Nos. 2019 / 0144601 and 2020 / 0190253, both of which are incorporated herein in their entireties.

[0088] The polymeric dyes having the structure of formula (I) can utilize dihydrophenanthrene (DHP), fluorene, carbazole and / or binaphthyl monomers, and combinations of DHP, fluorene, carbazole and / or binaphthyl monomers. The polymeric dyes having the structure of formula (I) can utilize monomer A having a structure according to formula (II), where each "X" can be independently C, N or Si, and each "Y" can be independently CH2, CR 1 R 2 , SiR 1 R 2 Or it may be a bond. When Y is a bond, X is directly attached to both rings.

[0089] For example, the polymeric dye may have the structure of formula (VIII): [ka] may have the following structure:

[0090] The polymeric dye has the structure of formula (IX): [ka] may have the following structure:

[0091] In some cases, the polymer has a structure of formula (X): [ka] may have the following structure:

[0092] In some cases, the polymer has a structure of formula (XI): [ka] may have the following structure:

[0093] In some cases, the polymer is a copolymer and has the structure of formula (XII): [ka] has.

[0094] In some cases, the polymer is a copolymer and has the structure of formula (XIII): [ka] has.

[0095] In some cases, the polymer is a copolymer and has the structure of formula (XIV): [ka] It has During the ceremony, each B is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, band gap modifying monomers, optionally substituted ethylene and ethynylene; each E is an independently selected chromophore, functional moiety, or binding partner; the subscripts n and m are independently integers ranging from 1 to 10,000; The subscript p is an integer ranging from 0 to 10,000; the sum of the subscripts n, m and p is in the range of 2 to 10,000; Each t is an integer ranging from 1 to 20.

[0096] The polymers described herein can be characterized by a minimum number average molecular weight (Mn) of greater than 5,000 g / mol, greater than 10,000 g / mol, greater than 15,000 g / mol, greater than 20,000 g / mol, greater than 25,000 g / mol, greater than 30,000 g / mol, greater than 40,000 g / mol, greater than 50,000 g / mol, greater than 60,000 g / mol, greater than 70,000 g / mol, greater than 80,000 g / mol, greater than 90,000 g / mol, or greater than 100,000 g / mol.

[0097] The polymers described herein can be characterized by a minimum weight average molecular weight (Mw) of greater than 5,000 g / mol, greater than 10,000 g / mol, greater than 15,000 g / mol, greater than 20,000 g / mol, greater than 25,000 g / mol, greater than 30,000 g / mol, greater than 40,000 g / mol, greater than 50,000 g / mol, greater than 60,000 g / mol, greater than 70,000 g / mol, greater than 80,000 g / mol, greater than 90,000 g / mol, or greater than 100,000 g / mol.

[0098] The term "Mw" refers to weight average molecular weight and "Mn" refers to number average molecular weight. Number average and weight average molecular weight values ​​can be determined by gel permeation chromatography (GPC) using polymeric standards (e.g., polystyrene or similar materials).

[0099] Conjugated Polymer The polymer having the structure of formula (I) can be conjugated with functional groups, including acceptor dyes, quenching moieties and / or binding partners. Conjugation can occur at various positions of the polymer, such as monomers A, L, G1 or G2 in the polymer structure of formula (I).

[0100] For example, as disclosed in U.S. Application Publication No. 2019 / 0144601, the entirety of which is incorporated herein by reference, the acceptor dye can be attached to the polymer via a linker, L: [ka]

[0101] Tandem polymer dyes and other functionalized polymers can be prepared by post-polymerization modification of polymer intermediates, as described in U.S. Patent Publication No. 2020 / 0190253, the entirety of which is incorporated herein by reference. For example, a pendant solubilizing group having formula (XVI) may be attached to monomer A: [ka] Functionalized solubilizing groups according to formula (XVII): [ka] where W is a water-solubilizing moiety and L 1 and L 2 is a linking moiety. In some examples, the L1-W group having the structure of formula (XVI) is 1 or R 2 In some embodiments, each E is an independently selected functional moiety, such as a chromophore, a quenching moiety, or a binding partner. In some embodiments, each E is an independently selected chromophore (e.g., and an independently selected fluorophore). In some embodiments, all of the E moieties in the polymer have the same structure. In some embodiments, the E moieties in the polymer have different structures. The water-solubilizing moiety W in the groups according to formula (XVI) and formula (XVII) can be, for example, an ammonium alkyl salt, an ammonium alkyloxy salt, an ammonium oligoether salt, a sulfonate alkyl salt, a sulfonate alkoxy salt, a sulfonate oligoether salt, a sulfonamide oligoether, an oligo(ethylene glycol), or a poly(ethylene glycol).

[0102] Connecting part L 1 and L 2 are independently selected from the group consisting of, but not limited to, a covalent bond, C 1~8It may be an alkylene, 2-8 membered heteroalkylene. In some embodiments, the linker is a single atom, linear chain, branched chain, cyclic moiety. In some embodiments, the linker is a chain of 2-100 main chain atoms (e.g., carbon atoms) in length, such as between 2-50 main chain atoms in length, or between 2-20 main chain atoms in length. In certain cases, one, two, three, four, or five or more carbon atoms of the linker backbone may be optionally replaced by sulfur, nitrogen, or oxygen. The bonds between the main chain atoms may be saturated or unsaturated, and typically no more than one, two, or three unsaturated bonds are present in the linker backbone. The linker may include one or more substituents (e.g., alkyl or aryl groups). The linker may include, but is not limited to, oligo(ethylene glycol); ether; thioether; tertiary amine; and alkylene groups (i.e., divalent alkyl radicals), which may be linear or branched. The linker backbone can include a cyclic group, e.g., a divalent aryl radical, a divalent heterocyclic radical, or a divalent cycloalkyl radical, in which case two or more atoms of the cyclic group, e.g., 2, 3 or 4 atoms, are included in the backbone.

[0103] L 1 can include sulfonamides, sulfinamides, disulfonamides, disulfinamides, sultams, amides, secondary amines, phosphonamides, phosphinamides, phosphonamidates, selenone amides, or selenin amides. In some embodiments, L 1 In some such embodiments, L comprises a sulfonamide, an amide, a secondary amine, or a phosphonamide. 2 is a linear or branched, saturated or unsaturated C 1~30 Contains an alkylene group, C 1~30 One or more of the carbon atoms in an alkylene group may be O, S, NR a Replaced as necessary and independently by C 1~30 Any two or more groupings of adjacent carbon atoms in an alkylene may optionally and independently be represented by -NR a(CO)- or -(CO)NR a - has been replaced by R a are H and C, respectively. 1~6 alkyl.

[0104] The polymer is prepared in a first step by adding L in a pendant solubilizing group according to formula (XVI) 1 The internal position of the linker moiety L 2 and then in a second step, a dye or other functional group or binding partner E is covalently attached to the first end of the linker moiety L 2 In some embodiments, L can be functionalized by covalently attaching it to the second end of L. 1 The nitrogen atom in (e.g., the amide nitrogen, sulfonamide nitrogen, or phosphonamide nitrogen) may be linked to a linker moiety L 2 At the first end of the linker moiety L 2 In some embodiments, for example, the leaving group is a halogen (e.g., chloro, bromo, or iodo). In some embodiments, the leaving group is a sulfonate ester (i.e., -OS(O)2R, where R is alkyl, haloalkyl, aryl, or substituted aryl). Suitable sulfonate esters include, but are not limited to, mesylate (methanesulfonate), triflate (trifluoromethanesulfonate), besylate (benzene-sulfonate), tosylate (p-toluenesulfonate), and brosylate (4-bromobenzenesulfonate).

[0105] Any suitable solvent may be used for the alkylation step during functionalization of the polymer with E groups. Suitable solvents include, but are not limited to, toluene, methylene chloride, ethyl acetate, acetonitrile, tetrahydrofuran, benzene, chloroform, diethyl ether, dimethylformamide, dimethylsulfoxide, petroleum ether and mixtures thereof. The alkylation reaction is typically carried out at a temperature ranging from about 25° C. to about 100° C. by attaching a linking moiety L to one or more pendant groups in the polymer. 2or linked functional group -L 2 The reaction is carried out for a period of time sufficient to introduce -E. The reaction can be carried out for a period ranging from a few minutes to a few hours or longer, depending on the polymer and reagents used in the reaction. For example, the reaction can be carried out for about 10 minutes, or about 30 minutes, or about 1 hour, or about 2 hours, or about 4 hours, or about 8 hours, or about 12 hours, at about 40°C, or about 50°C, or about 60°C, or about 70°C, or about 80°C.

[0106] Connecting part L 2 The second terminus of can contain a functional group (e.g., an amine or a carboxylic acid) that is used in protected form during the first step (e.g., the alkylation step) and is then deprotected prior to covalently attaching the dye or other functional group or binding partner E to the second terminus of the linking moiety. Examples of amine protecting groups include, but are not limited to, benzyloxycarbonyl; 9-fluorenylmethyloxycarbonyl (Fmoc); tert-butyloxycarbonyl (Boc); allyloxycarbonyl (Alloc); p-toluenesulfonyl (Tos); 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc); 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf); mesityl-2-sulfonyl (Mts); 4-methoxy-2,3,6-trimethylphenylsulfonyl (Mtr); acetamide; phthalimide, and the like. Such and other protecting groups for amines, carboxylic acids, alcohols and further functional groups are described, for example, in Green and Wuts (Protective Groups in Organic Synthesis, 4 th Ed 2007, Wiley-Interscience, New York The polymers of the present disclosure can be added and removed using known techniques as described above.

[0107] Attachment of dyes, binding partners and functional groups, such as quenching moieties, can be accomplished using any suitable method. For example, an amide linkage can be attached to L2 and a carboxylate-functionalized dye. The dye may be used in an activated form, for example, EC(O)X' may be used as the reagent, where X' is a leaving group. Activated carboxylate-functionalizing reagents include, but are not limited to, acid anhydrides (including symmetric mixed acid anhydrides or cyclic anhydrides), activated esters (e.g., p-nitrophenyl esters, pentafluorophenyl esters, N-succinimidyl esters, etc.), acyl azoles (e.g., acylimidazoles prepared using carbonyldiimidazole, etc.), acyl azides, and acid halides (e.g., acid chlorides). Alternatively, a coupling agent may be used to couple L to L. 2An amide linkage may be formed between the deprotected primary amine group of and the carboxylate-functionalized chromophore EC(O)OH. A coupling agent may be used to form the activated dye reagent prior to reaction with the polymeric amine group. Any suitable coupling agent may be used. In some embodiments, the coupling agent is a carbodiimide, a guanidinium salt, a phosphonium salt, or a uronium salt. Examples of carbodiimides include, but are not limited to, N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), and the like. Examples of phosphonium salts include, but are not limited to, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP); bromotris(dimethylamino)phosphonium hexafluorophosphate (BroP), and the like. Examples of guanidinium / uronium salts include, but are not limited to, N,N,N',N'-tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU); O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyl-uronium hexafluorophosphate (HBTU); 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate (HATU); 1-[(1-(cyano-2-ethoxy-2-oxoethylidene-aminooxy)dimethylaminomorpholino)]uronium hexafluorophosphate (COMU), and the like. Solvents, reaction times and other reaction conditions can vary as described above, depending on factors such as the nature of the specific polymer and dye / functional group.

[0108] For example, the attachment of dyes, binding partners, functional groups and quenching moieties can be accomplished by the addition of a polymer according to formula (XVIIIa): [ka] a polymer according to formula (XVIII) [ka]

[0046] During the ceremony, each A is independently an aromatic or heteroaromatic comonomer; L1 a are covalent bonds, C 1~8 Alkylene, C 1~8 Alkoxy, 2-8 membered heteroalkylene, -NHC(O)L a -,-C(O)NHL a - and -C(O)L a - independently selected from the group consisting of: L 2 is a covalent bond, C 1~8 Alkylene, 2-8 membered heteroalkylene, -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 is C 1~8 independently selected from the group consisting of alkylene and 2-8 membered heteroalkylene; W is a water-solubilizing moiety; each E is an independently selected chromophore, functional moiety, quenching moiety, or binding partner; each B is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, band gap modifying monomers, optionally substituted ethylene and ethynylene; G 1 and G 2 is independently selected from an unmodified polymer end and a modified polymer end; R 8 is R as defined herein above. 1 , H or an amine protecting group; the subscripts n and m are independently integers ranging from 1 to 10,000; The subscript p is an integer ranging from 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 1 or 2, A and B are randomly or non-randomly distributed in the fluorescent polymer.

[0109] The conversion of a polymer of formula (XVIIIa) to a polymer according to formula (XVIII) may involve one or more alkylation steps or one or more amide formation steps, as described above.

[0110] In some embodiments, the polymer dye conjugate is a tandem dye conjugate according to formula (XIX): [ka] It is.

[0111] Any suitable chromophore or fluorophore can be used to functionalize the polymer. Generally, suitable chromophores and fluorophores can be covalently attached to a pendant solubilizing group (e.g., the linking moiety L described above). 2) and reactive groups (e.g., carboxylate moieties, amino moieties, haloalkyl moieties, etc. Examples of suitable chromophores and fluorophores include, but are not limited to, those described in U.S. Patents Nos. 7,687,282, 7,671,214, 7,446,202, 6,972,326, 6,716,979, 6,579,718, 6,562,632, 6,399,392, 6,316,267, 6,162,931, 6,130,101, 6,005,113, 6,004,5 36, 5,863,753, 5,846,737, 5,798,276, 5,723,218, 5,696,157, 5,658,751, 5,656,449, 5,582,977, 5,576,424, 5,573,909, and 5,187,288, which are incorporated herein by reference in their entirety. Polymeric dyes and monomers are also described in US2020 / 0190253, which is incorporated herein by reference in its entirety.

[0112] For example, E can be FITC, CY3B, Cy55, Alexa 488, Texas red, Cy5, Cy7, Alexa 750 or 800CW.

[0113] For example, the chromophore E may have the following structure: [ka] and the like, may be a boron-dipyrromethene moiety having the formula: During the ceremony, R 6a , R 6b , R 6c , R 6d , R 6e , R 6f and R 6g Six of them are H, halogens, and C. 1~6 Alkyl, C 3~8 Cycloalkyl, C 6~10 Aryl, C 7~16Aryl alkyl, C 1~6 R is independently selected from acyl and -SOH; 6a , R 6b , R 6c , R 6d , R 6e , R 6f and R 6g One of them is the linking moiety -L 2 -It is. 6a and R 6c are independently selected C 1~6 alkyl (e.g., methyl or ethyl), R 6e , R 6f and R 6g One of them is the linking moiety -L 2 In some embodiments, R 6a and R 6c is methyl, R 6g is the connecting part -L 2 -It is.

[0114] Chromophore E has the following structure: [ka] and the cyanine moiety may have the formula: During the ceremony, R 6h and R 6i , H, C 1~6 Alkyl, (CH2) t COOH, (CH2) t SO3H and linking moiety L 2 are independently selected from each subscript t is independently an integer from 1 to 10; R 6j and R 6k H, halogen, C 1~6 Alkyl, optionally substituted fused C 6~10 Aryl (e.g., optionally substituted benzo), -SO3H, -PO3H2, -OPO3H2, -COOH and linking moiety L 2 and Y is independently selected from O, S, C(R 6l )2, -CH=CH- and NR6l are independently selected from R 6l are each independently H or C 1~6 is alkyl, The subscript n is an integer from 1 to 6, where R 6h , R 6i , R 6j and R 6k Only one of the links -L 2 - This is subject to the condition that

[0115] Chromophore E has the following structure: [ka] and the coumarin moiety may have the formula: During the ceremony, W is N or CR 6p and Z is O, S or NR 6q and R 6m , R 6n , R 6o , R 6p are H, halogen, and C, respectively. 1~6 Alkyl, -CN, -CF3, -COOR 3v , -CON(R 3v )2, -OR 3v and the linking portion-L 2 - selected independently from R 6n -OR 3v and -N(R 3v )2 is selected, R 6q are H and C, respectively. 1~6 Alkyl and Linking Moiety-L 2 - independently selected from, where R 6m , R 6n , R 6o , R 6p and R 6q Only one of the linking moieties -L 2 - This is subject to the condition that

[0116] Chromophore E has the following structure: [ka] and the xanthene moiety has the formula: During the ceremony, T is O, S, C(R 6u )2 and NR 6u and U is selected from O or N(R 6u )2, R 6r are H, halogen, and C, respectively. 1~6 Alkyl, -SO3H and linking moiety -L 2 - selected independently from R 6s is H, -OH, -OR 6u , -N(R 6u ) 2 and the linking portion-L 2 - selected from R 6t , H, C 1~6 Alkyl, R 6v and the linking portion-L 2 - selected from R 6u are each independently H or C 1~6 is alkyl, R 6v teeth, [ka] is selected from During the ceremony, R 6w are H and the linking moiety -L, respectively. 2 - selected independently from However, R 6r , R 6s , R 6t and R 6v Only one of the linking moieties -L 2 - This is subject to the condition that

[0117] The xanthene moiety can be fluorescein, T and U are O, and R 6s is OH and R 6t teeth, [ka] It is.

[0118] The xanthene moiety can be eosin, where T and U are O and R 6s is OH and R 6r are each halogen (e.g., bromo), and R 6t teeth, [ka] It is.

[0119] The xanthene moiety can be rhodamine, T is O and U is N(R 6u )2 (e.g., =NH2 + ) and R is -N(R 6u )2 (e.g., -NH2), and R 6t teeth, [ka] It is.

[0120] The xanthene moiety has the following structure: [ka] and the rhodamine may be a rhodamine having the formula: In the formula, R 6v teeth, [ka] is selected from R 6w One of them is H and the other is R 6w is the connecting part -L 2 -It is.

[0121] In addition to chromophores, other functional moieties can be attached to functionalized polymers using the methods provided herein. For example, functional moiety "E" can be biotin, digoxigenin, a peptide tag (such as FLAG peptide), an oligonucleotide or a polynucleotide. As used herein, the term "FLAG peptide" refers to an oligopeptide or polypeptide that contains the amino acid sequence Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Asp-Lys (i.e., DYKDDDDK). FLAG peptides and variants thereof are described, for example, in U.S. Patent No. 4,703,004 to Hopp et al., which is incorporated herein by reference. Other peptides that can be used in place of the FLAG peptide include, but are not limited to, the HA peptide tag containing the sequence Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala (i.e., YPYDVPDYA), the His6 peptide tag containing the sequence His-His-His-His-His-His (i.e., HHHHHH), and the Myc peptide tag containing the sequence Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu (i.e., EQKLISEEDL). The peptide tag can be recognized by an antibody or other binding moiety for use with colorimetric reagents, chemiluminescent reagents, etc., for convenient identification and / or quantification. Nucleotides (e.g., RNA, single-stranded DNA, or double-stranded DNA) can be recognized by a complementary primer or other complementary nucleotides, for example, as described in WO2016 / 019929 (Navratil et al.), the publication of which is incorporated herein by reference. As used herein, the term "digoxigenin" refers to 3-[(3S,5R,8R,9S,10S,12R,13S,14S,17R)-3,12,14-trihydroxy-10,13-dimethyl-1,2,3,4,5,6,7,8,9,11,12,15,16,17-tetradecahydrocyclopenta[a]-phenanthren-17-yl]-2H-furan-5-one (CAS Registry Number 1672-46-4) and its substituted analogs.As used herein, the term "biotin" refers to 5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoic acid (CAS Registry Number 58-85-5) and its substituted analogs.

[0122] To synthesize binding partner-dye conjugates such as CD19-SN v428, CD20-SN v605, etc., polymeric dyes may be conjugated to binding partners of differing specificity, e.g., target analyte-specific antibodies.

[0123] As used herein, "binding partner" refers to any molecule or complex of molecules that can specifically bind to a target analyte. A binding partner can be, for example, a protein (e.g., an antibody or an 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, etc. In some embodiments, the binding partner is an antibody or a fragment thereof. In some embodiments, the binding partner is an antibody or an antigen-binding fragment thereof that specifically binds to the target analyte. Specific binding, in the context of the present invention, refers to a binding reaction that determines the presence of the target analyte in the presence of a heterogeneous population. Thus, under certain assay conditions, a designated binding partner preferentially binds to a particular protein, or isoform of a particular protein, and does not bind in significant amounts to other proteins or other isoforms present in the sample.

[0124] In some cases, the antibodies include intravenous immunoglobulin (IVIG) and / or antibodies derived from IVIG (e.g., enriched from IVIG, purified from IVIG, e.g., affinity purified from IVIG). IVIG is a blood product containing IgG (immunoglobulin G) pooled from plasma (e.g., in some cases free of any other proteins) from a large number (e.g., sometimes ranging from 1,000 to 60,000) of normal, healthy blood donors. IVIG is commercially available. Aspects of IVIG are described, for example, in U.S. Patent Application Publication Nos. 2010 / 0150942; 2004 / 0101909; 2013 / 0177574; 2013 / 0108619; and 2013 / 0011388, which are incorporated herein by reference.

[0125] When the binding partners are antibodies, they may be monoclonal or polyclonal antibodies. The term "antibody" as used herein refers to immunoglobulin molecules and, for example, immunologically active portions of immunoglobulin (Ig) molecules that specifically bind to an antigen in a target analyte. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monospecific polyclonal antibodies, antibody mimetics, chimeric, single chain, Fab, Fab' and F(ab')2 fragments, Fv and Fab expression libraries. In some cases, the antibodies are monoclonal antibodies 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 different subclasses. For example, the antibodies may be IgG1 antibodies. In some embodiments, the monoclonal antibodies are humanized. Antibody fragments can include molecules such as Fab, scFv, F(ab')2 and Fab' molecules. Antibody derivatives include antibodies or fragments thereof with additions or substitutions, such as chimeric antibodies. Antibodies can be derived from human or animal sources, from hybridomas, by recombinant methods, or any other method known in the art.

[0126] Binding partners other than antibodies or target analyte-specific antibody fragments or derivatives can also be used in the present systems and methods. For example, the binding partner can be a nucleic acid or a 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 a preselected target, 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 can also be used. Examples of such binding partner pairs include carbohydrates and lectins, biotin and avidin or streptavidin, folate and folate binding protein, vitamin B12 and intrinsic factor, protein A and immunoglobulins, and protein G and immunoglobulins. Similarly, binding partners that form covalent bonds with the target analyte are included.

[0127] The polymer dye conjugates can include any known polymer dye conjugated to a binding partner using techniques known to those of skill in the art. In some embodiments, the polymer dyes can be conjugated to a binding partner to form the polymer dye conjugates using the methods of direct modification of the core polymer described in U.S. Application Publication No. 2020 / 0190253, which is incorporated herein by reference in its entirety.

[0128] In some examples, the polymeric dye can be conjugated to a binding partner to form a polymeric dye conjugate using the methods described in U.S. Application Publication No. 2019 / 0144601, which is incorporated by reference in its entirety. The methods can be illustrated as follows: [ka]

[0129] Target Analysis The present disclosure also relates to a method for detecting a target analyte in a sample, the target analyte can be a substance, e.g., a molecule, including a target antigen, whose abundance / concentration is determined by some analytical procedure. The present invention is designed to detect the presence, and in some cases, the amount, of a specific target analyte. The term "target analyte" refers to a target molecule, e.g., peptides, proteins, polynucleotides, organic molecules, sugars and other carbohydrates, lipids and small molecules, that contains the target antigen to be detected in a biological sample. It is an important aspect of the present disclosure that the target analyte is contained in a liquid sample and is accessible or made accessible at some point in time to bind to the target analyte-specific binding partner of the present invention. The target analyte can be observed in a biological sample, such as a blood sample, a cell line growth sample, a tissue culture sample, etc.

[0130] The target analyte can be, for example, a nucleic acid (DNA, RNA, mRNA, tRNA or rRNA), a peptide, a polypeptide, a protein, a lipid, an ion, a monosaccharide, an oligosaccharide, a polysaccharide, a lipoprotein, a glycoprotein, a glycolipid, or a fragment thereof. The target analyte can be a protein, for example, structural microfilament, microtubule and intermediate filament proteins, organelle-specific markers, proteasomes, transmembrane proteins, surface receptors, nuclear pore proteins, protein / peptide translocases, protein folding chaperones, signaling scaffolds, ion channels, etc. The protein can be an activatable protein or a protein that is differentially expressed or activated in diseased or abnormal cells, including, but not limited to, transcription factors, DNA and / or RNA binding and modifying proteins, nuclear import and export receptors, regulators of apoptosis or survival, etc.

[0131] The target analyte can be present on the surface of the cell and can be accessible. Illustrative examples of useful analytes include, but are not limited to: 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 permeabilized cells, including abnormal DNA or RNA sequences, or abnormal amounts of specific messenger RNA. Detection of these analytes can be particularly useful in situations where the analyte is contained in and / or is an identifier of rare cells, such as in the early detection of various cancers.

[0132] In some examples, 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-alpha, IFN-beta, TNF-alpha, or ZAP-70, or other target analyte of interest.

[0133] Non-fluorescent components of polymer dyes The composition according to the present disclosure can include at least one or at least two non-fluorescent components of the polymer dye. The non-fluorescent components of the polymer dye can be selected from the group consisting of monomeric components of the polymer dye, non-fluorescent polymer dyes, photobleached polymer dyes, and polymer dyes that include a quenching moiety. In some embodiments, the non-fluorescent components of the polymer dye do not include a binding partner.

[0134] Monomer components of polymer dyes The compositions described herein include, among other components, monomeric components of the polymeric dyes described herein (also referred to herein as "monomers", e.g., Monomer A and / or Monomer B). The monomeric components of the polymeric dyes may be water-soluble monomers.

[0135] The water-soluble monomers may be monomeric units that contain an aryl or heteroaryl moiety, each of which optionally has a water-solubilizing moiety attached thereto. The water-solubilizing moieties may be one or more poly(ethylene glycol) moieties.

[0136] The monomer components of the polymeric dye may include dihydrophenanthrene (DHP)-based monomers, fluorene-based monomers and / or carbazole-based monomers. For example, the water-soluble monomer may be a monomer unit comprising an aryl or heteroaryl moiety, such as a monomer of the present disclosure having a structure according to (II), (III), (IV), (Va), (Vb), (VIa), (VIb), (VIc), (VIIa), (VIIb), (VIIc), (VIId), (XXII) and / or (VVIII), where both ends of the monomer represented by the wavy line are halogen atoms, boronic esters or acids, silyl, diazonium salts, triflates, acetyloxy, sulfonates, or phosphates that can undergo Pd or nickel salt catalyzed polymerization reactions, either independently or both. The DHP monomers of the present disclosure may be as taught in U.S. Application Publication No. 2019 / 0144601 and U.S. Application Publication No. 2020 / 0190253, both of which are incorporated herein by reference in their entirety. The monomeric components of the polymeric dye are dihydrophenanthrene (DHP) based monomers having a chemical structure according to formula (XXII): [ka] may be During the ceremony, G1, G2 are each independently selected from the group consisting of halo (F, Cl, Br, I), C1-C6 alkyl and PEG; R 2 , R 4 , R 5 , Z, n and f are each as defined herein above.

[0137] The monomeric components of the polymeric dyes are fluorene-based or carbazole-based monomers having a chemical structure according to formula (XXIII): [ka] may be During the ceremony, Each of G1 and G2 is independently selected from the group consisting of halo (F, Cl, Br, I), C1-C6 alkyl, and PEG; Each X is C, N or Si; R 2 , R 4 , R 5 , Z, n and f are each as defined herein above. In some embodiments, f is 1-50, 2-40, or 5-20. In some embodiments, n is 1-10, 2-5, or 3, respectively. In some embodiments, each m is 11-12.

[0138] As used herein, examples of monomeric components of polymeric dyes include the following: [ka] [ka] These are referred to as exemplary "Monomer A" and exemplary "Monomer B."

[0139] The various monomers A and B, and derivatives thereof, can be prepared by any suitable method known in the art, for example, as provided in U.S. Application Publication No. 2019 / 0144601 or U.S. Application Publication No. 2020 / 0190253, both of which are incorporated by reference herein in their entireties.

[0140] For example, 2,7-dibromo-trans-9,10-dihydrophenanthrene-9,10-diol (DHP-OH) can be prepared as follows: In a conical flask (2000 L), about 26 g of NaBH4 is added to a stirred water-ethanol mixture (120 mL + 780 mL). To this solution, about 24 g of 2,7-dibromophenanthrene, 9,10-dione is added in small portions but quickly (within 5 minutes). The reaction mix is ​​stirred for 1 day. By the end of the reaction, the color of the solution changes from red-orange to pale yellow to pale yellow to white. The reaction is stopped and the reaction mixture is neutralized with dilute HCl acid. After neutralization, the white precipitate is filtered and washed with excess water. The white precipitate thus obtained is washed with very cold (<-15°C) ethanol (100 mL) and methanol (100 mL).

[0141] DHP-O-alkyl-SO3H can be prepared as follows: In a two-neck round bottom flask, DHP-OH (3.6 g) and 18C6 (500 mg) were dissolved in 120 mL of THF. The solution was purged with nitrogen (20 min) and NaH (2 g) was added while the nitrogen purging continued. The color of the solution changed from colorless to light pink to dark pink to brown to dark green over 10-15 min. In a separate RB, 12 g of 1,3 propane sultone was dissolved in 20 mL of THF and purged with nitrogen. The sultone solution was added to the DHP-OH solution via a dropping funnel over 20-30 min. The reaction was stirred at room temperature for 4-5 h. The solvent was evaporated and the precipitate was dissolved in water. Acetone was added to give a white precipitate of DPS in the form of the disodium salt. The precipitate was filtered, redissolved in water (minimal amount), neutralized with HCl, and precipitated again in acetone. The precipitation was repeated (2-3 times) followed by centrifugation to give DPS as a white solid.

[0142] DHP-O-alkyl-SO2Cl can be prepared as follows: 5 g of DHP-O-alkyl-SO3H was taken in a round bottom flask and mixed with 25 mL of DMF. To this, about 10 mL of SOCl2 was added dropwise and the mixture was stirred overnight. The next morning, the reaction mixture was poured into 200 mL of water and the precipitate was filtered and dried.

[0143] DHP-sulfonamide PEG can be prepared as follows: DHP-O-alkyl-SO2Cl was mixed with 2.2 equivalents of PEG amine in dichloromethane / TEA mixture. After 3 hours of ultrasonication reaction, the crude product was extracted into dichloromethane and then subjected to column chromatography (silica gel, MeOH-CHCl3).

[0144] The diboronic ester of DHP-sulfonamide PEG can be prepared as follows: The dibromo compound was mixed with DMSO under nitrogen, to which 3 equivalents of bis-pinacolatodiboron were added. The reagent was reacted with 12 equivalents of potassium acetate and 4 equivalents of Pd(dppf)Cl2 catalyst at 80°C for 5 hours. The reaction mixture was cooled and extracted with CHCl3 / water. The organic layer was concentrated and purified by column chromatography (silica gel, MeOH-CHCl3).

[0145] Similarly, the fluorene monomers of the present disclosure can be made as described below. For example, FL-O-alkyl-SO3H can be prepared as follows: 5 g of fluorene was mixed in 70 DMSO in a two-neck round bottom flask. The solution was purged with nitrogen (20 min) and 50% NaOH (12 eq.) was added while the nitrogen purging continued. The solution changed color from colorless to dark brown. Propane sultone (3 eq.) was weighed and dissolved in DMSO. This was added dropwise to the fluorene reaction mixture over a period of 5 min. The reaction was stirred at room temperature for 4-5 hours. The solvent was evaporated and the precipitate was dissolved in water. Acetone was added to give a white precipitate of DPS in the form of the disodium salt. The precipitate was filtered, redissolved in water (minimal amount), neutralized with HCl, and precipitated again in acetone. The precipitation was repeated (2-3 times) followed by centrifugation to give FL-OSO3H as a white solid.

[0146] FL-O-alkyl-SO2Cl can be prepared as follows: 5 g of FL-O-alkyl-SO3H was taken in a round bottom flask and mixed with 25 mL of DMF. To this, about 10 mL of SOCl2 was added dropwise and the mixture was stirred overnight. The next morning, the reaction mixture was poured into 200 mL of water and the precipitate was filtered and dried.

[0147] FL-sulfonamide PEG can be prepared as follows: FL-O-alkyl-SO2Cl was mixed with 2.2 equivalents of PEG amine in dichloromethane / TEA mixture. After 3 hours of ultrasonication reaction, the crude product was extracted into dichloromethane and then subjected to column chromatography (silica gel, MeOH-CHCl3).

[0148] The diboronic ester of FL-sulfonamide PEG can be prepared as follows: The dibromo compound was mixed with DMSO under nitrogen, to which 3 equivalents of bis-pinacolatodiboron were added. These reagents were reacted with 12 equivalents of potassium acetate and 4 equivalents of Pd(dppf)Cl2 catalyst at 80°C for 5 hours. The reaction mixture was cooled and extracted with CHCl3 / water. The organic layer was concentrated and purified by column chromatography (silica gel, MeOH-CHCl3).

[0149] Adding monomer components of the polymer dye, such as Exemplary Monomer A and / or Exemplary Monomer B, at 100-400ug / test to a test staining buffer composition comprising a biological buffer and a non-ionic surfactant has been found to be highly efficient in reducing non-specific interactions between the polymer dye conjugates. The staining buffer composition of the present disclosure may comprise 10-500mg / mL, 20-400mg / mL or 30-300mg / mL of the monomer components of the fluorescent dye. A working concentration staining buffer composition (1x) may comprise 10-50mg / mL, 20-40mg / mL or about 30mg / mL of the monomer components of the fluorescent polymer dye. A concentrated concentration (10x) staining buffer composition may comprise 100-500mg / mL, 200-400mg / mL or about 300mg / mL of the monomer components of the fluorescent polymer dye. The compositions of the present disclosure can include monomeric components of the fluorescent polymer dye in an amount sufficient to provide 100-400 ug / test, 200-400 ug / test, or about 300 ug / test.

[0150] Quenched polymer dye The non-fluorescent component of the polymer dye may be a polymer dye that includes a quenching moiety, i.e., a quenched polymer dye ("quenched polymer"). The quenched polymer may include a polymer dye according to the present disclosure that includes one or more, or a number of, quenching moieties, e.g., 1-50, 2-25, or 5-8 quenching moieties. In some embodiments, the quenched polymer exhibits a quantum yield (QY) with a Φ of 0.1 or less, or 0.06 or less, or 0.056 or less, 0.05 or less, 0.02 or less, or 0.015 or less. The quenched polymer may exhibit a fluorescence profile of less than 50 AFU, less than 40 AFU, or 30 AFU when excited with a 405 nm laser. In some embodiments, the quenched polymer dye may exhibit >95% quenching of the original maximum emission intensity, >98% quenching of the original maximum emission intensity, up to 100% quenching when excited at 405 nm, compared to the parent fluorescent polymer.

[0151] In some embodiments, the quenched polymer dye emits ≦50 AFU at 450 nm at 10 micrograms / mL (slit excitation / emission is 6 nm / 4 nm; 1 cm cuvette). In some embodiments, the quenched polymer dye exhibits <47 AFU at 10 ug / mL when excited with a 405 nm laser using an AFU slit excitation / emission 6 nm / 4 nm on a Fluorometer LS50B Perkin Elmer.

[0152] The quenching moiety can be a non-fluorescent quenching moiety. The non-fluorescent quenching moiety can be a dark quencher, which can absorb excitation energy from a fluorophore and dissipate it as heat.

[0153] The quenching moiety may be selected from, for example, DABCYL, DABSYL, DYQ425 Black Hole Quencher 1 (BHQ1), QSY7, QSY9, QSY35, and TAMRA (carboxytetramethylrhodamine) moieties. Quenching moieties are commercially available, for example, as N-hydroxysuccinimide active esters (NHS esters), for conjugation to polymeric dyes, for example, from Thermo Scientific (e.g., DYQ425; DyLight 425Q NHS ester). Other quenching moieties are available as active esters, for example, Dabcyl Q, Dabcyl plus, Anaspec 490Q, Dyomics 425Q, Dynomics 505Q, etc. Preferably, the quenching moiety is capable of quenching fluorescence emission within the range of about 400 to 550 nm, about 480 to about 580 nm, or about 500 to about 600 nm. Examples of quenching moieties include, for example: [ka] may include.

[0154] For example, the quenched polymers of the present disclosure include a polymeric dye having a structure according to formula (I): [ka] You can use wherein each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers; each L is a linker moiety; each M is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, band gap modifying monomers, optionally substituted ethylene and ethynylene; and G 1 and G 2are independently selected from unmodified polymer ends and modified polymer ends; a, c, and d independently define the mol % of each unit in the structure, each of which may be repeated evenly or randomly; each a is a mol % of 10-100%, each c is a mol % of 0-90%, each d is a mol % of 0-25%, each b is independently 0 or 1; m is an integer from 1 to about 10,000; and the quenched polymeric dye exhibits a quantum yield (QY) of 0.1 or less, or 0.06 or less, or 0.056 or less.

[0155] In some embodiments, the quenched polymer dye (I) exhibits a quantum yield (QY) of 0.1 or less, or 0.06 or less, or 0.056 or less; optionally, a fluorescence profile of less than 50 AFU, less than 40 AFU, or less than 30 AFU when excited by a 405 nm laser. In some embodiments, the quenched polymer dye (I) may exhibit >95% quenching of the original maximum emission intensity, >98% quenching of the original maximum emission intensity, or up to 100% quenching when excited at 405 nm, compared to the parent fluorescent polymer. The term "parent fluorescent polymer" refers to a polymer dye that does not have a quenching moiety.

[0156] For example, the quenched polymer of the present disclosure can be, for example, a polymer having the formula (VIII): [ka] Dihydrophenanthrene (DHP), fluorene, carbazole, and combinations of DHP, carbazole and fluorene monomers can be utilized, as shown in

[0157] In the formula, G1, G2, R 1 , R 2, X, Y, M, L, a, b, c, d and m are as previously defined herein, and the polymer exhibits a quantum yield (QY) of 0.1 or less, or 0.06 or less, or 0.056 or less. In some embodiments, the quenched polymer dye (VIII) exhibits a fluorescence profile of less than 90 AFU, less than 50 AFU, less than 40 AFU, or less than 30 AFU when excited with a 405 nm laser. In some embodiments, the quenched polymer dye (VIII) may exhibit >95% quenching of the original maximum emission intensity, >98% quenching of the original maximum emission intensity, or up to 100% quenching when excited at 405 nm, compared to the parent fluorescent polymer.

[0158] The quenched polymer according to formula (VIII) is 1 , R 2 , L, G 1 or G 2 In the formula (I), the quenching moiety may comprise 1 to 50, 2 to 25, or 5 to 8 quenching moieties.

[0159] The quenched polymer may comprise a structure according to formula (XX), where the quenching moiety is L 2 It is connected to: [ka] In the formula, A, B, E, G 1 , G 2 , L 1 , L 2 , L 3 , W, n, m, p, q, r, s, and t are each defined herein above;

[0160] A and B are randomly or non-randomly distributed in the conjugated polymer, and the polymer exhibits a quantum yield (QY) of 0.1 or less, or 0.06 or less, or 0.056 or less. In some embodiments, the quenched polymer dye (XX) exhibits a fluorescence profile of less than 90 AFU, less than 50 AFU, less than 40 AFU, or less than 30 AFU when excited with a 405 nm laser. In some embodiments, the quenched polymer dye (XX) may exhibit >95% quenching of the original maximum emission intensity, >98% quenching of the original maximum emission intensity, or up to 100% quenching when excited at 405 nm, compared to the parent fluorescent polymer.

[0161] In some embodiments, each E may be an independently selected chromophore, functional moiety, or quenching moiety, and at least one or more E, or at least two or more E, are quenching moieties.

[0162] In some examples, the quenched polymeric dyes of the present disclosure can utilize polymeric dyes having a structure according to formula (I), (XVIII)-(XIV), (XVIII), (XIX), (XX) and / or (XXIV).

[0163] In some embodiments, the quenched polymer may comprise 2 to 20, 3 to 15, or 5 to 8 quenching moieties. In some embodiments, m=2 to 20, 3 to 15, or 5 to 8.

[0164] In some embodiments, the quenching moiety may be selected from a Dabcyl, Dabsyl, BHQ1, DYQ425, DYQ505, QSY7, QSY9, QSY35, or TAMRA quenching moiety.

[0165] In some embodiments, the quenched polymer does not comprise a binding partner.

[0166] The quenched polymer may be prepared according to any suitable method, for example, the quenching moiety is commercially obtained in the form of an active ester, such as an NHS ester, and exposed to the polymeric dye according to the methods of the present disclosure.

[0167] The staining buffer of the present disclosure may comprise 0.2-15 mg / mL, 0.3-12 mg / mL, or 0.5-10 mg / mL of the quenched polymer. In some embodiments, the working concentration (1×) staining buffer composition of the present disclosure may comprise 0.2-2.0 mg / mL, 0.3-1.5 mg / mL, 0.5-1.2 mg / mL, or about 1 mg / mL of the quenched polymer dye. In some embodiments, the concentrated staining buffer composition (10×) may comprise 3-15 mg / mL, 5-12 mg / mL, or about 10 mg / mL of the quenched polymer. The staining buffer composition of the present disclosure may comprise the quenched polymer dye in an amount sufficient to provide 2-20 ug / test, 3-15 ug / test, or about 10 ug / test.

[0168] Photobleached polymer dye The compositions described herein may include at least one photobleached polymeric dye having a structure according to the present disclosure. For example, the photobleached polymeric dye may include a structure according to any of Formulas I, VIII-XIV, XVIII, XIX and / or XX according to the present disclosure. Such compounds have been previously described in PCT Publication No. WO2017 / 180998 and U.S. Publication No. 2020 / 0190253(A1), which are incorporated by reference in their entirety as if fully set forth herein.

[0169] The term "photobleached dye" refers to a dye that originally contains a fluorophore that has been subjected to high intensity irradiation such that it can no longer fluoresce or exhibits a quantum yield (QY) of 0.1 or less. In some embodiments, the photobleached polymer exhibits a quantum yield (QY) of 0.1Φ or less, 0.06Φ or less, 0.056Φ or less, 0.02Φ or less, or 0.015Φ or less. In some embodiments, the photobleached polymer dye exhibits less than about 50 arbitrary units of fluorescence (AFU) when excited by a 405 nm laser. In some embodiments, the photobleached polymer dye may exhibit >95% quenching of the original maximum emission intensity, >98% quenching of the original maximum emission intensity, and up to 100% quenching when excited at 405 nm, compared to the parent fluorescent polymer. The term "parent fluorescent polymer" refers to the polymer dye prior to photobleaching.

[0170] Fluorophores can undergo the fluorescence process repeatedly. This means that a fluorophore molecule can theoretically generate a signal multiple times. In practice, due to the structural instability of the fluorophore during its excited lifetime, it can be prone to decomposition. High-intensity irradiation can cause the fluorophore to change its structure, so that it can no longer emit fluorescence, which is called photobleaching.

[0171] As used herein, the term "photobleached polymer dye" generally refers to a polymer dye, e.g., a violet-excitable polymer dye, that exhibits a quantum yield (QY) of 0.1 or less, or 0.06 or less, or 0.056 or less after being photobleached. In some embodiments, the photobleached dye exhibits an arbitrary fluorescence unit (AFU) of less than about 50, less than about 45 AFU, less than about 40 AFU, less than about 35 AFU, less than about 30 AFU, less than about 25 AFU, less than about 10 AFU, less than about 5 AFU, less than about 1 AFU, between about 1 AFU and about 50 AFU, between about 5 AFU and about 25 AFU, between about 20 AFU and about 40 AFU, between about 15 AFU and about 30 AFU, between about 15 AFU and about 40 AFU, or between about 20 AFU and about 36 AFU.

[0172] In some embodiments, the photobleached dye emits ≦50 AFU at 450 nm at 10 micrograms / mL (slit excitation / emission is 6 nm / 4 nm; 1 cm cuvette). In some embodiments, the photobleached polymer dye exhibits <47 AFU at 10 ug / mL when excited with a 405 nm laser using an AFU slit excitation / emission of 6 nm / 4 nm on a fluorometer LS50B Perkin Elmer. For example, the photobleached polymer dye may be a violet dye that originally contains a fluorophore that can no longer fluoresce or has a quantum yield (QY) of 0.1 or less, or 0.06 or less, or 0.056 or less; optionally, the fluorophore may be exposed to high intensity UV irradiation to exhibit less than about 50 arbitrary fluorescence units (AFU) when excited by a 405 nm laser. In some embodiments, the photobleached polymer dye may exhibit >95% quenching of the original maximum emission intensity, >98% quenching of the original maximum emission intensity, up to 100% quenching when excited at 405 nm, compared to the parent fluorescent polymer.

[0173] For example, the photobleached polymeric dyes of the present disclosure include polymeric dyes having a structure according to formula (I): [ka] You can use wherein each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers; each L is a linker moiety; each M is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, band gap modifying monomers, optionally substituted ethylene and ethynylene; and G 1 and G 2are independently selected from unmodified polymer ends and modified polymer ends; a, c, and d each independently define the mol % of each unit in the structure, which may be repeated evenly or randomly; each a is a mol % of 10-100%, each c is a mol % of 0-90%, each d is a mol % of 0-25%, each b is independently 0 or 1; m is an integer from 1 to about 10,000; and the photobleached polymer dye is 0.1 or less, or 0.06 or less. or a quantum yield (QY) of 0.056 or less, and optionally the photobleached polymer dye exhibits less than about 50 arbitrary units of fluorescence (AFU) when excited by a 405 nm laser, and / or optionally exhibits >95% quenching of the original maximum emission intensity, >98% quenching of the original maximum emission intensity, up to 100% quenching when excited at 405 nm, compared to the parent fluorescent polymer.

[0174] For example, the photobleached polymeric dye of the present disclosure can be, for example, a polymer having the formula (VIII): [ka] Dihydrophenanthrene (DHP), fluorene, and combinations of DHP and fluorene monomers can be utilized, as shown in wherein G1, G2, R1, R2, X, Y, M, L, a, b, c, d and m are as described herein above, and the photobleached polymer dye is prepared by exposing the fluorescent polymer dye according to formula (VIII) to irradiation with UV light so as to exhibit a quantum yield (QY) of 0.1 or less, or 0.06 or less, or 0.056 or less; optionally an arbitrary fluorescence unit (AFU) of less than about 50 when excited by a 405 nm laser, and or optionally a quenching of >95% of the original maximum emission intensity, a quenching of >98% of the original maximum emission intensity, up to 100% quenching when excited at 405 nm, compared to the parent fluorescent polymer.

[0175] For example, the photobleached polymeric dye of the present disclosure may be a polymeric dye having a structure according to formula (XX): [ka] You can use In the formula, A, B, E, G 1 , G 2 , L 1 , L 2 , L 3 , W, n, m, p, q, r, s, and t are each defined herein above; A and B are randomly or non-randomly distributed in the conjugated polymer, and the photobleached polymer dye is prepared by exposing a fluorescent polymer dye according to formula (XX) to irradiation with UV light such that the dye exhibits a quantum yield (QY) of 0.1 or less, or 0.06 or less, or 0.056 or less; optionally, an arbitrary fluorescence unit (AFU) of less than about 50 when excited by a 405 nm laser. In some embodiments, the photobleached polymer dye may exhibit >95% quenching of the original maximum emission intensity, >98% quenching of the original maximum emission intensity, up to 100% quenching when excited at 405 nm, compared to the parent fluorescent polymer.

[0176] For example, the photobleached polymer dyes of the present disclosure may utilize a polymer dye having a structure according to formula (XVIII), which is prepared by exposing a fluorescent polymer dye according to formula (XVIII) to irradiation with UV light such that the polymer dye exhibits a quantum yield (QY) of 0.1 or less, or 0.06 or less, or 0.056 or less, and optionally exhibits an arbitrary fluorescence unit (AFU) of less than about 50 when excited by a 405 nm laser, and optionally exhibits >95% quenching of the original maximum emission intensity, >98% quenching of the original maximum emission intensity, up to 100% quenching when excited at 405 nm, compared to the parent fluorescent polymer.

[0177] For example, the photobleached polymer dye of the present disclosure may be prepared from a fluorescent polymer dye, such as a violet-excitable fluorescent polymer dye, e.g., SuperNova™ ("SN") v428 (Beckman Coulter, Inc.), which is a fluorescent polymer dye optimally excited by a violet laser (405 nm), and the photobleached polymer dye is prepared by exposing the fluorescent polymer dye according to formula (I) to irradiation with UV light such that it exhibits an arbitrary unit of fluorescence (AFU) of less than about 50. In some embodiments, the photobleached dye does not include a binding partner.

[0178] The compositions of the present disclosure may include the photobleached dye at a working concentration (1×) of about 0.2-0.8 mg / mL, 0.3-0.7 mg / mL, or about 0.5 mg / mL. The compositions of the present disclosure may include the photobleached dye at about 2-8 mg / mL, 3-7 mg / mL, or about 5 mg / mL in a concentrated composition (10×). The compositions of the present disclosure may include the photobleached dye in the range of about 0.1 to about 10 mg / mL, 0.2-8 mg / mL, 0.3-7 mg / mL, or 0.5-5 mg / mL. The compositions of the present disclosure may include the photobleached dye in an amount sufficient to provide 2-8 ug / test, 3-7 ug / test, or about 5 ug / test.

[0179] Nonionic Surfactants The composition may include one or more non-ionic surfactants. A sufficient amount of non-ionic surfactant may be included to prevent aggregation of the polymer-dye conjugate. Non-limiting examples of non-ionic surfactants include poloxamer surfactants such as PLURONIC™ F-68 (PF-68), polysorbates including TWEEN® 20 and TWEEN® 80, and ether-linked non-ionic surfactants such as polyoxyethylene glycol alkyl ethers (BRIJ®), polyoxyethylene glycol octylphenol ethers (TRITON®) or polyoxyethylene nonylphenyl ethers (IGEPAL) surfactants. In some embodiments, the surfactant is a poloxamer non-ionic surfactant.

[0180] The term poloxamer nonionic surfactant refers to polyethylene oxide-polypropylene oxide-polyethylene oxide (PEG-PPG-PEG) nonionic triblock copolymers. The term poloxamer nonionic surfactant includes PLURONIC® nonionic surfactants. Examples of PLURONIC® surfactants include, for example, PLURONIC® F68, F77, F87, F98, F108, F127, P103, P104, P105 and P123.

[0181] In some embodiments, the surfactant is a polyoxypropylene-containing surfactant, such as a poloxamer surfactant. Poloxamer surfactants include non-ionic triblock copolymers, such as polyoxyethylene oxide-polyoxypropylene oxide-polyoxyethylene oxide (PEO-PPO-PEO), characterized in that a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) is sandwiched between two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). An exemplary non-ionic triblock copolymer has the structure according to formula (XXI): [ka] may include wherein each a is independently an integer ranging from 2 to 130, and b is an integer ranging from 15 to 67. In some embodiments, a is in the range of 50 to 100, and b is in the range of 20 to 40. In some embodiments, a is in the range of 70 to 90, and b is in the range of 25 to 30. The non-ionic surfactant may be poloxamer 188. Non-limiting examples of poloxamers may include poloxamer 188, also known as Pluronic® F-68 or KOLLIPHOR® P188, for example, having a=80 and b=27. Other poloxamers include poloxamer 338, also known as Synperonic™ PE / F108, poloxamer 407, also known as Synperonic™ PE / F127, and poloxamer 331, also known as Synperonic™ PE / L101.

[0182] The term "PLURONIC® F68", "Pluronic® F-68" or "PF-68", also known as Poloxamer 188, refers to a poly(ethylene glycol)-block-poly(propylene glycol)-block poly(ethylene glycol) copolymer having an average molecular weight or average Mn of 8350-8400.

[0183] The term "PLURONIC® F127", also known as Poloxamer 407, refers to a triblock copolymer consisting of a central hydrophobic block of polypropylene glycol flanked by two hydrophilic blocks of polyethylene glycol (PEG). The two PEG blocks are approximately 101 repeat units long, while the propylene glycol block is approximately 56 repeat units long. It is also known by the Croda trade name Synperonic PE / F127, with an average of 12,600 g / mol.

[0184] The term "PLURONIC® F108" refers to a poly(ethylene glycol)-block-poly(propylene glycol)-block poly(ethylene glycol) having an average Mn of about 14,600.

[0185] The term "PLURONIC® P103" refers to a poly(ethylene glycol)-block-poly(propylene glycol)-block poly(ethylene glycol) having an average Mw of about 4,950.

[0186] The term "PLURONIC® P104" refers to a poly(ethylene glycol)-block-poly(propylene glycol)-block poly(ethylene glycol) having an average Mw of about 5,900.

[0187] The term "PLURONIC® P123" refers to a poly(ethylene glycol)-block-poly(propylene glycol)-block poly(ethylene glycol) having an average Mn of about 5,800.

[0188] The length of the polymer block can be customized, so there are many different poloxamers with slightly different properties. Poloxamer copolymers are commonly named using the letter "P" (for poloxamer) followed by three digits, the first two digits x 100 indicate the approximate molecular mass of the polyoxypropylene core, and the last digit x 10 indicates the percentage of polyoxyethylene content (e.g., P407 = poloxamer with a polyoxypropylene molecular mass of 4,000 g / mol and 70% polyoxyethylene content). In the case of the trade names of Pluronic® and Synperonic poloxamers, the coding of these copolymers begins with a letter that defines their physical form at room temperature (L = liquid, P = paste, F = flake (solid)), followed by two or three digits. The first digit (or two digits in a three-digit number) multiplied by 300 in the numerical designation represents the approximate molecular weight of the hydrophobic chain, and the last digit x 10 indicates the percentage of polyoxyethylene content (e.g., F-68 indicates a polyoxypropylene molecular mass of 1,800 g / mol and a polyoxyethylene content of 80%). Exemplary poloxamer surfactants include, but are not limited to, Pluronic® F-68. PF-68 is a non-ionic triblock copolymer polyoxyethylene oxide-polyoxypropylene oxide-polyoxyethylene oxide (PEO-PPO-PEO). The concentration of surfactant used can be determined empirically (i.e., titrated to avoid precipitation of the conjugate). In some embodiments, the staining buffer composition may include a non-ionic surfactant, such as a poloxamer surfactant. The non-ionic surfactant may be Pluronic® F-68 (Poloxamer 188).The non-ionic surfactant may be present in the staining buffer composition at a working concentration (1×) of 0.01-10%, 0.02-8%, 0.05-7%, 0.1-5%, 0.2-2%, 0.1-0.4%, or about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (weight / volume), or any value in between. The non-ionic surfactant may be present in the dark stain buffer composition (10X) at 0.1-40%, 0.2-30%, 0.5-25% or 10-20% (weight / volume). In some embodiments, the stain buffer composition of the present disclosure may contain 0.01-40%, 0.01-20%, 0.02-10% (weight / volume) of the non-ionic surfactant.

[0189] biological buffer The term "biological buffer" refers to a physiologically compatible aqueous solution containing one or more biological buffering agents that maintains a pH in a biological range of 6 to 8, 6.5 to 8, or 7 to 8 in a cell-free system. In certain embodiments, the biological buffering agent is selected from the group consisting of N-(2-acetamido)-aminoethanesulfonic acid (ACES), acetate, N-(2-acetamido)-iminodiacetic acid (ADA), 2-aminoethanesulfonic acid (AES), ammonia, 2-amino-2-methyl-1-propanol (AMP), 2-amino-2-methyl-1,3-propanediol (AMPD), N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonate, among others. Acid (AMPSO), N,N-Bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), Bicarbonate, N,N'-Bis-(2-hydroxyethyl)-glycine, [Bis-(2-hydroxyethyl)-imino]-tris-(hydroxymethylmethane) (BIS-Tris), 1,3-Bis[tris(hydroxymethyl)-methylamino]propane (BIS-Tris-propane), Boric acid, Dimethylarsinic acid, 3-(Cyclohexylamino)-propanesulfonic acid (CAP) S), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO), carbonate, cyclohexylaminoethanesulfonic acid (CHES), citrate, 3-[N-bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), formate, glycine, glycylglycine, N-(2-hydroxyethyl)-piperazine-N'-ethanesulfonic acid (HEPES), lactate, N-(2-hydroxyethyl)-piperazine-N'-3-propanesulfonic acid Phenol acid (HEPPS, EPPS), N-(2-hydroxyethyl)-piperazine-N'-2-hydroxypropanesulfonic acid (HEPPSO), imidazole, malate, maleate, 2-(N-morpholino)-ethanesulfonic acid (MES), 3-(N-morpholino)-propanesulfonic acid (MOPS), 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO), phosphate, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), piperazine-N,N'-Bis(2-hydroxypropanesulfonic acid) (POPSO), pyridine, polyvinylpyrrolidone (PVP), succinate, 3-{[tris(hydroxymethyl)-methyl]-amino}-propanesulfonic acid (TAPS), 3-[N-tris(hydroxymethyl)-methylamino]-2-hydroxypropanesulfonic acid (TAPSO), 2-aminoethanesulfonic acid, AES (taurine), trehalose, triethanolamine (TEA), 2-[tris(hydroxymethyl)-methylamino]-ethanesulfonic acid (TES), N-[tris(hydroxymethyl)-methylamino]-ethanesulfonic acid Representative buffering agents may include one or more of the following: tyl]-glycine (tricine), tris(hydroxymethyl)-aminomethane (Tris), glyceraldehyde, mannose, glucosamine, mannoheptulose, sorbose-6-phosphate, trehalose-6-phosphate, iodoacetate, sodium citrate, sodium acetate, sodium phosphate, sodium tartrate, sodium succinate, sodium maleate, magnesium acetate, magnesium citrate, potassium phosphate, magnesium phosphate, ammonium acetate, ammonium citrate, ammonium phosphate. Representative buffering agents may 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, i.e., tromethamine hydrochloride, or phosphate. Specific examples of conventional biological buffers include phosphate buffered saline (PBS), N-2-hydroxyethylpiperazine-N'-2-hydroxypropanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), 2-([2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)ethanesulfonic acid (TES), 3-[N-tris(hydroxymethyl)ethylamino]-2-hydroxyethyl]-1-piperazinepropanesulfonic acid (EPPS), tris[hydroxymethyl]-aminomethane (THAM), 1,Buffers may include 4-piperazinediethanesulfonic acid (PIPES) and tris[hydroxymethyl]methylaminomethane (TRIS). Conventional biological buffers may have a pK in the physiological range and function most efficiently in this range. Biological buffers may be present in aqueous solution at concentrations of, for example, 10-100 mM or 5-25 mM.

[0190] The term "PBS" refers to phosphate buffered saline, an aqueous buffer that can contain sodium chloride, disodium hydrogen phosphate, potassium chloride, and / or potassium dihydrogen phosphate. For example, PBS can contain milliQ water or deionized water, and 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4. The pH can be about pH 7.0-7.4. PBS may or may not be stored with an azide, such as sodium azide. PBS may be an isotonic solution. The buffer can be a PBA buffer. The PBA buffer can include PBS, BSA, and sodium azide. The PBA buffer can include 1×PBS, about 2 mg / mL BSA, and about 0.1% sodium azide.

[0191] Additional Components The compositions of the present disclosure can be used, for example, as staining buffer compositions in flow cytometry sample analysis, and thus may include additional components, including, but not limited to, one or more of any suitable carriers, stabilizers, salts, chelating agents (e.g., EDTA), colorants, or preservatives. The compositions can also include one or more additional surfactants (e.g., ionic surfactants and zwitterionic surfactants).

[0192] The term "protein stabilizer" refers to a protein that acts to reduce non-specific binding, e.g., to reduce cell-cell interactions or to help prevent non-specific binding between an antibody and a non-target molecule. A composition according to the present disclosure may include a protein stabilizer. The protein stabilizer may be selected from one or more of the group consisting of serum albumin, e.g., bovine serum albumin (BSA), casein, or gelatin. The protein stabilizer may be BSA. In some embodiments, the protein stabilizer is present at 0.1-100 mg / mL, 0.2-50 mg / mL, 1-20 mg / mL, or 1-10 mg / mL. The protein stabilizer may be present in the working composition (1×) of the present disclosure at a concentration of 0.1-10 mg / mL, 0.5-5 mg / mL, 1-3 mg / mL, or about 2 mg / mL. The protein stabilizer may be present in concentrated compositions (10X) of the present disclosure at a concentration of 1-100 mg / mL, 5-50 mg / mL, 10-30 mg / mL, or about 20 mg / mL.

[0193] The carrier can be water, an aqueous solution such as saline, alcohol, or a biological buffer such as PBS, Hank's solution, Ringer's solution, or physiological saline buffer.

[0194] The carrier may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. For example, the staining buffer composition can contain a carrier such as water or a solvent, for example, DMSO as a solubilizing agent.

[0195] The compositions may also include suitable biological buffers and / or pH adjusting agents; typically the buffers are salts prepared from organic acids or bases.

[0196] The compositions of the present disclosure may include any suitable preservative. The preservative may be an antioxidant, a biocide, or an antimicrobial. The preservative may be an inorganic salt. For example, the preservative may be sodium azide, 2-chloroacetamide, 2-methylisothiazolinone, salicylic acid, ProClin™, Kathon™ CG, 5-chloro-2-methyl-4-isothiazolin-3-one, or 2-methyl-4-isothiazolin-3-one. The preservative may be present in the compositions of the present disclosure at 0.01-0.5%, 0.05-0.3%, or about 0.1%.

[0197] The composition of the present disclosure may include an additional surfactant. Suitable additional surfactants that may be used as needed in accordance with the methods described herein include zwitterionic surfactants such as betaines, such as alkyl betaines, alkyl amido betaines (amidazolinium betaines), amidazolinium betaines, sulfo betaines (INCI sultaines), and phospho betaines. Examples of suitable zwitterionic surfactants include those having the general formula R 1’ [CO-X(CH2) j ] g -N + (R 2’ )(R 3’ )-(CH2) f -[CH(OH)CH2] h -Y - wherein R 1’ is C 8~18 Alkyl, saturated C 10~16 Alkyl or saturated C 12~14 Saturated or unsaturated C such as alkyl 6~22 alkyl, and X is NH, NR 4’ and R 4’ is C 1~4 alkyl, O or S; j is an integer from 1 to 10, such as 2 to 5 and 3; g is 0 or 1; R 2’ and R 3’ are each independently 1~4alkyl, optionally hydroxy (substituted by hydroxyethyl or methyl); f is an integer from 1 to 4, such as 1, 2 or 3; h is 0 or 1; and Y is COO, SO3, OPO(OR 5’ )O or P(O)(OR 5’ )O and R 5’ is H or C 1~4 It is an alkyl.

[0198] Examples of suitable zwitterionic surfactants include those having the following 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 - These include alkyl betaines such as those listed below.

[0199] Examples of suitable betaines and sulfobetaines are given below (according to INCI): almondamidopropyl betaine, apricotamidopropyl betaine, avocadoamidopropyl betaine, babassuamidopropyl betaine, behenamidopropyl betaine, behenyl betaine, canolaamidopropyl betaine, capryl / capramidopropyl betaine, carnitine, cetyl betaine, cocamidoethyl betaine, cocamidopropyl betaine, cocamidopropyl hydroxysultaine, coco betaine, coco hydroxysultaine, Coco / Oleamidopropyl Betaine, Coco Sultaine, Decyl Betaine, Dihydroxyethyl Oleyl Glycinate, Dihydroxyethyl Soy Glycinate, Dihydroxyethyl Stearyl Glycinate, Dihydroxyethyl Tallow Glycinate, Dimethicone Propyl PG-Betaine, Dolucamidopropyl Hydroxysultaine, Hydrogenated Tallow Betaine, Isostearamidopropyl Betaine, Lauramidopropyl Betaine, Lauryl Betaine, Lauryl Hydroxysultaine, Lauryl Sultaine, Milkamidopropyl Betaine amidopropyl betaine, milkamidopropyl betaine, myristamidopropyl betaine, myristyl betaine, oleamidopropyl betaine, oleamidopropyl hydroxysultaine, oleyl betaine, olivamidpropyl betaine, palmamidopropyl betaine, palmitamidpropyl betaine, palmitoyl carnitine, palm kernelamidopropyl betaine, polytetrafluoroethylene acetoxypropyl betaine, ricinoleamidopropyl betaine, sesamidopropyl betaine, soyamidopropyl betaine, stearamidopropyl betaine, stearyl betaine, tallowamidopropyl betaine, tallowamidopropyl hydroxysultaine, tallow betaine, tallow dihydroxyethyl betaine, undecylenamidopropyl betaine and wheat germ amidopropyl betaine.

[0200] For example, coconut dimethyl betaine is commercially available from Seppic under the trade name AMONYL 265®, and lauryl betaine is commercially available from Sigma-Aldrich under the trade name EMPIGEN BB®. A further example betaine is lauryl-imino-dipropionate, sold by Rhodia under the trade name MIRATAINE H2C-HA®. The presence of an optional zwitterionic surfactant in the staining buffer composition can reduce non-specific binding in biological samples, for example, to monocytes. The optional zwitterionic surfactant can be present in the composition at 0-0.5%, 0.05-0.3%.

[0201] composition A staining buffer composition is provided for reducing polymer-polymer interactions between polymer-dye conjugates in a biological sample and for reducing precipitation of the dye conjugates. A staining buffer composition is provided for reducing polymer-polymer interactions between polymer-dye conjugates in a multicolor panel comprising two or more polymer-dye conjugates.

[0202] Compositions according to the present disclosure may be added to mixtures of dye conjugates, including one or more, two or more, or three or more polymer-dye conjugates, prior to, simultaneously with, or after addition to a biological sample to reduce, substantially reduce, and / or prevent non-specific binding, such as polymer-polymer interactions, between the dye conjugates.Mixtures of dye conjugates include, for example, fluorescein, coumarin, cyanine, and rhodamine dye conjugates, such as FITC (fluorescein isothiocyanate), PE (phycoerythrin), ECD (phycoerythrin-Texas Red®-X), PC5 (phycoerythrin-cyanine 5.5), PC5.5 (phycoerythrin-cyanine 5.5), PC7 (phycoerythrin-cyanine 7), APC (allophycocyanin), AA700, AA750, PBE, Alexa Fluor® 488 (AF488), AF532, AF647, AF700, AF750, Atlantis Bioscience CF® 350 dye, CF® 405S, CF® 405, CF® 405L, CF® 430, CF® 440, CF® 450, CF® 488A, CF® 514, AAT Bioquest iFluor™ 488, iFluor™ 350, iFluor™ 405, mFluor™ Blue 570, mFluor™ Blue 580, mFluor™ Blue 590, mFluor™ Blue 620, mFluor™ Blue 630, mFluor™ Blue 660, ThermoFisher Scientific NovaFluor Blue 510, NovaFluor Blue 530, NovaFluor Blue 555, NovaFluor Blue 585, NovaFluor Blue 610 / 30S, NovaFluor Blue 660 / 40S, NovaFluor The dyes or dye conjugates may include one or more, two or more, or three or more polymer dye conjugates, such as BioLegend® Kiravia Blue520™, KrO dye conjugates, and optionally one or more, two or more, three or more conventional fluorescent dye conjugates. Other dyes or dye conjugates may include Super Bright polymer dyes (Invitrogen, ThermoFisher Scientific).The Super Bright dye can be excited by a violet laser (405 nm). The Super Bright dye can be Super Bright 436 (excitation maximum 414 nm, emission maximum 436 nm, 450 / 50 band pass filter), Super Bright 600 (emission maximum 600 nm, 610 / 20 band pass filter), Super Bright 645 (emission maximum 645 nm, 660 / 20 band pass filter) or Super Bright 702 (emission maximum 702 nm, 710 / 50 band pass filter).

[0203] The present disclosure provides a composition comprising at least one non-fluorescent component of a first polymer dye; and a biological buffer. The composition can comprise at least one non-fluorescent component of a first polymer dye; a biological buffer; and a non-ionic surfactant. The non-ionic surfactant can be a poloxamer non-ionic surfactant. For example, the composition can comprise a non-fluorescent component of a first polymer dye selected from one or more of a monomer component of a polymer dye, a photobleached polymer dye, and a polymer dye comprising a quenching moiety.

[0204] A staining buffer composition is provided that includes a monomer component of a polymer dye, a photobleached polymer dye, and a biological buffer. A staining buffer composition is provided that includes a monomer component of a polymer dye, a photobleached polymer dye, a non-ionic surfactant, and a biological buffer. In some embodiments, a staining buffer composition is provided that includes 10-40 mg / mL of monomer A, 0.2-1.5 mg / mL of photobleached dye, and a working concentration (1x) of a biological buffer, and optionally includes a protein stabilizer and a preservative. In some embodiments, a staining buffer composition is provided that includes 10-40 mg / mL of monomer A, 0.2-1.5 mg / mL of photobleached dye, 0.01-10% (weight / volume) of non-ionic surfactant, and a working concentration (1x) of a biological buffer, and optionally includes a protein stabilizer and a preservative. In some embodiments, a staining buffer composition is provided that includes 20-40 mg / mL of monomer A, 0.2-0.8 mg / mL of photobleached dye, 0.01-4% (wt / vol) non-ionic surfactant, and a working concentration (1x) of biological buffer, optionally including a protein stabilizer and a preservative.

[0205] A staining buffer composition is provided that includes a monomer component of a polymer dye, a non-ionic surfactant, and a biological buffer. In some embodiments, a staining buffer composition is provided that includes 10-40 mg / mL of a monomer component of a polymer dye, 0.01-4% (weight / volume) of a non-ionic surfactant, and a working concentration (1×) of a biological buffer, and optionally includes a protein stabilizer and a preservative.

[0206] A dyeing buffer composition is provided that includes a polymeric dye that includes a quenching moiety, a biological buffer, and a non-ionic surfactant. A dyeing buffer composition is provided that includes a monomer component of a polymeric dye, a polymeric dye that includes a quenching moiety, a biological buffer, and a non-ionic surfactant. Optionally, the composition may include a protein stabilizer. Optionally, the composition may include a preservative.

[0207] In some embodiments, a staining buffer composition is provided that includes 10-40 mg / mL of monomer A, 0.2-1.5 mg / mL of quenched polymer, 0.01-4% (weight / volume) of a non-ionic surfactant and a biological buffer, and optionally includes a protein stabilizer and a preservative. In some embodiments, a staining buffer composition is provided that includes 20-40 mg / mL of monomer A, 0.5-1.5 mg / mL of quenched polymer, 0.01-1% (weight / volume) of a non-ionic surfactant and a biological buffer, and optionally includes a protein stabilizer and a preservative.

[0208] The dyeing buffer composition according to the present disclosure reduces, substantially reduces, or eliminates non-specific polymer-polymer interactions between fluorescent polymer dye conjugates when compared to the fluorescent polymer dye conjugates in the absence of the composition.The dyeing buffer composition according to the present disclosure reduces, substantially reduces, or eliminates non-specific polymer-polymer interactions of at least one fluorescent polymer dye conjugate when compared to the at least one fluorescent polymer dye conjugate in the absence of the composition.

[0209] method The present disclosure also relates to a method for detecting an analyte in a sample, comprising contacting a composition described herein with a sample suspected of containing the analyte. For example, a binding partner present in a polymer dye conjugate described herein can interact with the analyte to form a polymer dye conjugate complex with the analyte. A light source capable of exciting the polymer dye conjugate complex with the analyte is applied to the sample, and emission from the conjugated polymer conjugate is detected. In some embodiments, the polymer dye conjugates described herein can be excited using light having a wavelength within a range of about 340 nm to about 800 nm, between about 340 nm to about 450 nm (e.g., between about 395 nm to about 415 nm). In some embodiments, the emitted light is typically between about 400 nm to about 800 nm (e.g., between about 400 nm to about 500 nm or between about 415 nm to about 475 nm). Alternatively, the excitation light can have a wavelength between about 340 nm to about 370 nm, and the emitted light can be between about 390 nm to about 420 nm.

[0210] The sample in the disclosed method can be, for example, blood, bone marrow, spleen cells, lymphocytes, bone marrow aspirate (or any cell obtained from bone marrow), urine (lavage), serum, saliva, cerebrospinal fluid, urine, amniotic fluid, interstitial fluid, stool, mucus, or tissue (e.g., tumor sample, disaggregated tissue, disaggregated solid tumor). The sample can be a blood sample. The blood sample can be whole blood. Whole blood can be obtained from a subject using standard clinical procedures. The sample can be a subset of one or more cells of whole blood (e.g., red blood cells, white blood cells, lymphocytes (e.g., T cells, B cells, or NK cells), phagocytes, monocytes, macrophages, granulocytes, basophils, neutrophils, eosinophils, platelets, or any cell that contains one or more detectable markers). The whole blood sample can be a processed whole blood sample. The sample can be derived from a cell culture.

[0211] The subject may be a human (e.g., a patient suffering from a disease), a commercially important mammal (including, for example, a monkey, a cow or a horse). Samples may also be obtained from domestic pets, including, for example, a dog or a cat. The subject may be an experimental animal used as an animal model of a disease or an animal model for drug screening, such as a mouse, a rat, a rabbit or a guinea pig.

[0212] "Analyte," as used herein, refers to a substance, e.g., a molecule, whose abundance / concentration is determined by an analytical procedure. For example, in the present invention, an analyte can be a protein, peptide, nucleic acid, lipid, carbohydrate, or small molecule.

[0213] Assay systems that utilize binding partners and fluorescent labels to quantitate bound molecules are well known. Examples of such systems include flow cytometers, scanning cytometers, imaging cytometers, fluorescent microscopes, and confocal fluorescent microscopes.

[0214] Flow cytometry is used to detect fluorescence. Several devices suitable for such use are available and known to those skilled in the art. Examples include the BCI Navios, Gallios, Aquios and CytoFLEX flow cytometers.

[0215] The assay can be an immunoassay. Examples of immunoassays useful in the present invention include, but are not limited to, fluoroluminescence assays (FLA), etc. The assay can also be performed on a protein array.

[0216] When the binding partner is an antibody, an antibody or multiple antibody sandwich assay can also be used. Sandwich assay refers to the use of sequential recognition events that build layers of different binding partners and reporting elements to signal the presence of a specific analyte. Examples of sandwich assays are disclosed in U.S. Patent No. 4,486,530 and the references cited therein.

[0217] kit The present disclosure provides a kit comprising a staining buffer composition according to the present disclosure. The kit may comprise one or more containers comprising a staining buffer composition comprising one or more non-fluorescent components of a polymer dye and a biological buffer, and optionally one or more separate containers comprising one or more fluorescent polymer dye conjugates. The kit may comprise one or more non-fluorescent components of a first polymer dye and a non-ionic surfactant in one container; and at least one fluorescent polymer dye conjugate in another container. The kit may comprise two or more non-fluorescent components of a first polymer dye in one container; and at least one fluorescent polymer dye conjugate in another container. The kit may comprise one or more containers comprising a staining buffer according to the present disclosure, and a plurality of separate containers, each of which comprises a different polymer dye conjugate.

[0218] The kit may include one or more components suitable for lysing cells. One or more additional components of the kit may be supplied in separate containers (e.g., separate tubes, bottles, or wells in a multi-well strip or plate).

[0219] The kit may also include one or more cell fixation reagents, such as paraformaldehyde, glutaraldehyde, methanol, acetone, formalin, or any combination or buffer thereof. Additionally, the kit may include a cell permeabilizing agent, such as methanol, acetone, or a detergent, such as triton®, NP-40, saponin, tween® 20, digitonin, leucoperm, or any combination or buffer thereof. The kit may include instructions for using the staining buffer composition of the present disclosure, for example in conjunction with a multicolor panel of polymer-dye conjugates. The instructions may be in printed form, on the kit packaging, in a insert, or at a website address.

[0220] definition The abbreviations used herein have their conventional meaning within the chemical and biological arts.

[0221] As used herein, the term "ammonium" refers to a cation having the formula NHR3+, where each R group is independently hydrogen, or a substituted or unsubstituted alkyl, aryl, aralkyl, or alkoxy group. Preferably, each R group is hydrogen.

[0222] As used herein, "oligoether" is understood to mean an oligomer that includes structural repeat units having ether functional groups. As used herein, "oligomer" is understood to mean a molecule that includes one or more identifiable structural repeat units of the same or different formula.

[0223] The term "sulfonate functional group" or "sulfonate," as used herein, refers to both the free sulfonate anion (-S(=O)2O-) and its salts. Thus, the term sulfonate encompasses sulfonate salts such as sodium sulfonate, lithium sulfonate, potassium sulfonate, and ammonium sulfonate.

[0224] The term "sulfonamide," as used herein, refers to a group of formula -SO2NR-, where R is hydrogen, alkyl, or aryl.

[0225] The term "alkyl" as used herein refers to a straight 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 can contain any number of carbons, such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6, and 5-6. Alkyl groups are usually monovalent, but can be divalent, such as when an alkyl group is a group of two moieties linked together.

[0226] The term "comonomer" or "comonomer group" refers to a structural unit of a polymer that may itself be part of a repeat unit of the polymer.

[0227] The term "cycloalkyl" as used herein refers to a saturated or partially unsaturated monocyclic, fused bicyclic, or bridged polycyclic ring assembly containing 3 to 12 ring atoms or the number of atoms indicated, where monocyclic rings include, 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.

[0228] The term "haloalkyl" as used herein refers to an alkyl as defined above in which some or all of the hydrogen atoms are replaced by halogen atoms. Halogen (halo) preferably represents chloro or fluoro, but may also be bromo or iodo. For example, haloalkyl includes trifluoromethyl, flouromethyl, 1,2,3,4,5-pentafluoro-phenyl, etc. The term "perfluoro" defines a compound or radical in which at least two available hydrogens are replaced by 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.

[0229] As used herein, the term "halogen" refers to fluorine, chlorine, bromine and iodine.

[0230] The term "alkoxy" as used herein refers to an alkyl group as defined above with an oxygen atom connecting the alkyl group to the point of attachment. Alkoxy groups include, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, and the like. Alkoxy groups can be further substituted with various substituents as described herein. For example, alkoxy groups can be substituted with halogen to form "halo-alkoxy" groups.

[0231] The term "alkene" as used herein refers to either a straight-chain or branched hydrocarbon having at least one double bond. Examples of alkene groups include, but are not limited to, vinyl, propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl. Alkene groups are usually monovalent, but can be divalent, such as when an alkenyl group links two moieties together.

[0232] The term "alkyne" as used herein refers to either a straight-chain or branched hydrocarbon having at least one triple bond. Examples of alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl. Alkynyl groups are usually monovalent, but can be bivalent, such as when the alkynyl group links two moieties together.

[0233] The term "aryl" or "aromatic" as used herein refers to a monocyclic or fused bicyclic, tricyclic or higher cyclic aromatic ring assembly containing 6 to 16 ring carbon atoms. For example, aryl can be phenyl, benzyl, naphthyl, dihydrophenanthrenyl (DHP), 9,10-dihydrophenanthrenyl or fluorenyl. "Arylene" means a divalent radical derived from an aryl group. The aryl group can be mono-, di- or tri-substituted with one, two or three radicals selected from alkyl, alkoxy, aryl, hydroxy, halogen, cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy and oxy-C2-C3-alkylene, all of which are optionally further substituted, e.g., as defined hereinbefore, or 1- or 2-naphthyl; or 1- or 2-phenanthrenyl. Alkylenedioxy is a divalent substitute attached to two adjacent carbon atoms of phenyl, such as methylenedioxy or ethylenedioxy. Oxy-C2-C3-alkylene is also a divalent substitute attached to two adjacent carbon atoms of phenyl, such as oxyethylene or oxypropylene. An example of oxy-C2-C3-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

[0234] Preferred as aryl are dihydrophenanthrenyl (DHP), 9,10-dihydrophenanthrenyl, naphthyl, phenyl or phenyl mono- or di-substituted by alkoxy, phenyl, halogen, alkyl or trifluoromethyl, especially phenyl or phenyl mono- or di-substituted by alkoxy, halogen or trifluoromethyl, and especially phenyl.

[0235] The term "aryloxy" as used herein refers to an O-aryl group, where aryl is as defined above. The aryloxy group can be unsubstituted or substituted with one or two suitable substituents. The term "phenoxy" refers to an aryloxy group, where the aryl moiety is a phenyl ring. The term "heteroaryloxy" as used herein refers to an -O-heteroaryl group, where heteroaryl is as defined below. The term "(hetero)aryloxy" is used to indicate that the moiety is either an aryloxy group or a heteroaryloxy group.

[0236] The term "AFU" refers to arbitrary fluorescence units.

[0237] The term "AUF" refers to arbitrary units of fluorescence.

[0238] The term "polyethylene glycol" or "PEG," as used herein, refers to a family of biocompatible, water-solubilizing linear polymers based on ethylene glycol monomer units.

[0239] The term "heteroaryl" or "heteroaromatic" as used herein refers to a monocyclic or fused bicyclic or tricyclic heteroaromatic ring assembly containing 5 to 16 ring atoms, with 1 to 4 of the ring atoms being heteroatoms, each of which is N, O or S. For example, heteroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, carbazolyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any other radical substituted, especially mono- or di-substituted, for example by alkyl, nitro or halogen. Pyridyl represents 2-, 3- or 4-pyridyl, advantageously 2- or 3-pyridyl. Thienyl represents 2- or 3-thienyl. Quinolinyl preferably represents 2-, 3- or 4-quinolinyl. Isoquinolinyl preferably represents 1-, 3- or 4-isoquinolinyl. Benzopyranyl, benzothiopyranyl preferably represent 3-benzopyranyl or 3-benzothiopyranyl, respectively. Thiazolyl preferably represents 2- or 4-thiazolyl, and most preferably 4-thiazolyl. Triazolyl is preferably 1-, 2- or 5-(1,2,4-triazolyl). Tetrazolyl is preferably 5-tetrazolyl.

[0240] Preferably, heteroaryl is pyridyl, indolyl, quinolinyl, pyrrolyl, thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl, benzothienyl, oxazolyl, indazolyl, or any of these radicals which are substituted, especially mono- or di-substituted.

[0241] Similarly, substituents on the aryl and heteroaryl groups are varied and include -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(N H2)=NR', -S(O)R', -S(O)2R', -S(O)2NR'R", -N3, -CH(Ph)2, perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl, where R', R" and R''' are independently selected from hydrogen, (C1-C5)alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4)alkyl and (unsubstituted aryl)oxy-(C1-C4)alkyl.

[0242] Two of the substituents on adjacent atoms of an aryl or heteroaryl ring may optionally be of the formula -TC(O)-(CH2) q Each of the substituents on adjacent atoms of the aryl or heteroaryl ring may be replaced by a substituent of formula -U-, where T and U are independently -NH-, -O-, -CH2- or a single bond, and q is an integer from 0 to 2. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may be replaced by a substituent of formula -A-(CH2) r A and B are independently -CH2-, -O-, -NH-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'- or a single bond, and r is an integer from 1 to 3. One of the single bonds of the new ring thus formed may be optionally replaced by a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may be of the formula -(CH2) s -X-(CH2) twhere s and t are independently an integer of 0 to 3, and X is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. The substituent R' in -NR'- and -S(O)2NR'- is selected from hydrogen or unsubstituted (C1 to C6) alkyl.

[0243] The term "(hetero)arylamino" as used herein refers to an amine radical substituted with an aryl group (e.g., -NH-aryl). Arylamino may also be an aryl radical substituted with an amine group (e.g., -aryl-NH2). Arylamino may be substituted or unsubstituted.

[0244] The term "amine" as used herein refers to an alkyl group, as defined herein, having one or more amino groups. The amino group can be primary, secondary or tertiary. The alkylamine can be further substituted with a hydroxy group. Amines useful in the present invention include, but are not limited to, ethylamine, propylamine, isopropylamine, ethylenediamine and ethanolamine. The amino group can link the alkylamine to the point of attachment to the remainder of the compound, can be in the omega position of the alkyl group, or can link at least two carbon atoms of the alkyl group together. Those skilled in the art will appreciate that other alkylamines are useful in the present invention.

[0245] The term "carbamate," as used herein, refers to a functional group having the structure -NR"CO2R', where R' and R" are independently selected from hydrogen, (C1-C8)alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4)alkyl, and (unsubstituted aryl)oxy-(C1-C4)alkyl. Examples of carbamates include t-Boc, Fmoc, benzyloxy-carbonyl, alloc, methyl carbamate, ethyl carbamate, 9-(2-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.

[0246] The term "carboxylate" as used herein refers to the conjugate base of a carboxylic acid, which can generally be represented by the formula RCOO. For example, the term "magnesium carboxylate" refers to the magnesium salt of a carboxylic acid.

[0247] The term "activated ester" as used herein refers to a carboxyl active group used in peptide chemistry to facilitate the easy condensation of the carboxyl group of an amino acid derivative with a free amino group. Descriptions of these carboxyl active groups can be found in the general texts of peptide chemistry, such as KD Kopple, "Peptides and Amino Acids", WA Benjamin, Inc., New York, 1966, pp. 50-51 and E. Schroder and K. Lubke, "The Peptides"; Vol. 1, Academic Press, New York, 1965, pp. 77-128.

[0248] The terms "hydrazine" and "hydrazide" refer to a compound containing single-bonded nitrogens, one of which is a primary amine functional group.

[0249] The term "aldehyde," as used herein, refers to a chemical compound that has a --CHO group.

[0250] The term "thiol" as used herein refers to a compound that contains a functional group consisting of a sulfur-hydrogen bond. The general chemical structure of a thiol functional group is R-SH, where R represents an alkyl, alkene, aryl, or other carbon-containing group.

[0251] The term "silyl" as used herein means Si(R z )3 refers to 、 R z Each is independently alkyl, aryl or other carbon-containing group.

[0252] The term "diazonium salt" as used herein means a diazonium salt represented by R-N2 + X - where R can be any organic residue (e.g., alkyl or aryl) and X is an inorganic or organic anion (e.g., a halogen).

[0253] The term "triflate," also called trifluoromethanesulfonate, is a group having the formula CF3SO3.

[0254] The term "boronic acid" as used herein refers to the structure -B(OH). It will be appreciated by those skilled in the art that boronic acids may be present as boronic esters at various stages in the synthesis of a quencher. Boronic acids are intended to include such esters. The term "boronic ester" or "boronate ester" as used herein refers to the structure -B(Z 1 )(Z 2 ) moiety, Z 1 and Z2 taken together form a moiety in which the atom bonded to boron in each case is an oxygen atom. The boronic ester moiety can be a 5-membered ring. The boronic ester moiety can be a 6-membered ring. The boronic ester moiety can be a mixture of 5- and 6-membered rings.

[0255] The term "DABCYL" is an acronym for 4-(dimethylaminoazo)benzene-4-carboxylic acid. DABCYL may be used as a quenching moiety. DABCYL has an absorption maximum at about 474 nm.

[0256] The term "DABSYL" refers to 4-(dimethylaminoazo)benzene-4"-sulfonyl chloride. DABSYL may be used as a quenching moiety.

[0257] The term "Black Hole Quencher 1" (BHQ-1) refers to a quenching moiety that has an absorption maximum at about 534 nm.

[0258] Values ​​expressed in range format should be interpreted flexibly to include not only the numerical values ​​explicitly stated as the boundaries of the range, but also all of the individual numerical values ​​or subranges subsumed within the range as if each numerical value and subrange were explicitly stated. For example, the range "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not only about 0.1% to about 5%, but also the individual values ​​(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%) within the indicated range. The description "about X to Y" has the same meaning as "about X to about Y" unless otherwise indicated. Similarly, the description "about X, Y or about Z" has the same meaning as "about X, about Y or about Z" unless otherwise indicated.

[0259] The term "CD" refers to cluster of differentiation.

[0260] The term "compensation" in flow cytometry refers to the mathematical process of correcting for fluorescence spillover (spectral overlap in multiparameter flow cytometry data). For example, compensation can be performed by removing any signal of a given fluorochrome from all detectors except the detector intended for measurement of such dye. Because fluorochromes can have a wide range of spectra, they can overlap and cause undesirable confusion during data analysis.

[0261] The term "MdFl" or "MDFl" refers to median fluorescence intensity.

[0262] The term MFI = mean fluorescence intensity.

[0263] The term "% recruitment" refers to the gated cell numbers of the relevant population.

[0264] The term "multicolor dye conjugate panel" or "multicolor antibody panel" refers to a cocktail containing multiple different fluorochrome conjugates (e.g., CD4-FITC, CD8-PE, CD20-APC, CD3-PC5.5, CD16-FITC, CD25-PE, CD3-ECD, CD38-PC5.5, CD27-PC7, CD10-APC, CD14-APCA700, CD45-AA750, CD8-KRO, CD56-SNv428, CD20-SNv605, CD4-SNv786, etc.) that can be used directly to stain blood and analyze it on a flow cytometer.

[0265] The term "multiplexed," as used herein, refers to an assay or other analytical method in which multiple analytes can be assayed simultaneously.

[0266] Water-solubilizing moieties can be included in polymeric dyes to provide increased water solubility. The increase in solubility can vary, but in some cases the increase can be at least 2-fold or more, such as 5-fold, 10-fold, 25-fold, 50-fold, 100-fold or more, compared to a polymeric dye that does not include the water-solubilizing moiety.

[0267] The term "water-solubilizing moiety" refers to a group that is sufficiently solvated in an aqueous environment, e.g., under physiological conditions, and that confers improved water solubility to the molecule to which it is attached. The water-solubilizing moiety can be any suitable hydrophilic group that is sufficiently solvated in an aqueous environment. In some cases, the hydrophilic water-solubilizing group is charged, e.g., positively or negatively charged. In certain cases, the hydrophilic water-solubilizing group is a neutral hydrophilic group. In some embodiments, the water-solubilizing moiety is a hydrophilic polymer, e.g., polyethylene glycol, cellulose, chitosan, or derivatives thereof. Water solubilizing moieties can include, but are not limited to, carboxylates, phosphonates, phosphates, sulfonates, sulfates, sulfinates, sulfonium, esters, polyethylene glycols (PEGs) and modified PEGs, hydroxyls, amines, ammonium, guanidinium, pyridinium, polyamines and sulfonium, polyalcohols, linear or cyclic saccharides, primary, secondary, tertiary or quaternary amines and polyamines, phosphonate groups, phosphinate groups, ascorbate groups, glycols. In some embodiments, the water solubilizing moiety is PEG.

[0268] The term "PEG" refers to a PEG having the formula -(CH2-CH2-O-) n- or its derivatives. The water-solubilizing moiety can provide a solubility in water of at least 10 mg / mL. PEG moieties may be used as water-solubilizing moieties. In some embodiments, "n" is 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less (such as 3-15 or 10-15). It is understood that the PEG polymer group may be of any convenient length and may include a variety of end groups and / or further substituents, including but not limited to alkyl, aryl, hydroxyl, amino, acyl, carboxylic acid, carboxylic ester, acyloxy, and amide end groups and / or substituents. The number following "PEG" refers to the average molecular weight, Mw refers to the weight average molecular weight, and Mn refers to the number average molecular weight.

[0269] The term "non-fluorescent component of the polymer dye" refers to a monomer unit of the polymer dye, a photobleached polymer dye, a polymer dye containing a quenching moiety, or a non-fluorescent polymer dye, where the non-fluorescent component of the polymer dye exhibits little or no ability to re-emit upon photoexcitation. The non-fluorescent component of the polymer dye may exhibit a quantum yield of about 0.1 or less, 0.06 or less, or 0.056 or less. The non-fluorescent component of the polymer dye may emit ≦50 AFU at 450 nm (slit excitation / emission is 6 nm / 4 nm; 1 cm cuvette) for 10 micrograms / mL. The non-fluorescent component of the polymer dye may have less than about 50 AFU when excited by a 405 nm laser. The non-fluorescent component of the polymer dye may exhibit >95% quenching of the original maximum emission intensity, >98% quenching of the original maximum emission intensity, up to 100% quenching when excited at 405 nm, compared to the parent fluorescent polymer.

[0270] The term "non-fluorescent polymer dye" refers to a polymer dye according to formula (I) that exhibits a quantum yield of about 0.1, 0.06, or 0.056 or less without photobleaching, and does not include a quenching moiety. The non-fluorescent polymer dye may have less than about 50 AFU when excited by a 405 nm laser. The non-fluorescent polymer dye may exhibit >95% quenching of the original maximum emission intensity, >98% quenching of the original maximum emission intensity, or up to 100% quenching when excited at 405 nm, compared to the parent fluorescent polymer.

[0271] The term "non-specific binding", as used herein, generally refers to any binding that is not caused by specific binding, and more specifically refers to the binding of a polymer-dye conjugate by means other than the specific binding of the binding partner to the target analyte. Non-specific binding can be due to several factors, including the hydrophobicity of the polymer, immunocomplexing agents, charged proteins, and antibody-interfering proteins that may be present in the staining buffer or biological sample. One type of non-specific binding is polymer-polymer interactions that may occur between one or more, or two or more fluorescent polymer-dye conjugates. Non-specific binding in a test staining buffer composition can be evaluated, for example, according to the methods provided herein, by comparing the FCA dot plot of a mixture of multi-color fluorescent polymer-dye conjugates in a biological sample with the FCA dot plot of individual single-color fluorescent polymer-dye conjugates of the mixture in the same sample. For example, if a solution is highly efficient at blocking non-specific binding polymer-polymer interactions, individual cell populations will appear to be well compensated for staining similar to that obtained with the single-color conjugates used individually. In contrast, if the solution is inefficient, the population will appear misaligned and tilted.

[0272] An alternative method of measuring the efficiency of a staining buffer composition according to the present disclosure to reduce non-specific binding, such as polymer-polymer interactions, uses the MFI of the negative and positive populations of conjugates when they are used individually and in mixtures.

[0273] The term "photobleached dye" refers to a dye that originally contains a fluorophore that has been subjected to high-intensity irradiation so that it can no longer fluoresce. In some embodiments, the photobleached polymer exhibits a quantum yield (QY) of 0.1Φ or less, or 0.06Φ or less, or 0.056Φ or less, 0.05Φ or less, 0.02Φ or less, or 0.015Φ or less. In some embodiments, the photobleached polymer exhibits an arbitrary unit of fluorescence (AFU) of less than about 50 when excited by a 405 nm laser. The photobleached dye may exhibit >95% quenching of the original maximum emission intensity, >98% quenching of the original maximum emission intensity, or up to 100% quenching when excited at 405 nm, compared to the parent fluorescent polymer.

[0274] Fluorophores can undergo the fluorescence process repeatedly. This means that a fluorophore molecule can theoretically generate a signal multiple times. In fact, due to the structural instability of the fluorophore during its excited lifetime, it can be prone to decomposition. High-intensity irradiation can cause the fluorophore to change its structure, so that it can no longer emit fluorescence, which is called photobleaching.

[0275] The term "quantum yield" (QY) (Φ) or "fluorescence quantum yield" refers to the ratio of the number of photons emitted to the number of photons absorbed. Quantum yield is independent of instrument settings and describes how efficiently a fluorophore converts excitation energy into fluorescence. Experimentally, relative fluorescence quantum yield can be determined by measuring the fluorescence of a fluorophore of known quantum yield using the same experimental parameters (excitation wavelength, slit width, photomultiplier voltage, etc.) as the test dye. Quantum yield can be determined by any method known in the art. For example, QY can be determined at a selected excitation wavelength on a spectrofluorometer or spectrofluorimeter according to the manufacturer's instructions. For example, quantum yield (QY) can be determined on a Shimadzu Rf-6000 spectrofluorometer by measuring the emission intensity at 428 nm from a dilute PBS solution of staining buffer with an absorbance of 0.05 at 405 nm (excitation at 405 nm, excitation slit 1.5, emission slit 3.0, 1 cm quartz cuvette). The quantum yield can be calculated, for example, by comparing the intensity measured from a sample with that measured from a solution of NHS Pacific Blue (QY=0.78 in PBS 1x) under the same experimental conditions. In some embodiments, the QY can be determined, for example, by Lawson-Wood et al., Application Note-Fluorescence Spectroscopy, Determination of relative fluorescence quantum yield using the FL5600 fluorescence spectrometer, 2018, PerkinElmer, Inc. The excitation wavelength selected can be, for example, 405 nm. In some embodiments, the QY of a quenched or photobleached polymer can be compared to a parent fluorescent polymer in the absence of photobleaching and without a quenching moiety.

[0276] The term "fluorescent dye" refers to a dye that contains a light-excitable fluorophore that can be re-emitted upon light excitation. The term "fluorescent dye" encompasses both fluorescent polymeric dyes and fluorescent non-polymeric dyes, including fluorescent monomers and other conventional fluorescent dyes. The fluorescent polymeric dye may be any suitable fluorescent polymeric dye, including, for example, structures according to the present disclosure. Fluorescent polymeric dyes are also commercially available. For example, SuperNova™ ("SN") v428 (Beckman Coulter, Inc.) is a fluorescent polymeric dye that is optimally excited by a violet laser (405 nm) with an excitation maximum of 414 nm, an emission peak of 428 nm, and can be detected using a 450 / 50 bandpass filter or equivalent. SN v605 and SN v786 are tandem polymeric dyes derived from the core SN v428 polymeric dye. Both share the same absorbance characteristics, with an excitation maximum at 414 nm. Since the emission peaks of SN v605 and SN v786 are at 605 nm and 786 nm, respectively, they are optimally detected using 610 / 2 and 780 / 60 nm bandpass filters on the flow cytometer.

[0277] The term "fluorophore" refers to a fluorescent chemical compound that can re-emit light upon excitation with light. Fluorophores can usually contain some mixed aromatic groups, or planar and cyclic molecules with some p-pi bonds.

[0278] The terms "patient," "subject," or "subjects" include, but are not limited to, humans, and can also include other mammals, or domestic or exotic animals, such as dogs, cats, ferrets, rabbits, pigs, horses, cows, birds, or reptiles.

[0279] Unless otherwise specified, the term "room temperature" refers to 18-27°C.

[0280] Unless otherwise specified, the term "percent" or "%" refers to percent by weight.

[0281] The phrases "ready-to-use reagent", "ready-to-use reagent composition", "reagent at working concentration" and "reagent composition at working concentration" refer to a staining buffer composition made at about a 1× working concentration suitable for use in a mixture of polymer-dye conjugates to stain biological samples, e.g., for flow cytometric analysis (FCA).

[0282] The phrase "concentrated staining buffer" or "concentrated staining buffer composition" refers to a staining buffer composition that is prepared, for example, at about a ten-fold concentration factor (10x), for dilution with a diluent, such as a biological buffer or water, to obtain a working concentration of the staining buffer composition useful for reducing non-specific polymer interactions in a multicolor panel when staining biological samples for flow cytometry analysis. A concentrated staining buffer composition can be prepared at a concentration of 1x (1x) to at least 10x (10x), or at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x more concentrated than the working concentration of the staining buffer composition, and can be stable.

[0283] In some embodiments, the working strength dye buffer composition is stable for at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 18 months, or at least 24 months or longer from the date of manufacture when stored in its original sealed container at a temperature in the range of 2-8° C. (15-37° C., or with deviations to ambient temperatures of 19-27° C.). In some embodiments, the dark dye buffer composition is stable for at least 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, 30 months, or 36 months or longer from the date of manufacture when stored in its original sealed container at a temperature in the range of 2-8° C. (15-37° C., or with deviations to ambient temperatures of 19-27° C.).

[0284] The acronym "SN" refers to SuperNova™.

[0285] The acronym "SSC" refers to side scatter.

[0286] The term "WBC" refers to white blood cells.

[0287] The term "about," when referring to a measurable value, e.g., amount of compound, dosage, time, temperature, and the like, is intended to encompass variations of 10%, 5%, 1%, 0.5% or even 0.1% of the specified amount and at least the industry standard deviation in the testing method for determining the value.

[0288] In this specification, the terms "a", "an" or "the" are used to include one or more than one, unless the context clearly dictates otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise indicated. Furthermore, any expressions or terminology used herein and not specifically defined should be understood to be for descriptive purposes only and not limiting. Any use of section headings is intended to aid in the reading of this specification and should not be construed as limiting. Furthermore, information related to a section heading may appear within or outside such a particular section. Furthermore, all publications, patents and patent documents referred to herein are hereby incorporated by reference in their entirety as if individually incorporated by reference. In the event of any inconsistency in usage between this specification and any document so incorporated by reference, the usage in the incorporated references should be considered as supplementary to the usage in this specification, and in the event of any inconsistency, the usage in this specification shall prevail.

[0289] As used herein, "reducing" or "eliminating" non-specific binding of a polymer-dye conjugate can refer to when the mean fluorescence intensity (MFI) of "negative" (e.g., negative granulocyte, monocyte 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 higher; about 50% to about 95%, about 50% to about 75%, about 60% to about 80% or about 65% to about 90%) in % compared to when the non-fluorescent component of the first polymer dye is not used. In other words, the percent reduction in background staining of at least one of monocytes, granulocytes, and lymphocytes compared to when no surfactant is used 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 higher; about 50% to about 95%, about 50% to about 75%, about 60% to about 80%, or about 65% to about 90%).

[0290] In the methods described herein, steps may be performed in any order without departing from the principles of the invention, unless a chronological or operational order is explicitly recited. Moreover, unless express claim language recites that certain steps are performed separately, those steps may be performed simultaneously. For example, a claimed step of performing X and a claimed step of performing Y may be performed simultaneously in a single operation, and the resulting process would fall within the literal scope of the claimed process.

[0291] Each of the above embodiments is contemplated to be applicable in each combination with other embodiments described herein. For example, an embodiment corresponding to formula (I) is equally contemplated to be applicable to formulas (VIII)-(XIV), (XVIII), (XIX), and (XX). As another example, an embodiment corresponding to any of formulas (VIII)-(XIV), (XVIII), (XIX), and (XX) is equally contemplated to be applicable to formula (I).

[0292] The term "substantially" as used herein refers to a majority or majority, 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.

[0293] The terms "substantially free" or "substantially free" as used herein refer to less than about 1%, 0.5%, 0.1%, 0.05%, 0.001%, or about 0.0005% or less, about 0%, below the limit of quantification, below the limit of detectability, or 0%.

[0294] Those skilled in the art will recognize that numerous modifications to the embodiments described herein are possible without departing from the spirit and scope of the present disclosure. Thus, the present description is not intended to, and should not be construed as being limited to the examples shown, but should be accorded the full scope of protection afforded by the appended claims and their equivalents. Moreover, it is possible to use some of the features of the present disclosure without the corresponding use of other features. Thus, the above description of exemplary embodiments or exemplary embodiments are provided for the purpose of illustrating the principles of the present disclosure, and can include modifications and variations thereof, but not the limitations thereof. EXAMPLES

[0295] The present invention may be better understood by reference to the following examples, which are given by way of illustration and are not intended to be limiting of the examples set forth herein.

[0296] Example 1 Preparation of DHP polymer dyes [ka] Method 1: In a round bottom flask, both dibromoDHP and diboronDHP monomers (1:1) were taken in a (DMF-water) mixture and purged with nitrogen for 10 minutes. About 20 equivalents of CsF and 10% Pd(OAc)2 were mixed under nitrogen and heated at 80 degrees Celsius. Polymerization was monitored using UV-Vis spectroscopy and SEC chromatography. A capping agent containing an appropriate functional group (selected from G1) was then added to the reaction mixture, followed by a second capping agent (selected from G2) after 3 hours. After reaction, the crude reaction mixture was solvent 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. 20% ethanol was used to wash until the filtrate absorbance decreased.

[0297] Method 2: Alternatively, polymerization can be performed by self-polymerization of the bromo-boronic ester of the DHP molecule. In a round bottom flask, the DHP bromo-boronic ester was taken in a (DMF-water) mixture and purged with nitrogen for 10 minutes. Approximately 10 equivalents of CsF and 5% Pd(OAc)2 were mixed under nitrogen and heated to 80 degrees Celsius. Polymerization was monitored using UV-Vis spectroscopy and SEC chromatography. A capping agent containing an appropriate functional group (selected from G1) was then 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 solvent 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. 20% ethanol was used to wash until the filtrate absorbance decreased.

[0298] Method 3: In a round bottom flask, take both dibromodihydrophenanthrene and diborondihydrophanenthrene monomers (1:1) and add 10 equivalents of The crude polymer was dissolved in a THF-water (4:1) mixture containing K2CO3 and 3% Pd(PPh3)4. The reaction mixture was attached to a Schlenk line, degassed by three freeze-vacuum-thaw cycles, and then heated to 80 degrees Celsius under nitrogen with vigorous stirring for 18 hours. A capping agent (selected from G1) containing an appropriate functional group was then added to the reaction mixture via cannula under nitrogen overpressure, and after 3 hours, a second capping agent (selected from G2) was added. After the reaction, the crude reaction mixture was solvent 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. 20% ethanol was used to wash until the filtrate absorbance decreased.

[0299] Method 4: Alternatively, polymerization can be carried out by self-polymerization of the bromo-boronic ester of the dihydrophenanthrene molecule. In a round bottom flask, the dihydrophenanthrene bromo-boronic ester was taken and dissolved in a THF-water (4:1) mixture containing 10 equivalents of K2CO3 and 3% Pd(PPh3)4. The reaction mixture was attached to a Schlenk line, degassed by three freeze-vacuum-thaw cycles, and then heated to 80 degrees Celsius under nitrogen with vigorous stirring for 18 hours. A capping agent (selected from G1) containing an appropriate functional group was then added to the reaction mixture through a cannula under nitrogen overpressure, and after 3 hours, a second capping agent (selected from G2) was added. After the reaction, the crude reaction mixture was solvent 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. Wash with 20% ethanol until the absorbance of the filtrate decreases.

[0300] Example 2 Preparation of fluorene-DHP copolymer dyes [ka] Method 1: In a round bottom flask, both dibromoDHP and diboronfluorene monomers (1:1) were taken in a (DMF-water) mixture and purged with nitrogen for 10 minutes. About 20 equivalents of CsF and 10% Pd(OAc)2 were mixed under nitrogen and heated at 80 degrees Celsius. Polymerization was monitored using UV-Vis spectroscopy and SEC chromatography. A capping agent containing an appropriate functional group (selected from G1) was then 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 solvent 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. Washing was performed using 20% ​​ethanol until the absorption of the filtrate decreased.

[0301] Method 2: In a round bottom flask, both dibromofluorene and diboron DHP monomers (1:1) were taken in a (DMF-water) mixture and purged with nitrogen for 10 minutes. About 20 equivalents of CsF and 10% Pd(OAc)2 were mixed under nitrogen and heated at 80 degrees Celsius. Polymerization was monitored using UV-Vis spectroscopy and SEC chromatography. A capping agent containing an appropriate functional group (selected from G1) was then added to the reaction mixture, followed by a second capping agent (selected from G2) after 3 hours. After reaction, the crude reaction mixture was solvent 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. Washing was performed using 20% ​​ethanol until the absorption of the filtrate decreased.

[0302] Method 3: In a round bottom flask, both dibromodihydrophenanthrene and diboronfluorene monomers (1:1) were taken and dissolved in a THF-water (4:1) mixture containing 10 equivalents of K2CO3 and 3% Pd(PPh3)4. The reaction mixture was attached to a Schlenk line and degassed with three freeze-vacuum-thaw cycles, then heated to 80 degrees Celsius under nitrogen with vigorous stirring for 18 hours. A capping agent (selected from G1) containing an appropriate functional group was then added to the reaction mixture through a cannula under nitrogen overpressure, followed by a second capping agent (selected from G2) after 3 hours. After the reaction, the crude reaction mixture was solvent 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. 20% ethanol was used to wash until the filtrate absorbance decreased.

[0303] Method 4: In a round bottom flask, dibromofluorene and diborondihydrophenanthrene monomers (1:1) were taken and dissolved in a THF-water (4:1) mixture containing 10 equivalents of K2CO3 and 3% Pd(PPh3)4. The reaction mixture was attached to a Schlenk line and degassed with three freeze-vacuum-thaw cycles, then heated to 80 degrees Celsius under nitrogen with vigorous stirring for 18 hours. A capping agent (selected from G1) containing an appropriate functional group was then added to the reaction mixture through a cannula under nitrogen overpressure, and after 3 hours, a second capping agent (selected from G2) was added. After the reaction, the crude reaction mixture was solvent 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. 20% ethanol was used to wash until the filtrate absorbance decreased.

[0304] Example 3 Comparison of fluorescence emission spectra A comparison of the fluorescence emission spectra of fluorene (Fl-Fl), dihydrophenanthrene (DHP-DHP) and fluorene-DHP (DHP-Fl) polymers was performed. The DHP-containing polymers show a significant difference in their fluorescence maxima, which lie between 426 and 428 nm, while the fluorene-based polymers show a maximum at 421 nm.

[0305] Example 4 Comparison of absorption spectra The absorption spectra of both fluorene (Fl-Fl) and dihydrophenanthrene (DHP-DHP) polymers were measured. The graph shows the absorption of the DHP-DHP polymer (black curve) at 390 and 410 nm, while the Fl-Fl (grey curve) polymer shows a maximum at about 400 nm. The samples were measured under different concentrations.

[0306] Example 5 CD4 signal-to-noise ratio Flow cytometric analysis of lysed whole blood stained with the novel polymer-conjugated anti-human CD4 and Pacific Blue-conjugated CD4 was performed. The positive signal intensity of the polymer dye was approximately 5-fold stronger than that of Pacific Blue.

[0307] Example 6 Surface staining procedure with co-immobilization buffer during sample preparation for flow cytometry In this procedure, the dye conjugate is added after the staining buffer according to the present disclosure is added to the test tube to avoid any possible non-specific interactions that may occur between the dye conjugates over time. Fixation is a step that allows the storage of the white blood cell preparation for several hours after staining with fluorescent antibodies without deterioration. The lysis solution may be used to lyse red blood cells in the preparation of biological samples for flow cytometry.

[0308] 1. Quickly prepare a "fixation and lysis" mixture by adding 25 μL of undiluted IOTest 3 10X Fixative Solution (AO7800, Beckman Coulter, Inc.) to 1 mL of VersaLyse™ Lysis Solution (AO9777, Beckman Coulter, Inc.). Prepare a sufficient amount of this "fixation and lysis" mixture depending on the number of biological test samples to be lysed (1 mL of mixture per tube). 2. Add 10 μL of staining buffer according to the present disclosure to each tube. This staining buffer is not required for tubes that do not contain a polymer-dye conjugate mix. 3. Add the appropriate amount of dye conjugate. Gently vortex the tube. 4. Add 100 μL of test sample to each tube. Gently vortex the tubes. 5. Incubate for 15-20 minutes at room temperature (18-25°C) protected from light. Next, lysis of red blood cells is performed. 6. Add 1 ml of the quickly prepared "fixation and lysis" mixture and vortex immediately for 1 second. 7. Incubate for 10 minutes at room temperature, protected from light. 8. Centrifuge at 150 x g for 5 minutes at room temperature. 9. Remove the supernatant by aspiration. 10. Resuspend the cell pellet using 3 mL of PBS. 11. Centrifuge at 150 x g for 5 minutes at room temperature. 12. Remove the supernatant by aspiration. 13. Resuspend the cell pellet using 0.5 mL of PBS and 0.1% formaldehyde (0.1% formaldehyde PBS can be obtained by diluting 12.5 μL of IOTest 3 Fixative Solution (for PN, see catalog) at its 10× concentration in 1 mL of PBS).

[0309] These preparations can be kept protected from light for 24 h between 2-8 °C prior to analysis by flow cytometry.

[0310] Example 7 Procedure for photobleaching polymer dyes The photobleached polymer dye was prepared as follows. Briefly, the process involves thawing violet polymer dye 428, diluting the polymer dye at 1 mg / mL in PBA / PF-68 0.02%, placing the diluted dye in a Roux glass flask, and placing the flask in a UV chamber (Bio-Link-BLX). The diluted polymer dye is exposed to UV light until a fluorescence value of less than or equal to 50 AFU is reached. The residual fluorescence of the photobleached dye is measured by fluorometry (Fluorometer LS50B, Perkin Elmer). The photobleached dye emits ≦50 AFU at 450 nm (slit excitation / emission is 6 nm / 4 nm; 1 cm cuvette) at 10 micrograms / mL, passing the fluorometry criteria. It was found that polymer dye 428, when used in compositions according to the present disclosure, needs to be effectively photobleached in order to avoid visible non-specific staining in FCA of blood samples. Residual fluorescence of photobleached dye was found to have a direct impact on spillover of the conjugate and therefore on the flow cytometry results. Figure 6 shows two FCA dot plots of stained and lysed samples treated with polymer dye conjugate CD56-SNv428 / CD4-SN786. The left panel shows the impact of insufficiently photobleached polymer dye, showing undesired spillover of the conjugate (arrow). The right panel shows a two-dimensional flow cytometry dot plot of two polymer-dye conjugates CD56-SNv428 / CD4-SN786, where the sample is prepared with a composition according to the present disclosure that contains effectively photobleached polymer dye 428 that exhibits a QY of 0.056 or less and <47 AFU at 10ug / mL when excited with a 405nm laser (AFU slit excitation / emission 6nm / 4nm, fluorometer LS50B Perkin Elmer). Bleed-over between the conjugates was substantially reduced.

[0311] Example 8 Dyeing buffer composition containing monomeric components of a polymeric dye and a photobleached polymeric dye - Patent Application 20070229633 Initial efforts to design a staining buffer composition included testing individual candidate components in PBS biological buffer with PF-68 using a mix of two violet polymer dye conjugates and obtaining FCA dot plots to evaluate performance. Exemplary monomer A (100-800ug / test), maleimide violet polymer dye 428 (m428) (3.1-100ug / test), carboxyl violet polymer dye 428 (3.1-100ug / test), amino polymer dye 428 (3.1-100ug / test), photobleached maleimide 428 (m428) dye (with and without cysteamine HCl), photobleached carboxyl 428 (c428) dye, photobleached amino polymer dye 428 (a428), PEG, or Empigen zwitterionic surfactants were evaluated individually. Exemplary monomer A and the photobleached dye were individually found to be effective in reducing non-specific interactions between dye conjugates.

[0312] Based on the results with the individual components, a test staining buffer composition was developed using exemplary monomer A from Example 7 and photobleached violet dye 428.

[0313] PF-68 detergent was quickly added to the PBS / BSA / NaN3 to reach a final concentration of 0.02%. The mix was stored at room temperature until used to formulate the buffer. The compositions were formulated by mixing the components to obtain the compositions shown in Table 1.

[0314] [Table 1]

[0315] Example 9 Performance of test staining buffer compositions containing monomeric components of polymeric dyes and photobleached polymeric dyes Polymer-dye-antibody conjugates are antibodies conjugated to polymer dyes that may interact non-specifically when mixed together. To allow customers to perform multi-color experiments using more than one polymer-dye conjugate in a panel, the staining buffer composition was designed to reduce, substantially reduce, or eliminate non-specific polymer-dye conjugate interactions.

[0316] In this example, unlike other staining buffer manufacturers, quenching of dye fluorescence is performed by exposing the dye to UV light, according to the present disclosure. Briefly, photobleaching was performed by exposing the dye to UV light (365 nm).

[0317] Photobleached violet dye 428 was prepared by thawing maleimide violet dye 428, diluting it to 1 mg / mL in PBS / PF-68 (0.02%), placing the diluted dye in a Roux glass flask, and placing the flask in a UV chamber (Bio-Link-BLX) for three cycles of 10 hour UV exposure (365 nm). The photobleached dye exhibited a quantum yield (QY) of 0.056 or less. Residual fluorescence of the photobleached dye was also measured by fluorimetry and was ≦47 AFU when excited with a 405 nm laser (10ug / mL, AFU slit excitation / emission 6nm / 4nm, Fluorometer LS50B Perkin Elmer).

[0318] A test staining buffer composition was prepared containing photobleached violet dye 428, exemplary monomer A (subunit of dye 428), PF-68 (0.02%), 1×PBS / BSA (2 mg / mL) / NaN3 (0.1%).

[0319] FCA dot plot comparisons of stained and lysed whole blood samples using a mixture of three SuperNova (SNv) dye-antibody conjugates, CD56-SNv428, CD20-SNv605, and CD4-SNv786, with or without pre-addition of test staining buffer, are shown in Figure 1. Gating is on lymphocytes (LY). Acquisition was on a CytoFLEX LX flow cytometer using CytExpert acquisition software. Analysis was by Kaluza analysis software. As shown in the top three panels of Figure 1, staining anomalies can occur in the absence of test staining buffer and data can appear under-corrected. In the presence of test staining buffer, a substantial reduction in conjugate-to-conjugate spillover was demonstrated, as shown in the three lower panels.

[0320] The efficiency of the test staining buffer can be assessed by FCA analysis, where the median spillover for the positive population is divided by the median spillover for the negative population on the same axis. The top panel of FIG. 1 shows an FCA dot blot of a mix without staining buffer, showing that there is spillover indicative of nonspecific interactions between the polymer-dye conjugates. In contrast, the FCA dot plots show that this test staining buffer is efficient in reducing nonspecific polymer-dye conjugate interactions, as shown by the bottom three panels of FIG. 1, where this buffer was used.

[0321] In another experiment, a test staining buffer was prepared by mixing exemplary monomer A and photobleached dye 428 in a solution of PBS / BSA / NaN3 / PF-68, as described herein (100 tests / vial; 10 μL / test). The polymer dye conjugate was added on top of the test staining buffer before adding the whole blood biological sample. A multicolor panel was used that included a mixture of three SuperNova polymer dye conjugates, including CD56-SNv428+CD19-SNv605+CD4-SNv786. Figure 2A shows the FCA dot plot of a stained and lysed whole blood sample using the multicolor panel without staining buffer. Figure 2B shows the FCA dot plot and multicolor panel of a stained and lysed whole blood sample using comparative BD Horizon™ Brilliant stain buffer (BD Biosciences). Figure 2C shows the FCA dot plot and multicolor panel of a whole blood sample using test staining buffer. The comparative buffer composition and the test staining buffer composition showed reduced bleeding compared to the multicolor panel without buffer. The test staining buffer composition showed some low degree of reduced bleeding when compared to the comparative buffer of the prior art, indicating reduced non-specific polymer-polymer interactions.

[0322] Example 10 Quenched polymer Polymer dyes containing various quenching moieties were prepared and the possibility of using staining buffers to prevent non-specific polymer-polymer interactions was investigated. Fluorescent polymer dyes were conjugated to quenching moieties to form quenched polymers where fluorescence was reduced or eliminated. It was found that the addition of a quenched polymer to a mixture of two or more polymer-dye conjugates reduced or eliminated non-specific polymer-polymer interactions.

[0323] Various violet-excitable fluorescent polymer dyes were conjugated to various quencher moieties. Approximately 15 different quenched polymers were prepared from violet-excitable fluorescent polymer dyes and approximately 10-15 fold molar excess of various commercially available quencher moieties by methods similar to those of the present disclosure. Three different violet-excitable fluorescent polymers (Polymer 1, Polymer 2, Polymer 3) containing structures according to the present disclosure were selected from various MW ranges of 80-150 kDa or 90-120 kDa. The commercially available quencher moieties included Dabcyl Q, Dabcyl plus, Anaspec 490Q, Dylight 425Q, Dyomics 425Q, and Dyomics 505Q. Exemplary quenched polymers are shown in Table 2 along with their quantum yields.

[0324] [Table 2]

[0325] Figure 3 shows a graph of the fluorescence profile in wavelength (nm) vs. AFU for the quenched polymers: polymer 1 Dabcyl (QY 0.01), polymer 2 Dabcyl (QY 0.005), polymer 2 DY425Q (QY 0.01), polymer 3 Dabcyl (QY 0.005), polymer 3 Dabcyl plus (QY 0.015) and polymer 3 DY425Q (QY 0.009). The inset in Figure 3 shows a graph of a representative parent fluorescent polymer (polymer 3 QY 0.54) before conjugation to a quenching moiety and after conjugation to a quenching moiety to obtain quenched polymer 3, which shows a substantial decrease in fluorescence QY when excited by a 405 nm laser. QY refers to quantum yield. Each of the quenched polymers in Table 2 exhibits a quantum yield (QY) of <0.02, ≦0.015, or ≦0.01 when excited at 405 nm, in contrast to the unquenched polymer 3 (QY 0.54).

[0326] Example 11 Characterization of quenched polymers The quenched polymers in Table 2 were evaluated using a single dye conjugate or a panel of dye conjugates, and whole blood samples in the FCA assay as follows: The quenched polymer was added in 10 uL aliquots (500 ug / mL) to 100 uL of whole blood.

[0327] Quenched polymers were used at 5 micrograms (5ug) for 1 conjugate and 10 micrograms (10ug) for 2 conjugates in a buffer containing PBS / BSA / NaN3 / PF-68 (if required). Antibody conjugates (1ug each) were added on top of the additive mix before adding whole blood. After 20 minutes of incubation, 1mL of VersaFix was added, followed by incubation for 15 minutes. A final wash with 3mL of 1x PBS was performed. The pellet was resuspended in 0.5mL of 1x PBS or 1x PBS / 0.1% FA.

[0328] The ability of quenched polymers to reduce non-specific binding and to reduce population spreading was examined.

[0329] Figure 4 shows FCA dot plots (top row, left to right) of stained and lysed whole blood samples after staining with a mixture of two polymer dye conjugates, CD4-UV-excitable polymer dye (CD4-UVEPD) and CD20-violet-excitable polymer dye (CD20-VEPD) (both Beckman Coulter Life Sciences, top left panel), and a mixture of two polymer dye conjugates, CD4-BUV395 (BD Biosciences) and CD20-VEPD (Beckman Coulter Life Sciences, top right panel), when gated on lymphocytes without the use of a quenched polymer staining buffer additive. When the two different dye conjugates are mixed without a quenched polymer, the dot plots show nonspecific interactions and associated spillover. Addition of a quenched polymer according to the present disclosure (quenched polymer 2 DYQ425) during staining with CD4-UVEPD and CD20-VEPD (lower left panel) or BUV395-CD4 and CD20-VEPD (lower right panel) reduced nonspecific interactions and spillover.

[0330] Similar FCA improvement results were demonstrated using test staining buffer containing quenched polymer 3 DYQ425 (10 uL) (data not shown).

[0331] Figure 5 shows FCA dot plots of stained and lysed whole blood samples after staining without buffer with a mixture of two polymer dye conjugates, CD4-UV-excitable polymer dye (CD4-UVEPD) and CD20-violet-excitable polymer dye (CD20-VEPD) (both Beckman Coulter Life Sciences, top left panel), and a mixture of two polymer dye conjugates, CD4-BUV395 (BD Biosciences) and CD20-VEPD (Beckman Coulter Life Sciences, top right panel), when gated on lymphocytes without the use of a quenched polymer staining buffer additive. When each of the mixtures of two different dye conjugates is mixed without the quenched polymer, the dot plots show nonspecific interactions and associated spillover. Addition of a quenched polymer according to the present disclosure (quenched polymer 2-Dabcyl) during staining with CD4-UVEPD and CD20-VEPD (lower left panel) or BUV395-CD4 and CD20-VEPD (lower right panel) reduced nonspecific interactions and spillover.

[0332] Similar FCA improvement results were shown with other quenched polymers, i.e., Polymer 1-Dabcyl, Q / P5.6 (10 uL), Polymer 3-Dabcyl, Q / P7.14 (10 uL) and Polymer 3-Dabcyl plus, Q / P5.4 (10 uL) (data not shown). The Polymer-Dabcyl quenched polymer was selected for further development because it showed a very good ability to reduce spillover and polymer-polymer interactions in polymer-dye conjugates.

[0333] Example 12 Exemplary dyeing buffer compositions containing quenched polymers A dyeing buffer composition was developed using the quenched polymer according to Example 10. As shown in Example 10, dyeing buffer composition B was found to be suitable for reducing bleeding and non-specific polymer-polymer interactions in a multicolor polymer-dye conjugate panel.

[0334] PF-68 detergent is quickly added to the PBS / BSA / NaN3 to reach a final concentration of 0.02%. This mix is ​​stored at room temperature until used to formulate the buffer. The composition is formulated by mixing the components to obtain the composition shown in Table 3.

[0335] [Table 3]

[0336] Example 13 Exemplary dyeing buffer compositions including monomeric components of polymeric dyes and quenched polymeric dyes A composition was developed that includes an exemplary monomer A (a partial component of Violet 428 SuperNova dye) and a poly-Dabcyl tandem dye. The poly-Dabcyl tandem dye was made by conjugating a Violet 428 dye monomer to a Dabcyl molecule. Dabcyl is a quencher molecule that absorbs the fluorescence emitted by Violet 428 SuperNova dye when excited by a 405 nm laser. As a result, the poly-Dabcyl tandem dye emits almost no fluorescence.

[0337] A typical polydabcyl tandem dye has a structure according to formula (XXIV): There were approximately 5-8 dabcyl dyes in each polymer backbone. [ka]

[0338] Prior to addition to the biological sample, the two SuperNova polymer-dye conjugates (CD3-SN428 and CD19-SN605) were pre-formulated individually, i.e., without the exemplary Monomer A + Poly-Dabcyl additive. Other buffers and protocols were similar to those in Example 10. Figure 7 shows the two-dimensional FCA dot plots of CD3-SN428 / CD19-SN605 without additive (left panel) and with the exemplary Monomer A + Poly-Dabcyl additive (right panel). It was found that in the presence of additive, non-specific interactions between the polymer-dye conjugates were substantially reduced.

[0339] The quenched polymer of poly-Dabcyl violet polymer-dabcyl tandem (XXIV) was found to be effective in reducing non-specific interactions between various polymer dyes.

[0340] PF-68 detergent is quickly added to the PBS / BSA / NaN3 to reach a final concentration of 0.02%. This mix is ​​stored at room temperature until used to formulate the buffer. Staining buffer composition C is formulated by mixing the components to obtain the composition shown in Table 4.

[0341] [Table 4]

[0342] Example 14 Performance of quenched polymer compositions containing protein stabilizers and non-ionic surfactants The performance of quenched polymer dye compositions containing the quenched violet polymer 2-Dabcyl was evaluated in the presence and absence of the non-ionic surfactant Pluronic® F-68 (1%) compared to the commercially available BD Horizon™ Brilliant stain buffer Plus. FCA of stained lysed blood samples from donor A was performed using a mixture of dye conjugates SuperNova SN v428-CD19 (Beckman Coulter Life Sciences) and BV650-CD4 (Brilliant Violet 650™ anti-human CD4 antibody, BioLegend, Inc.) in the processed blood sample from donor A.

[0343] Figure 8 shows FCA dot plots of mixtures of dye conjugates SuperNova SN v428-CD19 and BV650-CD4 in blood samples without quenched polymer (upper left), with 1% PF-68 (upper right), with 10ug quenched polymer 2-Dabcyl (lower left), with 10ug quenched polymer 2-Dabcyl and 1% PF-68 non-ionic surfactant (lower right). Test sample compositions with 10ug quenched polymer 2-Dabcyl (MFI 2172) and 1% PF-68 (MFI 2400) showed reduced non-specific binding when compared to the control sample without buffer (MFI 7804). A test sample composition containing 10 ug of quenched polymer 2-Dabcyl and 1% PF-68 (MFI 1327) showed improved reduction in MFI, improved reduction in non-specific binding, and improved reduction in polymer-polymer interactions compared to the control sample.

[0344] Example 15 Flow cytometric performance of a non-ionic surfactant alone in a mixture of two different polymer-dye conjugates. Non-ionic surfactants were found to be desirable additives in staining buffer compositions to reduce non-specific polymer-dye conjugate interactions. A mixture of two different polymer-dye conjugates was used to evaluate the effect of various concentrations of non-ionic surfactants alone on the FCA of stained and lysed blood cells. Figure 9 shows FCA dot plots of cells stained and lysed with a mixture of CD4-BV650 (BD Biosciences) and CD19-SNv428 (Beckman Coulter Life Sciences) without buffer (top panel), with 0.1% PF-68 (lower left panel), with 0.5% PF-68 (lower middle panel), and with 1% PF-68 (weight / volume) (lower right panel). The presence of increasing concentrations of PF-68 (0.1-1% weight / volume) was accompanied by a reduction in non-specific interactions in the mixture, as evidenced by improved separation compared to the absence of PF-68.

[0345] Example 16 NON-FLUORESCENT POLYMERS FOR USE IN DYEING BUFFER COMPOSITIONS - Patent application Staining buffer compositions containing non-fluorescent components of polymeric dyes, including photobleached polymeric dyes, quenched polymeric dyes, and / or monomeric components of polymeric dyes, were shown to be effective in reducing spillover and non-specific polymer-polymer interactions in two multicolor panels of more polymer-dye conjugates in FCA of stained biological samples, as demonstrated in this disclosure.

[0346] In this example, non-fluorescent polymeric dyes with QY of 0.1 or less were prepared, and emission spectra and QYs were measured and tested in staining buffer compositions. Figure 10 shows graphs of emission spectra in the range of 415-700 nm, and quantum yields of two non-fluorescent polymeric dyes. The structures of DHP-pyrrole polymer (QY 0.043) and DHP-nitro-capped polymer (QY 0.092) are also shown. In FCA studies of lysed and stained cells using at least two polymeric dye conjugates, DHP-pyrrole polymer and DHP-nitro-capped polymer were found to be effective in reducing spillover. (Data not shown). Non-fluorescent polymeric dyes with quantum yields (QY) of 0.1 or less, or 0.06 or less, or 0.056 or less were found to be useful in reducing non-specific interactions and spillover in FCA analysis in staining buffer compositions. The present invention provides, for example, the following items. (Item 1) 1. A composition for use in staining a biological sample, for use with at least one fluorescent polymer dye conjugated to a binding partner, comprising: At least one non-fluorescent component of the first polymeric dye non-ionic surfactants, and biological buffer Including, A composition that reduces non-specific binding of said at least one fluorescent polymer-dye conjugate when compared to said at least one fluorescent polymer-dye conjugate in the absence of said composition. (Item 2) The non-fluorescent component of the first polymer dye is selected from the group consisting of: Quantum yield (QY) of 0.1 or less, 0.06 or less, or 0.056 or less less than about 50 arbitrary fluorescence units (AFU) when excited by a 405 nm laser; and >95% quenching of the original maximum emission intensity compared to the parent fluorescent polymer when excited at 405 nm The composition according to item 1, which exhibits at least one of the following: (Item 3) 3. The composition according to item 1 or 2, wherein the non-fluorescent component of the first polymeric dye is selected from the group consisting of a monomeric component of a polymeric dye, a photobleached polymeric dye, a polymeric dye comprising a quenching moiety and a non-fluorescent polymeric dye. (Item 4) 4. The composition of claim 3, comprising a monomeric component of a polymeric dye and a photobleached polymeric dye. (Item 5) 4. The composition according to item 3, comprising a monomer component of a polymeric dye and a polymeric dye comprising a quenching moiety. (Item 6) 6. The composition according to items 1 to 5, wherein the non-ionic surfactant is a poloxamer non-ionic surfactant. (Item 7) 6. The composition according to items 1 to 5, wherein the at least one polymeric dye comprises two or more polymeric dyes, each conjugated to a binding partner. (Item 8) 8. The composition according to item 7, wherein the two or more polymer-dye conjugates comprise different polymer dyes or comprise the same polymer dye. (Item 9) The at least one fluorescent polymer dye has a structure according to formula (I). [ka] is a polymer having During the ceremony, Each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers; each L is a linker moiety; each M is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, band gap modifying monomers, optionally substituted ethylene and ethynylene; G 1 and G 2 is independently selected from an unmodified polymer end and a modified polymer end; a, c, and d independently define the mol % of each unit in said structure, which may each be evenly or randomly repeated, where a is each mol % of 10-100%, c is each mol % of 0-90%, and d is each mol % of 0-25%; Each b is independently 0 or 1; m is an integer from 1 to about 10,000; 9. The composition according to any one of items 1 to 8. (Item 10) The at least one fluorescent polymeric dye has a structure according to formula (XX): [ka] is a polymer having During the ceremony, Each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers; each B is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, band gap modifying monomers, optionally substituted ethylene and ethynylene; each E is an independently selected chromophore, functional moiety, or binding partner; G 1 and G 2 is independently selected from an unmodified polymer end and a modified polymer end, L 1 , L 2 and L 3 is a linker moiety, W is a water-solubilizing moiety; the subscripts n and m are independently integers ranging from 1 to 10,000; The subscript p is an integer ranging from 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 1 or 2, the sum of the subscripts r and s ranges from 1 to 4; A and B are randomly or non-randomly distributed in the conjugated polymer; 10. The composition according to any one of items 1 to 9. (Item 11) L 1 is selected from the group consisting of sulfonamides, sulfonamides, sultams, disulfinamides, amides, phosphonamides, phosphonamidates, phosphinamides and secondary amines. (Item 12) 12. The composition according to any one of items 1 to 11, wherein the binding partner conjugated to the at least one polymeric dye is a molecule or a complex of molecules capable of specifically binding to a target analyte. (Item 13) 13. The composition according to any one of the preceding claims, wherein the binding partner conjugated to the at least one polymeric dye is selected from the group consisting of a protein, an affinity ligand, an antibody, an antibody fragment and an oligonucleotide. (Item 14) 14. The composition of claim 13, wherein the binding partner is a monoclonal or polyclonal antibody. (Item 15) 15. The composition according to any one of items 1 to 14, wherein the binding partner is an immunoglobulin or an immunologically active part of an immunoglobulin. (Item 16) 16. The composition of any one of items 1 to 15, wherein the binding partner is a single chain antibody, Fab, Fab' and F(ab')2 fragments, Fv or a Fab expression library. (Item 17) 17. The composition of any one of the preceding claims, wherein the monomeric component of at least one polymeric dye comprises a moiety selected from the group consisting of a dihydrophenanthrene (DHP) moiety and a fluorene moiety. (Item 18) The monomeric component of the at least one polymeric dye has a chemical structure according to formula (XXII): [ka] The dihydrophenanthrene (DHP)-based water-soluble monomer has the formula: During the ceremony, Each of G1 and G2 is independently selected from the group consisting of halo (F, Cl, Br, I), C1-C6 alkyl, and PEG; R 2 respectively represent water-solubilizing moieties, alkenes, alkynes, cycloalkyls, haloalkyls, (hetero)aryloxys, (hetero)arylaminos, sulfonamido-PEGs, phosphoramido-PEGs, ammonium alkyl salts, ammonium alkyloxy salts, ammonium oligoether salts, sulfonate alkyl salts, sulfonate alkoxy salts, sulfonate oligoether salts, sulfonamide oligoethers, sulfonamides, sulfinamides, phosphonamidates, phosphinamides, [ka] are independently selected from the group consisting of: R 3 are the water-solubilized moieties, R 4 are each independently selected from the group consisting of H, alkyl, PEG, a water solubilizing moiety, a linker moiety, a chromophore, a carboxylamine, an amine, a carbamate, a carboxylic acid, a carboxylic ester, a maleimide, an activated ester, N-hydroxysuccinimidyl, a hydrazine, a hydrazide, a hydrazone, an azide, an alkyne, an aldehyde, or a thiol, or a protected group thereof; R 5 are H, hydroxyl, C1-C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, C3-C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 Alkoxy, C2-C18 (Hetero)aryloxy, C2-C 18 (Hetero)arylamino, C2-C 12 Carboxylic acids and C2-C 12 carboxylic acid esters; Each Q is independently a bond, NR 4 or -CH2, Each Z is independently CH, O, or NR 4 and Each f is independently an integer from 0 to 50; Each n is independently an integer from 1 to 20. 18. The composition according to any one of items 3 to 17. (Item 19) 19. The composition according to item 18, wherein G1 and G2 are each halo, Z is each O, R2 is each H, n is each independently 2 to 4, f is each independently 5 to 20, and optionally n is each 3. (Item 20) The monomeric component of the at least one polymeric dye has a chemical structure according to formula (XXIII): [ka] is a fluorene-based or carbazole-based water-soluble monomer having the formula During the ceremony, Each of G1 and G2 is independently selected from the group consisting of halo (F, Cl, Br, I), C1-C6 alkyl, and PEG; Each X is C, N or Si; R 4 are each independently selected from the group consisting of H, alkyl, PEG, a water solubilizing moiety, a linker moiety, a chromophore, a carboxylamine, an amine, a carbamate, a carboxylic acid, a carboxylic ester, a maleimide, an activated ester, N-hydroxysuccinimidyl, a hydrazine, a hydrazide, a hydrazone, an azide, an alkyne, an aldehyde, or a thiol, or a protected group thereof; R5 is H, hydroxyl, C1-C 12 Alkyl, C2-C 12 Alkenes, C2-C 12 Alkynes, C3-C 12 Cycloalkyl, C1-C 12 Haloalkyl, C1-C 12 Alkoxy, C2-C 18 (Hetero)aryloxy, C2-C 18 (Hetero)arylamino, C2-C 12 Carboxylic acids, C2-C 12 Carboxylic acid esters and C1-C 12 independently selected from the group consisting of alkoxy; Each Z is independently CH, O, or NR 4 and Each f is independently an integer from 0 to 50; Each n is independently an integer from 1 to 20. 20. The composition according to any one of items 3 to 19. (Item 21) 21. The composition according to item 20, wherein G1 and G2 are each halo, Z is each O, R2 is each H, n is each independently 2 to 4, f is each independently 5 to 20, and optionally n is each 3. (Item 22) 4. The photobleached polymer dye comprising: Quantum yield (QY) of 0.1 or less, 0.06 or less, or 0.056 or less Optionally, an arbitrary fluorescence unit (AFU) of less than about 50 when excited by a 405 nm laser, and >95% quenching of the original maximum emission intensity compared to the parent fluorescent polymer when excited at 405 nm 22. The composition according to any one of items 3 to 21, wherein the composition is prepared by exposing a fluorescent polymer dye according to formula (I), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV), (XVIII), (XIX) or (XX) to irradiation with UV light such that the polymer dye exhibits at least one of the following: (Item 23) The polymeric dye comprising a quenching moiety has a structure according to formula (XX): [ka] Including, During the ceremony, Each A is independently selected from the group consisting of aromatic comonomers and heteroaromatic comonomers; each B is independently selected from the group consisting of aromatic comonomers, heteroaromatic comonomers, band gap modifying monomers, optionally substituted ethylene and ethynylene; each E is an independently selected chromophore, functional moiety, or quenching moiety, and at least one or more E is a quenching moiety; G 1 and G 2 is independently selected from an unmodified polymer end and a modified polymer end, L 1 , L 2 and L 3 is a linker moiety, W is a water-solubilizing moiety; The subscript n is an integer ranging from 1 to 10,000; The subscript m is an integer ranging from 1 to 25; The subscript p is an integer ranging from 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 1 or 2, the sum of the subscripts r and s ranges from 1 to 4; A and B are randomly or non-randomly distributed in the conjugated polymer; E is a quenching moiety; as needed, the polymeric dye containing a quenching moiety exhibits a quantum yield of 0.1 or less, and optionally, the polymeric dye containing a quenching moiety exhibits a fluorescence profile with an AFU of less than 50; 23. The composition according to any one of items 3 to 22. (Item 24) 24. The composition of claim 23, wherein the quenching moiety is selected from the group consisting of Dabcyl, Dabsyl, DYQ425, DYQ505, BHQ1, QSY7, QSY9, QSY35, and TAMRA quenching moieties, and optionally m=2-20, 3-15, or 5-8. (Item 25) 25. The composition according to any one of items 5 to 24, wherein the nonionic surfactant is a polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer. (Item 26) The nonionic surfactant has a structure according to formula (XXI): [ka] 26. The composition according to item 25, comprising: (Item 27) 27. The composition of any one of the preceding claims, wherein the composition comprises a plurality of fluorescent polymer-dye conjugates, and wherein the composition substantially reduces the non-specific binding between the plurality of fluorescent polymer-dye conjugates. (Item 28) 28. The composition according to any one of the preceding claims, further comprising a zwitterionic surfactant. (Item 29) 1. A method for detecting an analyte in a sample, comprising: Adding at least one polymer-dye conjugate to the composition according to any one of items 1 to 28 to form a polymer-dye conjugate composition. contacting said polymer-dye conjugate composition with a biological sample suspected of containing the analyte to form a fluorescent polymer-dye conjugate complex with said analyte. applying to the sample a light source capable of exciting at least one fluorescent polymer-dye conjugate complex; and Detecting light emitted from the fluorescent polymer-dye conjugate complex. A method comprising: (Item 30) 30. The method of claim 29, wherein the biological sample is selected from the group consisting of blood, bone marrow, spleen cells, lymphocytes, bone marrow aspirate, urine, serum, saliva, cerebrospinal fluid, urine, amniotic fluid, interstitial fluid, stool, mucus or tissue. (Item 31) 31. The method according to item 29 or 30, wherein the biological sample is a blood sample. (Item 32) 32. The method of any one of items 29 to 31, wherein the biological sample is whole blood. (Item 33) 33. The method of claim 32, wherein the biological sample comprises one or more cells of whole blood. (Item 34) 34. The method of claim 33, wherein the one or more cells of whole blood are red blood cells, white blood cells, lymphocytes, phagocytes, monocytes, macrophages, granulocytes, basophils, neutrophils, eosinophils, platelets, or any cell that contains one or more detectable markers. (Item 35) 35. The method of any one of items 29 to 34, wherein the biological sample is derived from a cell culture. (Item 36) 36. The method of any one of items 29 to 35, wherein the light from the light source has a wavelength between about 340 nm and about 800 nm or between about 340 nm and about 450 nm. (Item 37) Item 37. The method of item 36, wherein the emitted light has a wavelength between about 400 nm and about 800 nm. (Item 38) the step of detecting light further comprises analyzing by flow cytometry to obtain a first flow cytometry plot; the first flow cytometry plot, when compared to a second flow cytometry plot obtained by contacting the biological sample with a composition that does not include the non-ionic surfactant and the non-fluorescent component of the first polymeric dye, Reducing non-specific interactions of polymer-dye conjugates, and Reducing Aggregation of Polymer-Dye Conjugates 38. The method of any one of items 29 to 37, wherein the method further comprises one or more of the group consisting of: (Item 39) 29. A kit comprising the composition of any one of items 1 to 28, comprising separate containers containing the one or more non-fluorescent components of the first polymer dye; and the at least one fluorescent polymer-dye conjugate. (Item 40) 40. The kit of claim 39, further comprising a non-ionic surfactant in the container containing the one or more non-fluorescent components. (Item 41) 29. A kit comprising the composition of any one of items 1 to 28, comprising in one container the one or more non-fluorescent components of the first polymer dye and the non-ionic surfactant; and in another container the at least one fluorescent polymer dye conjugate. (Item 42) 29. A kit comprising the composition of any one of items 1 to 28, comprising in one container the two or more non-fluorescent components of a first polymeric dye; and in another container the at least one fluorescent polymeric dye conjugate.

Claims

[Claim 1] The invention described in the specification.

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