Polymer tandem dyes with pendant narrow emission acceptors

Abstract

The invention provides a polymer tandem dye. The polymeric tandem dye includes a narrow emission fluorophore configured to access the pendant group acceptor of the donor hydrosolvated light harvesting multi-chromophore upon energy reception. The narrow emission acceptor fluorophore of the present invention is capable of providing a polymer tandem dye having a narrow emission spectrum suitable for multiplexing. The present invention also provides a composition for multicolor fluorescent signal generation, the composition comprising two or more polymeric tandem dyes. Furthermore, the present invention provides methods of assessing the presence of a target analyte in a sample and methods of labeling a target molecule wherein a target polymeric dye is used in the methods. The invention also provides systems and kits for implementing the subject methods.

Description

[0001] This application is a divisional application of the patent application for invention titled "Polymer Tandem Dyes with Side-Group Narrow-Emission Acceptors", with the application date of July 26, 2019, the application number of 201980060591.2.

[0002] Cross-reference to Related Patent Applications

[0003] This patent application claims the benefit of the filing date of U.S. Provisional Patent Application No. 62 / 715,737, filed on August 7, 2018, under 35 U.S.C. § 119(e); the content of which is incorporated herein by reference. Technical Field

[0004] This application relates to polymer tandem dyes and compositions for generating multicolor fluorescence signals. Background Art

[0005] Fluorescent dyes are compounds that emit (usually) light of a different wavelength when irradiated with light of the wavelength they absorb. Fluorescent dyes can be used in various biochemical, biological, and medical applications, for example, in diagnostic kits, microscopy, or drug screening. Fluorescent dyes are characterized by many parameters so that users can select suitable dyes according to the intended use. The target parameters include the maximum excitation wavelength, the maximum emission wavelength, the Stokes shift, the extinction coefficient, the fluorescence quantum yield, and the fluorescence lifetime. For example, in order to enable the excitation radiation to penetrate a biological sample, minimize background fluorescence, and / or obtain a high signal-to-noise ratio, the dye can be selected according to the target application.

[0006] Molecular recognition involves the specific binding of two molecules. Molecules that have binding specificity for a target biomolecule can be used in various research and diagnostic applications, for example, analyte labeling and separation, flow cytometry, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), western blot analysis, magnetic cell sorting, and chromatography. The target biomolecule can be detected by labeling it with a fluorescent dye. Summary of the Invention

[0007] The present invention provides narrow emission fluorophore dyes and polymeric tandem dyes comprising the narrow emission fluorophore dyes. The polymeric tandem dyes comprise a narrow emission fluorophore configured to be proximal to a side chain acceptor of a donor hydrosolvated light harvesting multi-chromophore upon energy receipt. The polymeric tandem dyes provide a variety of narrow emission spectra suitable for multiplexing applications. The present invention also provides compositions for multicolor fluorescence signal generation, the compositions comprising two or more polymeric tandem dyes. In addition, the present invention also provides methods for assessing the presence of a target analyte in a sample and methods for labeling a target molecule, wherein the labeled polymeric dyes are used in the methods. The present invention also provides systems and kits for practicing the labeled methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] It should be understood that the following drawings are for illustrative purposes only and are not intended to limit the scope of the teachings of the present invention in any way.

[0009] Figure 1 The relationship between the full width at half maximum (FWHM) and the maximum emission wavelength of exemplary narrow emission dyes and various commercially available BODIPY dyes is shown. While the emission peaks of the commercially available BODIPY dyes become broader when moving to longer wavelengths, the peak widths are substantially the same as those of the exemplary dyes.

[0010] Figures 2A-2B Absorption ( Figure 2A ) and emission ( Figure 2B ) spectra of four exemplary narrow emission BODIPY dyes (exhibiting small Stokes shifts) are shown.

[0011] Figure 3 The structure of an exemplary polymeric tandem dye (Violet-D2, see Table 2 for spectral characteristics) comprising a narrow emission spectral acceptor fluorophore substituted with a water-soluble group is illustrated. G 1 is the end group.

[0012] Figure 4 Absorption and narrow emission spectra of an exemplary acceptor fluorophore (D6) with a Stokes shift of about 5 nm are shown.

[0013] Figures 5A-5B Absorption and narrow emission spectra of an exemplary acceptor fluorophore D6 itself ( Figure 5A ) and a polymeric tandem dye (comprising an ultraviolet-excitable polymeric dye linked to the acceptor fluorophore D6) ( Figure 5B ) are compared.

[0014] Figures 6A-6B Absorption and narrow emission spectra of an exemplary acceptor fluorophore D2 itself ( Figure 6A)Comparison of the absorption and narrow emission spectra of polymer tandem dyes (including purple-light-excitable polymer dyes linked to the acceptor fluorophore D2). Figure 6B )

[0015] Figures 7A-7B Exemplary acceptor fluorophore D5 itself is shown ( Figure 7A )Comparison of the absorption and narrow emission spectra of polymer tandem dyes (including purple-light-excitable polymer dyes linked to the acceptor fluorophore D5). Figure 7B )

[0016] Figures 8A-8B Exemplary acceptor fluorophore D8 itself is shown ( Figure 8A )Comparison of the absorption and narrow emission spectra of polymer tandem dyes (including purple-light-excitable polymer dyes linked to the acceptor fluorophore D8). Figure 8B )

[0017] Figure 9 Five different narrow emission curves of multiple polymer tandem dyes based on a single purple-light-excitable polymer dye donor are shown, each polymer tandem dye having a different narrow emission acceptor fluorophore D2, D4, D5, D6, or D8, respectively. DETAILED DESCRIPTION

[0018] As described above, the present invention provides narrow emission fluorophore dyes and polymer tandem dyes including the narrow emission fluorophore dyes. The polymer tandem dyes include a narrow emission fluorophore configured to be adjacent to a side-chain acceptor of a donor water-solvated light-harvesting multi-chromophore upon energy reception. The polymer tandem dyes provide various narrow emission spectra suitable for multiplexing applications. The present invention also provides a composition for generating multi-color fluorescence signals, the composition including two or more polymer tandem dyes. In addition, the present invention also provides a method for evaluating the presence of a target analyte in a sample and a method for labeling a target molecule, wherein the labeled polymeric dye is used in the method. The present invention also provides a system and a kit for implementing the labeled method.

[0019] Before describing the present invention in more detail, it should be understood that the present invention is not limited to the specific embodiments described, as there will surely be differences in actual implementation. It should also be understood that the terms used herein are only for describing specific embodiments and are not intended to limit the inventive concept of the present invention, and the scope of the present invention will only be defined by the appended claims.

[0020] Where a numerical range is provided, it is to be understood that each intermediate value between the upper and lower limits of the range, as well as any other specified value or intermediate value within the range, is included within the scope of the present invention. Unless the context clearly dictates otherwise, each intermediate value shall be as low as one-tenth of the unit of the lower limit. The upper and lower limits of these smaller ranges may independently be included within the smaller ranges and are also included within the present invention, subject to any requirements of any specifically excluded limit values within the stated range. Where the range includes one or both of the limits, ranges excluding any one or both of the included limits are also included within the present invention.

[0021] Certain ranges presented herein are preceded by the term "about" in front of the numerical value. The purpose of using the term "about" herein is to provide literal support for the exact number that follows as well as numbers that are close to or approximate the number that follows the term. When determining whether a number is close to or approximate to a specifically recited number, the unrecited number that is close to or approximate may, in the context in which it appears, be a number that is substantially equivalent to the specifically recited number.

[0022] Unless otherwise defined, all technical and scientific terms used in this patent have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative, exemplary methods and materials are described below.

[0023] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference, and the purpose of incorporating by reference herein is to disclose and describe the methods and / or materials related to the cited publications. Any citation of a publication is for its content that was publicly available prior to the filing date, and it should not be construed as an admission that the present invention is not entitled to antedate such publication due to a prior invention. Additionally, the provided publication dates may differ from the actual publication dates and may require separate verification.

[0024] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional elements. Accordingly, this statement is intended as a basis for using exclusive terms such as "solely", "only", etc. in relation to the recitation of claim elements or for using a "negative" limitation.

[0025] After reading this invention, the following will be apparent to those skilled in the art. Each individual embodiment described and listed herein has hierarchical components and features that can be quickly disassembled or combined with the features of any one of several other embodiments without departing from the scope and spirit of this invention. Any stated method can be implemented in the order of the stated events or any other order that is logically possible.

[0026] Although the device and method have been or will be described for grammatical fluency and functionally, it should be clearly understood that unless specifically provided in Title 35 of the United States Code, in no case shall the claims be construed as necessarily limited by "means" or "step", but shall conform to the meaning defined in the claims and the full scope of equivalents in accordance with the judicial doctrine of equivalents. When the claims are specifically written in accordance with the provisions of Section 112 of Title 35 of the United States Code, the claims shall conform exactly to the statutory equivalents in Section 112 of Title 35 of the United States Code.

[0027] In further describing this invention, narrow emission fluorophore dyes for conjugating with target molecules are also described. Next, polymer tandem dyes including such narrow emission acceptor fluorophores as side groups are described in more detail. Next, labeled specific binding members including the subject polymer dyes are described. Then, multi-color fluorescence signal generation achieved using two or more polymer tandem dyes is described. The methods of use of the subject polymer dyes are also reviewed. In addition, systems and kits that can be used to implement the methods described herein are also described.

[0028] Narrow emission fluorophore dye

[0029] Fluorescence detection depends on an excitation source and a signal detector. If the detector collects some light from the excitation source, an erroneous signal may be generated. When the emission signal is laser-induced fluorescence, the intensity of the excitation light source can introduce a background signal at the detector that is significantly higher than the fluorescence emission signal from the fluorophore itself. Detectors suitable for laser-induced systems (such as photomultiplier tubes) may be damaged when irradiated by the laser source due to their high sensitivity. Therefore, in a laser-induced detection system, the excitation wavelength is shifted by a minimum spectral space relative to the wavelength at which the detector collects the emitted light. For example, in flow cytometry, the minimum difference between the excitation wavelength and the emission wavelength detection value can be 10 nm or greater.

[0030] As described above, the present invention provides narrow emission fluorophores and polymeric tandem dyes comprising the narrow emission acceptor fluorophores as side group acceptor groups. In some cases, the narrow emission spectrum is an emission spectrum having an emission peak with a full width at half maximum (FWHM) of 35 nm or less, such as 25 nm or less, 20 nm or less, 15 nm or less, 12 nm or less, 10 nm or less, e.g., a spectrum measured in ethanol or phosphate buffered saline (PBS).

[0031] The subject narrow emission fluorescent dyes may have a small Stokes shift. The Stokes shift is the wavelength or frequency difference between the maximum absorption and the maximum emission of a particular fluorescent dye. A small Stokes shift is about 10 nm or less, such as 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm or even less. Due to the small difference between the absorption maximum and the emission maximum, it may be difficult to effectively excite the dye and collect the fluorescence.

[0032] Aspects of the present invention include narrow emission fluorescent dyes and polymeric tandem dyes comprising the emission fluorescent dyes. The subject narrow emission fluorescent dyes have a narrow emission spectrum which can provide important advantages when used in a multiplexed signal format. Due to the narrow emission spectrum, a greater number of dyes can be simultaneously combined and used within the same spectral range compared to multiplexed systems based on broad-spectrum emitters. By keeping the detection window suitable for each dye to a small size, multiple smaller emission ranges can be included in a multiplex assay, thereby increasing the number of independent fluorescence signals observed in a given system.

[0033] For the absorption and emission spectra of exemplary narrow emission fluorescent dyes, see Figure 4 .. The narrow emission of the dye makes it suitable for efficient signal collection in a narrow detection window and thus for multiplex assays, but the small Stokes shift may make it difficult to excite and detect dyes with good signal throughput and maximum achievable excitation and detection. Note that in the Figure 4 emission spectrum shown, emission data located on the blue side of the emission maximum are not collected. This is because the detector wavelength is close to the excitation wavelength of the light source. An example of this problem is the signal used in flow cytometry in laser-induced fluorescence. Since in some cases the minimum safe difference between excitation and emission is 10 nm, if the dye is excited at its absorption maximum, many signals from narrow emission dyes with a small Stokes shift (e.g., as defined herein) will not be collected.

[0034] The subject narrow emission fluorescent dyes can be used as FRET acceptors with a donor, whose absorption wavelength is significantly different from the maximum emission wavelength of the acceptor dye, thus providing a system capable of discrete excitation and emission detection and maximum signal collection. While maintaining the narrow emission spectrum of such tandem dyes, an effective Stokes shift of dozens of nanometers to hundreds of nanometers can be achieved. The spectra of exemplary narrow emission acceptor dyes with small Stokes shifts and correspondingly tandem with polymer light-harvesting multi-chromophores as donors can be seen in Figures 5A-5B . The individual narrow emission acceptor dye exhibits an 8 nm Stokes shift in PBS. The polymer tandem dye including the narrow emission acceptor dye donor exhibits a shift of at least 150 nm from the maximum absorption wavelength to the maximum emission wavelength. The present invention provides a number of different narrow emission tandem dyes, all of which have a large effective Stokes shift.

[0035] Any suitable fluorophore dye with a narrow emission spectrum can be used in the tandem dyes of the present invention. The target narrow emission acceptor fluorophores include but are not limited to fluorescent semiconductor nanocrystals, quantum dots, and small molecule fluorophores. The full width at half maximum (FWHM) of the spectrum of the narrow emission fluorophore for use in the aspects of the present invention can be 35 nm or less, such as 30 nm or less, 25 nm or less, 20 nm or less, 15 nm or less, or 10 nm or less. In some cases, the FWHM of the narrow emission fluorophore is 25 nm or less. In some cases, when directly exciting the fluorophore, the FWHM of the narrow emission fluorophore can be measured. In some cases, such as when indirectly exciting the fluorophore through a polymer donor dye, the FWHM of the narrow emission fluorophore can be measured.

[0036] In some cases, the small molecule fluorophore having a narrow emission spectrum is a BODIPY dye. In some cases, BODIPY dyes are a class of dyes having a narrow emission spectrum suitable for multiplexing applications. However, the small Stokes shift of such dyes can offset the advantage of narrow emission and reduce the utility of such dyes. The present invention provides tandem dyes intermediate between polymer donor dyes and narrow emission dyes, which have a large effective Stokes shift and a high signal flux within a narrow spectral emission window. In some cases, conventional BODIPY dyes have low solubility, which makes them unsuitable as acceptors, for example, for use in combination with conventional proteins. Any suitable BODIPY dye can be applicable to the subject dyes and polymer tandem dyes. Exemplary BODIPY dyes include, but are not limited to, the dyes described in the following publications: Loudet and Burgess ("BODIPY Dyes and Their Derivatives: Synthesis and Spectral Properties," Chemical Reviews, 2007, 107, 4891–4932); or Suzuki et al., U.S. Patent No. 8,193,350; Ulrich et al., U.S. Patent No. 8,476,461; and Ulrich et al., U.S. Patent No. 7,897,786, the contents of which are incorporated herein by reference in their entirety.

[0037] Aspects of the invention include BODIPY dyes (including water-soluble groups, BODIPY dye precursors, and BODIPY dye reagents) adapted to be linked to a target molecule; and polymer tandem dyes comprising the BODIPY dyes. In some cases, the subject BODIPY dyes are substituted with groups or substituents including chemoselective functional groups that promote conjugation of the BODIPY dyes to a target molecule such as a polymer donor dye. It should be understood that for any BODIPY dye described herein (e.g., the dyes represented by formulas (I)-(IXa)), the invention includes novel BODIPY dyes and their BODIPY dye reagent derivatives (including chemoselective functional groups) that are not conjugated to the polymeric dye backbone. Such dyes and dye reagents can be used in a variety of settings. Exemplary BODIPY dye reagents can be conjugated to any suitable target molecule (e.g., a biomolecule suitable for a second dye such as a polymeric dye). Accordingly, the invention also includes polymer tandem dyes comprising the linked BODIPY dyes described herein (e.g., polymer tandem dyes comprising a dye represented by one of formulas (I)-(IXa)).

[0038] In certain embodiments, the BODIPY dye has a structural formula as shown in formula (I):

[0039]

[0040] Wherein:

[0041] Q is C or N;

[0042] R 1 -R 7 Each independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG) and –L 1 -Z 1 , or

[0043] Optionally, selected from R 6 and R 7 、R 2 and R 3 、R 5 and R 6 、R 3 and R 4 、R 4 and R 1 as well as R 5 and R 1 Any one or more pairs of substituents together form a divalent group, connect to form a cyclic structure, and together with the carbon atom to which it is attached form a 5- or 6-membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring (e.g., a 5- or 6-membered ring containing a carbon atom and 0-3 heteroatoms selected from O, S and N), where the ring may be unsubstituted or may be further substituted by a substituent independently selected from the following groups: alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG) and –L 1 -Z 1 ;

[0044] L 1 is a linker;

[0045] Z 1 is a light-harvesting multi-chromophore or a linked chemoselective functional group; and

[0046] Y 1 and Y 2 are independently selected from F, OH, H, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl and WSG;

[0047] where Y 1 、Y 2 and R 1 -R 7One of which is connected to the repeating unit of the light-harvesting multi-chromophore or to a chemically selective functional group that has been connected (e.g., –L 1 -Z 1 , for conjugating with a target molecule having a compatible chemically selective functional group). In formula (I), it should be understood that the substituents Y 1 、Y 2 and R 1 -R 7 can be selected from groups that do not inhibit the fluorescence emission of the BODIPY dye. In certain cases of formula (I), Q is C. It should be understood that for any formula described herein, the corresponding formula can be included, in which the atom represented by Q in formula (I) is a nitrogen atom. In certain embodiments of formula (I), one or more of Y 1 、Y 2 and R 1 -R 7 include WSG. In certain cases of formula (I), Y 1 and Y 2 each include WSG. In certain cases of formula (I), the linker of –L 1 -Z 1 includes WSG. In some cases of formula (I), one of Y 1 、Y 2 and R 1 -R 7 is connected when approaching the light-harvesting multi-chromophore that has been connected during energy reception.

[0048] In formula (I), the substituent pairs R 6 and R 7 and / or R 2 and R 3 can be connected to form a cyclic structure, forming a 5- or 6-membered fused ring, and the ring is unsubstituted or substituted. In some cases of formula (I), the 5- or 6-membered fused ring is an aryl or heteroaryl ring selected from furan, thiophene, pyrrole, oxazole, isoxazole, thiazole, isothiazole, imidazole, and pyrazole. In some cases of formula (I), the structural formula of the BODIPY dye is as shown in formula (IIa) or formula (IIb):

[0049]

[0050] Wherein:

[0051] Z 11 and Z 12 are independently the fused 5- or 6-membered fused heterocyclic, carbocyclic, aryl, or heteroaryl ring;

[0052] Each “i” is independently 0-3; and

[0053] Each R 20 is independently a substituent as defined for R in formula (I) 2 -R 7 as defined herein.

[0054] In certain embodiments of formulas (IIa)-(IIb), Z 11 and Z 12 are independently 5- or 6-membered fused aryl or heteroaryl rings. In some cases, Z 11 and Z 12 are independently selected from furan, thiophene, pyrrole, oxazole, isoxazole, thiazole, isothiazole, imidazole, and pyrazole. In some cases of formulas (IIa)-(IIb), Z 11 and / or Z 12 is furan or thiophene. In certain cases of formula (IIa), none of the R 1 -R 5 are cyclic linking groups. In some cases of formulas (IIa)-(IIb), Z 11 and Z 12 are independently selected from the following rings:

[0055]

[0056] wherein:

[0057] X is O or S;

[0058] Y is O, S, or NR, where R is H, alkyl, substituted alkyl, or a substituent as defined for R in formulas (IIa)-(IIb); and 20 wherein

[0059] R 21 -R 23 is independently selected from H and a substituent as defined for R in formulas (IIa)-(IIb). In certain cases of formulas (IIa)-(IIb), Z 20 and Z 11 and Z 12 are independently selected from the following rings:

[0060] as described above.

[0061] In certain cases of formula (IIb), Z 11 and / or Z 12 are the same ring. In certain cases, Z 11 and Z 12 comprise different rings.

[0062] In certain embodiments of formula (IIb), the structure of the BODIPY dye is as follows:

[0063]

[0064] In certain embodiments of formula (IIa), the structural formula of the BODIPY dye is as shown in formula (IIIa) or formula (IIIb):

[0065]

[0066] Wherein:

[0067] X is O or S; and

[0068] R 16 and R 17 are substituents as defined for R 6 and R 7 in formula (I).

[0069] In certain cases of formula (IIIa) or (IIIb), neither R 1 -R 5 is a cyclic linking group.

[0070] In certain embodiments of formula (I) or (IIb), the structural formula of the BODIPY dye is as shown in formula (IVa) or (IVb):

[0071]

[0072] Wherein:

[0073] Each X is O or S; and

[0074] R 12 、R 13 、R 16 and R 17 are substituents as defined for R 2 、R 3 、R 6 and R 7 in formula (I).

[0075] In certain embodiments of formula (I)-(IVb), the BOBIPY dye comprises a chemically selective functional group or a target molecule that has been attached, for example, a light-harvesting multi-chromophore (e.g., –L 1 -Z 1 ). In some cases, one of R 1 -R 7 includes –L 1 -Z 1 . In certain cases, Y 1 or Y 2 includes –L 1 -Z 1。In certain embodiments of formulas (I)-(IVb), R 1 is –L 1 -Z 1 , where L 1 is a linker, and Z 1 is a chemoselective functional group or a light-harvesting multi-chromophore. In certain embodiments of formulas (I)-(IVb), R 4 or R 5 is –L 1 -Z 1 . In certain embodiments of formulas (I), (IIa), and (IIIa)-(IIIb), R 2 or R 7 is –L 1 -Z 1 . In certain embodiments of formulas (I), (IIa), and (IIIa)-(IIIb), R 3 or R 6 is –L 1 -Z 1 . In certain embodiments of formulas (IIa)-(IIb), the R 20 substituent is –L 1 -Z 1 . In certain embodiments of formulas (IIIa)-(IIIb), R 16 or R 17 is –L 1 -Z 1 . In certain embodiments of formulas (IVa)-(IVb), R 12 , R 13 , R 16 , or R 17 is –L 1 -Z 1 .

[0076] In some cases, R 1 is -L 1 -Z 1 , where L 1 is a linker having a backbone of 20 atoms or fewer in length (e.g., as described herein), and Z 1 is the chemoselective functional group. In some cases of R 1 , L 1 is selected from alkyl or substituted alkyl linkers, alkoxy or substituted alkoxy linkers, PEG linkers, sulfonamido-alkyl or substituted sulfonamido-alkyl linkers, amido-alkyl or substituted amido-alkyl linkers, and alkyl-amido-alkyl or substituted alkyl-amido-alkyl linkers. The linker can be substituted with a WSG (e.g., a PEG group). In certain cases of R 1 , L1 Selected from C 1 -C 12 alkyl or substituted alkyl linker, C 1 -C 12 alkoxy or substituted alkoxy linker, C 1 -C 12 amido-alkyl or substituted amido-alkyl linker and C 1 -C 12 alkyl-amido-alkyl or substituted alkyl-amido-alkyl linker. In certain cases of R 1 , Z 1 is a carboxylic acid or its activated ester.

[0077] In certain cases of formula (I)-(IVb), R 1 includes an optionally substituted carbocyclic or heterocyclic group linked to the repeating unit or chemo-selective functional group of the light-harvesting multi-chromophore. In certain cases, R 1 is an optionally substituted aryl or heteroaryl linked to the repeating unit or chemo-selective functional group of the light-harvesting multi-chromophore. The target divalent carbocyclic or heterocyclic groups include but are not limited to 1,4-cyclohexyl, 1,3-cyclohexyl, piperidinyl (e.g., 1,4-piperidinyl), piperazinyl (e.g., 1,4-piperazinyl), etc. The target divalent aryl or heteroaryl groups include but are not limited to 1,4-phenyl, 1,3-phenyl, 2,5-pyridinyl, 2,6-pyridinyl, 3,5-pyridinyl, etc. The divalent carbocyclic or heterocyclic group or the divalent aryl or heteroaryl group of R 1 can be linked to –L 2 -Z 1 , where L 2 is a linking group, e.g., the linking groups described in any of the embodiments herein.

[0078] In certain cases of formula (I), the structural formula of the BODIPY dye is as shown in formula (V):

[0079]

[0080] Wherein:

[0081] L 3 is a covalent bond, oxo (-O-), alkylene (e.g., C 1 -C 6 -alkylene), -O-alkylene or its substituted form;

[0082] R 11 is the group defined for 1 ;

[0083] Each R 9Each is an optional substituent selected from halogen, hydroxyl, cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; and t is 0 - 4. In certain cases of formula (V), L 3 is a covalent bond. In some cases of formula (V), L 3 is oxo. In certain cases of formula (V), L 3 is a covalent bond.

[0084] In certain cases of formula (V), the structural formula of the BODIPY dye is as shown in formula (Va):

[0085]

[0086] Wherein: R 11 is the group defined for R 1 ; each R 9 is an optional substituent selected from halogen, hydroxyl, cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; and t is 0 - 4. In some cases of formula (V)-(Va), R 11 is L 1 -Z 1 (for example, as described herein). In some cases of formula (V)-(Va), R 11 includes a sulfonamido-alkyl or substituted sulfonamido-alkyl linker, an amido-alkyl or substituted amido-alkyl linker, or an alkyl-amido-alkyl or substituted alkyl-amido-alkyl linker. In certain cases of formula (V)-(Va), R 2 , R 4 , R 5 and R 7 are each independently H, alkyl, or substituted alkyl. In certain cases of formula (V)-(Va), R 3 and R 6 are each independently H, alkyl, or substituted alkyl. In some cases, R 3 and R 6 are both H. In certain cases of formula (V)-(Va), R 2 , R 4 , R 5 and R 7 are each independently C 1 -C 6 alkyl or substituted C 1 -C 6 alkyl.

[0087] In certain cases of formula (V), the structural formula of the BODIPY dye is as shown in formula (VIa) or (VIb):

[0088]

[0089] Wherein:

[0090] * is a bonding point for a repeating unit or a chemoselective functional group that bonds to the light-harvesting multi-chromophore; and

[0091] Y 1 and Y 2 are independently selected from the group consisting of F, OH, H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkynyl, substituted alkynyl, and WSG.

[0092] In certain cases of formula (I), the structural formula of the BODIPY dye is as shown in formula (VIIa) or (VIIb):

[0093]

[0094] Wherein: n is 0 - 12, Z 1 is a chemoselective functional group (e.g., as described herein) or a linker molecule of interest. In certain cases of formula (VIIa)-(VIIb), n is 1 - 12 or 1 - 6, for example, 1, 2, 3, 4, 5, or 6. In certain cases of formula (VIIa)-(VIIb), R 2 -R 4 and R 5 -R 7 are each independently selected from H, C 1 -C 6 alkyl and substituted C 1 -C 6 alkyl. In certain cases of formula (VIIa)-(VIIb), R 2 and R 4 and / or R 5 and R 7 are each independently selected from C 1 -C 6 alkyl and substituted C 1 -C 6 alkyl. In certain cases of formula (VIIa)-(VIIb), R 3 and / or R 6 is H. In certain cases of formula (VIIa)-(VIIb), R 3 and / or R 6 is alkyl or substituted alkyl.

[0095] In certain embodiments of formula (VIIa), the structure of the BODIPY dye is as follows:

[0096]

[0097] In certain cases of formula (IIIa), the structural formula of the BODIPY dye is as shown in formula (VIII):

[0098]

[0099] Wherein: L 1 is a linker, and Z 1 is a chemoselective functional group (e.g., as described herein) or a linker molecule of interest. In certain cases of formula (VIII), the structural formula of the BODIPY dye is as shown in formula (VIIIa):

[0100]

[0102] Wherein: L2 is a linker (e.g., the linking group component of L 1 ); each R 9 is an optional substituent selected from halogen, hydroxyl, cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; and t is 0 - 4.

[0103] In certain cases of formula (IVa), the structural formula of the BODIPY dye is as shown in formula (IX):

[0104]

[0105] Wherein: R 12 and R 13 are the substituents defined for R 2 and R 3 in formula (I); L 1 is a linker, and Z 1 is a chemoselective functional group (e.g., as described herein) or a linker molecule of interest. In certain cases of formula (IX), the structural formula of the BODIPY dye is as shown in formula (IXa):

[0106]

[0107] Wherein: L2 is a linker (e.g., the linking group component of L 1 ); each R 9are each independently selected from the group consisting of halogen, hydroxyl, cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; and t is from 0 to 4.

[0108] In any of the embodiments of Formulas (I)-(IXa) described herein, the BODIPY dye may include a specific –L 1 -Z 1 group as described in one of the following embodiments.

[0109] L 1 can be a linker having a backbone of 20 atoms or less in length (e.g., as described herein). In some cases, L 1 is selected from an alkyl or substituted alkyl linker, an alkoxy or substituted alkoxy linker, a PEG linker, a sulfonamido-alkyl or substituted sulfonamido-alkyl linker, an amido-alkyl or substituted amido-alkyl linker, and an alkyl-amido-alkyl or substituted alkyl-amido-alkyl linker. The linker may be substituted with a WSG (e.g., a PEG group). In certain cases, L 1 is selected from C 1 -C 12 alkyl or substituted alkyl linkers, C 1 -C 12 alkoxy or substituted alkoxy linkers, C 1 -C 12 amido-alkyl or substituted amido-alkyl linkers, and C 1 -C 12 alkyl-amido-alkyl or substituted alkyl-amido-alkyl linkers. The linker L may include multiple linked components, e.g., one or more linking groups independently selected from the group consisting of lower alkylene, substituted lower alkylene, alkenylene (-CH═CH-), substituted alkenylene, alkynylene (-C≡C-), substituted or unsubstituted amido (e.g., -NRCO- or –CONR-, where R is H, alkyl, or substituted alkyl), substituted or unsubstituted sulfonamido (e.g., -NRSO 2 - or -SO 2 NR-, where R is H, alkyl, or substituted alkyl), oxo (-O-), thio (-S-), ethylene glycol (-OCH 2 CH 2 O-), polyethylene glycol (e.g., -(CH 2 CH 2 O) n-, where n is from 2 to 20, for example, from 2 to 10 or from 2 to 6, or 2, 3, 4, 5 or 6), arylene, substituted arylene, heteroarylene, substituted heteroarylene, α - amino acid residue, β - amino acid residue, etc.

[0110] Z 1 can be a chemoselective functional group suitable for conjugating with a target molecule having compatible functional groups. The chemoselective functional groups for the target BODIPY dyes include, but are not limited to, amine groups (e.g., -NH 2 ), carboxylic acids (-CO 2 H), activated esters (e.g., NHS or sulfo - NHS esters), thiols, maleimides, iodoacetamides, hydroxyl groups, hydrazide groups, hydrazine groups, aldehydes, ketones, azides, alkynes, tetrazines, alkenes, phosphines, and epoxides. It should be understood that in some cases, the chemoselective functional group of the BODIPY dye is a synthetic precursor or a protected form of the target functional group, which can be converted into a reactive functional group capable of conjugating with the target molecule. For example, a carboxylic acid is a chemoselective functional group that can couple with an amine group on the target molecule. The carboxylic acid can be converted into an activated ester for coupling with the amine group in situ or before coupling. In certain embodiments, Z 1 is a protected hydroxy acid, such as an ester - protected carboxylic acid. In some cases, Z 1 is a synthetic precursor of a chemoselective functional group (e.g., an azide that can be reduced to an amine, or a cyano group that can be hydrolyzed to a hydroxy acid) by any suitable method.

[0111] In certain embodiments of formula (IXa), the structure of the BODIPY dye is as follows:

[0112]

[0113] In certain embodiments of -L 1 -Z 1 in formulas (I)-(IVb), L 1 is an alkylene or a substituted alkylene, and Z 1 is a carboxylic acid or a carboxylic acid activated ester. In some cases, -L 1 -Z 1 is -(C 1 -C 12 ) alkylene - CO 2 H, for example –(C 1 -C 6 ) alkylene - CO 2 H. In certain embodiments of -L 1 -Z 1 in formulas (I)-(IVb), L 1 is an arylene or a substituted arylene, and Z 1is a carboxylic acid or a carboxylic acid activated ester. -L in formulas (I)-(IVb) 1 -Z 1 In certain embodiments of, L 1 is heteroarylene (e.g., divalent pyridine) or substituted heteroarylene, and Z 1 is a carboxylic acid or a carboxylic acid activated ester. In some cases, -L 1 -Z 1 is selected from – phenyl - O - (C 1 -C 12 ) alkylene - CO 2 H and – substituted phenyl - O - (C 1 -C 12 ) alkylene - CO 2 H.

[0114] In some embodiments of formulas (I)-(IXa), L 1 comprises an optionally substituted carbocyclic or heterocyclic group linked to a repeating unit or a chemoselective functional group (Z 1 ) of the light - harvesting multi - chromophore. In certain cases, L 1 is an optionally substituted aryl or heteroaryl. The target divalent carbocyclic or heterocyclic groups include but are not limited to 1,4 - cyclohexyl, 1,3 - cyclohexyl, piperidinyl (e.g., 1,4 - piperidinyl), piperazinyl (e.g., 1,4 - piperazinyl), etc. The target divalent aryl or heteroaryl groups include but are not limited to 1,4 - phenyl, 1,3 - phenyl, 2,5 - pyridinyl, 2,6 - pyridinyl, 3,5 - pyridinyl, etc. The divalent carbocyclic or heterocyclic group or divalent aryl or heteroaryl group of L 1 can be linked to –L 2 -Z 1 , where L 2 is a linking group, e.g., a linking group described in any embodiment herein. In some cases of formulas (I)-(IXa), the structure of -L 1 -Z 1 is shown as one of the following structures:

[0115]

[0116] Wherein:

[0117] R 11 is L 2 -Z 1 ;

[0118] L 2 is a linker, Z 1 is a chemoselective group or a target molecule;

[0119] t is 0 - 4; and each R 9Each is independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, sulfonic acid, and water-soluble group (WSG). In certain embodiments, -L 1 -Z 1 has a structure shown in one of the following structures:

[0120]

[0121] Wherein:

[0122] L 2 is a linker, and Z 1 is a chemoselective group or a target molecule;

[0123] t is 0 - 4; and each R 9 is independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, sulfonic acid, and water-soluble group (WSG). In certain embodiments, -L 1 -Z 1 has a structure shown in one of the following structures:

[0124]

[0125] Wherein:

[0126] L 2 is a linker, and Z 1 is a chemoselective group or a target molecule;

[0127] R 32 is H, alkyl, substituted alkyl, and water-soluble group (WSG);

[0128] L 3 is a linker selected from alkylene (e.g., C 1 -C 6 -alkylene), -O-alkylene, and its substituted forms;

[0129] t is 0 - 4; and each R 9 is independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, sulfonic acid, and water-soluble group (WSG).

[0130] In any of the embodiments of the formulas (I)-(IXa) described herein, the BODIPY dye may include a specific Y 1 and Y 2 group described in one of the following embodiments. In some cases of the formulas (I)-(IXa), Y1 comprises a water-soluble group (WSG). In some cases, Y 1 is an alkynyl group substituted with WSG. Y2 may be the same as or different from Y 1 . In some cases of formulas (I)-(IXa), Y 2 is selected from F, OH, H, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl and substituted alkynyl. In some cases, Y 2 is selected from F, CN, phenyl and substituted phenyl.

[0131] In some cases of formulas (I)-(IXa), Y 1 and Y 2 both comprise a water-soluble group (WSG). In some cases, Y 1 and / or Y 2 is an alkynyl group substituted with WSG (e.g., as described herein). In some cases, Y 1 and / or Y 2 is an alkynyl group substituted with polyethylene glycol (PEG) or modified (PEG). In some cases as described in any embodiment of formulas (I)-(IXa), the structural formula of Y 1 and / or Y 2 is as follows:

[0132]

[0133] wherein:

[0134] s is from 1 to 12;

[0135] q is from 0 to 50; and

[0136] R 21 is H, alkyl or substituted alkyl. In some cases, s is from 1 to 6, e.g., 1, 2 or 3. In some cases, q is from 1 to 50, from 1 to 30, from 2 to 30, from 4 to 30, from 6 to 30, from 8 to 30, from 10 to 30, from 10 to 20 or from 11 to 16. In some cases, q is from 10 to 50, e.g., from 10 to 30, from 10 to 20 or from 11 to 16.

[0137] In some embodiments of formulas (I) and (VIIa), the structure of the BODIPY dye is as follows:

[0138]

[0139] where Z 1 is a chemoselective functional group (e.g., hydroxy acid) or a donor polymeric dye; n is 0 - 6; each R, R 2 , R 4, R 5 and R 7 are each independently H, C 1 -C 6 alkyl or substituted C 1 -C 6 alkyl; and each q is from 6 to 20 (e.g., 10 to 20). In certain embodiments of formulas (I) and (VIIa), the structure of the BODIPY dye is shown as one of the following structures:

[0140]

[0141] In certain embodiments of formulas (V) and (Va), the structure of the BODIPY dye is as follows:

[0142]

[0143] wherein Z 1 is a chemoselective functional group (e.g., hydroxy acid) or a donor polymeric dye; n is 0 - 6; R, R 2 , R 4 , R 5 and R 7 are each independently H, C 1 -C 6 alkyl or substituted C 1 -C 6 alkyl; and each q is from 6 to 20 (e.g., 10 to 20). In certain embodiments of formulas (V), (Va) and (VIa) or (VIb), the structure of the BODIPY dye is shown as one of the following structures:

[0144]

[0145] In certain embodiments of formula (VIII), the structure of the BODIPY dye is as follows:

[0146]

[0147] wherein Z 1 is a chemoselective functional group (e.g., hydroxy acid) or a donor polymeric dye; n is 0 - 6; each R, R 2 and R 4 are each independently H, C1 - C 6 alkyl or substituted C 1 -C 6 alkyl; and each q is from 6 to 20 (e.g., 10 to 20). In certain embodiments of formula (VIII), the structure of the BODIPY dye is shown as one of the following structures:

[0148]

[0149] In certain embodiments of formula (IX)-(IXa), the structure of the BODIPY dye is as follows:

[0150]

[0151] wherein Z 1 is a chemoselective functional group (e.g., hydroxy acid) or a donor polymeric dye; n is 0 - 6; each R and R 13 are each independently H, C1 - C 6 alkyl or substituted C 1 -C 6 alkyl; and each q is 6 - 20 (e.g., 10 - 20). In certain embodiments of formula (IX)-(IXa), the structure of the BODIPY dye is as follows:

[0152]

[0153] In certain embodiments of formula (IIIa), the structure of the BODIPY dye is as follows:

[0154]

[0155] In certain embodiments of formula (IVa), the structure of the BODIPY dye is as follows:

[0156]

[0157] In certain embodiments of formula (IXa), the structure of the BODIPY dye is shown as one of the following structures:

[0158]

[0159] Water-soluble group

[0160] The terms "water - soluble enhancing group", "water - soluble group" and WSG used herein are used interchangeably and refer to a group or substituent that is effectively solvated in an aqueous environment (e.g., under physiological conditions) and can enhance the water solubility of the molecule to which it is attached. In some embodiments, compared to a control multi - chromophore without WSG, WSG can increase the solubility of the multi - chromophore in an aqueous solution. The water - soluble group can be any suitable hydrophilic group that is effectively solvated in an aqueous environment.

[0161] The water-soluble group can be attached to the comonomer and / or the narrow emission fluorophore (e.g., as described herein) at any suitable position. In some cases, the WSG provides a side chain group that protrudes away from the backbone of the conjugated polymer to exert a highly solvating effect in an aqueous environment. In some cases, the WSG is a branched nonionic water-soluble group (WSG) comprising two or more water-soluble polymers, each polymer having 6-100, such as 6-50, 6-40, or 6-30 monomer units. In some cases, the WSG is a branched nonionic water-soluble group.

[0162] The water-soluble polymer tandem dyes of the present invention can be dissolved in an aqueous environment, making them particularly suitable for various bioassays. The subject water-soluble polymers and their conjugates can resist unwanted polymerization, which can result in favorable fluorescence and spectral properties in various bioassays. Dye polymerization is unwanted because it can lead to a reduction in the fluorescence signal, e.g., dye fluorescence quenching caused by polymerization. The subject water-soluble conjugated polymer dyes can be used as fluorescence reporters for various biosensors and provide an unusually bright signal as well as a range of excitation and emission wavelength options suitable for various applications (e.g., flow cytometry and imaging).

[0163] In certain cases, the solubility of the water-soluble polymer tandem dye in water or buffer is 1 mg / mL or higher, e.g., 3 mg / mL or higher, 10 mg / mL or higher, 20 mg / mL or higher, 30 mg / mL or higher, 40 mg / mL or higher, 50 mg / mL or higher, 60 mg / mL or higher, 70 mg / mL or higher, 80 mg / mL or higher, 90 mg / mL or higher, 100 mg / mL or higher, or even higher. It should be understood that under certain conditions, the water-soluble polymeric dye can form discrete water-solvated nanoparticles in an aqueous system. In certain cases, the water-solvated nanoparticles have anti-polymerization properties and can be used in various bioassays.

[0164] The branched nonionic water-soluble group (WSG) contains a branched group attached to the comonomer to which it is attached and is further bonded to two, three or more nonionic water-soluble polymers. In some cases, the two or more water-soluble polymers used in the WSG are polyethylene glycol (PEG) groups or modified PEG groups. Any suitable branched group that provides three or more attachment points (e.g., four or more), such as one attachment point to the comonomer attached to the conjugate polymer backbone and two or more attachment points (e.g., two, three, four or more) to the nonionic water-soluble polymer, can be utilized. The branched group can be attached to the comonomer and the nonionic water-soluble polymer via an optional linker using any suitable linking functional group and linking chemistry. In certain cases, the branched group is a non-aromatic group. In certain cases, the branched group is a cyclic group. In some cases, the branched group is a substituted aryl or heteroaryl ring, e.g., a trisubstituted or tetrasubstituted aryl ring, such as a trisubstituted or tetrasubstituted benzene ring, or a trisubstituted or tetrasubstituted heteroaryl ring, such as a trisubstituted or tetrasubstituted pyridyl ring. In certain cases, the branched group is an acyclic group. In some cases, the branched group is an atom, e.g., C, Si, N. In certain cases, the branched group is a linking functional group, such as an amide group or a sulfonamide group. In certain cases, the branched group is an amino acid residue or a branched linker, such as glycerol or aminoglycerol.

[0165] The purpose water-soluble polymers include, but are not limited to, polyalkylene oxide-based polymers, e.g., polyethylene glycol (PEG) (see, e.g., “Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications”, J.M. Harris, editor, Plenum Press, New York, NY (1992); Poly(ethylene glycol) Chemistry and Biological Applications, J.M. Harris and S.Zalipsky, editor, ACS (1997); International Patent Applications: WO 90 / 13540, WO 92 / 00748, WO 92 / 16555, WO 94 / 04193, WO 94 / 14758, WO 94 / 17039, WO 94 / 18247, WO 94 / 28937, WO 95 / 11924, WO 96 / 00080, WO 96 / 23794, WO 98 / 07713, WO 98 / 41562, WO 98 / 48837, WO 99 / 30727, WO 99 / 32134, WO 99 / 33483, WO 99 / 53951, WO 01 / 26692, WO 95 / 13312, WO 96 / 21469, WO 97 / 03106, WO 99 / 45964; and U.S. Patents numbered as follows: 4,179,337; 5,075,046; 5,089,261; 5,100,992; 5,134,192; 5,166,309; 5,171,264; 5,213,891; 5,219,564; 5,275,838; 5,281,698; 5,298,643; 5,312,808; 5,321,095; 5,324,844; 5,349,001; 5,352,756; 5,405,877; 5,455,027; 5,446,090; 5,470,829; 5,478,805; 5,567,422; 5,605,976; 5,612,460; 5,614,549; 5,618,528; 5,672,662; 5,637,749; 5,643,575; 5,650,388; 5,681,567; 5,686,110; 5,730,990; 5,739,208; 5,756,593; 5,808,096; 5,824,778; 5,824,784; 5,840,900; 5,874,500; 5,880,131; 5,900,461; 5,902,588; 5,919,442; 5,919,455; 5,932,462; 5,965,119; 5,965,566; 5,985,263; 5,990,237; 6,011,042; 6,013,283; 6,077,939; 6,113,906; 6,127,355; 6,177,087; 6,180,095; 6,194,580; 6,214,966).

[0166] Examples of water-soluble polymers include, but are not limited to, water-soluble polymers containing polyalkylene oxides, polyamide alkylene oxides, or derivatives thereof, including polyalkylene oxides and polyamide alkylene oxides containing ethylene oxide repeating units represented by the formula -(CH 2 -CH 2 -O)-. Further examples of the target polymers include polyamides of the formula -[C(O)-X-C(O)-NH-Y-NH]n- or -[NH-Y-NH-C(O)-X-C(O)] n - having a molecular weight greater than 1,000 daltons, wherein X and Y are divalent groups, which may be the same or different, and may be branched or linear, and n is a discrete integer between 2 and 100, for example, 2 to 50; and wherein one or both of X and Y contain water-soluble repeating units that are biocompatible and substantially non-antigenic, and the repeating units may be branched or linear. Further examples of water-soluble repeating units include those represented by the formula -(CH 2 -CH 2 -O)- or -(O-CH 2 -CH 2 )- of ethylene oxide. The number of the water-soluble repeating units can vary widely, and the number of the units ranges from 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, 6 to 100, for example, 2 to 50 or 6 to 50. An example of an embodiment is an example in which one or both of X and Y are selected from the following: -((CH 2 ) n1 -(CH 2 -CH 2 -O) n2 -(CH 2 )- or -((CH 2 ) n1 -(O-CH 2 -CH 2 ) n2 -(CH 2 ) n-1 -), where n1 is 1 to 6, 1 to 5, 1 to 4, or 1 to 3; and where n2 is 2 to 50, 2 to 25, 2 to 15, 2 to 10, 2 to 8, or 2 to 5. A further example of an embodiment is where X is -(CH 2 -CH 2 )-, and where Y is -(CH 2 -(CH 2 -CH 2 -O) 3 -CH 2 -CH 2 -CH 2)- or -(CH 2 -CH 2 -CH 2 -(O-CH 2 -CH 2 ) 3 -CH 2 )- examples.

[0167] As used herein, the term "modified polymer" (e.g., modified PEG) refers to a water-soluble polymer that has been modified or derivatized at either or both ends, e.g., including end substituents (e.g., end alkyl, substituted alkyl, alkoxy or substituted alkoxy, etc.) and / or end-linked functional groups (e.g., amino or carboxylic acid groups suitable for attachment via amide bond formation), which is suitable for attaching the polymer to the multi-chromophore (e.g., via a branched group). The subject water-soluble polymer is suitable for including any suitable linking group.

[0168] It should be understood that in some cases, based on the preparation and / or purification method of the polymer starting material, the water-soluble polymer may have a certain dispersity with respect to the polymer length. In some cases, the subject water-soluble polymer is monodisperse. In some cases, the subject water-soluble polymer is substantially monodisperse, e.g., including 20 wt% or less of non-target species, such as 15 wt% or less, 10 wt% or less, 5 wt% or less, 2 wt% or less or 1 wt% or less.

[0169] The water-soluble polymer may include one or more spacers or linkers. Examples of spacers or linkers include linear or branched moieties (which contain one or more repeating units, diamino and / or diacid units, natural or unnatural amino acids or their derivatives used in the water-soluble polymer) and aliphatic moieties (which include alkyl, aryl, heteroalkyl, heteroaryl, alkoxy, etc., which may contain, e.g., up to 18 carbon atoms or even other polymer chains).

[0170] The water-soluble polymer moiety or one or more spacers or linkers (if any) of the polymer moiety may include a polymer chain or unit having biostability or biodegradability. For example, based on the instability of the bond, polymers with repeating bonds have different degrees of stability under physiological conditions. Polymers having such bonds can be classified according to the known hydrolysis rates of low molecular weight analogs, for example, from less stable to more stable, for example, polyurethane (-NH-C(O)-O-) > polyorthoester (-O-C((OR)(R'))-O-) > polyamide (-C(O)-NH-). Similarly, the bonding system that attaches the water-soluble polymer to the target molecule can be biostable or biodegradable, for example, from less stable to more stable: carbonate (-O-C(O)-O-) > ester (-C(O)-O-) > urethane (-NH-C(O)-O-) > orthoester (-O-C((OR)(R'))-O-) > amide (-C(O)-NH-). Generally, based on the instability of the sulfate group, it may be necessary to avoid using sulfated polysaccharides. In addition, it may be less necessary to use polycarbonates and polyesters. The bonds are provided as examples and are not intended to limit the types of bonds in the polymer chain or bonding system of the water-soluble polymer used in the modified aldehyde-labeled polypeptide disclosed in the present invention.

[0171] In some cases, the branched nonionic WSG is independently selected from the following: -Y 1 -(CH 2 CH 2 O) r -R’; -Y 1 -O-CH[(CH 2 ) q -O-(CH 2 CH 2 O) r -R’] 2 ; and -Y 1 -CH 2 -Ph(Y 1 -(CH 2 CH 2 O) r -R’) s ; where Y 1 is selected from a covalent bond, –O-, -CONH-, -NHCO-, NHSO 2 -, -SO 2 -NH-, -CONR-, -NRCO-, NRSO 2 -, -SO 2 -NR- (for example, where R is an alkyl or a substituted alkyl), -(CH 2 )q -SO 2 -NH-, -(CH 2 ) q -CONH- and -(CH 2 ) q -O-, q and r are each independently an integer between 1 and 50, s is 1, 2 or 3, and each R’ is independently H, alkyl (e.g., methyl) or substituted alkyl.

[0172] In some cases, the structural formula of the branched nonionic WSG is shown as one of the following formulas:

[0173]

[0174] Wherein:

[0175] Each B 1 and B 2 are each independently a branched group;

[0176] Each W 1 is each independently a nonionic water-soluble polymer, e.g., containing 6 or more monomer units;

[0177] T 3 is an optional linker connecting the fused 6-5-6 tricyclic comonomer; and

[0178] Each p and q are each independently 0 or 1, where each T 1 and each T 2 (if any) are each independently a linker. In some cases, each W 1 is each independently PEG or a modified PEG polymer. In some cases, each W 1 is each independently selected from substituted alkyl, PEG or modified PEG groups and WSG. In some cases, each W 1 is each independently a PEG or modified PEG polymer containing 6-30 monomer units, e.g., 6-24 or 10-30, 10-24 or 10-20, 12-24, 12-20, 12-16 or 16-20 monomer units.

[0179] In some cases, the structural formula of the branched nonionic WSG is shown as follows:

[0180]

[0181] Wherein:

[0182] Each B 1 is a branched group;

[0183] Each W1 are each independently a nonionic water-soluble polymer, for example, comprising 6 or more monomer units;

[0184] T 3 is an optional linker connecting the fused 6-5-6 tricyclic comonomer; and

[0185] each p is independently 0 or 1, wherein each T 1 (if any) is independently a linker. In some cases, each W 1 is independently PEG or a modified PEG polymer. In some cases, each W 1 is independently selected from a substituted alkyl group, PEG or a modified PEG group, and WSG. In some cases, each W 1 is independently a PEG or a modified PEG polymer having 6-30 monomer units, for example, 6-24 or 10-30, 10-24 or 10-20, 12-24, 12-20, 12-16 or 16-20 monomer units. In some embodiments of the branched nonionic WSG, B 1 is a tetrasubstituted aryl group (for example, 1,3,4,5-phenyl).

[0186] In some embodiments of the branched nonionic WSG (for example, as shown in the above formula), B 1 is selected from CH, N, C(=O)N, SO 2 N, a trisubstituted aryl group (for example, 1,3,5-phenyl), a tetrasubstituted aryl group, and a trisubstituted heteroaryl group. In some embodiments of the branched nonionic WSG, each p is 0. In some embodiments of the branched nonionic WSG, p is 1; each T 1 is selected from –(CH 2 ) n -O–, –O-(CH 2 ) n –, –(CH 2 ) n –, and –O–, wherein n is from 1 to 12, for example, from 1 to 6. In some embodiments of the branched nonionic WSG, each T 2 and / or T 3 is independently a C1-C12-alkyl linker, for example, a C1-C6-alkyl linker, wherein one or more backbone atoms are optionally substituted with heteroatoms.

[0187] In some embodiments, the structure of the branched nonionic WSG is selected from one of the following structures:

[0188]

[0189]

[0190] Wherein:

[0191] T 5 is an optional linker connecting the comonomer (e.g., the fused 6-5-6 tricyclic comonomer);

[0192] T 6 is a linker;

[0193] Each s is independently an integer between 6 and 100 (e.g., 6 or 50); and

[0194] Each R 11 is independently hydrogen, alkyl, or substituted alkyl.

[0195] In some cases, each s is independently 6 to 30, e.g., 6 to 24, 6 to 20, 11 to 20, 12 to 20, 12 to 18, or 12 to 16. In some cases, each s is independently 6 to 30, e.g., 6 to 24, 8 to 24, 10 to 24, 12 to 24, 13 to 24, 14 to 24, 15 to 22, or 16 to 20. In some cases, each s is independently 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. In some embodiments, each s is independently 7 or greater, e.g., 8, 9 or greater, 10 or greater, 11 or greater, 12 or greater, 13 or greater, 14 or greater, 15 or greater, even greater, and in some cases, having at most 50 monomer units, e.g., at most 40, at most 30, or at most 24 monomer units. In some cases, each s is independently 6 - 30 monomer units, e.g., 6 - 24 or 10 - 30, 10 - 24 or 10 - 20, 12 - 24, 12 - 20, 12 - 16, or 16 - 20 monomer units. In some cases, each s is the same. In some embodiments of the branched nonionic WSG, T 5 and / or T 6 is a C1-C12-alkyl linker, e.g., a C1-C6-alkyl linker, wherein one or more backbone atoms are optionally substituted by heteroatoms (e.g., –O-). In some embodiments of the branched nonionic WSG, each R 11 is H. In some embodiments of the branched nonionic WSG, each R 11 is methyl.

[0196] Any suitable water-soluble group (WSG) can be included in the polymeric tandem dyes described herein to improve water solubility. Although the amount of increased solubility may vary, in some cases, the increase (compared to the compound without WSG) can reach 2-fold or more, e.g., 5-fold, 10-fold, 25-fold, 50-fold, 100-fold or more. In some cases, the hydrophilic water-soluble group is charged, e.g., positively or negatively charged. In certain cases, the hydrophilic water-soluble group is a neutral hydrophilic group. In some embodiments, the WSG is branched (e.g., as described herein). In certain cases, the WSG is linear. In some embodiments, the WSG is a hydrophilic polymer, e.g., polyethylene glycol, modified PEG, peptide sequence, peptidomimetic, carbohydrate, oxazoline, polyol, dendrimer, dendriglycerol, cellulose, chitosan or derivatives thereof. Exemplary water-soluble groups include, but are not limited to, carboxylate, phosphonate, phosphate, sulfonate, sulfate, sulfinate, sulfonium, esters, polyethylene glycol (PEG) and modified PEG, hydroxyl, amine, amino acid, ammonium, guanidine, pyridinium, polyamine and sulfonium, polyol, linear or cyclic saccharides, primary amine, secondary amine, tertiary amine or quaternary amine and polyamine, phosphonate ester group, phosphinate ester group, ascorbate ester group, ethylene glycol, including polyethers, -COOM′, -SO 3 M′, -PO 3 M′, -NR 3 +, Y′, (CH 2 CH 2 O) p R and mixtures thereof, where Y′ can be any halogen, sulfate, sulfonate or oxyanion; p can be from 1 to 500; each R can independently be H or alkyl (e.g., methyl); and M′ can be a cationic counterion or hydrogen, --(CH 2 CH 2 O) yy CH 2 CH 2 XR yy 、--(CH 2 CH 2 O) yy CH 2 CH 2 X--, --X(CH 2 CH 2 O) yy CH 2 CH 2 --, ethylene glycol and polyethylene glycol, where yy is selected from 1 to 1000; X is selected from O, S and NR ZZ ; and R ZZ and R YY are independently selected from H and C 1-3 alkyl. In some cases, the WSG is (CH2 ) x (OCH 2 CH 2 ) y OCH 3 , wherein each x is independently an integer between 0 and 20, and each y is independently an integer between 0 and 50. In some cases, the water-solubilizing group includes a nonionic polymer (e.g., a PEG polymer) substituted at the end with an ionic group (e.g., a sulfonate group).

[0197] In some embodiments of the formula, the comonomer includes a substituent selected from (CH 2 ) x (OCH 2 CH 2 ) y OCH 3 (wherein each x is independently an integer between 0 and 20, and each y is independently an integer between 0 and 50) and optionally a benzyl group substituted with one or more halogens, hydroxyl groups, C 1 -C 12 alkoxy or (OCH 2 CH 2 ) z OCH 3 (wherein each z is independently an integer between 0 and 50). In some cases, the substituent is (CH 2 ) 3 (OCH 2 CH 2 ) 11 OCH 3 . In some embodiments, one or more of the substituents is a benzyl group substituted with at least one WSG group (e.g., one or two WSG groups), the WSG group being selected from (CH 2 ) x (OCH 2 CH 2 ) y OCH 3 , wherein each x is independently an integer between 0 and 20, and each y is independently an integer between 0 and 50.

[0198] Multiple WSGs can be included at a single position of the subject polymeric tandem dye via a branched linker. In certain embodiments, the branched linker is an aralkyl substituent that is further disubstituted with water-solubilizing groups. Thus, in some cases, the branched linker group is a substituent of the multi-chromophore that links the multi-chromophore to two or more water-solubilizing groups. In certain embodiments, the branched linker is an amino acid, e.g., lysine amino acid, which links to three groups via an amino group and a carboxylic acid group. In some cases, after introducing multiple WSGs via a branched linker, the desired solubility of the multi-chromophore can be achieved. In some cases, the WSG is a nonionic side-chain group that can achieve a solubility in water of more than 50 mg / mL. In some cases, the WSG is a nonionic side-chain group that can achieve a solubility in water of more than 100 mg / mL. In some embodiments, the multi-chromophore includes substituents selected from the group consisting of alkyl, aralkyl, and heterocyclic groups, and each group is further substituted with a hydrophilic polymer group including a water-solubilizing group, e.g., polyethylene glycol (PEG) (e.g., a PEG group containing 2 to 20 units).

[0199] In certain instances of any of the formulas described herein, one or more of the comonomers are substituted with a WSG and / or a linked signaling chromophore. Any suitable WSG can be adapted to be included in the comonomers of the subject polymeric dye. In certain instances of any of the formulas described herein, one or more of the comonomers are substituted with a WSG that is independently selected from one of the following structures:

[0200]

[0201]

[0202]

[0203] Wherein: each R 11 is H, alkyl (e.g., methyl), or substituted alkyl; T 5 is an optional linker; T 6is a linker; and each s is independently an integer between 1 and 100 (e.g., 1 to 50 or 6 to 50). In certain cases, each s is independently 1 to 50, such as 3 to 40, 3 to 30, 3 to 20, 3 to 15, 3 to 12 or 6 to 12. In certain cases, each s is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some cases, each s is 3. In some cases, each s is 4. In some cases, each s is 5. In some cases, each s is 6. In some cases, each s is 7. In some cases, each s is 8. In some cases, each s is 9. In some cases, each s is 10. In some cases, each s is 11. In some cases, each s is 12. In some cases, each s is 14. In some cases, each s is 16. In some embodiments, T 5 is an alkyl linker, such as a C1-C12 or C1-C6 alkyl linker. In some embodiments, T 5 is a substituted alkyl linker. In some embodiments, T 5 is an alkoxy linker (e.g., -O-alkyl-). In some embodiments, T 5 is a substituted alkoxy. It should be understood that in any of the WSG groups described herein, a hydroxyl-terminated PEG chain can be used instead of a methoxy-terminated PEG chain. In some embodiments, each R 11 is H. In some embodiments, each R 11 is methyl.

[0204] In certain cases of any of the formulas described herein, one or more of the comonomers are substituted with a WSG, and the WSGs are independently selected from one of the following structures:

[0205]

[0206]

[0207] wherein: T 5 is an optional linker; and each s is an integer between 1 and 50. In certain cases of the WSG, T 5Does not exist. In some cases, each s in the WSG is independently 1 to 20, such as 3 to 20, 3 to 15, 3 to 12, or 6 to 12. In some cases, each s in the WSG is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some cases, each s in the WSG is 3. In some cases, each s in the WSG is 4. In some cases, each s in the WSG is 5. In some cases, each s is 6. In some cases, each s in the WSG is 7. In some cases, each s in the WSG is 8. In some cases, each s in the WSG is 9. In some cases, each s in the WSG is 10. In some cases, each s in the WSG is 11. In some cases, each s in the WSG is 12. In some cases, each s in the WSG is 14. In some cases, each s in the WSG is 16. In some embodiments, T 5 is an alkyl linker, such as a C1-C12 or C1-C6 alkyl linker. In some embodiments, T 5 is a substituted alkyl linker. In some embodiments, T 5 is an alkoxy linker (e.g., -O-alkyl-). In some embodiments, T 5 is a substituted alkoxy. It should be understood that in any of the above WSG groups, a hydroxyl-terminated PEG chain can be used instead of a methoxy-terminated PEG chain. In certain cases of any of the formulas described herein, one or more of the comonomers are substituted with a WSG, which is a dendron having a structure selected from one of the following structures:

[0208]

[0209] In certain cases of any of the formulas described herein, one or more of the comonomers are substituted with a WSG, which is a polyol having a structure selected from one of the following structures:

[0210]

[0211] In certain cases of any of the formulas described herein, one or more of the comonomers are substituted with a WSG, which is an oxazoline having the following structure:

[0212]

[0213] In certain cases of any of the formulas described herein, one or more of the comonomers are substituted with a WSG, which is a peptidomimetic having a structure selected from one of the following structures:

[0214]

[0215] Polymeric tandem dyes

[0216] The subject polymeric tandem dyes include a water-soluble light-harvesting multi-chromophore and a narrow-emission acceptor fluorophore configured to be proximate to the multi-chromophore upon energy receipt. The narrow-emission acceptor fluorophore may be attached as a side group of the multi-chromophore. The term "side group" refers to a side-chain group that is attached to the backbone of the multi-chromophore but is not part of any portion of the conjugated polymer backbone. The side-group narrow-emission acceptor fluorophores do not undergo π-conjugation with each other, nor do they form a delocalized π-electron system with the water-soluble light-harvesting multi-chromophore.

[0217] The present invention provides polymeric tandem dyes that enable high signal throughput with narrow-emission acceptor dyes, and also provides high-signal dyes that exhibit fluorescence in a narrow spectral window remote from the excitation source. By shifting the excitation wavelength, the tandem dyes can be excited at the excitation maximum, and it may be possible to collect all the emission peaks. This system also provides the major advantage of multiplexing. By using dyes with extremely narrow emission, more tandem dyes can be used within a given spectral range, where all the dyes share a single excitation wavelength. For example, Figure 9 shows the emission spectra of five exemplary tandem dyes in a multiplexing system. Figure 9 The FWHM of the emission signals of all the tandem dyes shown is 25 nm or less, and provides a wavelength range that does not overlap with the donor polymeric dyes excitable by the same violet light. These tandem dyes exhibit an effective Stokes shift of 100 to 250 nm. The small peak at 421 nm is due to the residual emission from the donor.

[0218] In some cases, when the light-harvesting multi-chromophore is directly excited with incident light, the polymeric tandem dyes exhibit an effective Stokes shift of 100 nm or greater, such as 110 nm or greater, 120 nm or greater, 130 nm or greater, 140 nm or greater, 150 nm or greater, 160 nm or greater, 170 nm or greater, 180 nm or greater, 190 nm or greater, 200 nm or greater, 250 nm or greater. In some cases, the effective Stokes shift of the polymeric tandem dyes is at most about 300 nm, such as 100 - 300 nm, 100 - 250 nm, or 100 - 200 nm.

[0219] The water-soluble light-harvesting multi-chromophore itself can be fluorescent and capable of transferring energy to a narrow-emission acceptor fluorophore linked to a specific repeating unit of the multi-chromophore backbone. Thus, excitation of the multi-chromophore donor can cause energy transfer to the covalently linked narrow-emission acceptor fluorophore or cause the fluorophore to emit energy. The number of narrow-emission acceptor fluorophores linked to the donor water-solvated light-harvesting multi-chromophore can vary, where in some cases the number ranges from 1 mol% to 50 mol%, such as 5 mol% to 25 mol% or 10 mol% to 25 mol%, for example, as measured by the number of repeating units having side-group narrow-emission acceptor fluorophores attached.

[0220] Mechanisms for transferring energy from the fluorescent water-solvated light-harvesting multi-chromophore donor to the attached narrow-emission acceptor fluorophore include, for example, resonance energy transfer (e.g., (or fluorescence) resonance energy transfer, FRET), quantum charge transfer (Dexter energy transfer), etc. In some cases, the transfer distances of these energy transfer mechanisms are relatively short; that is, effective energy transfer can be achieved when the light-harvesting multi-chromophore system is very close to the acceptor. In some cases, under conditions of effective energy transfer, amplification of the acceptor emission occurs, where the emission intensity of the luminescent signal-conducting fluorophore is higher when the incident light (“pump light”) is at a wavelength absorbed by the light-harvesting multi-chromophore compared to directly exciting the narrow-emission acceptor fluorophore with the pump light.

[0221] “Effective” energy transfer means 10% or more, such as 20% or more or 30% or more, of the energy captured by the donor and transferred to the acceptor. “Amplification” means that the signal emitted by the narrow-emission acceptor fluorophore excited by energy transfer from the donor light-harvesting multi-chromophore is 1.5 times or more higher compared to directly exciting with incident light of equivalent intensity. The signal can be measured by any suitable method. In some cases, the 1.5 times or more signal refers to the intensity of the emitted light. In certain cases, the 1.5 times or more signal refers to the increased signal-to-noise ratio. In certain embodiments of the polymer tandem dye, the signal emitted by the signal-conducting fluorophore excited by the multi-chromophore is 1.5 times or more higher compared to directly exciting the narrow-emission acceptor fluorophore with incident light, such as 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 8 times or more, 10 times or more, 20 times or more, 50 times or more, 100 times or more, or even higher compared to directly exciting the narrow-emission acceptor fluorophore with incident light.

[0222] The quantum yield of the polymer tandem dye emission is 0.03 or more. For example, the quantum yield is 0.04 or more, 0.05 or more, 0.06 or more, 0.07 or more, 0.08 or more, 0.09 or more, 0.1 or more, 0.15 or more, 0.2 or more, 0.3 or more, or even more. In some cases, the extinction coefficient of the polymer tandem dye is 5×10 5 cm -1 M -1 or higher. For example, 6×10 5 cm -1 M -1 or higher, 7×10 5 cm -1 M -1 or higher, 8×10 5 cm -1 M -1 or higher, 9×10 5 cm -1 M -1 or higher, 1×10 6 cm -1 M -1 or higher, 1.5×10 6 cm -1 M -1 or higher, 2×10 6 cm -1 M -1 or higher, 2.5×10 6 cm -1 M -1 or higher, 3×10 6 cm -1 M -1 or higher, 4×10 6 cm -1 M -1 or higher, 5×10 6 cm -1 M -1 or higher, 6×10 6 cm -1 M -1 or higher, 7×10 6 cm -1 M -1 or higher or 8×10 6 cm -1 M -1 or higher. In some embodiments, the molar extinction coefficient of the polymer tandem dye is 5×10 5 M -1 cm -1 or higher. In certain embodiments, the molar extinction coefficient of the polymer tandem dye is 1×106 M -1 cm -1 or higher.

[0223] The subject polymeric tandem dye provides fluorescence emission from a narrow emission acceptor chromophore, and the brightness of the fluorescence emission is higher than that which may be from the emission of such fluorescent dyes in isolation. The brightness of the emission of the polymeric tandem dye is 50 mM -1 cm -1 or higher, such as 60 mM -1 cm -1 or higher, 70 mM -1 cm -1 or higher, 80 mM -1 cm -1 or higher, 90 mM -1 cm -1 or higher, 100 mM -1 cm -1 or higher, 150 mM -1 cm -1 or higher, 200 mM -1 cm -1 or higher, 250 mM -1 cm -1 or higher, 300 mM -1 cm -1 or higher, even higher. In some cases, the brightness of the emission of the polymeric tandem dye is at least 5-fold higher than the brightness of the directly excited narrow emission acceptor fluorophore, for example, at least 10-fold higher, at least 20-fold higher, at least 30-fold higher, at least 50-fold higher, at least 100-fold higher, at least 300-fold higher, even higher than the brightness of the directly excited narrow emission acceptor fluorophore.

[0224] The terms "light-harvesting multi-chromophore", "polymeric dye" and "conjugated polymer" as used herein are used interchangeably and refer to a conjugated polymer having a structure capable of capturing light having a specific maximum absorption wavelength and converting it into emitted light having a longer maximum emission wavelength. In some cases, the light-harvesting multi-chromophore itself is fluorescent. A conjugated polymer (CP) is characterized by a delocalized electronic structure, and its effective conjugation length is significantly shorter than the length of the polymer chain, since the backbone may contain a large number of closely adjacent conjugated segments. In some cases, the conjugated polymer can efficiently harvest light and provide optical amplification by transferring Forster energy to an acceptor.

[0225] As used herein, the term "unit" refers to a structural subunit of a polymer. The term "unit" is intended to include monomers, comonomers, co-blocks, conjugated segments, repeating units, and the like. A "repeating unit" is a subunit of a polymer that is defined by the minimum number of different structural features required for a unit to be considered a monomer, such that when the unit is repeated n times, the resulting structure describes the polymer or its block. In some cases, the polymer may include two or more different repeating units. For example, when the polymer is a multi-block polymer or has a random arrangement of units, each block may define a different repeating unit, e.g., an n-block and an m-block. It should be understood that the n and / or m repeating units or blocks may occur in a variety of arrangements, and in the formulas shown for the multi-chromophore targets described herein, n and m co-blocks of various lengths arranged in any suitable linear manner may be included in the structure of the entire polymer. It should be understood that the polymer may also be represented by a formula in terms of the mol% value of each unit, and such formulas may represent various arrangements of repeating units, e.g., a random or multi-block polymer. In some cases, the repeating unit of the polymer includes a single monomer group. In certain cases, the repeating unit of the polymer includes two or more monomer groups, i.e., comonomer groups, e.g., two, three, four, or more comonomer groups. As used herein, the term "comonomer" or "comonomer group" refers to a structural unit of a polymer that can itself be part of a polymer repeating unit. In some embodiments, the conjugated polymer includes a block copolymer composed of blocks of polymerized monomers. In such cases, the block copolymer can be described as having different repeating units, each repeating unit corresponding to a different co-block of the polymer. In some cases, the polymer is a diblock copolymer containing two different co-blocks. In such cases, the polymer can be described as including co-blocks, where each co-block can be composed of comonomers (e.g., one, two, three, or more comonomers).

[0226] The multi-chromophore may have any suitable length. In some cases, the specific number range of monomer repeating units or segments of the multi-chromophore may be from 2 to 500,000, such as 2 to 100,000, 2 to 30,000, 2 to 10,000, 2 to 3,000 or 2 to 1,000 units or segments, or for example 5 to 100,000, 10 to 100,000, 100 to 100,000, 200 to 100,000 or 500 to 50,000 units or segments. In some cases, the specific number range of monomer repeating units or segments of the multi-chromophore may be from 2 to 1,000, such as 2 to 500, 2 to 100, 3 to 100, 4 to 100, 5 to 100, 6 to 100, 7 to 100, 8 to 100, 9 to 100 or 10 to 100 units or segments.

[0227] The multi-chromophore may have any suitable molecular weight (MW). In some cases, the MW of the multi-chromophore may be expressed as an average molecular weight. In some cases, the average molecular weight range of the polymeric dye is from 500 to 500,000, for example, 1,000 to 100,000, 2,000 to 100,000, 10,000 to 100,000, and even the average molecular weight range reaches 50,000 to 100,000.

[0228] In some embodiments, the multi-chromophore comprises a specific aryl or heteroaryl comonomer, and the molar percentage of the specific aryl or heteroaryl comonomer in the multi-chromophore is 5% or higher (e.g., 5 mol%), for example, the molar percentage in the multi-chromophore is 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 40% or higher, 45% or higher, 50% or higher, 60% or higher, 70% or higher, or even higher. In such cases, the multi-chromophore may comprise 5 or more repeating units, such as 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more, 500 or more, 1000 or more, 10,000 or more, or even more repeating units. In such cases, the multi-chromophore may comprise 5 or more comonomer units, such as 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more, 500 or more, 1000 or more, 10,000 or more, or even more comonomer units. In certain embodiments, the molar percentage of the target aryl or heteroaryl comonomer in the multi-chromophore is 25% or higher, for example, the molar percentage in the multi-chromophore is 30% or higher, 40% or higher, 45% or higher, 50% or higher, or even higher, and the multi-chromophore comprises 5 or more repeating units, such as 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more repeating units.

[0229] In some embodiments, the multi-chromophore comprises a plurality of first optically active units forming a conjugated system, which has an absorption wavelength at which the first optically active unit absorbs light to become an excited state (e.g., as described herein). In certain cases, the multi-chromophore comprises a conjugated polymer segment or oligomer structure, which comprises n-conjugated repeating units with a reduced bandgap.

[0230] The subject multi-chromophores can have one or more desired spectral properties, such as a specific maximum absorption wavelength, a specific maximum emission wavelength, extinction coefficient, quantum yield, etc. The subject polymeric tandem dyes provide a variety of absorption and emission spectra, which depend on various factors, such as the selected comonomers, linking groups, substituents, and the narrow emission acceptor fluorophores that have been attached, which make up the polymeric tandem dyes. In some cases, the maximum absorption wavelength of the polymeric dyes ranges from 300 to 600 nm, such as from 350 nm to 560 nm or from 400 nm to 560 nm. In certain embodiments, the maximum absorption wavelength of the multi-chromophores is 600 nm or less, such as 575 nm or less, 550 nm or less, 525 nm or less, 500 nm or less, 475 nm or less, 450 nm or less, 440 nm or less, 430 nm or less, 420 nm or less, 410 nm or less, 400 nm or less, or even less. In certain embodiments, the multi-chromophores only absorb ultraviolet light. In certain cases, the maximum absorption wavelength of the multi-chromophores ranges from 300 nm to 400 nm. In certain cases, the maximum absorption wavelength of the multi-chromophores ranges from 300 nm to 350 nm. In certain cases, the maximum absorption wavelength of the multi-chromophores ranges from 350 nm to 400 nm. In certain cases, the maximum absorption wavelength of the multi-chromophores ranges from 400 nm to 600 nm, such as from 400 nm to 560 nm. In certain cases, the maximum absorption wavelength of the multi-chromophores ranges from 400 nm to 450 nm. In certain cases, the maximum absorption wavelength of the multi-chromophores ranges from 450 nm to 500 nm. In certain cases, the maximum absorption wavelength of the multi-chromophores ranges from 500 nm to 550 nm. In certain cases, the maximum absorption wavelength of the multi-chromophores ranges from 550 nm to 600 nm.

[0231] In some embodiments, the maximum emission wavelength of the multi-chromophore ranges from 300 to 900 nm, for example, 350 to 850 nm, 350 to 600 nm, 360 to 500 nm, 370 to 500 nm, 380 to 500 nm, 390 to 500 nm, or 400 to 500 nm. Specific examples of the target emission maximum include, but are not limited to: 395 nm ± 5 nm, 460 nm ± 5 nm, 490 nm ± 5 nm, 550 nm ± 5 nm, 560 nm ± 5 nm, 605 nm ± 5 nm, 650 nm ± 5 nm, 680 nm ± 5 nm, 700 nm ± 5 nm, 805 nm ± 5 nm. In some cases, the maximum emission wavelength of the multi-chromophore is selected from the group consisting of 395 nm, 460 nm, 490 nm, 550 nm, 560 nm, 605 nm, 650 nm, 680 nm, 700 nm, and 805 nm. In some cases, the maximum emission wavelength of the multi-chromophore is 395 nm ± 5 nm. In some cases, the maximum emission wavelength of the multi-chromophore itself ranges from 375 to 900 nm (for example, the range is 380 nm to 900 nm, 390 nm to 900 nm, or 400 nm to 900 nm).

[0232] In some cases, the extinction coefficient of the multi-chromophore is 5×10 5 cm -1 M -1 or higher, for example 6×10 5 cm - 1 M -1 or higher, 7×10 5 cm -1 M -1 or higher, 8×10 5 cm -1 M -1 or higher, 9×10 5 cm -1 M -1 or higher, 1×10 6 cm -1 M -1 or higher, 1.5×10 6 cm -1 M -1 or higher, 2×10 6 cm -1 M -1 or higher, 2.5×10 6 cm -1 M -1 or higher, 3×10 6 cm -1 M -1 or higher, 4×106 cm -1 M -1 or higher, 5 × 10 6 cm -1 M -1 or higher, 6 × 10 6 cm -1 M -1 or higher, 7 × 10 6 cm -1 M -1 or higher or 8 × 10 6 cm -1 M -1 or higher. In such cases, the multi-chromophore may have 5 or more repeating units, such as 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, and even more repeating units. In some embodiments, the molar extinction coefficient of the multi-chromophore is 5 × 10 5 M -1 cm -1 or higher. In certain embodiments, the molar extinction coefficient of the multi-chromophore is 1 × 10 6 M -1 cm -1 or higher.

[0233] In some cases, the extinction coefficient of the multi-chromophore is 40,000 cm -1 M -1 / repeating unit or higher, such as 45,000 cm -1 M -1 / repeating unit or higher, 50,000 cm -1 M -1 / repeating unit or higher, 55,000 cm -1 M -1 / repeating unit or higher, 60,000 cm -1 M -1 / repeating unit or higher, 70,000 cm -1 M -1 / repeating unit or higher, 80,000 cm -1 M -1 / repeating unit or higher, 90,000 cm -1 M -1 / repeating unit or higher, 100,000 cm -1 M -1 / repeating unit or higher, and even higher. In some cases, the 40,000 cm -1 M -1 / monomeric repeating unit or higher is the average extinction coefficient. In some cases, the repeating units of the multi-chromophore may include a single monomer, two comonomers, or three or more comonomers. In some cases, the extinction coefficient of the multi-chromophore is 40,000 cm -1 M -1 / comonomer or higher, such as 45,000 cm -1 M -1 / comonomer or higher, 50,000 cm -1 M -1 / comonomer or higher, 55,000 cm -1 M -1 / comonomer or higher, 60,000 cm -1 M -1 / comonomer or higher, 70,000 cm -1 M -1 / comonomer or higher, 80,000 cm -1 M -1 / comonomer or higher, 90,000 cm -1 M -1 / comonomer or higher, 100,000 cm -1 M -1 / comonomer or higher, and even higher. In some cases, the 40,000 cm -1 M -1 / comonomer or higher is the average extinction coefficient.

[0234] It should be understood that in some cases, the subject multiple chromophores can include co-blocks (e.g., n- and m-co-blocks) or randomly arranged n and m repeating units. The subject multiple chromophores can include n and m co-blocks of various lengths arranged in any suitable linear manner within the structure of the entire polymer. In some cases, the linear arrangement of the comonomers described in the structures herein is random. Additionally, the multiple chromophores can include comonomers arranged in any suitable manner within such n and / or m co-blocks. Unless otherwise specified to the contrary, comonomers arranged in all potential manners are intended to be included in the polymeric dyes described herein. In preparing the subject multiple chromophores, various polymer synthesis methods can be utilized to prepare the desired comonomers and co-blocks. It should be understood that in some cases, the polymerization method can generate a composition comprising a population of conjugated polymers that includes some variation with respect to the specific length and / or end groups (i.e., terminal groups) present in each conjugated polymer of the population. The formulas described herein can refer to a single compound or a population or sub-population of polymers. The * used herein indicates the site for covalent attachment to the unsaturated backbone of the conjugated polymer. It should be understood that the formulas of the subject polymers described herein are a representation of the structures, and other representations can also be used, such as a representation indicating the mol% ratio of specific comonomers in the conjugated polymer segments.

[0235] Any suitable water-soluble light-harvesting multiple chromophore can be adapted to include a side-group narrow-emission acceptor fluorophore (e.g., as described herein). The desired multiple chromophores include, but are not limited to, those described in WO2016 / 144653, WO2018 / 013389, WO2018 / 009581, WO2018 / 111774, US2017 / 0247504, WO2017 / 180998, WO2018 / 009861, and US9,896,538, the contents of which are incorporated herein by reference in their entireties. In some cases, the polymeric tandem dye includes a multiple chromophore that includes a conjugated segment having a structure as shown in formula (XI):

[0236]

[0237] Wherein:

[0238] F 1 is a fused tricyclic comonomer substituted with a water-soluble group (WSG) (e.g., as described herein);

[0239] M 1 is an aryl or heteroaryl comonomer;

[0240] n is an integer between 1 and 100,000; and

[0241] *Denotes a site for covalent attachment to an unsaturated backbone or end group of a conjugated polymer; and

[0242] A narrow emission acceptor fluorophore linked to the multiple chromophores that are proximal upon energy receipt. In some cases, the water-soluble group (WSG) of the polymeric tandem dye can render the multiple chromophores soluble in water (e.g., soluble in an aqueous buffer) at 10 mg / mL or higher, such as 20 mg / mL or higher, 30 mg / mL or higher, 40 mg / mL or higher, 50 mg / mL or higher, 60 mg / mL or higher, 70 mg / mL or higher, 80 mg / mL or higher, 90 mg / mL or higher, 100 mg / mL or higher, or even higher.

[0243] Exemplary aryl or heteroaryl comonomers include, but are not limited to, fused tricyclic comonomers such as fluorene comonomers, carbazole comonomers, silole comonomers, or bridged biphenyl comonomers. A fused tricyclic comonomer is a comonomer that includes a tricyclic aromatic group (having three fused rings in the configuration), where two aryl or heteroaryl 6-membered rings are fused to a central 5- or 6-membered carbon or hetero ring. In some cases, the fused tricyclic comonomer includes two benzo- or pyrido-rings fused to a central 5- or 6-membered carbon or hetero ring. The fused tricyclic comonomer can effect π-conjugation with an adjacent comonomer in the polymer backbone through any suitable ring atom on the fused rings. The central 5- or 6-membered ring can be an aromatic or partially saturated carbon or hetero ring and can further include side-chain substituents such as, for example, a WSG and / or a linker for attaching a chemoselective tag. A bridged biphenyl comonomer is a fused tricyclic comonomer having a biphenyl group, where the two benzene rings are further connected to each other through a central 6-membered ring. In some cases, the structure of the fused tricyclic comonomer is as shown in the following structures:

[0244]

[0245] wherein:

[0246] Y 1 is C(R 23 ) 2 、-C(R 23 ) 2 C(R 23 ) 2 -、-C(R 23 ) 2 Si(R 23 ) 2 -、NR 23 、Si(R 23 ) 2 or Se;

[0247] Each Z is independently CH, CR 22 or N;

[0248] Each R 23 is independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido, aralkyl, substituted aralkyl, PEG moiety, and WSG, or any two suitable R 3 groups are optionally joined to form a cyclic structure to provide an optionally substituted carbocyclic or heterocyclic ring;

[0249] Each R 22 is one or more optional substituents, each independently selected from hydrogen, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro, or bromo), sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide, and water-soluble group, or any two R 11 groups are optionally joined to form a cyclic structure (e.g., to provide a carbocyclic or heterocyclic ring); and

[0250] * represents the site for covalent attachment to the unsaturated backbone or end group of the conjugated polymer. In some cases, at least two Zs in each ring are CH or CR 22 . In some cases, there is exactly one Z that is N in each ring.

[0251] In some cases, the structure of the fused tricyclic comonomer is shown as one of the following structures:

[0252]

[0253] Wherein:

[0254] Y is C(R 23 ) 2 、-C(R 23 ) 2 C(R 23 ) 2 -、-C(R 23 ) 2 Si(R 23 ) 2 -、NR 23 、Si(R 23 ) 2 or Se;

[0255] Each R 23Each is independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amino, substituted amino, aralkyl, substituted aralkyl, a PEG moiety, -L 11 -Z 1 , where L 11 is a linker, and Z 1 is a chemoselective tag (e.g., a tag comprising a chemoselective functional group) and a WSG; and

[0256] Each R is independently H, R 3 or one or more aryl or heteroaryl substituents, and where any two suitable R groups are optionally joined to form a cyclic structure. In some cases, each R refers to one or two ring substituents independently selected from halogen, sulfonate, alkoxy, substituted alkoxy, alkyl, and substituted alkyl, and where any two suitable R groups are optionally joined to form a cyclic structure. The symbol "*" represents a site for covalent attachment to the unsaturated backbone of the conjugate polymer, e.g., a conjugated segment, a terminal group, a linker, and a specifically bound member that has been attached. It should be understood that for any formula described herein that includes a 2,7-π conjugated fused tricyclic comonomer, a similar formula including a similar 3,6-π conjugated comonomer can also be depicted. In certain cases, the fused tricyclic comonomer is a fluorene comonomer, where Y is C(R 3 ) 2 . In some cases, the fused tricyclic comonomer is a carbazole comonomer, where Y is NR 3 . In some cases, the fused tricyclic comonomer is a silole comonomer, where Y is Si(R 3 ) 2 . In some cases, the fused tricyclic comonomer is a bridged biphenyl comonomer, where Y is -C(R 3 ) 2 C(R 3 ) 2 -. In some cases, the fused tricyclic comonomer is a bridged biphenyl comonomer, where Y is -CHR 3 CHR 3 -. In certain cases of any of the fused tricyclic comonomers described herein, each R is independently selected from H, halogen, alkoxy, substituted alkoxy, alkyl, and substituted alkyl. In certain cases, each R is independently selected from H, fluoro, chloro, methoxy, substituted alkoxy, alkyl, and substituted alkyl.

[0257] In certain embodiments, the fused tricyclic comonomer is selected from:

[0258]

[0259]

[0260] Wherein:

[0261] Y 3 is C or Si; and

[0262] Each R 23 and R 24 are each independently hydrogen, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro or bromo), sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide and water-soluble group, and wherein two R 14 groups are optionally linked to form a cyclic structure to form an optionally substituted fused carbocyclic or heterocyclic ring (e.g., a partially saturated seven-membered heterocyclic ring).

[0263] In certain embodiments of the fused tricyclic comonomer, the comonomer comprises two R substituents that are linked to form a cyclic structure to provide a carbocyclic or heterocyclic ring A that is optionally further substituted:

[0264]

[0265] Wherein Y is C(R 3 ) 2 , -C(R 3 ) 2 C(R 3 ) 2 -, -C(R 3 ) 2 Si(R 3 ) 2 , NR 3 , Si(R 3 ) 2 or Se; and each R 3 is independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amino, substituted amino, aralkyl, substituted aralkyl, PEG moiety, -L 11 -Z 1 , wherein L 11 is a linker, Z 1 is a chemoselective tag (e.g., a tag comprising a chemoselective functional group) and WSG. In some cases, the structure of the fused tricyclic comonomer is as follows:

[0266]

[0267] Each R 8 -R 9 is independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amino, substituted amino, aralkyl, substituted aralkyl, PEG moiety, -L 11 -Z 1 , where L 11 is a linker and Z 1 is a chemoselective tag (e.g., a tag including a chemoselective functional group) and a WSG. In some cases of the comonomer, Y is C(R 3 ) 2 .

[0268] In some cases, the structure of the fused tricyclic comonomer is shown as one of the following structures:

[0269]

[0270] Wherein:

[0271] Each R 3 is independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amino, substituted amino, aralkyl, substituted aralkyl, PEG moiety, -L 11 -Z 1 , where L 11 is a linker and Z 1 is a chemoselective tag (e.g., a tag including a chemoselective functional group) and a WSG (e.g., as described herein); and

[0272] Each R is independently H, R 3 or one or more aryl or heteroaryl substituents. In some cases, each R refers to one or two ring substituents independently selected from halogen, sulfonate, alkoxy, substituted alkoxy, alkyl, and substituted alkyl. In some cases, each R is fluoro or methoxy. In some cases, at least one R 3 is a WSG. In some cases, at least one R 3 is -L 11 -Z 1 .

[0273] In some cases, the structure of the fused tricyclic comonomer is shown as the following structure:

[0274]

[0275] wherein each WSG is a branched or linear water-soluble group, and each R 3 is H, an alkyl group, or a substituted alkyl group. In some cases, each WSG is a linear WSG (e.g., as described herein). In some cases, the structure of the fused tricyclic comonomer is as shown in the following structures:

[0276]

[0277] wherein T 5 is an optional linker; each s is an integer between 6 and 100 (e.g., 6 to 50); and each R and R 11 are each independently hydrogen, an alkyl group, or a substituted alkyl group.

[0278] A fused 6-5-6 tricyclic comonomer is a comonomer comprising a tricyclic aromatic group (having three fused rings in the 6-5-6 configuration, i.e., two benzo rings fused to a central 5-membered ring). The 5-membered ring can be a carbocyclic or heterocyclic ring and can further include side chain substituents on the ring atoms not fused to the benzo rings. In some cases, the structure of the fused 6-5-6 tricyclic comonomer is as shown in the following structures:

[0279]

[0280] wherein: Z is –C(R 1 ) 2 –, Si(R 1 ) 2 – or –N(R 1 )–; each R is independently H or one or more aryl substituents; and each R 1 is independently selected from branched nonionic water-soluble groups (WSGs), alkyl groups, substituted alkyl groups, aralkyl groups, substituted aralkyl groups, PEG moieties, and -L 11 -Z 1 where L 11 is a linker, Z 1 is a chemoselective tag (e.g., a tag comprising a chemoselective functional group), or a WSG. When used in any of the formulas described herein, * represents the site for covalent attachment to the unsaturated backbone or end group of the conjugated polymer. In some embodiments, when Z is –N(R 1 )–, the fused 6-5-6 tricyclic comonomer is a carbazole comonomer. Any suitable carbazole comonomer can be used in the subject multi-chromophore. In some embodiments, when Z is –C(R 1 ) 2– When, the fused 6-5-6 tricyclic comonomer is a fluorene comonomer. Any suitable fluorene comonomer can be used in the subject multiple chromophores. In certain cases of the fused 6-5-6 tricyclic comonomer, each R 1 is independently selected from benzyl groups substituted with one, two or more PEG moieties or alkyl groups substituted with two or more PEG moieties. In some embodiments, Z is –Si(R 1 ) 2 –. It should be understood that for any fluorene comonomer described herein, the present invention also includes the corresponding comonomer in which the C atom in Z is replaced by Si.

[0281] A fluorene comonomer is a comonomer comprising an aromatic group having a 9H-fluorene nuclear structure, the aromatic group being substituted at the 9-position with any suitable side chain substituent. In some cases, the fluorene comonomer is a 9,9-disubstituted fluorene. The fluorene comonomer is conjugated to adjacent groups of the polymer backbone through any suitable position of the fluorene nuclear structure, such as any two positions among positions 1-8 (see the following numbering scheme). In some embodiments, the fluorene nuclear structure is connected to adjacent groups of the polymer backbone through the 2- and 7-positions. In certain embodiments, the structure of the fluorene comonomer is as shown in the following structure:

[0282]

[0283] Wherein: each R 1 is independently selected from branched nonionic water-soluble groups (WSGs), alkyl groups, substituted alkyl groups, aralkyl groups, substituted aralkyl groups, PEG moieties, and -L 11 -Z 1 , wherein L 11 is a linker and Z 1 is a chemoselective tag (e.g., a tag comprising a chemoselective functional group) or a WSG. In certain cases of the fluorene comonomer, each R 1 is a branched nonionic water-soluble group (WSG). In certain cases of the fluorene comonomer, each R 1 is independently selected from benzyl groups substituted with one, two or more PEG moieties or alkyl groups substituted with two or more PEG moieties. The Z 1 functional group can be used to covalently link the multiple chromophore to a receptor chromophore (e.g., as described herein). In certain cases, Z 1 comprises an amino group for covalent linking to the receptor chromophore. In certain cases, Z 1 comprises a carboxylic acid group or a derivative thereof for covalent linking to the receptor chromophore. In some embodiments, L 11 is a linker to two or more Z 1A branched linker to which a group (e.g., WSG) is attached. In some cases, the fluorene comonomer is further substituted by R 5 and / or R 6 substituents at one, two or more positions selected from positions 1, 3, 4, 5, 6 and 8, where R 5 and R 6 are independently selected from water - soluble groups (WSG) and aryl substituents (e.g., as described herein).

[0284] In some cases, the structure of the fluorene comonomer is as shown in the following structure:

[0285]

[0286] Wherein: each R 1 is as defined above; and R 5 and R 6 are independently selected from H, water - soluble groups and aryl substituents (e.g., as described herein).

[0287] In some cases, the structure of the fluorene comonomer is as shown in the following structure:

[0288]

[0289] Where each R 2 is an alkyl group substituted by a water - soluble group or a branched linker attached to two or more water - soluble groups (e.g., PEG - disubstituted benzyl or PEG - substituted alkyl). In some cases of the fluorene comonomer, each R 2 is a benzyl group substituted by one, two or three PEG moieties (e.g., - O(CH 2 CH 2 O) n R’, where R’ is H or alkyl and n is 1 - 20, e.g., 3 - 16, e.g., n is 8 - 16). In some cases of the fluorene comonomer, each R 2 is a benzyl group substituted by one - O(CH 2 CH 2 O) n R’ group (e.g., at the 2, 3 or 4 position), where R’ is H or alkyl and n is 1 - 20, e.g., 3 - 16, e.g., n is 8 - 16. In some cases of the fluorene comonomer, each R 2 is a benzyl group substituted by two - O(CH 2 CH 2 O) nA benzyl group substituted with an R' group (e.g., at the 2,4-, 3,4- or 3,5-position), where each R' is independently H or alkyl, and each n is independently 1 - 20, e.g., 3 - 16, e.g., n is 8 - 16. In certain cases of the fluorene comonomer, each R 2 is substituted with three -O(CH 2 CH 2 O) n A benzyl group substituted with an R' group (e.g., at the 2,4,6-, 2,4,5- or 3,4,5-position), where each R' is independently H or alkyl, and each n is independently 1 - 20, e.g., 3 - 16, e.g., n is 8 - 16. In certain cases of the fluorene comonomer, each R 2 is substituted with a trivalent branched group (each substituted with two PEG moieties) (e.g., -CO-NR” 2 or -O(CH 2 R”) 2 A trivalent branched group, where each R” is independently a PEG moiety (e.g., -O(CH 2 CH 2 O) n R’, where R’ is H or alkyl, and n is 1 - 20, e.g., 3 - 16, e.g., n is 8 - 16)) substituted lower alkyl group. In certain cases of the fluorene comonomer, each R 2 is a branched nonionic water-soluble group (WSG), e.g., as described herein.

[0290] In certain embodiments, the structure of the fluorene comonomer is as shown in the following structure:

[0291]

[0292] where R 3 is an alkyl group substituted with a water-solubilizing group (e.g., alkyl substituted with PEG) or a branched nonionic water-soluble group (WSG) (e.g., as described herein), R 4 is L 2 -Z 2 where L 2 is a linker, Z 2 is a chemoselective tag (e.g., a tag for conjugation with a receptor chromophore). In some cases, the structure of the fluorene comonomer is as shown in the following structure:

[0293]

[0294] where: R 3is a substituent comprising a water-soluble group (e.g., as described herein) or a branched nonionic water-soluble group (WSG) (e.g., as described herein);

[0295] R 4 is L 2 -Z 2 wherein L 2 is a linker and Z 2 is a chemoselective label (e.g., a label for conjugating with an acceptor chromophore); and R 5 and R 6 are independently selected from H, water-soluble groups, and aryl substituents (e.g., alkyl, substituted alkyl, alkoxy, substituted alkoxy, halogen, or nitro). In certain cases of the fluorene comonomer, each R 3 is a trivalent branched group (each substituted with two PEG moieties) (e.g., -CO-NR” 2 or -O(CH 2 R”) 2 trivalent branched group, wherein each R” is a PEG moiety (e.g., -O(CH 2 CH 2 O) n R’, where R’ is H or alkyl, and n is 6 - 20, e.g., 8 - 16, e.g., n is 12 - 16)) substituted lower alkyl group. In certain cases of the fluorene comonomer, R 3 is a branched nonionic water-soluble group (WSG), e.g., as described herein.

[0296] Any of the fluorene comonomers described herein can be used in the target multiple chromophores, e.g., the multiple chromophores represented by formulas (XI) - (XIV). In some cases, the multiple chromophores include one of the following structures as part of the polymer backbone:

[0297]

[0298] wherein each R 3 is independently a water-soluble group connected by an optional linker or an optionally substituted alkyl, aralkyl, or aryl group; Ar is an optionally substituted aryl or heteroaryl group; n is an integer between 1 and 100,000; the terminus is a site for covalently connecting to an unsaturated backbone or end group of a conjugated polymer. In certain embodiments, each R 3 is independently a substituted alkyl group. In certain embodiments, each R 3 is independently a substituted aralkyl group. In some cases, each R 3Each Ar is independently substituted by a water-soluble group, a receptor chromophore, a chemoselective functional group, or a specific binding moiety (through an optional linker).

[0299] In certain cases of formula (XI), F 1 has the following structure:

[0300]

[0301] Wherein: Z is –C(R 1 )(R 2 )–, –Si(R 1 )(R 2 )– or –N(R 1 )–; and R 1 and R 2 are independently nonionic WSGs, such as linear or branched nonionic WSGs.

[0302] In certain cases of formula (XI), F 1 has the following structure:

[0303]

[0304] Wherein: Z is –C(R 1 )(R 2 )–, –Si(R 1 )(R 2 )– or –N(R 1 )–; and R 1 and R 2 are independently branched nonionic WSGs containing two or more water-soluble polymers (e.g., each having 6 - 30 monomer units).

[0305] In some embodiments of formula (XI), F 1 is a fluorene comonomer substituted by two branched nonionic WSG groups (e.g., as described herein) (e.g., disubstituted at the 9-position). In some embodiments of formula (XI), F 1 is a fluorene comonomer substituted by two linear nonionic WSG groups (e.g., as described herein) (e.g., disubstituted at the 9-position).

[0306] In some embodiments of formula (I), F 1 has the following structure:

[0307]

[0308] Where s is 6 - 60;

[0309] t is 0 - 10 (e.g., 0 - 6, e.g., 0, 1, 2, or 3);

[0310] R 11 is H, an alkyl group or a substituted alkyl group. In some cases, each R 11 is a methyl group.

[0311] In some embodiments of formula (I), F 1 is selected from one of the following comonomers:

[0312]

[0313]

[0314] In some embodiments of formula (I), n is 5 or greater, for example, 10 or greater, 30 or greater, 100 or greater, 300 or greater, 1000 or greater, 3000 or greater, or even greater. It should be understood that the conjugated polymer population can be represented by the subject formula including some variations relative to the specific length. In some cases, the molar percentage of the conjugated segment in the multi-chromophore is 25 mol% or higher, for example, 30 mol% or higher, 40 mol% or higher, 50 mol% or higher, 60 mol% or higher, 70 mol% or higher, 80 mol% or higher, or 90 mol% or higher.

[0315] In some embodiments of formula (XI), the structure of the multi-chromophore is as shown in formula (XII):

[0316]

[0317] Wherein:

[0318] M 1 、M 2 and L 1 are independently an aryl or heteroaryl comonomer;

[0319] Z 1 is a connected narrow emission acceptor fluorophore;

[0320] x and y represent the molar percentages of the units in the multi-chromophore; and

[0321] G 1 and G 2Each independently selected from a terminal group, a π-conjugated segment, a linker, and a specifically binding member that has been attached. In some cases of formula (II), the mole percentage of x in the multi-chromophore is 5% or higher (e.g., 5 mol%), such as 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or even higher, where x + y is 100%. In some cases of formula (II), the mole percentage of y in the multi-chromophore is 5% or higher (e.g., 5 mol%), such as 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or even higher.

[0322] In some embodiments of formulas (XI)-(XII), the structure of the polymeric dye is as shown in formula (XIIa):

[0323]

[0324] where F 1 is a fused tricyclic comonomer substituted with a water-soluble group (WSG) (e.g., as described herein); M 1 , M 2 and L 1 are independently aryl or heteroaryl comonomers (e.g., as described herein); N 1 is the narrow emission acceptor fluorophore connected to L 1 (e.g., via a non-conjugated linker); G 1 and G 2Each independently selected from a terminal group, a π-conjugated segment, a linker, and a specifically binding member that has been attached; and w, x, y, and z represent the mole percentages of the units in the multi-chromophore (e.g., z > 0 mol%, w > 0 mol%). In some cases of formula (XIIa), the mole percentage of w in the multi-chromophore is 5% or higher (e.g., 5 mol%), such as 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or even higher, where w + x + y + z = 100%. In some cases of formula (IIa), the mole percentage of x in the multi-chromophore is 5% or higher (e.g., 5 mol%), such as 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or even higher, where w + x + y + z = 100%. In some cases of formula (XIIa), the mole percentage of y in the multi-chromophore is 5% or higher (e.g., 5 mol%), such as 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or even higher. In some cases of formula (IIa), the mole percentage of z in the multi-chromophore is 5% or higher (e.g., 5 mol%), such as 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or even higher, where w + x + y + z = 100%.

[0325] In some embodiments, the structure of the polymeric dye is shown in formula (XIIb):

[0326]

[0327] where u, v, w, x, y, and z represent the mol% values of each comonomer in the multi-chromophore; Y 1-3The Z group of the fused 6-5-6 tricyclic comonomer is each independently, for example, as described herein; M 1 , M 2 and M 3 are each an aryl or heteroaryl comonomer; N 1 is the narrow emission acceptor fluorophore linked to Y 3 (e.g., achieved through a non-conjugated linker); and G 1 and G 2 are each independently selected from a terminal group, a conjugated segment, a linker, and a linked specific binding member. It should be understood that any polymeric dye described herein can be represented by formula (IIb). In some cases of formula (XIIb), the mole percentage of u + w + y in the multi-chromophore is 5% or higher (e.g., 5 mol%), such as 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or even higher. In certain cases, z is 0. In some cases of formula (IIb), the mole percentage of v + x + z in the multi-chromophore is 80% or lower (e.g., 80 mol%), such as 75% or lower, 70% or lower, 65% or lower, 60% or lower, 55% or lower, 50% or lower, 45% or lower, 40% or lower, 35% or lower, 30% or lower, 25% or lower, 20% or lower, 15% or lower, 10% or lower, or even lower.

[0328] In some cases of formula (XIIb), u is 5% or higher, such as 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or even higher. In some cases of formula (IIb), w is 5% or higher, such as 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or even higher. In some cases of formula (XIIb), y is 1% or higher, such as 2% or higher, 5% or higher, 10% or higher, 15% or higher, 20% or higher, 25% or higher, 30% or higher, 35% or higher, 40% or higher, 45% or higher, 50% or higher, 55% or higher, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, or even higher. In some cases of formula (XIIb), v is 80% or lower, such as 75% or lower, 70% or lower, 65% or lower, 60% or lower, 55% or lower, 50% or lower, 45% or lower, 40% or lower, 35% or lower, 30% or lower, 25% or lower, 20% or lower, 15% or lower, 10% or lower, or even lower. In some cases of formula (XIIb), x is 80% or lower, such as 75% or lower, 70% or lower, 65% or lower, 60% or lower, 55% or lower, 50% or lower, 45% or lower, 40% or lower, 35% or lower, 30% or lower, 25% or lower, 20% or lower, 15% or lower, 10% or lower, or even lower. In some cases of formula (XIIb), z is 80% or lower, such as 75% or lower, 70% or lower, 65% or lower, 60% or lower, 55% or lower, 50% or lower, 45% or lower, 40% or lower, 35% or lower, 30% or lower, 25% or lower, 20% or lower, 15% or lower, 10% or lower, or even lower.

[0329] In some embodiments of formula (XI), the structure of the multi-chromophore is as shown in formula (XIII):

[0330]

[0331] Wherein:

[0332] F1 is the fused 6-5-6 tricyclic comonomer such as replaced by WSG;

[0333] M 1 、M 2 and L 1 are each independently an aryl or heteroaryl comonomer optionally substituted by WSG;

[0334] N 1 is the narrow emission acceptor fluorophore connected to L 1 ;

[0335] a-d and f are each independently 0 or 1;

[0336] e is 1;

[0337] Each of n and m is an integer between 1 and 10,000;

[0338] p is an integer between 1 and 100,000; and

[0339] G 1 and G 2 are each independently selected from a terminal group, a π-conjugated chain segment, a linker, and a conjugated specific binding member.

[0340] In certain embodiments of formula (XIII), the structure of the multi-chromophore is as shown in formula (XIVa) or (XIVb):

[0341]

[0342]

[0343] In some cases of formulas (XII)-(XIV), L 1 is a fused 6-5-6 tricyclic comonomer substituted by Z 1 and a branched nonionic water-soluble group (WSG) (e.g., as described herein). In certain cases of formulas (XII)-(XIV), the structure of L 1 is as follows:

[0344]

[0345] Wherein: R 3 is a branched nonionic water-soluble group (WSG) (e.g., as described herein); and R 4 is L 2 -Z 2 wherein L 2 is a linker and Z 2 is a narrow emission acceptor fluorophore. In certain embodiments of L 1 , R3 The structure is selected from one of the following structures:

[0346]

[0347] In some cases of L 1 in some cases, T 5 is C 1 -C 6 linker. In some cases, each s is from 6 to 30, such as from 6 to 20, from 11 to 20, from 12 to 20, or from 12 to 16. In some cases, each s is independently 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some cases, each s is 11. In some cases, each s is 12. In some cases, each s is 13. In some cases, each s is 14. In some cases, each s is 15. In some cases, each s is 16. In some cases, each s is 17. In some cases, each s is 18. In some cases, each s is 19. In some cases, each s is 20. In L 1 in certain embodiments of L 4 R 2 is L 2 -Z 2 where L 2 is a linker (for example, a C1-C20 linker, which optionally includes one or more heteroatoms or linking functional groups in the linker backbone (for example, -CONH- or SO 2 NH-); Z 1 is a chemoselective functional group (for example, an amino group or a protected amino group, or a carboxylic acid or its active ester, or a protected carboxylic acid). In some cases, the structure of L

[0348]

[0349] In some embodiments of formulas (XI)-(XIV), the structure of the polymeric dye is as shown in formula (XV) or (XVI):

[0350]

[0351]

[0352] where each WSG is independently a branched or linear WSG (for example, as described herein); Z 1 is a linked chemoselective functional group or a linked narrow emission acceptor fluorophore (N 1 ); G 1 and G 2each independently selected from a terminal group, a conjugated segment, a linker, and a conjugated specific binding member that has been conjugated; and x, y, and z represent the mole percentages of the units in the multi-chromophore. In some cases of formula (V)

[0353] In some embodiments of formulas (I)-(VI), the structure of the polymeric dye is shown in formula (VII) or (VIII):

[0354]

[0355] wherein each s is independently an integer between 6 and 50, such as 6 to 30, 6 to 20, 11 to 20, 12 to 20, or 12 to 16; each R 11 is independently hydrogen, an alkyl group, or a substituted alkyl group; the dye is a conjugated narrow emission acceptor fluorophore; r is 0-6 (e.g., 0, 1, 2, 3, or 4); and q is 1-12, such as 1-6 (e.g., 1, 2, 3, or 4).

[0356] Any suitable comonomer can be adapted to include side chain substituents that can conjugate the polymeric dye with a moiety of interest (e.g., a signal-conducting chromophore or a biomolecule). Any comonomer can include substituents that make the polymeric dye more water-soluble.

[0357] In some embodiments of any of the formulas described herein, n, m, and p are selected such that the multi-chromophore includes a total of 2 to 100,000 repeating units (i.e., monomer repeating units), wherein the multi-chromophore can include various different monomer repeating units. In some cases, when m is 0, p is 1, and n is 2 to 100,000. It should be understood that the conjugated polymers of the formulas described herein can also be represented by formulas that provide the mol% values of each comonomer in the polymer. It should be further understood that in some cases, the polymerization method can produce a composition comprising a population of conjugated polymers that includes some variation with respect to the specific length and / or the terminal groups (i.e., end groups) present in each conjugated polymer of the population. The formulas described herein can refer to a single compound or a population or sub-population of polymers.

[0358] Aryl or heteroaryl comonomer

[0359] As described above, the subject polymeric dye includes a conjugated segment of aryl or heteroaryl comonomers connected by covalent bonds, vinylene groups, or ethynylene groups. In addition to the comonomers described in the above exemplary formulas, various aryl and heteroaryl comonomers can also be used in the subject polymeric dye. Any suitable aryl and heteroaryl comonomers can be used in the subject multi-chromophore, e.g., in a bandgap modification unit, and can be distributed uniformly or randomly along the conjugated polymers described herein.

[0360] In some embodiments of the formulas (e.g., formulas (XI)-(XIV)) described herein, the aryl or heteroaryl comonomer (e.g., L 1 , M 1 , M 2 and M 3 ) is independently selected from one of the formulas (XXIII)-(XXVI):

[0361]

[0362] wherein

[0363] Cy 2-7 is an aryl or heteroaryl group containing 2 to 7 fused and / or unfused rings;

[0364] Y 2 , Y 3 and Y 4 are independently selected from –CR 3 -, NR 3 , N, O, S and –C(=O)-, and together form a 5- or 6-membered fused aryl or heteroaryl ring;

[0365] Each R 3 is one or more independently selected from H, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano, alkoxy-substituted alkoxy and –T 1 -Z 1 ring substituents;

[0366] R 1 and R 2 are independently selected from H, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano, alkoxy, substituted alkoxy and –T 1 -Z 1 , or R 1 and R 2 together form a 5- or 6-membered fused aryl, heteroaryl, cycloalkyl or heterocycle, which may optionally be substituted;

[0367] Y 5 is N or CR 5 , Y 7 is N or CR 7 ;

[0368] R 4 -R 7independently selected from H, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano, alkoxy, substituted alkoxy, and –T 1 -Z 1 ;

[0369] Z 1 is a chemoselective functional group or an attached signaling chromophore; and

[0370] T 1 is a linker.

[0371] Cy 2-7 is an aryl or heteroaryl group that can include 2, 3, 4, 5, 6, or 7 rings, which can be fused together to form a fused ring system or can be unfused (i.e., connected together by a single covalent bond). In some cases, Cy 2-7 includes 2 or 3 unfused aryl and / or heteroaryl rings. In certain cases, Cy 2-7 consists of 5- and / or 6-membered carbon and / or heterocyclic rings. In certain cases, Cy 2-7 is a bicyclic, tricyclic, or tetracyclic aryl or heteroaryl group, such as naphthalene, anthracene, acridine, or quinoline, etc.

[0372] In certain embodiments of formulas (XXIII)-(XXVI), the comonomer (e.g., M 1 , M 2 , and M 3 ) is independently selected from one of the following structures (a) to (x):

[0373]

[0374]

[0375]

[0376]

[0377] wherein:

[0378] each Y 8 is independently C(R 3 ) 2 , -C(R 3 ) 2 C(R 3 ) 2 -, -C(R 3 ) 2 Si(R 3 ) 2 -, NR 3 or Si(R3 ) 2 ;

[0379] X is S or O; each R 3 is independently H, a water-solubilizing group (e.g., as described herein), amino, substituted amino, halogen, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano, alkoxy, substituted alkoxy, and –T 1 -Z 1 ;

[0380] R 1 and R 2 are independently selected from H, halogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, sulfonic acid, cyano, alkoxy, substituted alkoxy, and –T 1 -Z 1 , or R 1 and R 2 together form a 5- or 6-membered fused aryl, heteroaryl, cycloalkyl, or heterocycle, which may optionally be substituted (e.g., substituted by R 3 groups);

[0381] Z 1 is a chemoselective functional group or a linked signaling chromophore; and

[0382] T 1 is a linker.

[0383] In some cases, Y 8 is C(R 3 ) 2 . In some cases, Y 8 is -C(R 3 ) 2 C(R 3 ) 2 -. In some cases, Y 8 is NR 3 . In some cases, Y 8 is Si(R 3 ) 2 . In certain embodiments of formulas (a)-(x), when the comonomer is a linking comonomer, at least one of R 1 -R 7 is –T 1 -Z 1 . In certain embodiments of formulas (a)-(x), the comonomer includes a water-solubilizing group (e.g., as described herein). In certain embodiments of formula (s), R 3is H, a halogen (e.g., F), or an alkoxy group (e.g., methoxy). It should be understood that in any of the structures shown in (a)-(x) described herein, an N heteroatom may be included to convert a phenyl or fused benzo ring into a pyridyl or fused pyrido ring. Such heteroatom-substituted forms of formulas (a)-(x) are intended to be included within the scope of the present invention. In some cases of formulas (XXIII)-(XXVI), the structure of one or more of the said comonomers is as shown in one of the following formulas (s), where the benzo ring is replaced by a pyrido ring:

[0384]

[0385] where Y 8 is independently C(R 4 ) 2 , -C(R 4 ) 2 C(R 4 ) 2 -, NR 4 or Si(R 4 ) 2 .

[0386] In some cases of formulas (XXIII)-(XXVI) and (a)-(x), the said comonomers (M 2 , M 3 and M 4 ) are independently selected from one of the following structures (ba) to (cd):

[0387]

[0388]

[0389]

[0390] where:

[0391] Y 9 is C(R 4 ) 2 , -C(R 4 ) 2 C(R 4 ) 2 - or Si(R 4 ) 2 ;

[0392] X is S or O;

[0393] Each R 4Each is independently H, a water - solubilizing group (e.g., as described herein), an alkyl group, a substituted alkyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, an acyl group, a substituted acyl group, a sulfonic acid group, a cyano group, an alkoxy group, a substituted alkoxy group, and -T 1 -Z 1 ; and

[0394] R 1 and R 2 are independently selected from H, a halogen, an alkyl group, a substituted alkyl group, an aryl group, a substituted aryl group, a heteroaryl group, a substituted heteroaryl group, an acyl group, a substituted acyl group, a sulfonic acid group, a cyano group, an alkoxy group, a substituted alkoxy group, and -T 1 -Z 1 , or R 1 and R 2 together form a 5 - or 6 - membered fused aryl or heteroaryl ring, which may be optionally substituted; in certain embodiments of formulas (ba)-(cd), when the comonomer is a linking comonomer, at least one R 4 is -T 1 -Z 1 . In certain embodiments of formulas (ba)-(cd), R 4 includes a water - solubilizing group (e.g., as described herein).

[0395] The aryl and heteroaryl comonomers described herein (e.g., the comonomers represented by formulas (XI)-(XIV), (a)-(x), and (ba)-(cd)) may include a water - solubilizing group WSG (e.g., as described herein).

[0396] In certain embodiments of the formulas described herein (e.g., formulas (XI)-(XIV)), the comonomer (e.g., M 1 , M 2 and M 3 ) is independently selected from one of the following structures:

[0397]

[0398]

[0399]

[0400]

[0401] where each s is independently from 1 to 30, such as from 6 to 30, such as from 6 to 20, from 11 to 20, from 12 to 20 or from 12 to 16. In some cases, each s is independently 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some cases, each s is 4. In some cases, each s is 5. In some cases, each s is 6. In some cases, each s is 7. In some cases, each s is 8. In some cases, each s is 9. In some cases, each s is 10. In some cases, each s is 11. In some cases, each s is 12. In some cases, each s is 13. In some cases, each s is 14. In some cases, each s is 15. In some cases, each s is 16. In some cases, each s is 17. In some cases, each s is 18. In some cases, each s is 19. In some cases, each s is 20.

[0402] In some cases, the multi-chromophore (e.g., the multi-chromophore represented by formulae (XI)-(XIV)) includes a comonomer (e.g., L 1 ) linked to a signal-conducting chromophore having one of the following structures:

[0403]

[0404] where WSG is an optional water-soluble group; T 2 is a linker; and the dye is the narrow emission acceptor fluorophore.

[0405] In certain embodiments, the aryl or heteroaryl comonomer is selected from 2,1,3-benzothiadiazole, 2,1,3-benzoxadiazole, benzoxadiazole, benzoselenadiazole, benzothiadiazole, naphthoselenadiazole, 4,7-bis(thiophen-2-yl)-2,1,3-benzothiadiazole, squaraine dyes, quinoxaline, perylene, perylene diimide, pyrrolopyrrole dione, thiophenopyrazine low band-gap commercial dyes, olefins and cyano-substituted olefins and their isomers. In some cases, the aryl and heteroaryl comonomers that can be used in the subject multi-chromophore are selected from a'-m' having the following structures:

[0406]

[0407] where *=site for covalent attachment to the unsaturated backbone; each R is independently H, a non-ionic side group having water solubility (e.g., WSG) or –L 2 -Z 2 where L 2 is a linker and Z 2is a chemoselective label or a conjugated metal complex. In certain instances of a'-m', each R is H or a water-soluble group (e.g., as described herein). In certain instances, each R is an alkyl or benzyl substituted with one or more (CH 2 ) x (OCH 2 CH 2 ) y OCH 3 , where each x is independently an integer between 0 and 20 and each y is independently an integer between 0 and 50. In certain instances of a'-m', each R is (CH 2 ) 3 (OCH 2 CH 2 ) 11 OCH 3 . In certain instances of a'-m', each R is H or a nonionic branched WSG (e.g., as described herein).

[0408] In some embodiments, the multi-chromophore comprises an absorbance-modified comonomer having the following structure:

[0409]

[0410] where X is O or S, R 41 and R 42 are each independently H, halogen, WSG, alkyl, substituted alkyl, alkoxy, and substituted alkoxy. In certain instances, X is O. In some instances, X is S. In certain embodiments, X is O and R 41 and R 42 are both H. In certain embodiments, X is S and R 41 and R 42 are both H.

[0411] In some instances, the aryl or heteroaryl comonomer is a substituted or unsubstituted phenyl, biphenyl, or pyridyl comonomer. In certain embodiments, the aryl or heteroaryl comonomer is selected from substituted or unsubstituted 1,4-phenyl, substituted or unsubstituted 1,3-phenyl, substituted or unsubstituted 4,4'-biphenyl, substituted or unsubstituted 2,5-pyridyl, and substituted or unsubstituted 2,6-pyridyl. In certain instances, the absorbance-modified comonomer is an optionally substituted aryl or heteroaryl comonomer having a structure selected from the following structures:

[0412]

[0413] where Z 2 -Z 5Each is independently CR or N, with at least one Z 2 -Z 5 is N; each R and each R 11 -R 16 are each independently selected from the group consisting of hydrogen, a water-soluble group, a halogen, a cyano group, an alkoxy group, a substituted alkoxy group, an alkyl group, and a substituted alkyl group. In certain embodiments, Z 2 -Z 5 has exactly one N. In certain embodiments, Z 2 -Z 5 has exactly two Ns. In some cases, R 11 、R 12 and R 14 are each H. In some cases, R 12 and R 14 are each H. In some cases, R 11 and R 13 are each H. In some cases, R 15 and R 16 are each H. In some cases, the halogen is fluoro.

[0414] In some cases, the aryl or heteroaryl comonomer is selected from one of the following:

[0415]

[0416] where n is 1-20 and R' is H or a lower alkyl. In some embodiments of the aryl or heteroaryl comonomer structure, n is an integer between 6 and 20, such as 12-20 or 12-16 or 16-20.

[0417] In some embodiments, the multi-chromophore includes a substituted aryl comonomer as described by the following structure:

[0418]

[0419]

[0420]

[0421]

[0422] In some embodiments, the multi-chromophore includes a substituted aryl comonomer as described by the following structure:

[0423]

[0424] where n is from 1 to 20, and R' is H or lower alkyl. In some cases, n is from 3 to 12. In some embodiments, the multi-chromophore comprises a substituted aryl comonomer as described in the following structure:

[0425]

[0426] where each n is from 1 to 20, and each R' is independently H or lower alkyl. In certain embodiments of the above substituted aryl or heteroaryl comonomer structures, n is 3. In some cases, n is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In certain cases, R' is methyl. In certain cases, R' is hydrogen. In some embodiments, the multi-chromophore comprises a substituted aryl comonomer as described in the following structure:

[0427]

[0428] In some embodiments, the multi-chromophore comprises a substituted aryl comonomer as described in the following structure:

[0429]

[0430] In some embodiments of any of the formulas described herein, the multi-chromophore comprises a substituted aryl comonomer (M1) having the following structure:

[0431]

[0432] where s is from 6 to 50 (e.g., 6 - 40, 6 - 30 or 6 - 20); each R 11 is independently H, alkyl or substituted alkyl. In some embodiments, the multi-chromophore comprises a substituted aryl comonomer as described in the following structure, which comprises a branched non-ionic WSG:

[0433]

[0434] Any of the above absorbance-corrected comonomers can be used in the target multi-chromophore, e.g., the multi-chromophores represented by formulas (XI)-(XIV).

[0435] In certain embodiments or formulas described herein, such as formulas (XI)-(XIV), (a)-(w) and (ba)-(cb), include a linked narrow emission acceptor fluorophore (N 1 )(e.g., as described herein), as a substituent of the comonomer. Any suitable narrow emission acceptor fluorophore (N 1) can be coupled via the coupling of compatible chemoselective functional groups to any suitable polymeric dye described herein. The narrow emission acceptor fluorophore (N 1 ) can be selected to provide the desired emission spectrum and maximum emission wavelength.

[0436] Any suitable linking comonomer can be introduced into the target multi-chromophore to provide a linking group on which any suitable moiety of interest (e.g., a linked signaling chromophore) can be attached. Exemplary linking comonomers include, but are not limited to, the comonomers described in the formulas herein, as well as fluorene comonomers, phenylene vinylene comonomers, phenylene ethynylene comonomers, carbazole comonomers, C 2 -C 12 alkynyl comonomers, arylene-ethynylene comonomers, heteroarylene-ethynylene comonomers, arylene comonomers, and heteroarylene comonomers. The terms aryl or heteroaryl comonomer and arylene or heteroarylene comonomer as used herein are used interchangeably. In some cases, the linking comonomer is a substituted aryl comonomer. In some cases, the linking comonomer is a substituted heteroaryl comonomer. In some cases, the linking comonomer is a substituted or unsubstituted 1,4-phenylene, a substituted or unsubstituted 1,3-phenylene, a substituted or unsubstituted 4,4'-biphenylene, a substituted or unsubstituted 2,5-pyridyl, and a substituted or unsubstituted 2,6-pyridyl.

[0437] In some cases of any of the formulas described herein, the narrow emission acceptor fluorophore (N 1 ) is linked to a comonomer that is present in the multi-chromophore in a molar percentage of 1% to 50%, such as a molar percentage of 1% to 20%, 1% to 10%, or 11 to 20%. In some cases, the multi-chromophore is a conjugated polymer comprising 5 or more repeating units.

[0438] Any suitable chemoselective functional group can be included in the target multi-chromophore (e.g., on –Z 1 , –Z 2 and / or on G 1 or G 2 termini), including but not limited to carboxylic acid, active esters (e.g., NHS or sulfo-NHS esters), amino, hydroxyl, thiol, maleimide, iodoacetyl, hydrazide, hydrazine, aldehyde, ketone, azide, alkyne, phosphine, and epoxide. It should be understood that in the polymeric tandem dye structures described herein, in some cases, the groups Z 1 and Z 2 appear at equivalent positions in the structure, where these groups can be used interchangeably, and refer to the linked narrow emission acceptor fluorophore (N 1) or is capable of subsequently conjugating with a conventional dye precursor to produce a linked narrow emission acceptor fluorophore (N 1 ) with a chemo - selective functional group.

[0439] In some embodiments, the selected narrow emission acceptor fluorophore (N 1 ) has a maximum emission wavelength range of 300 to 900 nm, for example, 350 to 850 nm, 350 to 600 nm, 360 to 500 nm, 370 to 500 nm, 380 to 500 nm, 390 to 500 nm, or 400 to 500 nm. Specific examples of the emission maxima of the desired narrow emission acceptor chromophore dyes include, but are not limited to: 395 nm ± 5 nm, 420 nm ± 5 nm, 430 nm ± 5 nm, 440 nm ± 5 nm, 450 nm ± 5 nm, 460 nm ± 5 nm, 470 nm ± 5 nm, 480 nm ± 5 nm, 490 nm ± 5 nm, 500 nm ± 5 nm, 510 nm ± 5 nm, 520 nm ± 5 nm, 530 nm ± 5 nm, 540 nm ± 5 nm, 550 nm ± 5 nm, 560 nm ± 5 nm, 570 nm ± 5 nm, 580 nm ± 5 nm, 590 nm ± 5 nm, 605 nm ± 5 nm, 650 nm ± 5 nm, 680 nm ± 5 nm, 700 nm ± 5 nm, and 805 nm ± 5 nm.

[0440] Terminal group

[0441] Any suitable end - group (e.g., G 1 and G 2 ) can be used at the ends of the subject multi - chromophore. The terms "end - group" and "terminal group" as used herein are used interchangeably and refer to a group located at the end of the polymeric structure of the multi - chromophore, e.g., as described herein. The desired G 1 and G 2 groups include, but are not limited to, capping groups, conjugated segments, linkers, and linked specific binding members. In some embodiments, the capping group is a monovalent group that conjugates with the backbone of the multi - chromophore after polymerization. In certain cases, the capping group is an aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, or substituted alkyl. In some cases, the terminal comonomer is directionally linked to a chemo - selective tag or linker. In certain cases, the capping group is derived from a monomer used in the polymerization method, such as an end - group capable of further coupling, such as a halogen (e.g., Br), boric acid, or borate ester. In some cases, G 1 and / or G 2is a π-conjugated chain segment. The π-conjugated chain segment used herein refers to any suitable chain segment of a conjugated polymer to which the multiple chromophores can be conjugated, i.e., allowing π-electrons to delocalize across adjacent units. In certain embodiments, G 1 and / or G 2 is a linker, e.g., a linker comprising a functional group suitable for conjugation to a specific binding moiety. It should be understood that a linker located at the G 1 and / or G 2 position of the multiple chromophores can be selected to be orthogonal to any other linker (which may be present on the side chain of the multiple chromophores, e.g., at Z 2 ) comprising a chemoselective tag (e.g., as described herein). In certain embodiments, an amino functional group or a derivative thereof is introduced at G 1 and / or G 2 , and a carboxylic acid functional group or a derivative thereof is introduced at Z 2 . In certain embodiments, a carboxylic acid functional group or a derivative thereof is introduced at G 1 and / or G 2 , and an amino functional group or a derivative thereof is introduced at Z 2 .

[0442] In some embodiments of the formulas described herein, at least one of G 1 and G 2 is -L 3 -Z 4 , where L 3 is a linker (e.g., as described herein) and Z 4 is a specific binding member (e.g., as described herein). In some embodiments of the formulas described herein, at least one of G 1 and G 2 is -L 3 -Z 3 , where L 3 is a linker (e.g., as described herein) and Z 3 is a chemoselective tag (e.g., as described herein). Any suitable chemoselective tag and conjugation chemistry can be used in the target multiple chromophores. Exemplary chemoselective tags include, but are not limited to, amines, activated esters, maleimides, thiols, sulfur(VI) fluoride exchange chemistry (SuFEX), fluorosulfonyl, Diers Alder cycloaddition click reagents and click chemistry, tetrazines, trans-cyclooctenes, aldehydes, alkoxyamines, alkynes, cyclooctynes, azides, etc. In some cases, Z 3 is selected from the group consisting of carboxylic acids, activated esters (e.g., N-hydroxysuccinimide ester (NHS) or sulfo-NHS), amines, maleimides, iodoacetyls, and thiols. In certain embodiments of the formulas described herein, G 1 and G 2At least one of them has a structure as shown in the following structure:

[0443] *-Ar-L-Z

[0444] Where Ar is a conjugated aryl group, L is a linker, and Z is a chemoselective label or a specific binding member. In some cases, the L-Z group can be directly linked to the terminal comonomer. In certain embodiments of the formulas described herein, G 1 and G 2 At least one of them has a structure as shown in the following structure:

[0445]

[0446] Wherein:

[0447] q is 0 or an integer between 1 and 12;

[0448] L is an optional linker; and

[0449] Z is a chemoselective label or a specific binding member.

[0450] In certain embodiments, Z is a biomolecule. Target biomolecules include, but are not limited to, polypeptides, polynucleotides, carbohydrates, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs thereof, and combinations thereof. In certain cases, Z is an antibody. In some cases, Z is an antibody fragment or a binding derivative thereof. In some cases, the antibody fragment or its binding derivative is selected from the group consisting of Fab fragments, F(ab') 2 fragments, scFv, diabodies, and triabodies.

[0451] It should be understood that for any structure and formula described herein, in some cases of the target multi-chromophore, the depicted terminal group can be located at the terminal opposite to the shown terminal group. For example, the terminal group G 1 can be swapped with –Ph-L-Z. In some embodiments of the multi-chromophore described herein, at least one terminal group (e.g., T 2 -Z 2 , G 1 , G 2 , L-Z, -L 3 -Z 3 ) or the structure of the side chain group is selected from one of the following structures:

[0452]

[0453] Where r is 0 or an integer between 1 and 50; k is 0 or an integer between 1 and 50 (e.g., 1-20); R 1 is defined for any fluorene comonomer described herein; and R16 Selected from H, OH, NH 2 , -NH(CH 2 )r-NH 2 and -NH(CH 2 ) r COOH. In certain cases, r is from 1 to 20, such as 3 to 20, 3 to 15, 3 to 12 or 6 to 12. In certain cases, r is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In some cases, r is 3. In some cases, r is 4. In some cases, r is 5. In some cases, r is 6. In some cases, r is 7. In some cases, r is 8. In some cases, r is 9. In some cases, r is 10. In some cases, r is 11.

[0454] In some embodiments, the multi-chromophore comprises one or more of the following groups, for example, as end groups or side chain groups, to conjugate with a compatible functional group on another molecule:

[0455]

[0456]

[0457] Labeled specific binding member

[0458] Aspects of the invention include labeled specific binding members. A labeled specific binding member is a conjugate of a target polymeric dye (e.g., as described herein) and a specific binding member. Any of the polymeric dyes or polymer tandem dyes described herein can be conjugated to a specific binding member. The specific binding member and the polymeric dye can be conjugated to each other (e.g., covalently linked) at any suitable position of the two molecules via an optional linker.

[0459] In some embodiments, the labeled specific binding member has anti-polymerization properties. As used herein, "anti-polymerization" means that a labeled specific binding member at a concentration of 1 mg / ml or higher in a target aqueous buffer can form a homogeneous aqueous composition without polymer precipitates, for example, 2 mg / ml or higher, 3 mg / ml or higher, 4 mg / ml or higher, 5 mg / ml or higher, 6 mg / ml or higher, 7 mg / ml or higher, 8 mg / ml or higher, 9 mg / ml or higher, 10 mg / ml or higher, or even higher concentrations of the labeled specific binding member. In certain embodiments, the anti-polymerization labeled specific binding member comprises: a polymer tandem dye comprising a light-harvesting multi-chromophore (e.g., as described herein) and a narrow emission acceptor fluorophore linked to the multi-chromophore that is proximal upon energy reception; and a specific binding member covalently linked to the multi-chromophore.

[0460] As used herein, the term "specific binding member" refers to a member of a pair of molecules that have binding specificity for each other. One member of the pair of molecules can have a region on its surface or in a cavity that specifically binds to a corresponding region on the surface or in a cavity of the other member of the pair of molecules. Thus, the members of the pair of molecules have the property of specifically binding to each other to produce a binding complex. In some embodiments, the affinity between specific binding members in the binding complex is characterized by a K d (dissociation constant) of 10 -6 M or lower, such as 10 -7 M or lower, including 10 -8 M or lower, for example, 10 -9 M or lower, 10 -10 M or lower, 10 -11 M or lower, 10 -12 M or lower, 10 -13 M or lower, 10 -14 M or lower, including 10 -15 M or lower. In some embodiments, the specific binding member specifically binds with high affinity. High affinity means that the binding member specifically binds with an apparent affinity, and the apparent affinity is characterized by an apparent K d of 10×10 -9 M or lower, such as 1×10 -9 M or lower, 3×10 -10 M or lower, 1×10 -10 M or lower, 3×10 -11 M or lower, 1×10 -11 M or lower, 3×10 -12 M or lower or 1×10 -12 M or lower.

[0461] The specific binding member can be a protein moiety. As used herein, the term "proteinaceous" refers to a moiety composed of amino acid residues. The protein moiety can be a polypeptide. In some cases, the protein specific binding member is an antibody. In certain embodiments, the protein specific binding member is an antibody fragment, e.g., a binding fragment of an antibody that specifically binds to a polymeric dye. As used herein, the terms "antibody" and "antibody molecule" are used interchangeably and refer to a protein composed of one or more polypeptides that are substantially encoded by all or part of the well-known immunoglobulin genes. The well-known immunoglobulin genes (e.g., the corresponding genes in humans) include the κ (kappa), λ (lambda), and heavy chain loci, which together constitute numerous variable region genes, as well as the constant region genes μ (mu), δ (delta), γ (gamma), ε (epsilon), and α (alpha) that encode the IgM, IgD, IgG, IgE, and IgA isotypes, respectively. The variable region of an immunoglobulin light or heavy chain consists of "framework" regions (FRs) interrupted by three hypervariable regions (also referred to as "complementary determining regions" or "CDRs"). The ranges of the framework regions and CDRs have been precisely defined (see, for details, "Sequences of Proteins of Immunological Interest", E. Kabat et al., U.S. Department of Health and Human Services, (1991)). The numbering of all antibody amino acid sequences discussed herein conforms to the requirements of the Kabat system. The sequences of different light or heavy chain framework regions are relatively conserved within the same species. The framework regions of an antibody (i.e., the combined framework regions of the constitutive light and heavy chains) serve to position and align the CDRs. The CDRs are primarily responsible for binding to the antigen epitope. The term "antibody" is intended to include full-length antibodies and can refer to natural antibodies, engineered antibodies, or recombinantly produced antibodies from any organism for experimental, therapeutic, or other purposes further defined below.

[0462] Exemplary antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, or other antigen-binding sequences of an antibody, which are produced by modifying a whole antibody or de novo synthesis using recombinant DNA techniques. The antibody can be a monoclonal antibody or a polyclonal antibody and can have other specific activities against cells (e.g., antagonist, agonist, neutralizing antibody, inhibitory antibody, or stimulatory antibody). It should be understood that the antibody can undergo other conservative amino acid substitutions that have substantially no effect on antigen binding or other antibody functions.

[0463] In certain embodiments, the specific binding member is a Fab fragment, F(ab') 2 fragment, scFv, diabody, or triabody. In certain embodiments, the specific binding member is an antibody. In some cases, the specific binding member is a murine antibody or a binding fragment thereof. In certain cases, the specific binding member is a recombinant antibody or a binding fragment thereof.

[0464] In some embodiments, the labeled specific binding member includes: a water-solvated light-harvesting multi-chromophore (e.g., as described herein); a signaling chromophore covalently linked to the multi-chromophore that is proximal upon energy receipt (e.g., as described herein); and a specific binding member covalently linked to the multi-chromophore. In some instances of the labeled specific binding member, the multi-chromophore of any of the formulas (e.g., formula (V) and (VII)-(VIII)) described herein, wherein: G 1 and G 2 each independently is selected from the group consisting of an end group (e.g., terminal group), a conjugated segment, a linker, and a linked specific binding member, wherein G 1 and G 2 at least one of which is a linked specific binding member.

[0465] Multiplexed fluorescent composition

[0466] As described above, the polymeric tandem dyes provide a variety of narrow emission spectra suitable for multiplexing applications. The subject polymeric tandem dyes have narrow emission spectra that can provide a multiplexed signal format. Due to the narrow emission spectra, a variety of dyes can be simultaneously combined and used within the same spectral range as compared to multiplexing systems based on broad-spectrum emitters. By keeping the detection window suitable for each dye to a small size, multiple smaller emission ranges can be included in a multiplexed assay, thereby increasing the number of independent fluorescent signals observed in a given system. For example, Figure 9 shows the emission spectra of 5 exemplary tandem dyes in a multiplexing system. Figure 9 The FWHM of the emission signals of all the tandem dyes shown therein is 25 nm or less and provides wavelength ranges that do not overlap with the donor polymeric dyes excitable by the same violet light. These tandem dyes exhibit an effective Stokes shift of 100 to 250 nm. The small peak at 421 nm is due to the residual emission from the donor.

[0467] Aspects of the invention include compositions for multiplexed fluorescent signal generation. The composition can include: a first polymeric tandem dye having a first maximum emission wavelength and a first narrow emission spectrum (e.g., as described herein); and a second polymeric tandem dye having a second maximum emission wavelength orthogonal to the first narrow emission spectrum. The second polymeric tandem dye can be any suitable dye. In some cases, the width of the full width at half maximum (FWHM) of the first narrow emission spectrum is 25 nm or less, such as 20 nm or less, 15 nm or less, or 10 nm or less. The second maximum emission wavelength is different from the first maximum emission wavelength and the two differ by 20 nm or more in order to effect the desired separation of the emission signals.

[0468] Multiple dyes can be multiplexed in the subject compositions. In some cases, the compositions include one or more other polymeric tandem dyes, each having a different maximum emission wavelength. In some cases, each polymeric tandem dye in the compositions is independently a polymeric tandem dye having a narrow emission acceptor fluorophore (e.g., as described herein). In some embodiments, the compositions include three or more (4 or more, 5 or more, 6 or more, 10 or more, or even more) polymeric tandem dyes, each having a different and non-overlapping emission spectrum in the range of 480 nm to 700 nm (e.g., different emission spectra having non-overlapping full width at half maximum (FWHM) ranges). Each polymeric tandem dye in the compositions is capable of being excited with a single light source to provide orthogonally detectable fluorescence emissions. Figure 9 Shows the spectra of the multiplexed compositions where each tandem dye is excited with a single light source.

[0469] The polymeric tandem dyes in the compositions can be linked to specific binding members (e.g., as described herein). Accordingly, aspects of the invention include compositions for multiplexed detection of target molecules, the compositions including first and second labeled specific binding members. The first labeled specific binding member can include: (i) a first polymeric tandem dye having a first maximum emission wavelength and a first narrow emission spectrum, which includes: a water-soluble light-harvesting multi-chromophore; and a narrow emission acceptor fluorophore configured to be proximal to the light-harvesting multi-chromophore upon energy reception; and (ii) a first specific binding member that specifically binds to a first target molecule and is linked to the first polymeric tandem dye. The second labeled specific binding member can include: (i) a second polymeric tandem dye having a second maximum emission wavelength orthogonal to the first narrow emission spectrum; and (ii) a second specific binding member that specifically binds to a second target molecule and is linked to the second polymeric tandem dye. The narrow emission acceptor fluorophore can be a BODIPY dye (e.g., as described herein).

[0470] Methods

[0471] As described above, aspects of the present invention include methods for assessing the presence of a target analyte in a sample. Aspects of the method include contacting the sample with a polymeric dye conjugate that specifically binds to the target analyte to produce a labeled composition contacting the sample. The terms "polymeric dye conjugate" and "labeled specific binding member" as used herein are used interchangeably. Thus, the polymeric dye conjugate can include: (i) a water-solvated polymer tandem dye (e.g., as described herein); and (ii) a specific binding member (e.g., as described herein). In some cases, the polymer tandem dye conjugate includes a narrow emission acceptor fluorophore (e.g., as described herein) covalently linked to the multiple chromophores of the polymeric dye that are in proximity upon energy receipt.

[0472] Any suitable method can be employed to contact the sample with the polymeric dye conjugate that specifically binds to the target analyte to produce a labeled composition contacting the sample. In some cases, the sample is contacted with the polymeric dye conjugate under conditions where the specific binding member specifically binds to the target analyte, if any. To enable the specific binding member of the conjugate to specifically bind to the target analyte, an appropriate solution can be used to maintain the biological activity of the sample components and the specific binding member. The solution can be a balanced salt solution, e.g., physiological saline, PBS, Hank's balanced salt solution, etc., appropriately supplemented with fetal bovine serum, human platelet lysate or other factors and a low concentration (e.g., 5-25 mM) of an acceptable buffer. Suitable buffers include HEPES, phosphate buffer, lactate buffer, etc. A variety of media are commercially available and can be used depending on the nature of the target analyte, including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., and in some cases, fetal bovine serum or human platelet lysate needs to be supplemented. The final composition of the solution can be selected based on the sample components included.

[0473] The temperature at which the specific binding member of the conjugate specifically binds to the target analyte may vary, and in some cases, the temperature range can be from 5°C to 50°C, such as from 10°C to 40°C, from 15°C to 40°C, from 20°C to 40°C, for example, 20°C, 25°C, 30°C, 35°C or 37°C (for example, as described above). In some cases, the temperature for specific binding is selected to match the biological activity of the specific binding member and / or the target analyte. In certain cases, the temperature is 25°C, 30°C, 35°C or 37°C. In certain cases, the specific binding member is an antibody or a fragment thereof, and the temperature for specific binding is room temperature (for example, 25°C), 30°C, 35°C or 37°C. Any suitable incubation time for specific binding can be selected to obtain the desired amount of binding complex, and in some cases, the time can be 1 minute (min) or longer, for example, 2 minutes or longer, 10 minutes or longer, 30 minutes or longer, 1 hour or longer, 2 hours or longer, or even 6 hours or longer.

[0474] Any suitable specific binding member can be used in the conjugate. Exemplary specific binding members include, but are not limited to, reagents that specifically bind to cell surface proteins of various cell types, including but not limited to stem cells (e.g., pluripotent stem cells, hematopoietic stem cells), T cells, regulatory T cells, dendritic cells, B cells (e.g., memory B cells, antigen-specific B cells), granulocytes, leukemia cells, lymphoma cells, viral cells (e.g., HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythroid cells. Exemplary target cells include cells having suitable cell surface markers or antigens that can be captured by a suitable specific binding member conjugate. In some embodiments, the target cells are selected from cells containing HIV, Treg cells, antigen-specific T cell populations, tumor cells, or hematopoietic progenitor cells (CD34+) from whole blood, bone marrow, or cord blood. In the subject methods, any suitable cell surface protein or cell marker can be targeted for specific binding to the polymeric dye conjugate. In some embodiments, the target cells include cell surface markers selected from cell receptors and cell surface antigens. In some cases, the target cells can include cell surface antigens such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor α / β, T cell receptor γ / δ, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminus), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71.

[0475] Any suitable target can be selected for evaluation using the subject methods. Targets of interest include, but are not limited to, nucleic acids, e.g., RNA, DNA, PNA, CNA, HNA, LNA, or ANA molecules; proteins, e.g., fusion proteins, modified proteins (e.g., phosphorylated proteins, glycosylated proteins, ubiquitinated proteins, SUMOylated proteins, or acetylated proteins); or antibodies, peptides, polymeric biomolecules, cells, small molecules, vitamins, and drug molecules. The term "target protein" as used herein refers to all members of a target family and fragments thereof. The target protein can be any protein of interest, e.g., a therapeutic or diagnostic target, including but not limited to: hormones, growth factors, receptors, enzymes, cytokines, osteogenic inductive factors, colony stimulating factors, and immunoglobulins. The term "target protein" is intended to include recombinant and synthetic molecules, which can be prepared or obtained commercially using any suitable recombinant expression method or any suitable synthetic method. In some embodiments, the polymeric dye conjugate includes an antibody or an antibody fragment. In the subject methods, any suitable target analyte that specifically binds to the antibody or antibody fragment of interest can be targeted.

[0476] In some embodiments, the target analyte is associated with a cell. In some cases, the target analyte is a cell surface marker of the cell. In some cases, the cell surface marker is selected from the group consisting of cell receptors and cell surface antigens. In some cases, the target analyte is an intracellular target, and the method further includes lysing the cell.

[0477] In some embodiments, the sample can include a heterogeneous cell population from which target cells are isolated. In some cases, the sample includes whole peripheral blood, whole peripheral blood in which red blood cells have been lysed prior to cell isolation, cord blood, bone marrow, density gradient-purified peripheral blood mononuclear cells, or homogenized tissue. In some cases, the sample includes hematopoietic progenitor cells (e.g., CD34+ cells) in whole blood, bone marrow, or cord blood. In certain embodiments, the sample includes tumor cells in peripheral blood. In some cases, the sample is a sample that includes (or is suspected of including) viral cells (e.g., HIV).

[0478] The labeled specific binding member can be used in the subject methods, e.g., to label target cells, particles, targets or analytes with polymeric dyes or polymer tandem dyes. For example, the labeled specific binding member can be used to label cells to be processed (e.g., detected, analyzed, and / or sorted) in a flow cytometer. The labeled specific binding member can include an antibody that specifically binds to a cell surface protein such as various cell types (e.g., as described herein). The labeled specific binding member can be used to study various biological (e.g., cellular) properties or processes, such as the cell cycle, cell proliferation, cell differentiation, DNA repair, T cell signaling, apoptosis, cell surface protein expression and / or presentation, etc. The labeled specific binding member can be used in any application that includes (or may include) antibody-mediated labeling of cells, particles or analytes.

[0479] In certain instances of the method, the labeled specific binding member includes a multi-chromophore as described herein (e.g., a multi-chromophore according to any one of formulas (XI)-(XIV)). In certain instances, G 1 and G 2 are each independently selected from the group consisting of a terminal group, a π-conjugated segment, a linker and a conjugated specific binding member, wherein at least one of G 1 and G 2 is a conjugated specific binding member.

[0480] Aspects of the method include determining the presence of a polymeric dye conjugate-target analyte binding complex in a labeled composition contacting a sample to evaluate the presence of a target analyte in the sample. Once the sample is contacted with the polymeric dye conjugate, any suitable method can be employed to determine the presence of a polymeric dye conjugate-target analyte binding complex in the resulting labeled composition contacting the sample. The polymeric dye conjugate-target analyte binding complex is a binding complex formed when the specific binding member of the conjugate specifically binds to the target analyte, if any. Determining the labeled composition contacting the sample can include detecting a fluorescence signal (if any) from the binding complex. In some cases, the determination includes a separation step in which the target analyte, if any, is separated from the sample. Various methods can be utilized to separate the target analyte from the sample, e.g., by immobilization on a support. Exemplary determination methods include, but are not limited to, any suitable method and assay format that involves a specific binding member pair, e.g., avidin-biotin or hapten-anti-hapten antibody can be used. Exemplary methods and assay formats suitable for use in conjunction with the subject compositions include, but are not limited to, flow cytometry methods, in situ hybridization methods, enzyme-linked immunosorbent assay (ELISA), western blot analysis, magnetic cell sorting assays and fluorescent dye purification chromatography.

[0481] In certain embodiments, the method further comprises contacting the sample with a second specific binding member that specifically binds to the target analyte. In some cases, the second specific binding member is already bound to a support. Any suitable support can be used to immobilize the components (e.g., the second specific binding member) described in the subject method. In some cases, the support is a particle, e.g., magnetic powder. In some cases, the second specific binding member and the polymeric dye conjugate form a sandwich complex, which can be separated and detected (if any) by any suitable method. In some embodiments, the method further comprises analyzing the polymeric dye conjugate - target analyte binding complex, i.e., the fluorescently labeled target analyte, by flow cytometry. The determination of the presence of the polymeric dye conjugate - target analyte binding complex can provide corresponding assay results (e.g., qualitative or quantitative assay data), which can be used to evaluate the presence of the target analyte in the sample.

[0482] Any suitable support can be used in the subject method to immobilize any suitable component shown in the method, e.g., a labeled specific binding member, a target, a second specific binding member, etc. Exemplary supports include, but are not limited to: solid matrices, where the matrix can have various configurations, e.g., sheet-like, microspherical, or other structures, e.g., a well plate; microspheres, polymers, particles, fiber grids, hydrogels, porous substrates, needles, microarray surfaces, chromatography supports, etc. In some cases, the support is selected from the group consisting of: particles, planar solid matrices, fiber grids, hydrogels, porous substrates, needles, microarray surfaces, and chromatography supports. The support can be introduced into a system that can perform cell separation with the aid of any suitable method, e.g., a manually operated syringe, a centrifuge, or an automated liquid handling system. In some cases, the support can be used in an automated liquid handling system (e.g., a flow cytometer) to achieve high-throughput cell separation.

[0483] In some embodiments of the method, the separation step comprises applying an external magnetic field to immobilize the magnetic powder. Any suitable magnet can be used as the source of the external magnetic field (e.g., a magnetic field gradient). In some cases, the external magnetic field is generated by a magnetic source, e.g., by a permanent magnet or an electromagnet. In some cases, immobilizing the magnetic powder means causing the magnetic powder to accumulate near the surface closest to the source of the magnetic field gradient (i.e., the magnet).

[0484] The separation may further include one or more optional washing steps to remove unbound material of the sample from the support. Any suitable washing method may be employed, e.g., washing the immobilized support with a biocompatible buffer which may retain the specific binding interaction between the polymeric dye and the specific binding member. The unbound material of the sample is separated from the support and optionally washed to provide an enriched population of target cells, wherein undesired cells and materials may be removed.

[0485] In certain embodiments, the method further includes detecting the labeled target. Detecting the labeled target may include exciting the multiple chromophores with one or more lasers followed by detecting the fluorescence emission from the polymeric tandem dye with one or more optical detectors. The labeled target may be detected using any suitable instrument and method, including but not limited to flow cytometry, FACS systems, fluorescence microscopy; fluorescence, luminescence, ultraviolet, and / or visible light detection using a microplate reader; high performance liquid chromatography (HPLC); and mass spectrometry. When using fluorescently labeled components in the methods and compositions of the present invention, it is generally contemplated that different types of fluorescence detection systems may be used to practice the subject methods. In some instances, high throughput screening may be performed, e.g., using a system of a 96-well or greater microtiter plate. Various methods may be employed to measure the fluorescent species, e.g., the methods described in the following publications: Lakowicz, J.R., Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983); Herman, B., Resonance Energy Transfer Microscopy, in: Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, Volume 30, eds., Taylor, D.L. and Wang, Y.-L., San Diego: Academic Press (1989), pp. 219-243; Turro, N.J., Modern Molecular Photochemistry, Menlo Park: Benjamin / Cummings Publishing Col, Inc. (1978), pp. 296-361.

[0486] The fluorescence in a sample can be measured using a fluorometer. In some cases, excitation radiation from an excitation source having a first wavelength passes through excitation optics. The excitation optics cause the excitation radiation to excite the sample. In response, the radiation emitted by the fluorescently labeled target in the sample has a wavelength different from the excitation wavelength. Then, collection optics collect the emission from the sample. The associated equipment can include a temperature controller to keep the sample at a specific temperature while scanning the sample. In some cases, a multi-axis translation stage moves a microtiter plate containing multiple samples in order to position different wells to be exposed. The multi-axis translation stage, the temperature controller, the autofocus function, and the electronics associated with imaging and data acquisition can all be managed by a suitably programmed digital computer. The computer can also convert the data collected during the assay into another format for display.

[0487] In some embodiments, the method of assessing the presence of a target analyte in a sample further comprises detecting fluorescence in a flow cytometer. In some embodiments, the method of assessing the presence of a target analyte in a sample further comprises imaging the contact of a label composition with the sample by fluorescence microscopy. Fluorescence microscopy imaging can be used to identify polymer dye conjugate-target analyte binding complexes in the contact sample to assess the presence of the target analyte. The objective microscopy method used in the subject method includes laser scanning confocal microscopy.

[0488] The present invention also provides a method of labeling a target molecule. The subject polymeric tandem dyes can be used in a variety of labeling, separation, detection, and / or analysis methods. In some embodiments, the method comprises: contacting a target molecule with a polymeric tandem dye (e.g., as described herein) to produce a labeled target molecule, wherein the polymeric tandem dye comprises a conjugate tag covalently linked to the target molecule. In some cases of the method, the polymeric dye members comprise a multi-chromophore according to any one of formulas (XI)-(XIV) (e.g., as described herein), wherein G 1 and G 2 one of which is a terminal group, and G 1 and G 2 the other of which is a conjugate tag.

[0489] The term "conjugate label" refers to a group that includes a chemoselective functional group (e.g., as described herein), which can be covalently linked to a compatible functional group of a target molecule after optional activation and / or deprotection. Any suitable conjugate label can be used in the subject polymeric dyes to conjugate the dyes to the target molecule of interest. In some embodiments, the conjugate label includes a terminal functional group selected from the group consisting of: amino, carboxylic acid or its derivatives, thiol, hydroxyl, hydrazine, hydrazide, azide, alkyne, and protein-reactive groups (e.g., amino-reactive groups, thiol-reactive groups, hydroxyl-reactive groups, imidazole-reactive groups, or guanidine-reactive groups).

[0490] Any suitable method and reagent are suitable for use in the subject labeling method to covalently link the conjugate label to the target molecule. Methods and reagents for the purpose of labeling the target include, but are not limited to, those described in the following publications: Hermanson, Bioconjugate Techniques, Third Edition, Academic Press, 2013. The contacting step can be carried out in an aqueous solution. In some cases, the conjugate label includes an amino functional group and the target molecule includes an activated ester functional group, e.g., NHS ester or sulfo-NHS ester, and vice versa. In certain cases, the conjugate label includes a maleimide functional group and the target molecule includes a thiol functional group, and vice versa. In certain cases, the conjugate label includes an alkyne (e.g., cyclooctynyl) functional group and the target molecule includes an azide functional group, and vice versa, which can be conjugated by click chemistry.

[0491] Any suitable target molecule can be selected for labeling using the subject method. Target molecules of interest include, but are not limited to, nucleic acids, e.g., RNA, DNA, PNA, CNA, HNA, LNA, or ANA molecules; proteins, e.g., fusion proteins, modified proteins (e.g., phosphorylated proteins, glycosylated proteins, ubiquitinated proteins, SUMOylated proteins, or acetylated proteins); or antibodies, peptides, polymeric biomolecules, cells, small molecules, vitamins, and drug molecules. The term "target protein" as used herein refers to all members of a target family and their fragments. The target protein can be any protein of interest, e.g., a therapeutic or diagnostic target, including but not limited to: hormones, growth factors, receptors, enzymes, cytokines, osteogenic inductive factors, colony stimulating factors, and immunoglobulins. The term "target protein" is intended to include recombinant and synthetic molecules, which can be prepared or commercially available using any suitable recombinant expression method or any suitable synthetic method. In some embodiments, the target molecule is a specific binding member (e.g., as described herein). In certain cases, the specific binding member is an antibody. In some cases, the specific binding member is an antibody fragment or a binding derivative thereof. In some cases, the antibody fragment or a binding derivative thereof is selected from the group consisting of Fab fragments, F(ab') 2A group consisting of fragments, scFvs, diabodies, and triabodies.

[0492] In some cases, the method includes a separation step in which the labeled target molecule is separated from the reaction mixture, e.g., excess reagents or unlabeled targets. Various methods can be employed to separate the target in the sample, e.g., by immobilization on a support, precipitation, chromatography, etc.

[0493] In some cases, the method further includes detecting and / or analyzing the labeled target molecule. In some cases, the method further includes fluorescently detecting the labeled target molecule. Any suitable method as well as the subject methods and compositions can be utilized to detect and / or analyze the labeled target molecule. Methods for analyzing the target of interest used in the subject methods include, but are not limited to, flow cytometry, fluorescence microscopy, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), western blot analysis, magnetic cell sorting assay, and fluorescent dye purification chromatography. Subject detection methods include, but are not limited to, fluorescence spectroscopy, fluorescence microscopy, nucleic acid sequencing, fluorescence in situ hybridization (FISH), protein mass spectrometry, flow cytometry, etc.

[0494] Detection can be carried out directly by polymer tandem dyes or indirectly through a secondary detection system. The latter can be based on any one or a combination of several different principles, including but not limited to antibody-labeled anti-species antibodies and other forms of immune or non-immune bridging and signal amplification systems (e.g., biotin-streptavidin technology, protein-A and protein-G mediated technologies, or nucleic acid probes / anti-nucleic acid probes, etc.). Suitable reporter molecules can be those known in the fields of immunocytochemistry, molecular biology, light, fluorescence, and electron microscopy, cell immunophenotyping, cell sorting, flow cytometry, cell visualization, detection, enumeration, and / or signal output quantification. More than one antibody, specific and / or non-specific, can be labeled and used simultaneously or sequentially to enhance the detection, identification, and / or analysis of the target.

[0495] System

[0496] Aspects of the invention further include systems for carrying out processes related to the subject methods and compositions. A sample analysis system can include a sample viewing field or flow channel loaded with a sample and a labeled specific binding member. In some embodiments, the system is a flow cytometry system, which includes: a flow cytometer including a flow path; a composition in the flow path, wherein the composition includes: a sample; and a labeled specific binding member (e.g., as described herein).

[0497] In some embodiments, the system for analyzing a sample is a fluorescence microscopy system, which includes: a fluorescence microscope including a sample field of view; a composition disposed in the sample field of view, wherein the composition contains the sample; and a labeled specific binding member (e.g., as described herein).

[0498] In some cases of the system, the labeled specific binding member includes: a polymeric tandem dye (e.g., as described herein) and a specific binding member that specifically binds to a target analyte covalently linked to the multi-chromophore. In some cases of the subject system, the structural formulas of the labeled specific binding member and the polymeric tandem dye are shown as any one of Formulas (XI)-(XIV) (e.g., as described herein), wherein: G 1 and G 2 each independently selected from the group consisting of an end group, a π-conjugated segment, a linker, and a linked specific binding member, wherein G 1 and G 2 at least one of which is a linked specific binding member.

[0499] In certain embodiments of the system, the composition further includes a second specific binding member that is bound to a support and specifically binds to a target analyte. In some cases, the support includes magnetic powder. Thus, in certain cases, the system may further include a controllable external paramagnetic field that is configured to be applied to the measurement region of the flow channel.

[0500] The sample may include cells. In some cases, the sample is a biological sample containing cells. In some cases, the sample includes a labeled specific binding member that specifically binds to target cells. In certain cases, the target analyte that specifically binds to the specific binding member is a cell surface marker of the cell. In certain cases, the cell surface marker is selected from cell receptors and cell surface antigens.

[0501] In certain aspects, the system may further include a light source that is configured to direct light to the measurement region of the flow channel or the sample field of view. The system may include a detector that is configured to receive a signal from the measurement region of the flow channel or the sample field of view, wherein the signal is provided by a fluorescent composition. Optionally, the sample analysis system may further include one or more additional detectors and / or light sources to detect one or more additional signals.

[0502] In some aspects, the system may further include a computer-based system configured to detect the presence of a fluorescence signal. A "computer-based system" refers to a hardware device, a software device, and a data storage device for analyzing the information of the present invention. The minimum hardware configuration of the computer-based system described in the present invention includes a central processing unit (CPU), an input device, an output device, and a data storage device. It can be easily understood by those skilled in the art that any currently available computer-based system is applicable to the target system. The data storage device may include any product, including a record of the information of the present invention as described above, or a memory access device that can access the product.

[0503] To "record" data, the programming information or other information on a computer-readable medium refers to the process of storing information using any of the methods known in the art. Any suitable data storage structure can be selected based on the device used to access the stored information. Various data processor programs and formats can be used for storage, such as word processing text files, database formats, etc.

[0504] A "processor" refers to any combination of hardware and / or software that will perform its required functions. For example, any processor described herein can be a programmable digital microprocessor, such as provided in the form of an electronic controller, a mainframe, a server, or a personal computer (desktop or portable computer). If the processor is programmable, suitable programming can be transmitted to the processor from a remote location, or pre-stored in a computer program product (e.g., a portable or fixed computer-readable storage medium, whether based on magnetic, optical, or solid-state devices). For example, a magnetic medium or an optical disc can carry the programming and can be read by a suitable reader that communicates with each processor at its corresponding base station.

[0505] In addition to the sensor device and the signal processing module (e.g., as described above), the system described in the present invention may include a plurality of additional components, such as a data output device (e.g., a display and / or a speaker), a data input device (e.g., an interface, a keyboard, etc.), a fluid processing component, a power source, etc.

[0506] In some aspects, the system includes a flow cytometer. The intended flow cytometers include, but are not limited to, the devices described in the following U.S. patents: 4,704,891; 4,727,029; 4,745,285; 4,867,908; 5,342,790; 5,620,842; 5,627,037; 5,701,012; 5,895,922; and 6,287,791; the contents of these patents are incorporated herein by reference.

[0507] Other systems can be used to implement the subject methods. In some aspects, the system can be a fluorometer or microscope loaded with a sample (having a fluorescent composition as described in any of the embodiments discussed herein). The fluorometer or microscope can include a light source configured to direct light to an assay region of a flow channel or a field of view of a sample. The fluorometer or microscope can also include a detector configured to receive a signal from the assay region or field of view of the flow channel, wherein the signal is provided by the fluorescent composition.

[0508] Kit

[0509] Aspects of the invention further include kits for implementing the subject methods and processes related to the compositions. The compositions described herein can be included in the kits as reagents, which can be used as starting materials or provided for use in methods such as those described above.

[0510] The invention provides kits for multiplexed fluorescence signal generation. The kits can include a first polymeric tandem dye having a first maximum emission wavelength and a first narrow emission spectrum (e.g., as described herein); and one or more other polymeric tandem dyes having a maximum emission wavelength orthogonal to the first narrow emission spectrum. Each of the one or more other polymeric tandem dyes can have a different maximum emission wavelength. In some cases, the polymeric tandem dyes in the subject kits have different and non-overlapping emission spectra in the range of 480 nm to 700 nm (e.g., different emission spectra having non-overlapping full width at half maximum (FWHM) ranges).

[0511] The kits can include polymeric tandem dyes (including water-solvated light-harvesting multi-chromophores (e.g., as described herein)) and containers. Any suitable container can be used, e.g., a tube, bottle, well in a multi-well strip or plate, box, bag, insulated container, etc. The subject kits can further include one or more components selected from the group consisting of polymeric tandem dyes, fluorophores, specific binding members, specific binding member conjugates, support-bound specific binding members, cells, supports, biocompatible aqueous wash buffers, and instructions for use. In some embodiments of the kits, the multi-chromophores are covalently linked to specific binding members. In some cases, the specific binding member is an antibody. In certain cases, the specific binding member is an antibody fragment or a binding derivative thereof. In certain cases, the antibody fragment or binding derivative thereof is selected from the group consisting of Fab fragments, F(ab')2 fragments, scFv, diabodies, and triabodies.

[0512] In certain embodiments, the kit can be used to evaluate the presence of a target analyte in a sample, e.g., an intracellular target. Thus, in some cases, the kit includes one or more components adapted to lyse cells. One or more additional components of the kit can be provided in separate containers (e.g., separate tubes, bottles, or wells in a multi-well strip or plate).

[0513] In certain aspects, the kit further includes reagents for performing flow cytometry assays. The reagents include, but are not limited to, buffers for reconstitution and dilution, buffers for contacting cell samples with multiple chromophores, wash buffers, control cells, control microspheres, fluorescent microspheres for flow cytometer calibration, and combinations thereof. The kit can also include one or more cell fixation reagents, e.g., paraformaldehyde, glutaraldehyde, methanol, acetone, formalin, or any combination or buffer thereof. In addition, the kit can include cell permeabilization reagents, e.g., methanol, acetone, or detergents (e.g., Triton, NP-40, saponin, Tween 20, digitonin, leucoperm) or any combination or buffer thereof. Other protein transport inhibitors, cell fixation reagents, and cell permeabilization reagents familiar to those skilled in the art are within the scope of the subject kit.

[0514] The compositions of the kit can be provided in the form of a liquid composition (e.g., any suitable buffer). In addition, the compositions of the kit can be provided in the form of a dry composition (e.g., can be lyophilized), and the kit can optionally include one or more buffers for reconstituting the dry composition. In certain aspects, the kit can include aliquots of the compositions provided in separate containers (e.g., separate tubes, bottles, or wells in a multi-well strip or plate).

[0515] In addition, one or more components can be mixed and provided in a single container, e.g., a glass or plastic vial, tube, or bottle. In certain cases, the kit can further include a container (e.g., a box, bag, insulated container, bottle, tube, etc.) containing all the corresponding components (and their separate containers). The kit can further include a package separate from or attached to the kit container and bearing information printed thereon regarding the kit, the components of the kit, and / or instructions for using the kit.

[0516] In addition to the above components, the subject kit may further include instructions for practicing the subject method. These instructions may be present in the subject kit in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is printed information on a suitable medium or substrate (e.g., one or more sheets of paper on which information is printed), the kit packaging, package inserts, etc. Another form in which they may be present is a computer-readable medium on which information has been recorded, such as a floppy disk, CD, portable flash drive, etc. Yet another form in which these instructions may be present is a website address by which information on a remote website can be accessed via the Internet. Any suitable means may be present in the kit.

[0517] Utility

[0518] The narrow emission fluorophore dyes, polymeric tandem dyes, compositions, methods and systems described herein can be used in a variety of applications, including diagnostic and research applications where labeling, detecting and / or analyzing a target of interest is required. The applications include a variety of methods such as cell counting, microscopy, immunoassays (e.g., competitive or non-competitive), free analyte assessment, assessment of receptor-binding ligands, etc. The compositions, systems and methods described herein can be used to analyze any of a variety of samples, including but not limited to biological fluids, cell culture samples and tissue samples. In some aspects, the compositions, systems and methods described herein can be used in methods for detecting an analyte, if any, in a sample using a fluorescent label (e.g., in fluorescence-activated cell sorting or analysis, immunoassays, immunostaining, etc.). In some cases, the compositions and methods can be used in applications involving assessing the presence of a target analyte in a sample.

[0519] In some cases, the methods and compositions can be used in any assay format involving detecting and / or analyzing a target in a sample, including but not limited to flow cytometry, fluorescence microscopy, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), western blot analysis, magnetic cell sorting assays and fluorescence dye purification chromatography. In some cases, the methods and compositions can be used in any application involving fluorescent labeling of a target molecule. The subject compositions are suitable for any suitable application in which a specific binding member pair (e.g., biotin-streptavidin and hapten-anti-hapten antibody) can be used.

[0520] Definition

[0521] The present invention sets forth the following definitions in order to clarify and define the meaning and scope of the terms used in this specification.

[0522] Unless otherwise defined, all technical and scientific terms used in this patent have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Nevertheless, for clarity and for ease of reference, the definitions of certain terms are provided below.

[0523] It must be noted that, as used in this patent and the appended claims, the singular forms of "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "dye" refers to one or more dyes, i.e., a single dye and multiple dyes. It should also be noted that claims can be drafted to exclude any optional elements. Thus, this statement is intended as a basis for the use of special terms such as "solely", "only", etc. in relation to the recitation of claim elements or the use of "negative" limitations.

[0524] The term "sample" as used herein refers to a material or mixture of materials containing one or more analytes of interest, which material is in liquid form in some cases. In some embodiments, in a broad sense, a "sample" refers to any plant, animal, or bacterium containing cells or producing cell metabolites, e.g., tissue or fluid isolated from an individual (including but not limited to plasma, serum, cerebrospinal fluid, lymph, tears, saliva, and tissue sections) or in vitro cell culture components, as well as samples from the environment. The term "sample" can also refer to a "biological sample". The term "biological sample" as used herein refers to a whole organism or a subset of its tissues, cells, or components (e.g., body fluids, including but not limited to blood, mucus, lymph fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen). A "biological sample" can also refer to a homogenate, lysate, or extract prepared from a whole organism or a subset of its tissues, cells, or components or fragments or parts thereof (including but not limited to plasma, serum, spinal fluid, lymph fluid, outer skin sections, respiratory tract, intestinal tract, and urogenital tract, tears, saliva, milk, blood cells, tumors, and organs). In certain embodiments, the sample has been taken from an animal or a plant. A biological sample can include cells. By its conventional meaning, the term "cell" refers to the basic structural unit of eukaryotic and prokaryotic organisms that has at least a nucleus and a cell membrane. In certain embodiments, the cells include prokaryotic cells, e.g., prokaryotic cells from bacteria. In other embodiments, the cells include eukaryotic cells, e.g., cells obtained from a biological sample of an animal, a plant, or a fungus.

[0525] The terms "support-bound" and "linked to a support" as used herein are used interchangeably and refer to a moiety (e.g., a specific binding member) that is covalently or non-covalently linked to a target support. Covalent linkage may involve a chemical reaction of two compatible functional groups (e.g., two chemoselective functional groups, an electrophile and a nucleophile, etc.) to form a covalent bond between two target moieties (e.g., a support and a specific binding member). In some cases, non-covalent linkage may involve specific binding between two target moieties (e.g., two affinity moieties, such as a hapten and an antibody or a biotin moiety and streptavidin, etc.). In certain cases, non-covalent linkage may involve absorption to a substrate.

[0526] The term "polypeptide" as used herein refers to a polymeric form of amino acids of any length, including peptides of 2-50 amino acids in length and polypeptides of greater than 50 amino acids in length. The terms "polypeptide" and "protein" are used interchangeably herein. The term "polypeptide" includes polymers of coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified peptide backbone where the conventional backbone has been replaced by a non-naturally occurring or synthetic backbone. Polypeptides can have any suitable length, e.g., 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, 50 or more amino acids, 100 or more amino acids, 300 or more amino acids, e.g., up to 500 or 1000 or more amino acids. A "peptide" can be 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, e.g., up to 50 amino acids. In some embodiments, the length of the peptide ranges from 5 to 30 amino acids.

[0527] The term "isolated" as used herein refers to a target moiety that is at least 60% separated, at least 75% separated, at least 90% separated, at least 95% separated, at least 98% separated, or even 99% separated from other components (i.e., the components that were associated with the target moiety prior to purification).

[0528] "A plurality" contains at least 2 members. In certain cases, a plurality can have 10 or more, such as 100 or more, 1000 or more, 10,000 or more, 100,000 or more, 10 6 or more, 10 7 or more, 10 8 or more or 10 9 or more members.

[0529] A numerical range includes the numbers defining the range.

[0530] As used herein, the term "specifically binds" refers to the ability of a capture agent (or the first member of a specific binding pair) to preferentially bind to a specific analyte (or the second member of a specific binding pair) present in, for example, a homogeneous mixture of different analytes. In some cases, the specific binding interaction will distinguish between a desired and an undesired analyte in a sample, where the desired analyte is at least 10-fold more specific, such as at least 100-fold more specific or at least 1000-fold more specific, than the undesired analyte. In some cases, when the capture agent specifically binds to the analyte in the form of a capture agent / analyte complex, their affinity is at least 10 -8 M, at least 10 -9 M, such as up to 10 -10 M.

[0531] As used herein, the terms "affinity" and "avidity" have the same meaning and are used interchangeably herein. "Affinity" refers to the strength of binding, and an increase in binding affinity is associated with a decrease in Kd.

[0532] The methods described herein include multiple steps. Each step can be performed after a predetermined amount of time has elapsed between steps, as needed. Thus, the time between performing each step can be 1 second or longer, 10 seconds or longer, 30 seconds or longer, 60 seconds or longer, 5 minutes or longer, 10 minutes or longer, 60 minutes or longer, including 5 hours or longer. In certain embodiments, each subsequent step is performed immediately after the previous step is completed. In other embodiments, the step can be performed after an incubation or waiting period after the previous step is completed, for example, after a waiting period of from a few minutes to overnight.

[0533] As used herein, the terms "evaluate", "determine", "measure", "assess" and "assay" are used interchangeably and include both quantitative and qualitative determinations.

[0534] As used herein, the term "separation" refers to the physical separation of two elements (e.g., separation by size or affinity, etc.) and the degradation of one element while the other remains intact.

[0535] The terms "chemoselective functional group" and "chemoselective tag" are used interchangeably and refer to functional groups that, in some cases, can selectively react with another compatible functional group to form a covalent bond after optionally activating one of the functional groups. A variety of chemoselective functional groups, their synthetic precursors, and their protected forms are available and can be incorporated into the subject BODIPY dyes to conjugate the dyes to target molecules (e.g., as described herein). The term chemoselective functional group is intended to include any suitable synthetic precursor or masked or protected form of the desired functional group. Desired chemoselective functional groups include, but are not limited to, amino groups (e.g., -NH2 )), carboxylic acid (-CO 2 H), activated esters (e.g., NHS or sulfo-NHS esters), thiols, maleimides, iodoacetamides, hydroxyl groups, hydrazide groups, hydrazine groups, aldehydes, ketones, azides, alkynes, tetrazines, alkenes, phosphines, and epoxides, their protected forms, their masked forms, and their synthetic precursors. For example, a carboxylic acid can be considered as a synthetic precursor of an activated ester (e.g., NHS ester). Purpose-compatible chemoselective functional groups include but are not limited to thiols and maleimides or iodoacetamides, amines and carboxylic acids or their activated esters, and groups that can react with each other through click chemistry, such as azides and alkynes (e.g., cyclooctynyl), tetrazines and alkenes (e.g., trans-cyclooctene), etc.

[0536] The terms "linker", "linking group", and "bond" refer to a connecting portion that connects two groups and has a backbone with 100 or fewer atoms. The linker or bond can be a covalent bond connecting two groups or a chain with 1 to 100 atoms, for example, a chain with 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more carbon atoms, where the linker can be linear, branched, cyclic, or a single atom. In some cases, the linker is a branched linker, that is, a connecting portion that connects three or more groups. In certain cases, one, two, three, four, five, or more carbon atoms of the linker backbone can be optionally substituted by sulfur, nitrogen, or oxygen heteroatoms. In some cases, the linker backbone includes connecting functional groups, such as ether, thioether, amino, amide, sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester, or imine. The bonds between the backbone atoms can be saturated bonds or unsaturated bonds; in some cases, the number of unsaturated bonds in the linker backbone does not exceed one, two, or three. The linker can include one or more substituents, for example, substituted by alkyl, aryl, or alkenyl groups. The linker can include but is not limited to polyethylene glycol; ether; thioether; tertiary amine; alkyl, which can be linear or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), etc. The linker backbone can include cyclic groups, such as aryl, heterocyclic, or cycloalkyl groups, where the backbone includes 2 or more atoms of the cyclic group, for example, 2, 3, or 4 atoms. The linker can be cleavable or non-cleavable.

[0537] The terms "polyethylene oxide", "PEO", "polyethylene glycol", and "PEG" used herein are used interchangeably and refer to including, as in the chemical formula --(CH 2 --CH 2 --O--) n- The polymeric groups of the chains shown or derivatives thereof. In some embodiments, "n" is 5000 or less, for example, 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 to 15 or 10 to 15. It should be understood that the PEG polymeric groups can have any suitable length and can include various end groups and / or other substituents, including but not limited to alkyl, aryl, hydroxyl, amino, acyl, acyloxy, and amide end groups and / or substituents. PEG groups suitable for the subject multiple chromophores include the PEGs described in the following publications: S. Zalipsky, Functionalized poly(ethylene glycol) for the preparation of biologically relevant conjugates, Bioconjugate Chemistry, 1995, 6(2), pp. 150 - 165; and Zhu et al., Water-soluble conjugated polymers for imaging, diagnosis, and therapy, Chemical Reviews, 2012, 112(8), pp. 4687–4735.

[0538] As used herein, the term "alkyl" (by itself or as part of another substituent) refers to a saturated branched or straight-chain monovalent hydrocarbon group derived by removing one hydrogen atom from a single carbon atom of the parent alkane. - Representative alkyl groups include but are not limited to methyl; ethyl; propyl, such as propan-1-yl or propan-2-yl); and butyl, such as butan-1-yl, butan-2-yl, 2-methylpropan-1-yl, or 2-methylpropan-2-yl. In some embodiments, the alkyl group includes 1 to 20 carbon atoms. In some embodiments, the alkyl group includes 1 to 10 carbon atoms. In certain embodiments, the alkyl group includes 1 to 6 carbon atoms, for example, 1 to 4 carbon atoms. This term includes, for example, straight-chain and branched hydrocarbon groups, such as methyl (CH 3 -), ethyl (CH 3 CH 2 -), n-propyl (CH 3 CH 2 CH 2 -), isopropyl ((CH 3 ) 2 CH-), n-butyl (CH 3 CH 2 CH 2 CH 2 -), isobutyl ((CH 3 ) 2 CHCH 2 -), sec-butyl ((CH 3 )(CH 3 CH 2 )CH-), tert-butyl ((CH 3 ) 3 C-), n-pentyl (CH 3CH 2 CH 2 CH 2 CH 2 -), and neopentyl ((CH 3 )) 3 CCH 2 -).

[0539] The term "substituted alkyl" refers to an alkyl group as defined herein, wherein one or more carbon atoms in the alkyl chain have been optionally substituted with a heteroatom (e.g., -O-, -N-, -S-, -S(O) n -(where n ranges from 0 to 2), -NR- (where R is hydrogen or alkyl)), and has 1 to 5 substituents selected from the group consisting of: alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azide, cyano, halogen, hydroxy, oxo, thione, carboxyl, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocycloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocycloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -alkyl, -SO 2 -aryl, -SO 2 -heteroaryl and -NR a R b wherein R ’ and R ” may be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, and heterocyclic.

[0540] "Alkoxy" refers to the group -O-alkyl, where alkyl is as defined herein. By way of example, alkoxy includes methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, n-pentyloxy, and the like. The term "alkoxy" also refers to the groups alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0541] The term "substituted alkoxy" refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O-, where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0542] "Amino" refers to the group –NH 2。

[0543] The term "substituted amino" refers to the group -NRR, where each R is independently selected from the group consisting of: hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclic group, provided that at least one R is not hydrogen.

[0544] "Aryl" (by itself or as part of another substituent) refers to a monovalent aryl group derived by removing a hydrogen atom from a single carbon atom in an aromatic ring system. Aryl groups of interest include, but are not limited to, groups derived from the following: aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, heptacene, pyrene, picene, rubicene, triphenylene, trinaphthylene, etc. In certain embodiments, the aryl group includes 6 to 20 carbon atoms. In certain embodiments, the aryl group includes 6 to 12 carbon atoms. Examples of aryl groups are phenyl and naphthyl.

[0545] "Heteroaryl" (by itself or as part of another substituent) refers to a monovalent heteroaryl group derived by removing a hydrogen atom from a single atom in a heteroaromatic ring system. Heteroaryl groups of interest include, but are not limited to, groups derived from the following: acridine, arsenole, carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, pyridine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinazoline, quinoxaline, tetrazole, thiadiazole, thiazole, triazole, benzotriazole, thiophene, triazole, xanthene, benzodioxole, etc. In certain embodiments, the heteroaryl group is a 5-20 membered heteroaryl. In certain embodiments, the heteroaryl group is a 5-10 membered heteroaryl. In certain embodiments, the heteroaryl group is a group derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole, and pyrazine.

[0546] "Heterocycle", "heterocyclic", "heterocycloalkyl" and "heterocyclic group" refer to saturated or unsaturated groups having a monocyclic or multiple fused rings (including fused bridged rings and spiro ring systems) and having 3 to 20 ring atoms (including 1 to 10 heteroatoms). The ring atoms are selected from the group consisting of nitrogen, sulfur or oxygen, wherein, in a fused ring system, one or more rings may be cycloalkyl, aryl or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atoms of the heterocyclic group are optionally oxidized to provide nitrogen oxides, -S(O)- or -SO 2 - moiety.

[0547] Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinoxaline, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also known as thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, etc.

[0548] Unless the definition of the heterocyclic substituent is otherwise limited, such heterocyclic groups may be optionally substituted with 1 to 5 substituents or 1 to 3 substituents selected from the following: alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, amido, aminoacyloxy, oxyamido, azide, cyano, halogen, hydroxy, oxo, thione, carboxyl, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocycloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic group, heterocycloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -alkyl, -SO 2 -substituted alkyl, -SO 2 -aryl and -SO 2 -heteroaryl and fused heterocycles.

[0549] The term "alkaryl" or "aralkyl" refers to the groups -alkylene-aryl and substituted alkylene-aryl, wherein alkylene, substituted alkylene and aryl are as defined herein.

[0550] "Alkylene" refers to a divalent aliphatic hydrocarbon radical preferably having 1 to 6 (more preferably 1 to 3) straight-chain or branched carbon atoms, which is optionally interrupted by one or more groups selected from -O-, -NR 10 -, -NR 10 C(O)-, -C(O)NR 10 -, etc. This term includes, for example, methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), n-propylene (-CH 2 CH 2 CH 2 -), isopropylene (-CH 2 CH(CH 3 )-), (-C(CH 3 ) 2 CH 2 CH 2 -), (-C(CH 3 ) 2 CH 2 C(O)-), (-C(CH 3 ) 2 CH 2 C(O)NH-), (-CH(CH 3 )CH 2 -), etc. "Substituted alkylene" refers to an alkylene group in which 1 to 3 hydrogens are replaced by substituents as described in the definition of "substituted" below with respect to carbon.

[0551] "Substituted" means a group in which one or more hydrogen atoms are independently replaced by the same or different substituents. The intended substituents include, but are not limited to, alkylenedioxy (e.g., methylenedioxy), -M, -R 60 , -O - , =O, -OR 60 , -SR 60 , -S - , =S, -NR 60 R 61 , =NR 60 , -CF 3 , -CN, -OCN, -SCN, -NO, -NO 2 , =N 2 , -N 3 , -S(O) 2 O - , -S(O) 2 OH, -S(O) 2 R 60 , -OS(O) 2 O -, -OS(O) 2 R 60 , -P(O)(O - ) 2 , -P(O)(OR 60 )(O - ), -OP(O)(OR 60 )(OR 61 ), -C(O)R 60 , -C(S)R 60 , -C(O)OR 60 , -C(O)NR 60 R 61 , -C(O)O - , -C(S)OR 60 , -NR 62 C(O)NR 60 R 61 , -NR 62 C(S)NR 60 R 61 , -NR 62 C(NR 63 )NR 60 R 61 and -C(NR 62 )NR 60 R 61 , wherein M is a halogen; R 60 , R 61 , R 62 and R 63 are independently hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, or optionally, R 60 and R 61 together with the nitrogen atom to which they are attached form a cycloheteroalkyl or substituted cycloheteroalkyl ring; and R 64 and R 65 are independently hydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, or optionally, R 64 and R 65 together with the nitrogen atom to which they are attached form a cycloheteroalkyl or substituted cycloheteroalkyl ring. In certain embodiments, the substituents include -M, -R 60 , =O, -OR 60 , -SR 60 , -S - , =S, -NR 60 R 61 , =NR 60, -CF 3 , -CN, -OCN, -SCN, -NO, -NO 2 , =N 2 、-N 3 、-S(O) 2 R 60 、-OS(O) 2 O - 、-OS(O) 2 R 60 、-P(O)(O - ) 2 、-P(O)(OR 60 )(O - )、-OP(O)(OR 60 )(OR 61 )、-C(O)R 60 、-C(S)R 60 、-C(O)OR 60 、-C(O)NR 60 R 61 ,-C(O)O - 、-NR 62 C(O)NR 60 R 61 In certain embodiments, the substituents include -M, -R 60 ,=O,-OR 60 、-SR 60 、-NR 60 R 61 , -CF 3 、-CN、-NO 2 、-S(O) 2 R 60 、-P(O)(OR 60 )(O - )、-OP(O)(OR 60 )(OR 61 )、-C(O)R 60 、-C(O)OR 60 、-C(O)NR 60 R 61 、-C(O)O - In certain embodiments, the substituents include -M, -R 60 ,=O,-OR 60 、-SR 60 、-NR 60 R 61 , -CF 3 、-CN、-NO 2 、-S(O) 2 R 60 、-OP(O)(OR60 )(OR 61 )、-C(O)R 60 、-C(O)OR 60 ,-C(O)O - , where R 60 , R 61 and R 62 As defined above. For example, the substituted group may have a methylenedioxy substituent or one, two or three substituents selected from halogen atoms, (1-4C)alkyl groups and (1-4C)alkoxy groups. When the substituted group is an aryl or heteroaryl group, the substituent (e.g., as described herein) may be referred to as an "aryl substituent".

[0552] Additional definitions of terms may appear elsewhere in the specification.

[0553] Example

[0554] Example 1: Characterization of narrow emission acceptor fluorophores and polymer tandem dyes

[0555] BODIPY dyes can be prepared according to any suitable method, including those described in the following publication: Loudet and Burgess ("BODIPY dyes and their derivatives: synthesis and spectral properties," Chemical Reviews, 2007, 107, 4891-4932).

[0556] A variety of BODIPY dyes were prepared and coupled with polymeric dyes to prepare polymer tandem dyes. Figure 3 The structure of an exemplary polymer tandem dye is shown. The spectral properties of a specific BODIPY dye and a polymer tandem dye including the BODIPY dye were evaluated. The exemplary BODIPY dye of the present invention was observed to exhibit a quantum yield of 0.57 to 0.90, a maximum absorption wavelength range of 493 nm to 663 nm, and a maximum emission wavelength range of 504 nm to 673 nm. More detailed information of the exemplary BODPY dye and the tandem dye including the BODIPY dye is listed in Tables 1 and 2 below.

[0557] Table 1. Full width at half maximum of exemplary BODPY dyes and commercially available BODPY dyes

[0558]

[0559]

[0560] Aspects of the subject matter described above (including embodiments) may be used alone or in combination with one or more aspects or embodiments. Without limiting the content described herein, certain non-limiting content of the present invention is provided below. After reading the present invention, the following content will be apparent to those skilled in the art, and each individually numbered content may be used alone or in combination with any of the preceding or following individually numbered content. This is intended to provide support for all such combinations of the content, and is not limited to the combinations of the content explicitly provided below:

[0561] 1. A polymer tandem dye comprising:

[0562] a water-soluble light-harvesting multichromophore; and

[0563] A narrow emission acceptor fluorophore is configured to be proximal to the light-harvesting multichromophore upon energy reception.

[0564] 2. The polymer tandem dye according to 1, wherein the width of the full width at half maximum (FWHM) of the emission spectrum of the narrow emission acceptor fluorophore is 25 nm or less.

[0565] 3. The polymer tandem dye according to any one of 1-2, wherein the narrow emission acceptor fluorophore exhibits a Stokes shift of 20 nm or less upon direct excitation.

[0566] 4. The polymer tandem dye according to any one of 1-3, wherein the polymer tandem dye exhibits an effective Stokes shift of 100 nm or more (e.g., 100-300 nm) when the light-harvesting multichromophore is directly excited with incident light.

[0567] 5. The multichromophore according to any one of 1-4, wherein the narrow emission acceptor fluorophore is substituted with a water-soluble group.

[0568] 6. The polymer tandem dye according to any one of 1-5, wherein the narrow emission acceptor fluorophore is a pendant group covalently linked to the comonomer of the light-harvesting multichromophore via a non-conjugated linker.

[0569] 7. The polymer tandem dye according to any one of 1-6, wherein the narrow emission acceptor fluorophore is a fluorescent semiconductor nanocrystal or quantum dot.

[0570] 8. The polymer tandem dye according to any one of 1-7, wherein the narrow emission acceptor fluorophore is a small molecule fluorophore.

[0571] 9. The polymer tandem dye according to 8, wherein the small molecule fluorophore is a BODIPY dye.

[0572] 10. The polymer tandem dye according to 9, wherein the structural formula of the BODIPY dye is as shown in formula (I):

[0573]

[0574] in:

[0575] Q is C or N;

[0576] R 1 -R 7 independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, a water solubilizing group (WSG), and -L 1 -Z 1 ,or

[0577] Optionally, selected from R 6 and R 7 , R 2 and R 3 , R 5 and R 6 , R 3 and R 4 , R 4 and R 1 and R 5 and R 1 Any one or more pairs of substituents of are connected to form a cyclic structure, together forming a divalent radical, and together with the carbon atoms to which they are attached form a 5- or 6-membered fused heterocyclic ring, carbocyclic ring, aryl ring or heteroaryl ring (e.g., a 5- or 6-membered ring comprising carbon atoms and 0-3 heteroatoms selected from O, S and N), wherein the ring may be unsubstituted or further substituted with substituents independently selected from the following groups: alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water solubilizing group (WSG) and -L 1 -Z 1 ;

[0578] L 1 is a linker;

[0579] Z 1 is a light-harvesting multichromophore; and

[0580] Y 1 and Y 2 independently selected from F, OH, H, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and WSG;

[0581] wherein Y 1 、Y 2 and R 1 -R 7 one of which contains –L 1 -Z 1 .

[0582] 11. The polymeric tandem dye according to claim 10, wherein the structural formula of the BODIPY dye is as shown in formula (IIa) or (IIb):

[0583]

[0584] wherein:

[0585] Z 11 and Z 12 are independently a fused 5- or 6-membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring;

[0586] each i is independently 0-3; and

[0587] each R 20 is independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG) and -L 1 -Z 1 .

[0588] 12. The polymeric tandem dye according to claim 11, wherein Z 11 and Z 12 are independently selected from furan, thiophene, pyrrole, oxazole, isoxazole, thiazole, isothiazole, imidazole and pyrazole.

[0589] 13. The polymeric tandem dye according to claim 11 or 12, wherein Z 11 and Z 12 are independently selected from the following rings:

[0590]

[0591] wherein:

[0592] X is O or S;

[0593] Y is O, S or NR, where R is H, alkyl, substituted alkyl; and

[0594] R 21 -R 23independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG), and –L 1 -Z 1 .

[0595] 14. The polymeric tandem dye according to any one of 10 - 13, wherein the structural formula of the BODIPY dye is as shown in formula (IIIa) or (IIIb):

[0596]

[0597] wherein:

[0598] X is O or S; and

[0599] R 16 and R 17 are independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG), and –L 1 -Z 1 .

[0600] 15. The polymeric tandem dye according to any one of 10 - 13, wherein the structural formula of the BODIPY dye is as shown in formula (IVa) or (IVb):

[0601]

[0602] wherein:

[0603] each X is O or S; and

[0604] R 12 , R 13 , R 16 and R 17 are independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG), and –L 1 -Z 1 .

[0605] 16. The polymeric tandem dye according to any one of 10 - 15, wherein R 1 comprises a linker connecting the light-harvesting multi-chromophore (-L 1 -Z 1 ).

[0606] 17. The polymeric tandem dye according to claim 16, wherein L 1 comprises an optionally substituted aryl or heteroaryl group connecting the light-harvesting multi-chromophores (Z 1 ).

[0607] 18. The polymeric tandem dye according to any one of claims 16-17, wherein the structural formula of the BODIPY dye is as shown in formula (V):

[0608]

[0609] Wherein:

[0610] L 3 is a covalent bond, oxo (-O-), alkylene (e.g., C 1 -C 6 -alkylene), -O-alkylene or a substituted form thereof;

[0611] R 11 is selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG) and –L 1 -Z 1 ;

[0612] Each R 9 is independently selected from halogen, hydroxyl, cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl and substituted heteroaryl; and

[0613] t is 0-4.

[0614] 19. The polymeric tandem dye according to claim 18, wherein the structural formula of the BODIPY dye is as shown in formula (Va):

[0615]

[0616]

[0617] 20. The polymeric tandem dye according to claim 18 or 19, wherein:

[0618] R 2 、R 4 、R 6 and R 8 are each independently H, alkyl or substituted alkyl; and

[0619] R 11 is L 1 -Z 1 .

[0620] 21. The polymer tandem dye according to item 20, wherein the structural formula of the BODIPY dye is as shown in formula (VIa) or (VIb):

[0621]

[0622] Wherein:

[0623] * is the bonding point of the repeating unit or the chemo - selective functional group that bonds the light - harvesting multi - chromophore.

[0624] 22. The polymer tandem dye according to any one of items 10 - 15, wherein the structural formula of the BODIPY dye is as shown in formula (VIIa) or (VIIb):

[0625]

[0626]

[0627] Wherein: n is 0 - 12.

[0628] 23. The polymer tandem dye according to any one of items 10 - 14, wherein the structural formula of the BODIPY dye is as shown in formula (VIII):

[0629]

[0630] 24. The polymer tandem dye according to item 23, wherein the structural formula of the BODIPY dye is as shown in formula (VIIIa):

[0631]

[0632] Wherein:

[0633] L 2 is a linker;

[0634] Each R 9 is an optional substituent selected from halogen, hydroxyl, cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl and substituted heteroaryl; and

[0635] t is 0 - 4.

[0636] 25. The polymer tandem dye according to item 15, wherein the structural formula of the BODIPY dye is as shown in formula (IX):

[0637]

[0638] Wherein:

[0639] Each R9 Each is an optional substituent selected from halogen, hydroxyl, cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, and substituted heteroaryl; and

[0640] t is 0 - 4.

[0641] 26. The polymeric tandem dye according to item 25, wherein the structural formula of the BODIPY dye is as shown in formula (IXa):

[0642]

[0643] wherein L 2 is a linker.

[0644] 27. The polymeric tandem dye according to any one of items 10 - 26, wherein the structure of L 1 -Z 1 is shown as one of the following structures:

[0645]

[0646] wherein:

[0647] L 2 is a linker;

[0648] R 32 is H, alkyl, substituted alkyl, and a water - soluble group (WSG);

[0649] L 3 is a linker selected from alkylene (e.g., C 1 -C 6 -alkylene), -O-alkylene, and its substituted forms;

[0650] t is 0 - 4; and

[0651] each R 9 is independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halogen, sulfonic acid, and a water - soluble group (WSG).

[0652] 28. The polymeric tandem dye according to any one of items 10 - 27, wherein Y 1 and / or Y 2 is selected from fluoro, cyano, phenyl, and substituted phenyl.

[0653] 29. The polymeric tandem dye according to any one of items 10 - 28, wherein Y 1 and Y 2 contain WSG.

[0654] 30. The polymeric tandem dye according to claim 29, wherein Y 1 and Y 2 are each a substituted alkynyl group containing a WSG.

[0655] 31. The polymeric tandem dye according to claim 30, wherein Y 1 and Y 2 are independently structured as shown in the following formula:

[0656]

[0657] wherein:

[0658] s is 1 - 6;

[0659] q is from 0 to 50 (e.g., 1 to 50, 1 to 30, 2 to 30, 4 to 30, 6 to 30, etc.); and

[0660] R 21 is H, an alkyl group, or a substituted alkyl group.

[0661] 32. The polymeric tandem dye according to any one of claims 1 - 31, wherein the structural formula of the polymeric tandem dye is as shown in formula (XIIa):

[0662]

[0663] wherein:

[0664] F 1 is a fused tricyclic comonomer substituted with a water - soluble group (WSG);

[0665] M 1 、M 2 and L 1 are independently an aryl or heteroaryl comonomer optionally substituted with a WSG;

[0666] N 1 is the narrow - emission acceptor fluorophore connected to L 1 ;

[0667] G 1 and G 2 are each independently selected from a terminal group, a π - conjugated segment, a linker, and a conjugated specific binding member; and

[0668] w, x, y, and z represent the mole percentages of the units in the multi - chromophore.

[0669] 33. The polymeric tandem dye according to any one of claims 1 - 32, wherein the multi - chromophore includes a conjugated segment having a structure as shown in formula (XI):

[0670]

[0671] Wherein:

[0672] F 1 is a fused tricyclic comonomer substituted with a water-soluble group (WSG);

[0673] M 1 is an aryl or heteroaryl comonomer;

[0674] n is an integer between 1 and 100,000; and

[0675] * represents a site for covalently linking to an unsaturated backbone or end group of a conjugated polymer.

[0676] 34. The polymeric tandem dye according to any one of 32 - 33, wherein the structure of the polymeric tandem dye is as shown in formula (XIII):

[0677]

[0678] Wherein:

[0679] F 1 is a fused tricyclic comonomer substituted with WSG;

[0680] M 1 、M 2 and L 1 are independently aryl or heteroaryl comonomers optionally substituted with WSG;

[0681] N 1 is the narrow emission acceptor fluorophore linked to L 1 ;

[0682] a - d and f are each independently 0 or 1;

[0683] e is 1;

[0684] Each of n and m is an integer between 1 and 10,000;

[0685] p is an integer between 1 and 100,000; and

[0686] G 1 and G 2 are each independently selected from end groups, π - conjugated segments, linkers, and attached specific binding members.

[0687] 35. The polymeric tandem dye according to 34, wherein the structure of the polymeric tandem dye is as shown in formula (XIVa) or (XIVb):

[0688]

[0689]

[0690] 36. The polymeric tandem dye according to any one of 32 - 35, wherein the structural formula of the fused tricyclic comonomer is as follows:

[0691]

[0692] Wherein:

[0693] Y 1 is C(R 23 ) 2 、-C(R 23 ) 2 C(R 23 ) 2 -、-C(R 23 ) 2 Si(R 23 ) 2 -、NR 23 、Si(R 23 ) 2 or Se;

[0694] Each Z is independently CH, CR 22 or N;

[0695] Each R 23 is independently selected from H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, acyl, substituted acyl, alkoxy, substituted alkoxy, amido, substituted amido, aralkyl, substituted aralkyl, PEG moiety and WSG, or any two suitable R 3 groups are optionally joined to form a cyclic structure to provide an optionally substituted carbocyclic or heterocyclic ring;

[0696] Each R 22 is one or more optional substituents, each independently selected from hydrogen, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, alkoxy, substituted alkoxy, hydroxy, cyano, halogen (e.g., fluoro, chloro or bromo), sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide and water - soluble group, or any two R 11 groups are optionally joined to form a cyclic structure (e.g., to provide a carbocyclic or heterocyclic ring); and

[0697] * represents the site for covalent attachment to the unsaturated backbone or end - group of the conjugated polymer.

[0698] 37. The polymeric tandem dye according to 36, wherein the structural formula of the fused tricyclic comonomer is as follows:

[0699]

[0700] 38. The polymeric tandem dye according to 37, wherein the fused tricyclic comonomer is selected from the following:

[0701]

[0702] Wherein:

[0703] Y 3 is C or Si; and

[0704] Each R 23 and R 24 are each independently hydrogen, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxyl, cyano, halogen, sulfonate, sulfonamide, substituted sulfonamide, carboxyl, carboxamide, substituted carboxamide, and water-soluble groups, and wherein two R 24 groups are optionally linked to form a cyclic structure to form an optionally substituted fused carbocyclic or heterocyclic ring.

[0705] 39. The polymeric tandem dye according to any one of 32-38, wherein M 1 、M 2 and L 1 each independently contain comonomer groups selected from fluorene, carbazole, silole, biphenylene, 1,4-phenylene, 1,3-phenylene, 4,4'-biphenyl, 2,5-pyridyl, 2,5-thiophene, and 2,6-pyridyl.

[0706] 40. The polymeric tandem dye according to any one of 1-39, wherein the multiple comonomers of the multi-chromophore and / or the narrow emission acceptor fluorophore are substituted by one or more water-soluble groups (WSGs) independently selected from the following:

[0707]

[0708]

[0709] Wherein:

[0710] T 5 is an optional linker;

[0711] Each T 6 is a linker;

[0712] R 11R is independently H, alkyl or substituted alkyl; and

[0713] each s is an integer between 1 and 50.

[0714] 41. A composition for multiplexed fluorescence signal generation, the composition comprising:

[0715] a first polymeric tandem dye having a first maximum emission wavelength and a first narrow emission spectrum according to any one of claims 1 - 40; and

[0716] a second polymeric tandem dye having a second maximum emission wavelength orthogonal to the first narrow emission spectrum.

[0717] 42. The composition according to 41, wherein the width of the full width at half maximum (FWHM) of the first narrow emission spectrum is 25 nm or less.

[0718] 43. The composition according to 41 or 42, wherein the second maximum emission wavelength is different from the first maximum emission wavelength and the difference therebetween is 20 nm or more.

[0719] 44. The composition according to any one of 41 - 43, further comprising one or more other polymeric tandem dyes, each having a different maximum emission wavelength.

[0720] 45. The composition according to any one of 41 - 44, wherein the second and the one or more other polymeric tandem dyes are each independently a polymeric tandem dye according to any one of claims 1 - 40.

[0721] 46. The composition according to 45, wherein the composition comprises three or more polymeric tandem dyes, each having a different and non - overlapping emission spectrum in the range of 480 nm to 700 nm.

[0722] 47. The composition according to any one of 41 - 46, wherein each polymeric tandem dye in the composition is capable of being excited by a single light source to provide orthogonally detectable fluorescence emissions.

[0723] 48. The composition according to any one of 41 - 47, wherein the first and second polymeric tandem dyes are linked to specific binding members.

[0724] 49. The composition according to any one of 41 - 48, wherein the composition is an aqueous composition.

[0725] 50. A labeled specific binding member, comprising:

[0726] a polymeric tandem dye, comprising:

[0727] A water-soluble light-harvesting multi-chromophore; and

[0728] A narrow-emission acceptor fluorophore configured to be close to the light-harvesting multi-chromophore upon energy reception; and

[0729] A specific binding member linked to the polymeric tandem dye.

[0730] 51. The labeled specific binding member according to 50, wherein the specific binding member is an antibody.

[0731] 52. The labeled specific binding member according to 51, wherein the specific binding member is an antibody fragment or a binding derivative thereof.

[0732] 53. The labeled specific binding member according to 52, wherein the antibody fragment or the binding derivative thereof is selected from the group consisting of Fab fragments, F(ab') 2 fragments, scFv, diabodies, and triabodies.

[0733] 54. The labeled specific binding member according to any one of 50 - 53, wherein the narrow-emission acceptor fluorophore is a BODIPY dye.

[0734] 55. The labeled specific binding member according to any one of 50 - 54, wherein the polymeric tandem dye is a dye according to any one of claims 2 - 40.

[0735] 56. A composition for multiplex detection of a target molecule, the composition comprising:

[0736] (a) A first labeled specific binding member comprising:

[0737] (i) A first polymeric tandem dye having a first maximum emission wavelength and a first narrow emission spectrum, comprising:

[0738] A water-soluble light-harvesting multi-chromophore; and

[0739] A narrow-emission acceptor fluorophore configured to be close to the light-harvesting multi-chromophore upon energy reception; and

[0740] (ii) A first specific binding member that specifically binds to a first target molecule and is linked to the first polymeric tandem dye; and

[0741] (b) A second labeled specific binding member comprising:

[0742] (i) A second polymeric tandem having a second maximum emission wavelength orthogonal to the first narrow emission spectrum; and

[0743] (ii) A second specific binding member that specifically binds to a second target molecule and is linked to the second polymeric tandem dye.

[0744] 57. The composition according to 56, wherein the width of the full width at half maximum (FWHM) of the first narrow emission spectrum is 25 nm or less.

[0745] 58. The composition according to 56 or 57, wherein the second maximum emission wavelength is different from the first maximum emission wavelength, and the difference therebetween is 20 nm or more.

[0746] 59. The composition according to any one of 56-58, further comprising one or more other labeled specific binding members, each comprising a polymeric tandem dye having a different maximum emission wavelength.

[0747] 60. The composition according to any one of 56-59, wherein the second and the one or more labeled specific binding members each independently comprise a polymeric tandem dye according to any one of 1-40.

[0748] 61. The composition according to 60, wherein the composition comprises three or more labeled specific binding members, each independently comprising a polymeric tandem dye having different and non-overlapping emission spectra in the range of 480 nm to 700 nm.

[0749] 62. The composition according to any one of 56-61, wherein each polymeric tandem dye in the composition is capable of being excited by a single light source to provide orthogonally detectable fluorescence emissions.

[0750] 63. The composition according to any one of 56-62, wherein the first and second labeled specific binding members specifically bind to a first and a second target protein, respectively.

[0751] 64. The composition according to any one of 56-63, wherein the specific binding members are each an antibody.

[0752] 65. The composition according to any one of 56-64, wherein the specific binding members are each an antibody fragment or a binding derivative thereof.

[0753] 66. The composition according to 65, wherein the antibody fragment or the binding derivative thereof is selected from the group consisting of Fab fragments, F(ab') 2 fragments, scFv, diabodies, and triabodies.

[0754] 67. The composition according to any one of 56-66, wherein the composition is an aqueous composition.

[0755] 68. A method for assessing the presence of a target analyte in a sample, the method comprising:

[0756] (a) contacting the sample with a labeled specific binding member that specifically binds the target analyte to produce a labeled composition contacting the sample, wherein the labeled specific binding member comprises:

[0757] (i) a polymeric tandem dye according to any one of claims 1-40; and

[0758] (ii) a specific binding member linked to the polymeric tandem dye; and

[0759] (b) determining the presence of a labeled specific binding member-target analyte binding complex in the labeled composition contacting the sample to assess the presence of the target analyte in the sample.

[0760] 69. The method according to 68, further comprising contacting the sample with a second specific binding member, the second specific binding member being bound to a support and specifically binding to the target analyte.

[0761] 70. The method according to 69, wherein the support comprises magnetic powder.

[0762] 71. The method according to any one of 68-70, wherein the target analyte is associated with a cell.

[0763] 72. The method according to 71, wherein the target analyte is a cell surface marker of the cell.

[0764] 73. The method according to 72, wherein the cell surface marker is selected from the group consisting of cell receptors and cell surface antigens.

[0765] 74. The method according to 71, wherein the target analyte is an intracellular target, and the method further comprises lysing the cells.

[0766] 75. The method according to any one of 68-74, wherein the method further comprises analyzing the fluorescently labeled target analyte by flow cytometry.

[0767] 76. The method according to any one of 68-75, wherein the contacting step comprises contacting the sample with a multiplex composition, the composition comprising:

[0768] (a) a first labeled specific binding member, which comprises:

[0769] (i) A first polymeric tandem dye having a first maximum emission wavelength and a first narrow emission spectrum, comprising:

[0770] A water-soluble light-harvesting multi-chromophore; and

[0771] A narrow emission acceptor fluorophore configured to be close to the light-harvesting multi-chromophore upon energy reception; and

[0772] (ii) A first specific binding member that specifically binds to a first target analyte and is linked to the first polymeric tandem dye; and

[0773] (b) A second labeled specific binding member, comprising:

[0774] (i) A second polymeric tandem having a second maximum emission wavelength orthogonal to the first narrow emission spectrum; and

[0775] (ii) A second specific binding member that specifically binds to a second target analyte and is linked to the second polymeric tandem dye.

[0776] 77. The method according to 76, wherein the multiplex composition is the composition according to any one of 56 - 67.

[0777] 78. A method for labeling a target molecule, the method comprising:

[0778] Contacting the target molecule with a polymeric tandem dye to produce a labeled target molecule, wherein:

[0779] The polymeric tandem dye is the dye according to any one of claims 1 - 40 and comprises a conjugated tag covalently linked to the target molecule.

[0780] 79. The method according to 78, wherein the structural formula of the polymeric tandem dye is as follows:

[0781] Wherein the structural formula of the multi-chromophore is as shown in formula (XIII):

[0782]

[0783] Wherein:

[0784] F 1 Is a fused tricyclic comonomer substituted by WSG;

[0785] M 1 、M 2 And L 1 Are independently aryl or heteroaryl comonomers optionally substituted by WSG;

[0786] N 1 Is linked to L1 the linked narrow emission acceptor fluorophore;

[0787] a-d and f are each independently 0 or 1;

[0788] e is 1;

[0789] each n and m is an integer between 1 and 10,000;

[0790] p is an integer between 1 and 100,000;

[0791] G 1 is a terminal group, a polymer segment, a donor chromophore or a linker; and

[0792] G 2 is a linker containing a conjugate tag.

[0793] 80. The method according to 78 or 79, wherein the polymeric tandem dye has a first maximum emission wavelength and a first narrow emission spectrum, and the method further comprises:

[0794] contacting a second target molecule with a second polymeric tandem dye comprising a conjugate tag to produce a second labeled target molecule, wherein:

[0795] the second polymeric tandem dye has a second maximum emission wavelength orthogonal to the first narrow emission spectrum.

[0796] 81. The method according to 80, wherein the second polymeric tandem dye is a dye according to any one of claims 1-40 and comprises a conjugate tag covalently linked to the second target molecule.

[0797] 82. The method according to any one of 80-81, wherein the target molecules are each a protein.

[0798] 83. The method according to any one of 80-82, wherein the target molecules are each an antibody.

[0799] 84. The method according to any one of 80-82, wherein the target molecules are each an antibody fragment or a binding derivative thereof.

[0800] 85. The method according to 84, wherein the antibody fragment or binding derivative thereof is selected from the group consisting of Fab fragments, F(ab') 2 fragments, scFv, diabodies and triabodies.

[0801] 86. The method according to any one of 80-85, further comprising incorporating the labeled target molecule into an aqueous composition.

[0802] 87. A kit for multiplexed fluorescence signal generation, comprising:

[0803] a first polymeric tandem dye having a first maximum emission wavelength and a first narrow emission spectrum according to any one of claims 1 - 40; and

[0804] a second polymeric tandem dye having a second maximum emission wavelength orthogonal to the first narrow emission spectrum.

[0805] 88. The kit according to 87, wherein the full width at half maximum (FWHM) of the first narrow emission spectrum has a width of 25 nm or less.

[0806] 89. The kit according to 87 or 88, wherein the second maximum emission wavelength is different from the first maximum emission wavelength and the difference therebetween is 20 nm or more.

[0807] 90. The kit according to any one of 87 - 89, further comprising one or more other polymeric tandem dyes, each having a different maximum emission wavelength.

[0808] 91. The kit according to any one of 87 - 90, wherein the second and the one or more other polymeric tandem dyes are each independently a polymeric tandem dye according to any one of 1 - 40.

[0809] 92. The kit according to 91, wherein the kit comprises three or more (4 or more, 5 or more, or even more) polymeric tandem dyes, each having a different and non - overlapping emission spectrum in the range of 480 nm to 700 nm.

[0810] 93. The kit according to any one of 87 - 92, wherein each polymeric tandem dye in the kit is capable of being excited by a single light source to provide orthogonally detectable fluorescence emissions.

[0811] 94. The kit according to any one of 87 - 93, wherein the first and second polymeric tandem dyes are each linked to a specific binding member (specifically binding to a target molecule).

[0812] 95. The kit according to 94, wherein the specific binding members are each an antibody.

[0813] 96. The kit according to 94, wherein the specific binding members are each an antibody fragment or a binding derivative thereof.

[0814] 97. The kit according to 96, wherein the antibody fragment or the binding derivative thereof is selected from the group consisting of Fab fragments, F(ab') 2A group consisting of fragments, scFvs, diabodies, and triabodies.

[0815] In at least some of the foregoing embodiments, one or more elements used in one embodiment may be interchangeably used in another embodiment, unless such substitution is technically infeasible. Those skilled in the art should understand that various other omissions, additions, and modifications may be made to the above methods and structures without departing from the claimed subject matter. All such modifications and variations are intended to be covered by the scope of the subject matter defined in the appended claims.

[0816] Those skilled in the art should understand that, generally speaking, the terms used herein, especially the terms used in the appended claims (e.g., in the main body of the appended claims), should generally be understood as "open" terms (e.g., the term "comprising" should be interpreted as "comprising but not limited to", the term "having" should be interpreted as "having at least", etc.). Those skilled in the art should also understand that if it is intended to specify a specific number in the introduced claim, such intention will be clearly indicated in that claim, and in the absence of such clear indication, it is considered that such intention does not exist. For example, for the purpose of helping understanding, the appended claims may use guiding phrases such as "at least one" and "one or more" to introduce features in the claims. However, the use of such phrases should not be construed as implying that a claim feature introduced by the indefinite article "a" or "an" limits any particular claim containing that feature to only an embodiment containing one such feature, even if the claim includes both the guiding phrase "one or more" or "at least one" and the indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"); the same applies when using the definite article to introduce a claim feature. Additionally, even if the specific number of the introduced claim feature is clearly indicated, those skilled in the art should recognize that such enumeration should be interpreted as meaning at least the listed number (e.g., the phrase "two features" without other modifiers means at least two such features, or two or more such features). Additionally, in the case of using expressions such as "at least one of A, B, and C, etc.", generally it should be interpreted according to the meaning that those skilled in the art usually understand such expression (e.g., "a system having at least one of A, B, and C" should include but not be limited to a system having only A, only B, only C, having A and B, having A and C, having B and C, and / or having A, B, and C, etc.). In the case of using expressions such as "at least one of A, B, or C, etc.", generally it should be interpreted according to the meaning that those skilled in the art usually understand such expression (e.g., "a system having at least one of A, B, or C" should include but not be limited to a system having only A, only B, only C, having A and B, having A and C, having B and C, and / or having A, B, and C, etc.). Those skilled in the art should also understand that substantially any disjunctive conjunction and / or phrase representing two or more alternative items, whether in the specification, claims, or drawings, should be understood as presenting the possibility of including one of these items, either of these items, or both items. For example, the phrase "A or B" should be understood as including the possibility of "A" or "B" or "A and B".

[0817] In addition, in cases where the features or aspects of the present invention are described in terms of Markush groups, those skilled in the art should recognize that the present invention is thereby also described in terms of any individual member or subgroup of members within such Markush groups.

[0818] Those skilled in the art should understand that, for any and all purposes, such as to provide a written description, all ranges disclosed herein also include any and all possible subranges and combinations of such subranges. Any recited range can be readily regarded as sufficiently described and enabled such that the range can be at least bisected, trisected, quartered, quintupled, decupled, etc. As a non-limiting example, each range discussed herein can be readily divided into lower one-third, middle one-third, and upper one-third, etc. Those skilled in the art should understand that all language such as "up to", "at least", "greater than", "less than", etc. includes the recited numbers and refers to ranges that can then be divided into subranges as described above. Finally, those skilled in the art should understand that ranges include each individual number. Thus, for example, a group having 1 - 3 units refers to a group having 1, 2, or 3 units. Similarly, a group having 1 - 5 units refers to a group having 1, 2, 3, 4, or 5 units, and so on.

[0819] Although the foregoing invention has been described in detail by way of illustrations and examples for the purpose of clarity of understanding, it will be obvious to those of ordinary skill in the art that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0820] Accordingly, the foregoing merely illustrates the principles of the invention. It should be understood that those skilled in the art will be able to devise various structures which, although not explicitly shown or described herein, reflect the principles of the invention and fall within the spirit and scope of the invention. Moreover, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventor to further the art, and are to be construed as not being limited to such specifically recited examples and conditions. Also, all statements herein reciting principles, aspects, and embodiments of the invention and specific examples thereof are intended to cover both structural and functional equivalents thereof. Moreover, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function regardless of structure. And, regardless of whether the disclosure of the present invention is explicitly recited in the claims, no part of the disclosure will be dedicated to the public.

[0821] Accordingly, the scope of the present invention is not limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims. In the claims, only when the limitation of a claim expressly begins with the phrase "means for" or "step for", is 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) expressly invoked; if the phrase is not used in the limitation of a claim, then 35 U.S.C. § 112(f) or 35 U.S.C. § 112(6) is not invoked.

Claims

1. A tandem dye, comprising: a multi-chromophore; and a BODIPY dye covalently linked to the multi-chromophore and approaching the multi-chromophore upon energy reception, wherein the structural formula of the BODIPY dye is shown in Formula (I): Wherein: Q is C or N; R 1 -R 7 independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG), and -L 1 -Z 1 , or Optionally, selected from R 6 and R 7 、R 2 and R 3 、R 5 and R 6 、R 3 and R 4 、R 4 and R 1 and R 5 and R 1 any one or more pairs of substituents are joined to form a cyclic structure, together forming a divalent group, and together with the carbon atom to which they are attached form a 5- or 6-membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring (e.g., a 5- or 6-membered ring containing carbon atoms and 0-3 heteroatoms selected from O, S and N), wherein said ring may be unsubstituted or may be further substituted by substituents independently selected from the following groups: alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG) and -L 1 -Z 1 ; L 1 is a linker; Z 1 is a light-harvesting multi-chromophore; Y 1 and Y 2 are independently selected from F, OH, H, cyano, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, and WSG; and where Y 1 、Y 2 and R 1 -R 7 one of which contains -L 1 -Z 1 .

2. The tandem dye according to claim 1, wherein the full width at half maximum (FWHM) of the emission spectrum of the BODIPY dye is 25 nm or less than 25 nm.

3. The tandem dye according to any one of claims 1 to 2, wherein the Stokes shift exhibited by the BODIPY dye upon direct excitation is 20 nm or less than 20 nm.

4. The tandem dye according to any one of claims 1 to 3, wherein the effective Stokes shift exhibited by the tandem dye upon direct excitation of the multi-chromophore with incident light is 100 nm or greater than 100 nm.

5. The tandem dye according to any one of claims 1 to 4, wherein the structural formula of the BODIPY dye is shown in Formula (IIa) or (IIb): Wherein: Z 11 and Z 12 are independently a fused 5- or 6-membered fused heterocyclic, carbocyclic, aryl or heteroaryl ring; each i is independently 0 - 3; and Each R 20 is independently selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG), and -L 1 -Z 1 .

6. The tandem dye according to claim 5, wherein the structural formula of the BODIPY dye is shown in Formula (IVa) or (IVb): Wherein: each X is O or S; and R 12 、R 13 、R 16 and R 17 are independently selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG) and -L 1 -Z 1 。 7. The tandem dye according to any one of claims 1 to 4, wherein the structural formula of the BODIPY dye is shown in Formula (V): Wherein: L 3 is a covalent bond, oxo (-O-), alkylene (e.g., C 1 -C 6 alkylene), -O-alkylene or a substituted form thereof; R 11 selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, water-soluble group (WSG) and -L 1 -Z 1 ; Each R 9 is selected from halogen, hydroxy, cyano, nitro, alkyl, substituted alkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl and substituted heteroaryl; and t is from 0 to 4.

8. The tandem dye according to any one of claims 1 to 4, wherein the structural formula of the BODIPY dye is shown in Formula (VIIa) or (VIIb): Wherein: n is from 0 to 12.

9. The tandem dye according to any one of claims 1 to 4, wherein the structural formula of the BODIPY dye is shown in Formula (IXa): where L 2 is a linker.

10. The tandem dye according to any one of claims 1 to 9, further comprising one or more water-soluble groups (WSGs).

11. The tandem dye according to claim 10, wherein the WSGs are independently selected from: Wherein: T 5 is an optional linker; Each T 6 is a linker; R 11 R and, independently thereof, are each H, an alkyl group or a substituted alkyl group; and each s is an integer between 1 and 50.

12. A labeled specific binding member, comprising: the tandem dye according to any one of claims 1 to 11; and a specific binding member linked to the tandem dye.

13. The labeled specific binding member according to claim 12, wherein the specific binding member is an antibody, an antibody fragment, or a binding derivative thereof.

14. A method for evaluating the presence of a target analyte in a sample, the method comprising: (a) contacting the sample with the labeled specific binding member according to any one of claims 12 to 13; and (b) determining the presence of a labeled specific binding member - target analyte binding complex in the sample contacted with the labeling composition to evaluate the presence of the target analyte in the sample.

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