Ultraviolet excitable polyfluorene-based conjugates and their use in analyte detection methods

Abstract

The present invention provides UV excitable polyfluorene-based conjugates and their use in analyte detection methods.

Description

[0001] Relevant application materials

[0002] This application claims priority to U.S. Provisional Application No. 62 / 967,800, filed January 30, 2020, which is incorporated herein by reference for all purposes. Technical Field

[0003] This invention relates generally to fluorescent polymer conjugates and methods for detecting their analytes. Background Technology

[0004] Fluorescent probes are valuable reagents for the analysis and separation of molecules and cells, as well as for the detection and quantification of other materials. Under optimal conditions, very small amounts of fluorescent molecules can be detected. Barak and Webb used a SIT camera to make fewer than 50 fluorescent lipid analogs associated with cellular LDL reception visible, J. CELL BIOL., 90, 595-604 (1981). Fewer than 10,000 luciferin molecules associated with particles or certain cells can be detected using flow cytometry (Muirhead, Horan, and Poste, BIOTECHNOLOGY, 3, 337-356 (1985)). Some specific examples of the application of fluorescent probes are (1) the identification and separation of cell subpopulations in a mixture of cells using techniques such as fluorescence flow cytometry, fluorescence-activated cell sorting, and fluorescence microscopy; (2) the determination of the concentration of substances that bind to a second substance (e.g., antigen-antibody reaction) in techniques of fluorescence immunoassay; and (3) the localization of substances in gels and other insoluble supports using techniques of fluorescence staining. Herzenberg et al., "CELLULAR IMMUNOLOGY" 3rd edition, Chapter 22; Blackwell Scientific Publications (1978); and Goldman, "FLUORESCENCE ANTIBODY METHODS", Academic Press, New York, (1968); and Taylor et al., APPLICATIONS OF FLUORESCENCE IN THE BIOMEDICAL SCIENCES, Alan Liss Inc., (1986) describe these techniques.

[0005] When using fluorescent polymers for the aforementioned purposes, there are several limitations to the selection of the fluorescent polymer. One limitation is the absorption and emission characteristics of the fluorescent polymer, because many ligands, receptors, and materials in the tested samples (e.g., blood, urine, cerebrospinal fluid) fluoresce and interfere with the accurate determination of the fluorescence of the fluorescent label. This phenomenon is called autofluorescence or background fluorescence. Another consideration is the ability of the fluorescent polymer to conjugate with ligands and receptors, as well as other biological and non-biological materials, and the effect of such conjugation on the fluorescent polymer. In many cases, conjugation with another molecule can lead to a significant change in the fluorescence characteristics of the fluorescent polymer, and in some cases, significantly destroy or reduce the quantum efficiency of the fluorescent polymer. Conjugation with a fluorescent polymer may also inactivate the function of the labeled molecule. A third consideration is the quantum efficiency of the fluorescent polymer, which should be high for sensitive detection. A fourth consideration is the light absorption capacity or extinction coefficient of the fluorescent polymer, which should also be as large as possible. Also of concern is whether fluorescent molecules will interact when very close, leading to self-quenching. Another concern is whether the fluorescent polymer binds nonspecifically to other compounds or the container wall, whether it binds to itself or together with the compounds conjugated with the fluorescent polymer.

[0006] The applicability and value of the methods described above are closely related to the availability of suitable fluorescent compounds. Specifically, fluorescent substances with strong absorption and fluorescence emission in the ultraviolet range (e.g., 355 nm), and with large Stokes shifts, are needed because the excitation of these fluorophores produces less autofluorescence, and multiple chromophores emitting fluorescence at different wavelengths can be analyzed simultaneously if the entire visible and near-infrared regions of the spectrum can be utilized. In recent years, violet lasers (405 nm) have been increasingly installed in commercial fluorescence instruments because they offer a much larger emission wavelength window than other lasers (e.g., 488 nm argon lasers and 633 nm He-Ne lasers), and ultraviolet lasers are also beginning to be introduced.

[0007] Phycobiliproteins have made significant contributions due to their high extinction coefficients and high quantum yields. These fluorophore-containing proteins can be covalently linked to many other proteins and used in fluorescent antibody assays for microscopy and flow cytometry. However, phycobiliproteins have some drawbacks that limit their biological applications, such as (1) their relative complexity and easy dissociation in highly diluted solutions; (2) their extreme instability and rapid fading under light; and (3) their weak absorption by ultraviolet excitation.

[0008] Bright fluorescent polymers allow for the detection or localization of attached materials with high sensitivity. Certain polyfluorene polymers have demonstrated utility as labeling agents for immunological applications, as illustrated in U.S. Patents 8,158,444, 8,455,613, 8,354,239, 8,362,193, and 8,575,303 to Gaylord et al., and WO 2013 / 101902 to Chiu et al. Other biological applications of polyfluorene polymers have been well documented by Thomas III et al. (Chem. Rev. 2007, 107, 1339); Zhu et al. (Chem. Rev. 2012, 112, 4687); and Zhu et al. (Chem. Soc. Rev., 2011, 40, 3509). However, all existing water-soluble polyfluorene polymers are based on unsubstituted fluorene, due to the commercial unavailability of the required key intermediates. No effort has been made to explore the biological applications of substituted fluorene polymers. Unsubstituted polyfluorene polymers are known to have certain drawbacks, such as (1) the emission wavelengths of existing polyfluorene polymers are close to the ultraviolet edge of the visible wavelength range (400–800 nm); (2) existing polyfluorene polymers also exhibit a very strong tendency for self-aggregation (i.e., stacking), which can significantly reduce fluorescence quantum yield, as described in the extensive review in Mishra et al., CHEM.REV., 100, 1973 (2000); and (3) existing polyfluorene polymers allow two benzene units to freely rotate / vibrate around the central single bond, which significantly reduces the linearity and planarity of the polymer. This phenomenon is known as the "loose band effect," described in Chapters 5 and 6 of "Modern Molecular Pollution," by Nicholas J. Turro, University Science Books, Sausalito, CA (1991). There remains a need for fluorescent polymers with improved fluorescence properties that can be excited by ultraviolet light. Summary of the Invention

[0009] This invention addresses this need and is based on the discovery that the so-called “loose band effect” can be eliminated by crosslinking two benzene rings. It has been surprisingly found that rigid fluorene-based polymers unexpectedly produce the desired biological properties. These polymer conjugates possess (1) high fluorescence quantum yield; (2) redshifted emission; (3) high water solubility; (4) high linearity; (5) high planarity; (6) high fluorescence resonance energy transfer (FRET) efficiency when a second dye is coupled to the polymer; and (7) high photostability.

[0010] The core fluorene structure is as follows.

[0011]

[0012] Fluorene

[0013] The rigid bridged fluorene structure is as follows.

[0014]

[0015] Rigid bridged fluorene (A = N-, P-, O=P-, O=P-O-, -C-, -Si-, -S-, O=S-, O=S(O)-)

[0016] The present disclosure provides a polymer comprising monomer units of formula A

[0017]

[0018] A

[0019] where X is the number of monomer units of formula A in the polymer, where the monomer units of formula A are consecutive or non-consecutive and where X is from 10 to 200,

[0020] and one or more monomer units of formula B

[0021]

[0022] B

[0023] where Y is the number of monomer units of formula B in the polymer, where the monomer units of formula B are consecutive or non-consecutive and where Y is from 0 to 100,

[0024] and optionally one or more monomer units of formula C

[0025]

[0026] C

[0027] where Z is the number of monomer units of formula C in the polymer, where the monomer units of formula C are consecutive or non-consecutive and where Z is from 0 to 100, <C

[0028] where A is O, S, N or C;

[0029] where SG1, SG2, SG5, SG6, R1 and R2 are independently hydrogen, alkyl, amino, sulfo, polyethylene glycol (PEG), water-soluble group, receptor, linker (L) and / or a biological substrate conjugated via a linker (L-BS);

[0030] SG3, SG4, R3 and R4 are independently hydrogen, halogen, amino, PEG, linker (L) and / or biosubstrate (L-BS) conjugated via linker;

[0031] The polymer ends are independently hydrogen, alkyl, halogen, boron carbonyl, aryl, heteroaryl, or L-BS groups;

[0032] The ratio of X to Y+Z is 0.3-1.0, and

[0033] The sum of X+Y+Z is between 15 and 50.

[0034] In an exemplary embodiment, fluorenzooxetine, fluorenzoazaheptatriene, and fluorenzocycloheptane refer to the O, N, and C substitutions at the "A" substituent positions in the ring of the monomer unit of formula A.

[0035] In one embodiment, when Y is present in the polymer at least 40%, the polymer’s UV excitation is close to 350 nm.

[0036] In some embodiments, the individual units of Formula A, Formula B and Formula C are directly connected to each other.

[0037] In other embodiments, the receptor comprises a fluorophore or a fluorescent dye, and the ratio of the receptor to the polymer is 0.01-0.2.

[0038] In other embodiments, the connector includes alkyl, PEG, carboxamide, thioether, ester, imine, hydrazine, oxime, alkylamine, ether, arylamine, borate ester, N-acylurea or anhydride, platinum complex, aminotriazine, triazine ether, amidine, urea, urethane, thiourea, phosphite, silyl ether, sulfonamide, sulfonate ester, 1,2,3-triazole, pyridazine, thiazolidinyl, 2-diphenylphosphono-benzamide, isoxazole, or succinimide groups.

[0039] In some embodiments,

[0040] (i) SG1, SG2, SG5 and SG6 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, aminoalkyl or L-BS; and / or

[0041] (ii) SG3, SG4, R3, and R4 independently represent hydrogen, halogen, PEG, or connector (L), and / or

[0042] (iii) L is an alkyl chain or a PEG chain; and / or

[0043] (iv) BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; and / or

[0044] (v) hydrogen, alkyl, halogen, boron carbonyl, aryl, heteroaryl, or L-BS; and / or

[0045] (vi) X, Y and Z are each independent integers selected from 0 to 200, the ratio of X / Y+Z is >0.4 and the sum of X+Y+Z is between 20 and 200.

[0046] In some other embodiments, SG1, SG2, SG5 and SG6 are independently PEG3 to PEG30.

[0047] In other embodiments, SG1-SG6 and R1-R4 independently represent hydrogen, carboxyaryl, or L-BS.

[0048] In some embodiments, the monomer unit of formula B comprises

[0049]

[0050] B

[0051] Where Y is the number of monomer units of formula B in the polymer, wherein the monomer units of formula B are continuous or discontinuous and where Y is from 0 to 100; and

[0052] SG3, SG4, R3, and R4 are independently alkyl, fluorine, hydrogen, polyethylene glycol (PEG), or receptors.

[0053] In some other embodiments, A is C; and R1 and R2 are each polyethylene glycol (PEG).

[0054] In other embodiments, A is N; and R1 does not exist and R2

[0055] yes

[0056] or .

[0057] In some embodiments, the receptor further includes fluorescein, rhodamine, p-methaminophen, anthocyanin, BODIPY, squaric acid cyanide, coumarin, perylene diimide, diketopyrrolopyrrole, porphyrin, or phthalocyanine.

[0058] In some embodiments, formulas A, B, and C include

[0059] ,

[0060] ,

[0061] ,

[0062] ,

[0063] ,

[0064] ,

[0065] ,

[0066] or

[0067]

[0068] Where m and n range from 5 to 20.

[0069] In some embodiments, the receptor includes:

[0070]

[0071]

[0072]

[0073] or

[0074] .

[0075] This disclosure provides a polymer conjugate of formula I:

[0076]

[0077] Formula I

[0078] The polymer conjugate described above comprises the three monomer units indicated by the brackets x, y, and z, where the wavy lines represent monomer junctions, USU represents unsaturated units, double bonds, triple bonds, aryl or heteroaryl groups, SG represents water-soluble groups, and HG represents head groups. The monomer units are randomly distributed along the polymer backbone; where A = NR. 10 PR 11 O=PR 11 O=P-OR 11 R 11 -CR 12 R 11 -Si-R 12 R 11 -SR 12 O=SR 11 O=S(O)-R 11USU is an unsaturated unit, double bond, triple bond, aryl or heteroaryl; R1 to R12 independently represent hydrogen, alkyl, polyethylene glycol (PEG), aryl, heteroaryl group or linked biosubstrate (L-BS), wherein the linker (L) is alkyl or PEG; wherein SG1 to SG4 independently represent alkyl, water-soluble group or L-BS; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen or boron carbonyl, aryl, heteroaryl group or L-BS; and wherein x, y and z are integers from 0 to 100, provided that (1) the ratio of BS to polymer is 0.2-3, (2) the ratio of x / (y+z) is >1, and (3) the sum of x+y+z is >10.

[0079] In one embodiment, this disclosure provides a polymer conjugate of formula I, wherein R1 to R6 independently represent hydrogen, methyl, or ethyl; wherein SG1 to SG4 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, aminoalkyl, or L-BS; wherein L is an alkyl chain or a PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, or boron carbonyl; and wherein x, y, and z are integers from 0 to 80, provided that (1) the ratio of BS to polymer is 1-2, (2) the ratio of x / (y+z) is >1, and (3) the sum of x+y+z is >20.

[0080] In another embodiment, this disclosure provides a polymer conjugate of formula I, wherein R1 to R6 are hydrogen; wherein SG1 and SG2 are PEG; wherein SG3 to SG4 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, aminoalkyl, or L-BS; wherein L is an alkyl chain or a PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, or boron carbonyl; and wherein x, y, and z are integers from 0 to 80, provided that (1) the ratio of BS to polymer is 1-2, (2) the ratio of x / (y+z) is >1, and (3) the sum of x+y+z is >20.

[0081] In one embodiment, this disclosure provides a polymer conjugate of Formula I, wherein SG3 to SG4 independently represent PEG, alkyl, carboxyalkyl, or L-BS.

[0082] In another embodiment, this disclosure provides polymer conjugates of Formula I, wherein SG3 to SG4 independently represent PEG, alkyl, aminoalkyl, or L-BS.

[0083] In one embodiment, this disclosure provides a polymer conjugate of Formula I, wherein SG1 and SG2 are independently PEG6 to PEG18.

[0084] In another embodiment, this disclosure provides a polymer conjugate of formula I, wherein the ratio of BS to polymer is 1; and wherein the sum of x+y+z is 30-80.

[0085] In one embodiment, this disclosure provides a polymer conjugate of Formula I, wherein SG3 to SG4 independently represent PEG, methyl, carboxyalkyl, or L-BS.

[0086] In another embodiment, this disclosure provides polymer conjugates of Formula I, wherein SG3 to SG4 independently represent PEG, methyl, aminoalkyl, or L-BS.

[0087] In one embodiment, this disclosure provides a polymer conjugate of formula I, wherein R1 to R6 are hydrogen; wherein SG1 to SG4 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, or aminoalkyl; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, boron carbonyl, or L-BS; wherein L is an alkyl chain or a PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; and wherein x, y, and z are integers from 0 to 80, provided that (1) the ratio of BS to polymer is 1 to 2, (2) the ratio of x / (y+z) is >1, and (3) the sum of x+y+z is >20.

[0088] In another embodiment, this disclosure provides a polymer conjugate of formula I, wherein the ratio of BS to polymer is 1; and wherein the sum of x+y+z is 30-80.

[0089] In one embodiment, this disclosure provides a polymer conjugate of formula I, wherein HG1 and HG2 independently represent hydrogen, carboxyaryl, or L-BS.

[0090] In another embodiment, this disclosure provides polymer conjugates of Formula I, wherein HG1 and HG2 independently represent halogen, boron carbonyl, carboxyaryl, or L-BS.

[0091] This disclosure provides a polymer conjugate of formula II:

[0092]

[0093] Formula II

[0094] The polymer conjugate comprises the four monomer units indicated by the brackets w, x, y, and z above, where the wavy line represents the monomer junction, SG represents the water-soluble group, and HG represents the head group. The monomer units are randomly distributed along the polymer backbone. The fluorophore (FP) is a fluorescent dye with a maximum absorption wavelength greater than 370 nm, a maximum emission wavelength greater than 400 nm, and a fluorescence quantum yield greater than 5%. A = NR. 10 PR 11 O=PR 11 O=P-OR 11 R 11 -CR 12 R 11 -Si-R 12 R 11 -SR 12 O=SR 11 O=S(O)-R 11 R1 to R12 independently represent hydrogen, alkyl, PEG, aryl, heteroaryl groups or L-BS; where L is alkyl, PEG or FP; where SG1 to SG7 independently represent alkyl, water-soluble groups or L-BS; where HG1 and HG2 independently represent hydrogen, halogen, boron carbonyl, alkyl, aryl, heteroaryl groups or L-BS; where w, x and z are integers from 0 to 100; and where y is an integer from 1 to 20, provided that (1) the ratio of BS to polymer is 0.2-3, (2) the ratio of w / (x+y+z) is >1, and (3) the sum of w+x+y+z is >10.

[0095] In one embodiment, this disclosure provides a polymer conjugate of formula II, wherein FP is fluorescein, rhodamine, p-aminophenol, anthocyanin, BODIPY, squaric acid cyanide, coumarin, perylene diimide, diketopyrrolopyrrole, porphyrin, or phthalocyanine; wherein R1 to R6 independently represent hydrogen, methyl, or ethyl; wherein SG1 to SG7 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, aminoalkyl, or L-BS; wherein L is an alkyl chain, FP, or PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, or boron carbonyl; wherein w, x, and z are integers from 0 to 80; and wherein y is an integer from 1 to 10, provided that (1) the ratio of BS to polymer is 1-2, (2) the ratio of w / (x+y+z) is >1, and (3) the sum of w+x+y+z is >20.

[0096] In another embodiment, this disclosure provides polymeric conjugates of formula II, wherein R1 to R6 are hydrogen.

[0097] In one embodiment, this disclosure provides a polymer conjugate of formula II, wherein the ratio of BS to polymer is 1; and wherein the sum of w+x+y+z is 30-80.

[0098] In another embodiment, this disclosure provides polymer conjugates of Formula II, wherein FP is fluorescein, rhodamine, anthocyanin, BODIPY, squaric acid cyanine, perylene diimide, or phthalocyanine.

[0099] In one embodiment, this disclosure provides a polymeric conjugate of Formula II, wherein FP is rhodamine.

[0100] In another embodiment, this disclosure provides a polymer conjugate of formula II, wherein FP is anthocyanin.

[0101] In one embodiment, this disclosure provides a polymer conjugate of formula II, wherein FP is fluorescein, rhodamine, p-methylaminophenol, anthocyanin, BODIPY, squaric acid cyanide, coumarin, perylene diimide, diketopyrrolopyrrole, porphyrin, or phthalocyanine; wherein R1 to R6 are hydrogen; wherein SG1 to SG7 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, or aminoalkyl; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, boron carbonyl, or L-BS; wherein L is an alkyl chain, FP, or PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; wherein w, x, and z are integers from 0 to 80; and wherein y is an integer from 1 to 10, provided that (1) the ratio of BS to polymer is 1-2, (2) the ratio of w / (x+y+z) is >1, and (3) the sum of w+x+y+z is >20.

[0102] In another embodiment, this disclosure provides a polymer conjugate of formula II, wherein the ratio of BS to polymer is 1; and wherein the sum of x+y+z is 30-80.

[0103] In one embodiment, this disclosure provides a polymer conjugate of formula II, wherein HG1 and HG2 independently represent hydrogen, carboxyaryl, or L-BS.

[0104] In another embodiment, this disclosure provides polymer conjugates of formula II, wherein HG1 and HG2 independently represent halogen, boron carbonyl, carboxyaryl, or L-BS.

[0105] This disclosure provides a polymer conjugate of formula III:

[0106]

[0107] Formula III

[0108] The polymer conjugate described above comprises the three monomer units indicated by w, x, and z (highlighted in parentheses), where wavy lines represent monomer junctions, SG represents a water-soluble group, and HG represents a head group.

[0109] The monomer units are randomly distributed along the polymer backbone; the fluorophore (FP) is a fluorescent dye with a maximum absorption wavelength greater than 370 nm, a maximum emission wavelength greater than 400 nm, and a fluorescence quantum yield greater than 5%; where A = NR 10 PR 11 O=PR 11 O=P-OR 11 R 11 -CR 12 R 11 -Si-R 12 R 11 -SR 12 O=SR 11 O=S(O)-R 11 R1 to R12 independently represent hydrogen, alkyl, PEG, aryl, heteroaryl groups or L-BS; where L is alkyl, PEG or FP; where SG1 to SG6 independently represent alkyl, water-soluble groups or L-BS; where HG1 and HG2 independently represent hydrogen, halogen, boron carbonyl, alkyl, aryl, heteroaryl groups or L-BS; where w, x and z are integers from 0 to 100; and where z is an integer from 1 to 20, provided that (1) the ratio of BS to polymer is 0.2-3, (2) the ratio of w / (x+z) is >1, and (3) the sum of w+x+z is >10.

[0110] In one embodiment, this disclosure provides a polymer conjugate of formula III, wherein FP is fluorescein, rhodamine, p-methylaminophenol, anthocyanin, BODIPY, squaric acid cyanide, coumarin, perylene diimide, diketopyrrolopyrrole, porphyrin, or phthalocyanine; wherein R1 to R6 independently represent hydrogen, methyl, or ethyl; wherein SG1 to SG6 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, aminoalkyl, or L-BS; wherein L is an alkyl chain, FP, or PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, or boron carbonyl; wherein w, x, and z are integers from 0 to 80; and wherein z is an integer from 1 to 10, provided that (1) the ratio of BS to polymer is 1-2, (2) the ratio of w / (x+z) is >1, and (3) the sum of w+x+z is >20.

[0111] In another embodiment, this disclosure provides polymeric conjugates of formula III, wherein R1 to R6 are hydrogen.

[0112] In one embodiment, this disclosure provides a polymer conjugate of Formula III, wherein the ratio of BS to polymer is 1; and wherein the sum of w+x+z is 30-80.

[0113] In another embodiment, this disclosure provides polymeric conjugates of Formula III, wherein FP is fluorescein, rhodamine, anthocyanin, BODIPY, squaric acid cyanine, perylene diimide, or phthalocyanine.

[0114] In one embodiment, this disclosure provides a polymeric conjugate of Formula III, wherein FP is rhodamine.

[0115] In another embodiment, this disclosure provides a polymer conjugate of formula III, wherein FP is anthocyanin.

[0116] In one embodiment, this disclosure provides a polymer conjugate of formula III, wherein FP is fluorescein, rhodamine, p-methylaminophenol, anthocyanin, BODIPY, squaric acid cyanide, coumarin, perylene diimide, diketopyrrolopyrrole, porphyrin, or phthalocyanine; wherein R1 to R6 are hydrogen; wherein SG1 to SG6 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, or aminoalkyl; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, boron carbonyl, or L-BS; wherein L is an alkyl chain, FP, or PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; wherein w, x, and z are integers from 0 to 80; and wherein z is an integer from 1 to 10, provided that (1) the ratio of BS to polymer is 1-2, (2) the ratio of w / (x+z) is >1, and (3) the sum of w+x+z is >20.

[0117] In another embodiment, this disclosure provides a polymer conjugate of formula II, wherein the ratio of BS to polymer is 1; and wherein the sum of x+z is 30-80.

[0118] In one embodiment, this disclosure provides a polymer conjugate of formula II, wherein HG1 and HG2 independently represent hydrogen, carboxyaryl, or L-BS.

[0119] In another embodiment, this disclosure provides polymer conjugates of formula II, wherein HG1 and HG2 independently represent halogen, boron carbonyl, carboxyaryl, or L-BS.

[0120] This disclosure provides a polymer conjugate of formula IV:

[0121]

[0122] Formula IV

[0123] The polymer conjugate described above comprises the three monomer units indicated by the brackets x, y, and z, where the wavy line represents the monomer junction, SG represents the water-soluble group, and HG represents the head group.

[0124] The polymer conjugate comprises three monomer units randomly distributed along the polymer backbone; wherein A1, A2, or A3 = NR. 10 PR 11 O=PR 11 O=P-OR 11 R 11 -CR 12 R 11 -Si-R 12 R 11 -SR 12 O=SR 11 O=S(O)-R 11 R1 to R12 independently represent hydrogen, alkyl, polyethylene glycol (PEG), aryl, heteroaryl groups or linked biological substrates (L-BS); wherein the linker (L) is alkyl or PEG; wherein SG1 to SG6 independently represent alkyl, water-soluble groups or L-BS; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen or boron carbonyl, aryl, heteroaryl groups or L-BS; and wherein x, y and z are integers from 0 to 100, provided that (1) the ratio of BS to polymer is 0.2 to 3, and (2) the sum of x+y+z is >10.

[0125] In one embodiment, this disclosure provides a polymer conjugate of formula IV, wherein R1 to R4 independently represent hydrogen, methyl, or ethyl; wherein SG1 to SG6 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, aminoalkyl, or L-BS; wherein L is an alkyl chain or a PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, or boron carbonyl; and wherein x, y, and z are integers from 0 to 80, provided that (1) the ratio of BS to polymer is 1 to 2, and (2) the sum of x+y+z is >20.

[0126] In another embodiment, this disclosure provides a polymer conjugate of formula IV, wherein R1 to R4 are hydrogen; wherein SG1 and SG2 are PEG; wherein SG3 to SG6 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, aminoalkyl, or L-BS; wherein L is an alkyl chain or a PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, or boron carbonyl; and wherein x, y, and z are integers from 0 to 80, provided that (1) the ratio of BS to polymer is 1 to 2, and (2) the sum of x+y+z is >20.

[0127] In one embodiment, this disclosure provides polymer conjugates of formula IV, wherein SG3 to SG6 independently represent PEG, alkyl, carboxyalkyl, or L-BS.

[0128] In another embodiment, this disclosure provides polymer conjugates of formula IV, wherein SG3 to SG6 independently represent PEG, alkyl, aminoalkyl, or L-BS.

[0129] In one embodiment, this disclosure provides a polymer conjugate of formula IV, wherein the ratio of BS to polymer is 1; and wherein the sum of x+y+z is 30-80.

[0130] In another embodiment, this disclosure provides polymer conjugates of formula IV, wherein SG3 to SG6 independently represent PEG, methyl, carboxyalkyl, or L-BS.

[0131] In one embodiment, this disclosure provides polymer conjugates of formula IV, wherein SG3 to SG6 independently represent PEG, methyl, aminoalkyl, or L-BS.

[0132] In another embodiment, this disclosure provides a polymer conjugate of formula IV, wherein R1 to R4 are hydrogen; wherein SG1 to SG6 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, or aminoalkyl; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, boron carbonyl, or L-BS; wherein L is an alkyl chain or a PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; and wherein x, y, and z are integers from 0 to 80, provided that (1) the ratio of BS to polymer is 1-2, (2) the ratio of x / (y+z) is >1, and (3) the sum of x+y+z is >20.

[0133] In one embodiment, this disclosure provides a polymer conjugate of formula IV, wherein the ratio of BS to polymer is 1; and wherein the sum of x+y+z is 30-80.

[0134] In another embodiment, this disclosure provides polymeric conjugates of formula IV, wherein HG1 and HG2 independently represent hydrogen, carboxyaryl, or L-BS.

[0135] In another embodiment, this disclosure provides polymer conjugates of formula IV, wherein HG1 and HG2 independently represent halogen, boron carbonyl, carboxyaryl, or L-BS.

[0136] This disclosure provides a polymer conjugate of formula V:

[0137]

[0138] Formula V

[0139] The polymer conjugates described above contain the four monomer units indicated by the brackets w, x, y, and z, where the wavy line represents the monomer junction, SG represents the water-soluble group, and HG represents the head group.

[0140] The monomer units are randomly distributed along the polymer backbone; the fluorophore (FP) is a fluorescent dye with a maximum absorption wavelength greater than 370 nm and a maximum emission wavelength greater than 400 nm, and a fluorescence quantum yield greater than 5%; and A1, A2, A3, or A4 = NR. 10 PR 11 O=PR 11 O=P-OR 11 R 11 -CR 12 R 11 -Si-R 12 R 11 -SR 12 O=SR 11 O=S(O)-R 11 R1 to R12 independently represent hydrogen, alkyl, PEG, aryl, heteroaryl groups or L-BS; where L is alkyl, PEG or FP; where SG1 to SG7 independently represent alkyl, water-soluble groups or L-BS; where HG1 and HG2 independently represent hydrogen, halogen, boron carbonyl, alkyl, aryl, heteroaryl groups or L-BS; where w, x and z are integers from 0 to 100; and where y is an integer from 1 to 20, provided that (1) the ratio of BS to polymer is 0.2-3, and (2) the sum of w+x+y+z is >10.

[0141] In one embodiment, this disclosure provides a polymer conjugate of formula V, wherein FP is fluorescein, rhodamine, p-methylaminophenol, anthocyanin, BODIPY, squaric acid cyanide, coumarin, perylene diimide, diketopyrrolopyrrole, porphyrin, or phthalocyanine; wherein R1 to R4 independently represent hydrogen, methyl, or ethyl; wherein SG1 to SG7 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, aminoalkyl, or L-BS; wherein L is an alkyl chain, FP, or PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, or boron carbonyl; wherein w, x, and z are integers from 0 to 80; and wherein y is an integer from 1 to 10, provided that (1) the ratio of BS to polymer is 1-2, and (2) the sum of w+x+y+z is >20.

[0142] In another embodiment, this disclosure provides polymeric conjugates of formula V, wherein R1 to R4 are hydrogen.

[0143] In one embodiment, this disclosure provides a polymer conjugate of formula V, wherein the ratio of BS to polymer is 1; and wherein the sum of w+x+y+z is 30-80.

[0144] In another embodiment, this disclosure provides polymeric conjugates of formula V, wherein FP is fluorescein, rhodamine, anthocyanin, BODIPY, squaric acid cyanine, perylene diimide, or phthalocyanine.

[0145] In one embodiment, this disclosure provides a polymeric conjugate of formula V, wherein FP is rhodamine.

[0146] In another embodiment, this disclosure provides a polymer conjugate of formula V, wherein FP is anthocyanin.

[0147] In one embodiment, this disclosure provides a polymer conjugate of formula V, wherein FP is fluorescein, rhodamine, p-methylaminophenol, anthocyanin, BODIPY, squaric acid cyanide, coumarin, perylene diimide, diketopyrrolopyrrole, porphyrin, or phthalocyanine; wherein R1 to R4 are hydrogen; wherein SG1 to SG7 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, or aminoalkyl; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, boron carbonyl, or L-BS; wherein L is an alkyl chain, FP, or PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; wherein w, x, and z are integers from 0 to 80; and wherein y is an integer from 1 to 10, provided that (1) the ratio of BS to polymer is 1-2, and (2) the sum of w+x+y+z is >20.

[0148] In another embodiment, this disclosure provides a polymer conjugate of formula V, wherein the ratio of BS to polymer is 1; and wherein the sum of w+x+y+z is 30-80.

[0149] In one embodiment, this disclosure provides a polymer conjugate of formula V, wherein HG1 and HG2 independently represent hydrogen, carboxyaryl, or L-BS.

[0150] In another embodiment, this disclosure provides polymer conjugates of formula V, wherein HG1 and HG2 independently represent halogen, boron carbonyl, carboxyaryl, or L-BS.

[0151] This disclosure also provides a method for detecting an analyte in a sample, the method comprising:

[0152] a) Combining the sample with the detection reagent under conditions where the detection reagent comprising a polymer conjugate having the structure of Formula I will bind the analyte; and

[0153] b) Analytes bound to the reagents are detected by fluorescence detection.

[0154] The polymer conjugate comprises three monomer units randomly distributed along the polymer backbone; wherein R1 to R6 independently represent hydrogen, alkyl, polyethylene glycol (PEG), aryl, heteroaryl groups or linked biosubstrate (L-BS); wherein the linker (L) is alkyl or PEG; wherein SG1 to SG6 independently represent alkyl, water-soluble groups or L-BS; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen or boron carbonyl, aryl, heteroaryl groups or L-BS; and wherein x, y and z are integers from 0 to 100, provided that (1) the ratio of BS to polymer is 0.2-3, (2) the ratio of x / (y+z) is >1, and (3) the sum of x+y+z is >10.

[0155] This disclosure provides a method for detecting an analyte in a sample, the method comprising:

[0156] a) Combining the sample with the detection reagent under conditions where the detection reagent comprising a polymer conjugate having the structure of Formula II will bind the analyte; and

[0157] b) Analytes bound to the reagents are detected by fluorescence detection.

[0158] The polymer conjugate shown contains four monomer units randomly distributed along the polymer backbone; wherein the fluorophore (FP) is a fluorescent dye with a maximum absorption wavelength greater than 370 nm and a maximum emission wavelength greater than 400 nm and a fluorescence quantum yield greater than 5%; wherein R1 to R6 independently represent hydrogen, alkyl, PEG, aryl, heteroaryl groups or L-BS; wherein L is alkyl, PEG or FP; wherein SG1 to SG7 independently represent alkyl, water-soluble groups or L-BS; wherein HG1 and HG2 independently represent hydrogen, halogen, boron carbonyl, alkyl, aryl, heteroaryl groups or L-BS; wherein w, x and z are integers from 0 to 100; and wherein y is an integer from 1 to 20, provided that (1) the ratio of BS / polymer is 0.2-3, (2) the ratio of w / (x+y+z) is >1, and (3) the sum of w+x+y+z is >10.

[0159] This disclosure also provides a method for detecting an analyte in a sample, the method comprising:

[0160] a) Combining the sample with the detection reagent under conditions where the detection reagent comprising a polymer conjugate having the structure of Formula III will bind the analyte; and

[0161] b) Analytes bound to the reagents are detected by fluorescence detection.

[0162] The polymer conjugate comprises three monomer units randomly distributed along the polymer backbone; wherein R1 to R4 independently represent hydrogen, alkyl, polyethylene glycol (PEG), aryl, heteroaryl groups or linked biosubstrate (L-BS); wherein the linker (L) is alkyl or PEG; wherein SG1 to SG6 independently represent alkyl, water-soluble groups or L-BS; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen or boron carbonyl, aryl, heteroaryl groups or L-BS; and wherein x, y and z are integers from 0 to 100, provided that (1) the ratio of BS to polymer is 0.2-3, and (2) the sum of x+y+z is >10.

[0163] This disclosure provides a method for detecting an analyte in a sample, the method comprising:

[0164] a) Combining the sample with the detection reagent under conditions where the detection reagent comprising a polymer conjugate having the structure of Formula IV will bind the analyte; and

[0165] b) Analytes bound to the reagents are detected by fluorescence detection.

[0166] The polymer conjugate shown contains four monomer units randomly distributed along the polymer backbone; wherein the fluorophore (FP) is a fluorescent dye with a maximum absorption wavelength greater than 370 nm and a maximum emission wavelength greater than 400 nm and a fluorescence quantum yield greater than 5%; wherein R1 to R4 independently represent hydrogen, alkyl, PEG, aryl, heteroaryl groups or L-BS; wherein L is alkyl, PEG or FP; wherein SG1 to SG7 independently represent alkyl, water-soluble groups or L-BS; wherein HG1 and HG2 independently represent hydrogen, halogen, boron carbonyl, alkyl, aryl, heteroaryl groups or L-BS; wherein x, y and z are integers from 0 to 100; and wherein y is an integer from 1 to 20, provided that (1) the ratio of BS to polymer is 0.2-3, and (2) the sum of x+y+z is >10.

[0167] This disclosure provides a method for detecting an analyte in a sample, the method comprising:

[0168] a) Combining the sample with the detection reagent under conditions where the detection reagent comprising a polymer conjugate having the structure of Formula V will bind the analyte; and

[0169] b) Analytes bound to the reagents are detected by fluorescence detection.

[0170] The polymer conjugate shown contains four monomer units randomly distributed along the polymer backbone; wherein the fluorophore (FP) is a fluorescent dye with a maximum absorption wavelength greater than 370 nm and a maximum emission wavelength greater than 400 nm and a fluorescence quantum yield greater than 5%; wherein R1 to R4 independently represent hydrogen, alkyl, PEG, aryl, heteroaryl groups or L-BS; wherein L is alkyl, PEG or FP; wherein SG1 to SG7 independently represent alkyl, water-soluble groups or L-BS; wherein HG1 and HG2 independently represent hydrogen, halogen, boron carbonyl, alkyl, aryl, heteroaryl groups or L-BS; wherein w, x and z are integers from 0 to 100; and wherein y is an integer from 1 to 20, provided that (1) the ratio of BS to polymer is 0.2-3 and (2) the sum of w+x+y+z is >10.

[0171] In one embodiment, this disclosure provides polymer conjugates of formula I, II, III, IV or V, wherein BS is an antibody.

[0172] In another embodiment, this disclosure provides polymer conjugates of formula I, II, III, IV or V, wherein BS is an anti-digoxin antibody.

[0173] In yet another embodiment, this disclosure provides polymer conjugates of formula I, II, III, IV or V, wherein BS is a goat anti-mouse IgG antibody, a goat anti-rabbit IgG antibody, a goat anti-human IgG antibody, a donkey anti-mouse IgG antibody, a donkey anti-rabbit IgG antibody, a donkey anti-human IgG antibody, a chicken anti-mouse IgG antibody, a chicken anti-rabbit IgG antibody or a chicken anti-human IgG antibody.

[0174] In one embodiment, this disclosure provides polymer conjugates of formula I, II, III, IV or V, wherein BS is avidin, streptavidin, neutral avidin or avidin.

[0175] In another embodiment, this disclosure provides polymer conjugates of formula I, II, III, IV or V, wherein the analyte is a target protein expressed on a cell surface.

[0176] In another embodiment, the present invention provides polymer conjugates of formula I, II, III, IV or V, wherein the analyte is a target protein, which is an intracellular protein detected within cells.

[0177] Note that in this disclosure, and particularly in the claims and / or paragraphs, terms such as “comprises,” “comprised,” and “comprising” may have the meanings they are given under U.S. Patent Law; for example, they may mean “includes,” “included,” “including,” etc.; and terms such as “consisting essentially of” and “consistses essentially of” have the meanings they are given under U.S. Patent Law, for example, they allow elements not expressly listed but exclude elements found in the prior art or affecting the essential or novel features of the invention. These and other embodiments are disclosed or apparent from the following detailed description and are covered by the following detailed description. Attached Figure Description

[0178] The patent or application documents contain color-drawn drawings. A copy of this patent or patent application disclosure and the color-drawn drawings will be provided by the Patent Office upon request and after payment of the necessary fees. The above and other features and advantages of this embodiment will be more fully understood through the following detailed description of exemplary embodiments in conjunction with the accompanying drawings, wherein:

[0179] Figures 1-a to 1-eA typical synthesis of fluorene polymer bioconjugates is described. BS is a biological substrate (e.g., an antibody). w, x, y, and z are the number of monomer units. FG are the functional groups used for conjugation as listed in Table 2. FP are the fluorophores as listed in Table 1. L is the linker.

[0180] Figures 2A to 2C Using fluorene-based oxadiazine (FAP) Figure 2A ), fluorenecycloheptanine ( Figure 2B ) and fluorene-2-azine-heptanetriene ( Figure 2C Size exclusion purification of antibody conjugates prepared from polymers was performed. The crude conjugated reaction mixture was purified by passing it through a Superdex 200 incremental resin, and the purified conjugates were eluted in 7–10 mL volumes and collected separately. Unconjugated “free” antibodies were eluted in approximately 11.5 mL volumes and discarded.

[0181] Figures 3A to 3B Optical properties of fluorene-oxetane-heptane-based polymers with phenyl-based joint attachments. Figure 3A The absorption and emission spectra of the basic polymer. Figure 3B The same tandem absorption and emission spectra of the fluorene-oxetane-heptane-based polymers show energy transfer from the polymer to the acceptor dye emitting at approximately 563 nm.

[0182] Figures 4A to 4B It possesses the optical properties of fluorene-oxoheptatriene-based polymers with joiners based on aziridine monomers. Figure 4A The absorption and emission spectra of the basic polymer. Figure 4B The same tandem absorption and emission spectra of the fluorene-oxetane-heptane-based polymers show energy transfer from the polymer to the acceptor dye emitting at approximately 805 nm.

[0183] Figures 5A to 5B It possesses the optical properties of fluorenecycloheptane-based polymers with linker attachments based on aziridine monomers. Figure 5A The absorption and emission spectra of the basic polymer. Figure 5B The same tandem absorption and emission spectra of fluorenecycloheptanane-based polymers show energy transfer from the polymer to the acceptor dye emitting at approximately 563 nm.

[0184] Figures 6A to 6B Optical properties of fluorenecycloheptadecane-based polymers with phenyl-based joint attachments. Figure 6A The absorption and emission spectra of the basic polymer. Figure 6B The same tandem absorption and emission spectra of fluorenecycloheptanane-based polymers show energy transfer from the polymer to the acceptor dye emitting at approximately 805 nm.

[0185] Figures 7A to 7B It possesses the optical properties of fluorene-based polymers with joiners attached to aziridine monomers. Figure 7A The absorption and emission spectra of the basic polymer. Figure 7B The same tandem absorption and emission spectra of the fluorene-based polymers, with energy transferred from the polymer to the acceptor dye emitting at approximately 805 nm.

[0186] Figures 8A to 8B Optical properties of fluorene-aziridine-heptane-based polymers with phenyl-based linkers. Figure 8A The absorption and emission spectra of the basic polymer. Figure 8B The same tandem absorption and emission spectra of the fluorene-based polymers, with energy transferred from the polymer to the acceptor dye emitting at approximately 805 nm.

[0187] Figures 9A to 9D Performance of polymer conjugated antibodies based on fluorene-oxazine heptadiene in flow cytometry analysis. Figure 9A The basic polymer based on oxadiazine (1) linker was conjugated with an anti-mouse CD4 (clone RM4-5) monoclonal antibody and used to stain mouse spleen cells, which were then analyzed by flow cytometry. As shown in the upper right quadrant, the conjugated antibody recognized CD3-positive and CD4-positive cells. Figure 9B The fluorene-based heptadiene-based tandem polymer of linker 1 was conjugated with an anti-mouse CD4 (clone RM4-5) monoclonal antibody and used to stain mouse spleen cells, which were then analyzed by flow cytometry. As shown in the upper right quadrant, the conjugated antibody recognized CD3-positive and CD4-positive cells. Figure 9C The fluorene-based heptadiene-based polymer of linker 2 was conjugated with anti-human CD25 (clone BC96) and used to stain stimulated normal human peripheral blood cells, which were then analyzed by flow cytometry. As shown in the two quadrants above, the conjugated antibody recognizes CD25-positive cells. Figure 9D The tandem polymer based on oxadiazine, using linker 2, was conjugated to anti-human TNF α (clone MAb11) and used for intracellular staining of stimulated normal human peripheral blood cells. The stimulated normal human peripheral blood cells were analyzed by flow cytometry. As shown in the two quadrants above, the conjugated antibody recognizes TNF α-positive cells.

[0188] Figures 10A to 10D Performance of polymer-conjugated antibodies based on fluorenylcycloheptanine in flow cytometry analysis. Figure 10AThe fluorenylcycloheptanine-based polymer of linker 1 was conjugated with an anti-mouse CD4 (clone RM4-5) and used to stain mouse spleen cells, which were then analyzed by flow cytometry. As shown in the upper right quadrant, the conjugated antibody recognized CD3-positive and CD4-positive cells. Figure 10B The fluorenylcycloheptanane-based tandem polymer of linker 1 was conjugated with anti-human CD25 (clone BC96) and used to stain stimulated normal human peripheral blood cells, which were then analyzed by flow cytometry. As shown in the two quadrants above, the conjugated antibody recognizes CD25-positive cells. Figure 10C The fluorenylcycloheptanine-based polymer of linker 2 was conjugated with an anti-mouse CD4 (clone RM4-5) and used to stain mouse spleen cells, which were then analyzed by flow cytometry. As shown in the upper right quadrant, the conjugated antibody recognized CD3-positive and CD4-positive cells. Figure 10D The fluorenylcycloheptanyl tandem polymer of linker 2 was conjugated with anti-mouse CD4 (clone RM4-5) and used to stain mouse spleen cells, which were then analyzed by flow cytometry. As shown in the upper right quadrant, the conjugated antibody recognized CD3-positive and CD4-positive cells.

[0189] Figures 11A to 11D Performance of polymer conjugated antibodies based on fluorene-azacycloheptatriene in flow cytometry analysis. Figure 11A The fluorene-based heptadiene-based polymer of linker 1 was conjugated with an anti-mouse CD4 (clone RM4-5) and used to stain mouse spleen cells, which were then analyzed by flow cytometry. As shown in the upper right quadrant, the conjugated antibody recognized CD3-positive and CD4-positive cells. Figure 11B The fluorene-based tandem polymer of linker 1 was conjugated with anti-human CD20 (clone 2H7) and used to stain normal human peripheral blood cells, which were then analyzed by flow cytometry. The conjugated antibody, as shown in the upper left quadrant of the data plot, recognizes CD3-negative, CD20-bright cells, and as shown in the upper right quadrant, recognizes CD3-positive, CD20-dark cells. Figure 11C The fluorene-based polymer (based on heptadiene) of linker 2 was conjugated with an anti-human TNF α (clone MAb11) and used for intracellular staining of stimulated normal human peripheral blood cells. The stimulated normal human peripheral blood cells were analyzed by flow cytometry. As shown in the two quadrants above, the conjugated antibody recognizes TNF α-positive cells. Figure 11DThe fluorene-based tandem polymer of linker 2 was conjugated with anti-human Ki-67 (clone 20Raj1) and used for intracellular staining of stimulated normal human peripheral blood cells, which were then analyzed by flow cytometry. As shown in the upper left quadrant, the conjugated antibody recognized CD19-negative, Ki-67-positive cells. Detailed Implementation

[0190] Before describing the invention in further detail, it should be understood that the invention is not limited to the specific methods, apparatus, solutions, or devices described, as such methods, apparatus, solutions, or devices can certainly vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention.

[0191] Unless the context clearly indicates otherwise, the use of the singular forms “a,” “a,” and “the” includes plural indicators. Thus, for example, referring to “a probe” includes multiple probes, etc. Additionally, unless the context clearly indicates otherwise, specific plural indicators such as “two,” “three,” etc., are used when reading about a large number of the same topics.

[0192] Unless the context clearly indicates otherwise, terms such as “connection,” “attachment,” “combination,” and “linkage” are used interchangeably herein and cover both direct and indirect connections, attachments, links, or combinations; in one instance, the phrase “combined polymer” is used according to its common meaning in the art and refers to a polymer containing a series of extended unsaturated bonds, and the context indicates that the term “combination” should be interpreted as not merely direct or indirect connections, attachments, or links.

[0193] All publications mentioned herein are incorporated herein by reference for the purpose of disclosing and describing the specific materials and methods used in citing the references. The publications discussed herein are provided only for their publication prior to the filing date of this application. Nothing herein should be construed as an admission that the invention is not entitled to any prior disclosure by virtue of a prior invention.

[0194] Various chemical modifications of fluorene polymers have been explored to investigate their biological detection applications. It has been noted that any substitution at positions 1, 2, 3, 6, 7, and 8 significantly reduces the fluorescence intensity of the resulting polymer conjugates. Additionally, these substituted fluorene polymer conjugates also exhibit poor water solubility. Efforts focused on positions 4 and 5. Initial efforts with different substitutions and crosslinkings at positions 4 and 5 did not yield the desired polymers. Halogenation, alkylation, amination, and crosslinking of positions 4 and 5 with 5-membered, 6-membered, and 8-membered rings produced undesirable fluorene polymer conjugates. However, this current rigidly bridged fluorene polymer conjugate unexpectedly produced the desired biological properties, where positions 4 and 5 of fluorene are crosslinked via a 7-membered ring. These polymer conjugates have been found to possess the following properties: (1) high fluorescence quantum yield; (2) redshifted emission; (3) high water solubility; (4) high linearity; (5) high planarity; (6) high fluorescence resonance energy transfer (FRET) efficiency when a second fluorophore is coupled to the polymer; and (7) high photostability. It has been found that the rigid-bridging polyfluorene polymers described herein unexpectedly alleviate the problems discussed in the background section and produce fluorescent polymer conjugates with significantly enhanced fluorescence on proteins, nucleic acids, and other biopolymers. The enhanced fluorescence intensity of the polymer-biomolecule conjugates of the present invention results in higher assay sensitivity.

[0195] This disclosure provides polymer conjugates, including rigidly bridged fluorene-based polymer conjugates. These bioconjugates are used to locate or detect interactions or presence of analytes or ligands in a sample. Kits incorporating such polymers or polymer conjugates facilitate their use in such methods.

[0196] This disclosure provides polymer conjugates, including rigidly bridged fluorene-based polymer conjugates, comprising: 1) a polymer comprising a rigidly bridged fluorene monomer; and 2) a biosubstrate (BS). The polymer conjugates of this invention generally have the structure of Formula I:

[0197]

[0198] Formula I

[0199] The polymer conjugate comprises three monomer units randomly distributed along the polymer backbone; wherein A = NR. 10 PR 11 O=PR 11 O=P-OR 11 R 11 -CR 12 R 11 -Si-R 12 R 11 -SR12 O=SR 11 O=S(O)-R 11 USU is an unsaturated unit, double bond, triple bond, aryl or heteroaryl; R1 to R12 independently represent hydrogen, alkyl, polyethylene glycol (PEG), aryl, heteroaryl group or linked biosubstrate (L-BS), wherein the linker (L) is alkyl or PEG; wherein SG1 to SG4 independently represent alkyl, water-soluble group or L-BS; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen or boron carbonyl, aryl, heteroaryl group or L-BS; and wherein x, y and z are integers from 0 to 100, provided that (1) the ratio of BS to polymer is 0.2-3, (2) the ratio of x / (y+z) is >1, and (3) the sum of x+y+z is >10.

[0200] In a preferred embodiment, this disclosure provides a polymer conjugate of formula I, wherein R1 to R6 independently represent hydrogen, methyl, or ethyl; wherein SG1 to SG4 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, aminoalkyl, or L-BS; wherein L is an alkyl chain or a PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, or boron carbonyl; and wherein x, y, and z are integers from 0 to 80, provided that (1) the ratio of BS to polymer is 1-2, (2) the ratio of x / (y+z) is >1, and (3) the sum of x+y+z is >20.

[0201] In another preferred embodiment, this disclosure provides a polymer conjugate of formula I, wherein R1 to R6 are hydrogen; wherein SG1 and SG2 are PEG; wherein SG3 to SG4 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, aminoalkyl, or L-BS; wherein L is an alkyl chain or a PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, or boron carbonyl; and wherein x, y, and z are integers from 0 to 80, provided that (1) the ratio of BS to polymer is 1-2, (2) the ratio of x / (y+z) is >1, and (3) the sum of x+y+z is >20.

[0202] In another preferred embodiment, this disclosure provides polymer conjugates of Formula I, wherein SG3 to SG4 independently represent PEG, alkyl, carboxyalkyl, or L-BS.

[0203] In another preferred embodiment, this disclosure provides polymer conjugates of Formula I, wherein SG3 to SG4 independently represent PEG, alkyl, aminoalkyl, or L-BS.

[0204] In another preferred embodiment, this disclosure provides polymer conjugates of Formula I, wherein SG1 and SG2 are independently PEG6 to PEG18.

[0205] In another preferred embodiment, this disclosure provides a polymer conjugate of formula I, wherein the ratio of BS to polymer is 1; and wherein the sum of x+y+z is 30-80.

[0206] In another preferred embodiment, this disclosure provides polymer conjugates of Formula I, wherein SG3 to SG4 independently represent PEG, methyl, carboxyalkyl, or L-BS.

[0207] In another preferred embodiment, this disclosure provides polymer conjugates of Formula I, wherein SG3 to SG4 independently represent PEG, methyl, aminoalkyl, or L-BS.

[0208] In another preferred embodiment, this disclosure provides a polymer conjugate of formula I, wherein R1 to R6 are hydrogen; wherein SG1 to SG4 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, or aminoalkyl; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, boron carbonyl, or L-BS; wherein L is an alkyl chain or a PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; and wherein x, y, and z are integers from 0 to 80, provided that (1) the ratio of BS to polymer is 1-2, (2) the ratio of x / (y+z) is >1, and (3) the sum of x+y+z is >20.

[0209] In another preferred embodiment, this disclosure provides a polymer conjugate of formula I, wherein the ratio of BS to polymer is 1; and wherein the sum of x+y+z is 30-80.

[0210] In another preferred embodiment, this disclosure provides polymer conjugates of Formula I, wherein HG1 and HG2 independently represent hydrogen, carboxyaryl, or L-BS.

[0211] In another preferred embodiment, this disclosure provides polymer conjugates of Formula I, wherein HG1 and HG2 independently represent halogen, boron carbonyl, carboxyaryl, or L-BS.

[0212] A preferred embodiment is the polymer conjugate of formula II:

[0213]

[0214] Formula II

[0215] The polymer conjugate comprises four monomer units randomly distributed along the polymer backbone; wherein the fluorophore (FP) is a fluorescent dye with a maximum absorption wavelength greater than 370 nm, a maximum emission wavelength greater than 400 nm, and a fluorescence quantum yield greater than 5%; wherein A = NR 10 PR 11 O=PR 11 O=P-OR 11 R 11 -CR 12 R 11 -Si-R 12 R 11 -SR 12 O=SR 11 O=S(O)-R 11 R1 to R12 independently represent hydrogen, alkyl, PEG, aryl, heteroaryl groups or L-BS; where L is alkyl, PEG or FP; where SG1 to SG7 independently represent alkyl, water-soluble groups or L-BS; where HG1 and HG2 independently represent hydrogen, halogen, boron carbonyl, alkyl, aryl, heteroaryl groups or L-BS; where w, x and z are integers from 0 to 100; and where y is an integer from 1 to 20, provided that (1) the ratio of BS to polymer is 0.2-3, (2) the ratio of w / (x+y+z) is >1, and (3) the sum of w+x+y+z is >10.

[0216] In another preferred embodiment, this disclosure provides a polymer conjugate of formula II, wherein FP is fluorescein, rhodamine, p-methylaminophenol, anthocyanin, BODIPY, squaric acid cyanide, coumarin, perylene diimide, diketopyrrolopyrrole, porphyrin, or phthalocyanine; wherein R1 to R6 independently represent hydrogen, methyl, or ethyl; wherein SG1 to SG7 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, aminoalkyl, or L-BS; wherein L is an alkyl chain, FP, or PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, or boron carbonyl; wherein w, x, and z are integers from 0 to 80; and wherein y is an integer from 1 to 10, provided that (1) the ratio of BS to polymer is 1-2, (2) the ratio of w / (x+y+z) is >1, and (3) the sum of w+x+Y+z is >20.

[0217] In another preferred embodiment, this disclosure provides polymeric conjugates of formula II, wherein R1 to R6 are hydrogen.

[0218] In another preferred embodiment, this disclosure provides a polymer conjugate of formula II, wherein the ratio of BS to polymer is 1; and wherein the sum of w+x+y+z is 30-80.

[0219] In another preferred embodiment, this disclosure provides a polymer conjugate of Formula II, wherein FP is fluorescein, rhodamine, anthocyanin, BODIPY, squaric acid cyanine, perylene diimide, or phthalocyanine.

[0220] In another preferred embodiment, this disclosure provides a polymer conjugate of formula II, wherein FP is rhodamine.

[0221] In another preferred embodiment, this disclosure provides a polymer conjugate of formula II, wherein FP is anthocyanin.

[0222] In another preferred embodiment, this disclosure provides a polymer conjugate of formula II, wherein FP is fluorescein, rhodamine, p-methylaminophenol, anthocyanin, BODIPY, squaric acid cyanide, coumarin, perylene diimide, diketopyrrolopyrrole, porphyrin, or phthalocyanine; wherein R1 to R6 are hydrogen; wherein SG1 to SG7 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, or aminoalkyl; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, boron carbonyl, or L-BS; wherein L is an alkyl chain, FP, or PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; wherein w, x, and z are integers from 0 to 80; and wherein y is an integer from 1 to 10, provided that (1) the ratio of BS to polymer is 1-2, (2) the ratio of w / (x+y+z) is >1, and (3) the sum of w+x+y+z is >20.

[0223] In another preferred embodiment, this disclosure provides a polymer conjugate of formula II, wherein the ratio of BS to polymer is 1; and wherein the sum of x+y+z is 30-80.

[0224] In another preferred embodiment, this disclosure provides polymer conjugates of formula II, wherein HG1 and HG2 independently represent hydrogen, carboxyaryl, or L-BS.

[0225] In another preferred embodiment, this disclosure provides polymer conjugates of formula II, wherein HG1 and HG2 independently represent halogen, boron carbonyl, carboxyaryl, or L-BS.

[0226] A preferred embodiment is a polymer conjugate of formula III:

[0227]

[0228] Formula III

[0229] The polymer conjugate comprises four monomer units randomly distributed along the polymer backbone; wherein the fluorophore (FP) is a fluorescent dye with a maximum absorption wavelength greater than 370 nm, a maximum emission wavelength greater than 400 nm, and a fluorescence quantum yield greater than 5%; wherein A = NR 10 PR 11 O=PR 11 O=P-OR 11 R 11 -CR 12 R 11 -Si-R 12 R 11 -SR 12 O=SR 11 O=S(O)-R 11 R1 to R12 independently represent hydrogen, alkyl, PEG, aryl, heteroaryl groups or L-BS; where L is alkyl, PEG or FP; where SG1 to SG6 independently represent alkyl, water-soluble groups or L-BS; where HG1 and HG2 independently represent hydrogen, halogen, boron carbonyl, alkyl, aryl, heteroaryl groups or L-BS; where w, x and z are integers from 0 to 100; and where y is an integer from 1 to 20, provided that (1) the ratio of BS to polymer is 0.2-3, (2) the ratio of w / (x+z) is >1, and (3) the sum of w+x+z is >10.

[0230] In another preferred embodiment, this disclosure provides a polymer conjugate of formula III, wherein FP is fluorescein, rhodamine, p-methylaminophenol, anthocyanin, BODIPY, squaric acid cyanide, coumarin, perylene diimide, diketopyrrolopyrrole, porphyrin, or phthalocyanine; wherein R1 to R6 independently represent hydrogen, methyl, or ethyl; wherein SG1 to SG6 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, aminoalkyl, or L-BS; wherein L is an alkyl chain, FP, or PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, or boron carbonyl; wherein w, x, and z are integers from 0 to 80; and wherein y is an integer from 1 to 10, provided that (1) the ratio of BS to polymer is 1-2, (2) the ratio of w / (x+z) is >1, and (3) the sum of w+x+z is >20.

[0231] In another preferred embodiment, this disclosure provides polymeric conjugates of formula III, wherein R1 to R6 are hydrogen.

[0232] In another preferred embodiment, this disclosure provides a polymer conjugate of formula III, wherein the ratio of BS to polymer is 1; wherein the sum of w+x+z is 30-80.

[0233] In another preferred embodiment, this disclosure provides a polymer conjugate of Formula III, wherein FP is fluorescein, rhodamine, anthocyanin, BODIPY, squaric acid cyanine, perylene diimide, or phthalocyanine.

[0234] In another preferred embodiment, this disclosure provides a polymeric conjugate of formula III, wherein FP is rhodamine.

[0235] In another preferred embodiment, this disclosure provides a polymer conjugate of formula III, wherein FP is anthocyanin.

[0236] In another preferred embodiment, this disclosure provides a polymer conjugate of formula III, wherein FP is fluorescein, rhodamine, p-methylaminophenol, anthocyanin, BODIPY, squaric acid cyanide, coumarin, perylene diimide, diketopyrrolopyrrole, porphyrin, or phthalocyanine; wherein R1 to R6 are hydrogen; wherein SG1 to SG6 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, or aminoalkyl; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, boron carbonyl, or L-BS; wherein L is an alkyl chain, FP, or PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; wherein w, x, and z are integers from 0 to 80; and wherein y is an integer from 1 to 10, provided that (1) the ratio of BS to polymer is 1-2, (2) the ratio of w / (x+z) is >1, and (3) the sum of w+x+z is >20.

[0237] In another preferred embodiment, this disclosure provides a polymer conjugate of formula III, wherein the ratio of BS to polymer is 1; and wherein the sum of x+z is 30-80.

[0238] In another preferred embodiment, this disclosure provides polymer conjugates of Formula III, wherein HG1 and HG2 independently represent hydrogen, carboxyaryl, or L-BS.

[0239] In another preferred embodiment, this disclosure provides polymer conjugates of Formula III, wherein HG1 and HG2 independently represent halogen, boron carbonyl, carboxyaryl, or L-BS.

[0240] A preferred embodiment is a polymer conjugate of formula IV:

[0241]

[0242] Formula IV

[0243] The polymer conjugate comprises three monomer units randomly distributed along the polymer backbone; wherein A1, A2, or A3 = NR. 10 PR11 O=PR 11 O=P-OR 11 R 11 -CR 12 R 11 -Si-R 12 R 11 -SR 12 O=SR 11 O=S(O)-R 11 R1 to R12 independently represent hydrogen, alkyl, polyethylene glycol (PEG), aryl, heteroaryl groups or linked biological substrates (L-BS); wherein the linker (L) is alkyl or PEG; wherein SG1 to SG6 independently represent alkyl, water-soluble groups or L-BS; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen or boron carbonyl, aryl, heteroaryl groups or L-BS; and wherein x, y and z are integers from 0 to 100, provided that (1) the ratio of BS to polymer is 0.2 to 3, and (2) the sum of x+y+z is >10.

[0244] In another preferred embodiment, this disclosure provides a polymer conjugate of formula IV, wherein R1 to R4 independently represent hydrogen, methyl, or ethyl; wherein SG1 to SG6 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, aminoalkyl, or L-BS; wherein L is an alkyl chain or a PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, or boron carbonyl; and wherein x, y, and z are integers from 0 to 80, provided that (1) the ratio of BS to polymer is 1 to 2, and (2) the sum of x+y+z is >20.

[0245] In another preferred embodiment, this disclosure provides a polymer conjugate of formula IV, wherein R1 to R4 are hydrogen; wherein SG1 and SG2 are PEG; wherein SG3 to SG6 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, aminoalkyl, or L-BS; wherein L is an alkyl chain or a PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, or boron carbonyl; and wherein x, y, and z are integers from 0 to 80, provided that (1) the ratio of BS to polymer is 1 to 2, and (2) the sum of x+y+z is >20.

[0246] In another preferred embodiment, this disclosure provides polymer conjugates of formula IV, wherein SG3 to SG6 independently represent PEG, alkyl, carboxyalkyl, or L-BS.

[0247] In another preferred embodiment, this disclosure provides polymer conjugates of formula IV, wherein SG3 to SG6 independently represent PEG, alkyl, aminoalkyl, or L-BS.

[0248] In another preferred embodiment, this disclosure provides a polymer conjugate of formula IV, wherein the ratio of BS to polymer is 1; and wherein the sum of x+y+z is 30-80.

[0249] In another preferred embodiment, this disclosure provides polymer conjugates of formula IV, wherein SG3 to SG6 independently represent PEG, methyl, carboxyalkyl, or L-BS.

[0250] In another preferred embodiment, this disclosure provides polymer conjugates of formula IV, wherein SG3 to SG6 independently represent PEG, methyl, aminoalkyl, or L-BS.

[0251] In another preferred embodiment, this disclosure provides a polymer conjugate of formula IV, wherein R1 to R4 are hydrogen; wherein SG1 to SG6 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, or aminoalkyl; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, boron carbonyl, or L-BS; wherein L is an alkyl chain or a PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; and wherein x, y, and z are integers from 0 to 80, provided that (1) the ratio of BS to polymer is 1-2, (2) the ratio of x / (y+z) is >1, and (3) the sum of x+y+z is >20.

[0252] In another preferred embodiment, this disclosure provides a polymer conjugate of formula IV, wherein the ratio of BS to polymer is 1; and wherein the sum of x+y+z is 30-80.

[0253] In another preferred embodiment, this disclosure provides polymeric conjugates of formula IV, wherein HG1 and HG2 independently represent hydrogen, carboxyaryl, or L-BS.

[0254] In another preferred embodiment, this disclosure provides polymer conjugates of formula IV, wherein HG1 and HG2 independently represent halogen, boron carbonyl, carboxyaryl, or L-BS.

[0255] A preferred embodiment is a polymer conjugate of formula V:

[0256]

[0257] Formula V

[0258] The polymer conjugate comprises four monomer units randomly distributed along the polymer backbone; wherein the fluorophore (FP) is a fluorescent dye with a maximum absorption wavelength greater than 370 nm and a maximum emission wavelength greater than 400 nm, and a fluorescence quantum yield greater than 5%; wherein A1, A2, A3, or A4 = NR. 10 PR 11 O=PR 11 O=P-OR 11 R 11 -CR 12 R 11 -Si-R 12 R 11 -SR 12 O=SR 11 O=S(O)-R 11 R1 to R12 independently represent hydrogen, alkyl, PEG, aryl, heteroaryl groups or L-BS; where L is alkyl, PEG or FP; where SG1 to SG7 independently represent alkyl, water-soluble groups or L-BS; where HG1 and HG2 independently represent hydrogen, halogen, boron carbonyl, alkyl, aryl, heteroaryl groups or L-BS; where w, x and z are integers from 0 to 100; and where y is an integer from 1 to 20, provided that (1) the ratio of BS to polymer is 0.2-3, and (2) the sum of w+x+y+z is >10.

[0259] In another preferred embodiment, this disclosure provides a polymer conjugate of formula V, wherein FP is fluorescein, rhodamine, p-methylaminophenol, anthocyanin, BODIPY, squaric acid cyanide, coumarin, perylene diimide, diketopyrrolopyrrole, porphyrin, or phthalocyanine; wherein R1 to R4 independently represent hydrogen, methyl, or ethyl; wherein SG1 to SG7 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, aminoalkyl, or L-BS; wherein L is an alkyl chain, FP, or PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, or boron carbonyl; wherein w, x, and z are integers from 0 to 80; and wherein y is an integer from 1 to 10, provided that (1) the ratio of BS to polymer is 1-2, and (2) the sum of w+x+y+z is >20.

[0260] In another preferred embodiment, this disclosure provides polymeric conjugates of formula V, wherein R1 to R4 are hydrogen.

[0261] In another preferred embodiment, this disclosure provides a polymer conjugate of formula V, wherein the ratio of BS to polymer is 1; and wherein the sum of w+x+y+z is 30-80.

[0262] In another preferred embodiment, this disclosure provides a polymer conjugate of formula V, wherein FP is fluorescein, rhodamine, anthocyanin, BODIPY, squaric acid cyanine, perylene diimide, or phthalocyanine.

[0263] In another preferred embodiment, this disclosure provides a polymer conjugate of formula V, wherein FP is rhodamine.

[0264] In another preferred embodiment, this disclosure provides a polymer conjugate of formula V, wherein FP is anthocyanin.

[0265] In another preferred embodiment, this disclosure provides a polymer conjugate of formula V, wherein FP is fluorescein, rhodamine, p-aminophenol, anthocyanin, BODIPY, squaric acid cyanide, coumarin, perylene diimide, diketopyrrolopyrrole, porphyrin, or phthalocyanine; wherein R1 to R4 are hydrogen; wherein SG1 to SG7 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, or aminoalkyl; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen, boron carbonyl, or L-BS; wherein L is an alkyl chain, FP, or PEG chain; wherein BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; wherein w, x, and z are integers from 0 to 80; and wherein y is an integer from 1 to 10, provided that (1) the ratio of BS to polymer is 1-2, and (2) the sum of w+x+y+z is >20.

[0266] In another preferred embodiment, this disclosure provides a polymer conjugate of formula V, wherein the ratio of BS to polymer is 1; and wherein the sum of x+y+z is 30-80.

[0267] In another preferred embodiment, this disclosure provides polymer conjugates of formula V, wherein HG1 and HG2 independently represent hydrogen, carboxyaryl, or L-BS.

[0268] In another preferred embodiment, this disclosure provides polymer conjugates of formula V, wherein HG1 and HG2 independently represent halogen, boron carbonyl, carboxyaryl, or L-BS.

[0269] In another preferred embodiment, this disclosure also provides a method for detecting an analyte in a sample, the method comprising:

[0270] a) Combining the sample with the detection reagent under conditions where the detection reagent comprising a polymer conjugate having the structure of Formula I will bind the analyte; and

[0271] b) Analytes bound to the reagents are detected by fluorescence detection.

[0272] The polymer conjugate comprises three monomer units randomly distributed along the polymer backbone; wherein R1 to R6 independently represent hydrogen, alkyl, polyethylene glycol (PEG), aryl, heteroaryl groups or linked biosubstrate (L-BS); wherein the linker (L) is alkyl or PEG; wherein SG1 to SG6 independently represent alkyl, water-soluble groups or L-BS; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen or boron carbonyl, aryl, heteroaryl groups or L-BS; and wherein x, y and z are integers from 0 to 100, provided that (1) the ratio of BS to polymer is 0.2-3, (2) the ratio of x / (y+z) is >1, and (3) the sum of x+y+z is >10.

[0273] In another preferred embodiment, this disclosure provides a method for detecting an analyte in a sample, the method comprising:

[0274] a) Combining the sample with the detection reagent under conditions where the detection reagent comprising a polymer conjugate having the structure of Formula II will bind the analyte; and

[0275] b) Analytes bound to the reagents are detected by fluorescence detection.

[0276] The polymer conjugate shown contains four monomer units randomly distributed along the polymer backbone; wherein the fluorophore (FP) is a fluorescent dye with a maximum absorption wavelength greater than 370 nm and a maximum emission wavelength greater than 400 nm and a fluorescence quantum yield greater than 5%; wherein R1 to R6 independently represent hydrogen, alkyl, PEG, aryl, heteroaryl groups or L-BS; wherein L is alkyl, PEG or FP; wherein SG1 to SG7 independently represent alkyl, water-soluble groups or L-BS; wherein HG1 and HG2 independently represent hydrogen, halogen, boron carbonyl, alkyl, aryl, heteroaryl groups or L-BS; wherein w, x and z are integers from 0 to 100; and wherein y is an integer from 1 to 20, provided that (1) the ratio of BS / polymer is 0.2-3, (2) the ratio of w / (x+y+z) is >1, and (3) the sum of w+x+y+z is >10.

[0277] In another preferred embodiment, this disclosure also provides a method for detecting an analyte in a sample, the method comprising:

[0278] a) Combining the sample with the detection reagent under conditions where the detection reagent comprising a polymer conjugate having the structure of Formula III will bind the analyte; and

[0279] b) Analytes bound to the reagents are detected by fluorescence detection.

[0280] The polymer conjugate comprises three monomer units randomly distributed along the polymer backbone; wherein R1 to R4 independently represent hydrogen, alkyl, polyethylene glycol (PEG), aryl, heteroaryl groups or linked biosubstrate (L-BS); wherein the linker (L) is alkyl or PEG; wherein SG1 to SG6 independently represent alkyl, water-soluble groups or L-BS; wherein HG1 and HG2 independently represent hydrogen, aryl, halogen or boron carbonyl, aryl, heteroaryl groups or L-BS; and wherein x, y and z are integers from 0 to 100, provided that (1) the ratio of BS to polymer is 0.2-3, and (2) the sum of x+y+z is >10.

[0281] In another preferred embodiment, this disclosure provides a method for detecting an analyte in a sample, the method comprising:

[0282] a) Combining the sample with the detection reagent under conditions where the detection reagent comprising a polymer conjugate having the structure of Formula IV will bind the analyte; and

[0283] b) Analytes bound to the reagents are detected by fluorescence detection.

[0284] The polymer conjugate shown contains four monomer units randomly distributed along the polymer backbone; wherein the fluorophore (FP) is a fluorescent dye with a maximum absorption wavelength greater than 370 nm and a maximum emission wavelength greater than 400 nm and a fluorescence quantum yield greater than 5%; wherein R1 to R4 independently represent hydrogen, alkyl, PEG, aryl, heteroaryl groups or L-BS; wherein L is alkyl, PEG or FP; wherein SG1 to SG7 independently represent alkyl, water-soluble groups or L-BS; wherein HG1 and HG2 independently represent hydrogen, halogen, boron carbonyl, alkyl, aryl, heteroaryl groups or L-BS; wherein x, y and z are integers from 0 to 100; and wherein y is an integer from 1 to 20, provided that (1) the ratio of BS to polymer is 0.2-3, and (2) the sum of x+y+z is >10.

[0285] In another preferred embodiment, this disclosure provides a method for detecting an analyte in a sample, the method comprising:

[0286] a) Combining the sample with the detection reagent under conditions where the detection reagent comprising a polymer conjugate having the structure of Formula V will bind the analyte; and

[0287] b) Analytes bound to the reagents are detected by fluorescence detection.

[0288] The polymer conjugate shown contains four monomer units randomly distributed along the polymer backbone; wherein the fluorophore (FP) is a fluorescent dye with a maximum absorption wavelength greater than 370 nm and a maximum emission wavelength greater than 400 nm and a fluorescence quantum yield greater than 5%; wherein R1 to R4 independently represent hydrogen, alkyl, PEG, aryl, heteroaryl groups or L-BS; wherein L is alkyl, PEG or FP; wherein SG1 to SG7 independently represent alkyl, water-soluble groups or L-BS; wherein HG1 and HG2 independently represent hydrogen, halogen, boron carbonyl, alkyl, aryl, heteroaryl groups or L-BS; wherein w, x and z are integers from 0 to 100; and wherein y is an integer from 1 to 20, provided that (1) the ratio of BS to polymer is 0.2-3 and (2) the sum of w+x+y+z is >10.

[0289] In another preferred embodiment, this disclosure provides polymer conjugates of formula I, II, III, IV or V, wherein BS is an antibody.

[0290] In another preferred embodiment, this disclosure provides polymer conjugates of formula I, II, III, IV or V, wherein BS is an anti-digoxin antibody.

[0291] In yet another preferred embodiment, the present invention discloses polymer conjugates of Formula I, Formula II, Formula III, Formula IV or Formula V, wherein BS is a goat anti-mouse IgG antibody, a goat anti-rabbit IgG antibody, a goat anti-human IgG antibody, a donkey anti-mouse IgG antibody, a donkey anti-rabbit IgG antibody, a donkey anti-human IgG antibody, a chicken anti-mouse IgG antibody, a chicken anti-rabbit IgG antibody or a chicken anti-human IgG antibody.

[0292] In another preferred embodiment, this disclosure provides polymer conjugates of formula I, II, III, IV or V, wherein BS is avidin, streptavidin, neutral avidin or avidin.

[0293] In another preferred embodiment, this disclosure provides polymer conjugates of formula I, II, III, IV or V, wherein the analyte is a target protein expressed on a cell surface.

[0294] In another preferred embodiment, FP is a fluorescent dye selected from Table 1; R1 to R4 are hydrogen; SG1 to SG7 independently represent PEG, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, or aminoalkyl; HG1 and HG2 independently represent hydrogen, aryl, halogen, boron carbonyl, or L-BS; L is an alkyl chain, FP, or PEG chain; BS is an antibody, peptide, protein, oligonucleotide, nucleic acid, or carbohydrate; w, x, and z are integers from 0 to 80; and y is an integer from 1 to 10, provided that (1) the ratio of BS to polymer is 1, and (2) the sum of w+x+y+z is 30-80.

[0295] The fluorophores (FPs) attached to the polymers of this invention are typically fluorescent dyes with a maximum absorption wavelength greater than 370 nm and a maximum emission wavelength greater than 400 nm, and a fluorescence quantum yield greater than 10%. They are typically selected from coumarin, fluorescein, rhodamine, anthocyanins, bodily oil, or other polycyclic aromatic compounds. Many of them are commercially available, as some examples selectively listed in Table 1.

[0296] Table 1. Typical fluorophores that can be linked to fluorene polymers

[0297]

[0298]

[0299]

[0300]

[0301]

[0302] Many embodiments of the compounds of the present invention have a total electronic charge. It should be understood that when such electronic charges are present, they are balanced by the presence of appropriate counterions, which may or may not be explicitly identified. Preferred biocompatible counterions in some applications are non-toxic in biological applications and have no materially harmful effects on biomolecules. When the compounds of the present invention are positively charged, the counterions are generally selected from, but not limited to, anions of chloride, bromide, iodide, sulfate, alkyl sulfonate, aryl sulfonate, phosphate, perchlorate, tetrafluoroborate, tetraarylboron, nitrate, and aromatic or aliphatic carboxylic acids. When the compounds of the present invention are negatively charged, the counterions are generally selected from, but not limited to, alkali metal ions, alkaline earth metal ions, transition metal ions, ammonium ions, or substituted ammonium ions or pyridine ions. Preferably, any necessary counterions are biocompatible, non-toxic in use, and have no materially harmful effects on biomolecules. Counterions can be readily modified by methods well known in the art, such as ion exchange chromatography or selective precipitation.

[0303] It should be understood that the polymer conjugates of the present invention have been plotted as one or another specific electronic resonance structure. Each aspect of the invention is equally applicable to polymer conjugates plotted as other permissible resonance structures, since the electronic charge on the subject polymer conjugate is delocalized throughout the polymer conjugate itself.

[0304] In another preferred embodiment of the invention, the polymer conjugate contains at least one L-BS or L-FP-BS, wherein the BS is attached to the polymer via a known reaction as listed as an example in Table 2. In some embodiments, the covalent linking the polymer to the BS contains a plurality of inserted atoms acting as connectors (L). The polymer can be used to label a variety of biological, organic, or inorganic substances that contain or are modified to contain functional groups with suitable reactivity, resulting in the chemical attachment of the conjugate.

[0305] Table 2. Examples of covalently linked functional groups used to prepare L-BS or FP-BS

[0306]

[0307]

[0308] The choice of linker for attaching a polymer to a biological substrate typically depends on the functional groups on the substrate and the type or length of the desired covalent link. Types of functional groups commonly found on organic or inorganic biological substrates include, but are not limited to, amines, amides, thiols, alcohols, phenols, aldehydes, ketones, phosphonates, imidazoles, hydrazines, hydroxylamines, disubstituted amines, halogen groups, epoxy groups, carboxylic esters, sulfonates, purines, pyrimidines, carboxylic acids, olefins, azides, alkynes, tetrazines, or combinations thereof. A single type of reactive site may be available on the biological substrate (typically for polysaccharides), or multiple sites may be present (e.g., amines, thiols, alcohols, phenols), as is typical for proteins. The conjugated biological substrate can be conjugated to more than one polymer conjugate, which may be the same or different, or to another biological substrate modified with a hapten, such as biotin. Alternatively, multiple substrates can be conjugated to a single polymer. Although some selectivity can be obtained by carefully controlling the reaction conditions, the selectivity of the label is best obtained by selecting an appropriate reactive polymer conjugate.

[0309] Typically, the polymer will react with amines, thiols, alcohols, aldehydes, or ketones. Preferably, the polymer reacts with amine, thiol functional groups, or click-reactive groups. In some embodiments, the polymer reacts with acrylamide, reactive amines (including cadaverine or ethylenediamine), activated esters of carboxylic acids (typically succinimides of carboxylic acids), acyl azides, acyl nitriles, aldehydes, alkyl halides, acid anhydrides, aniline, aryl halides, azides, aziridines, borates, carboxylic acids, diazonides, haloacetamides, halotriazins, hydrazides (including acyl hydrazides), imino esters, isocyanates, isothiocyanates, maleimides, phosphoramides, reactive platinum complexes, sulfonyl halides, tetrazides, azides, alkynes, or thiols. "Reactive platinum complex" specifically means a chemically reactive platinum complex, such as those described in U.S. Patent Nos. 5,580,990, 5,714,327, and 5,985,566.

[0310] When the polymer is photoactivated, such as an azide, diazirinyl, azidoaryl, or psoralen derivative, the polymer only becomes chemically active upon irradiation with light of an appropriate wavelength. When the polymer is an activated ester of a carboxylic acid, the reactive polymer is particularly suitable for preparing polymeric conjugates of proteins, nucleotides, oligonucleotides, or haptens. When the polymer is a maleimide or haloacetamide, the reactive polymer is particularly suitable for conjugation to thiol-containing biological substrates. When the polymer is an acylhydrazine, the reactive polymer is particularly suitable for conjugation to periodate-oxidized carbohydrates and glycoproteins, and is also a polar tracer for aldehyde immobilization via cell microinjection. When the polymer is click-reactive, it is particularly suitable for conjugation to complementary click-reactive substrates. Preferably, the polymer is a carboxylic acid, a succinimide ester of a carboxylic acid, a haloacetamide, a hydrazine, an isothiocyanate, a maleimide group, a fatty amine, a perfluorobenzamide group, an azidoperfluorobenzamide group, or psoralen. More preferably, the polymer is a succinimide ester of a carboxylic acid, a maleimide, an iodoacetamide, or a reactive platinum complex.

[0311] Based on the above properties, suitable reactive polymers of the present invention are selected for the preparation of desired polymer conjugates, whose advantageous properties make them suitable for a wide variety of applications. Particularly useful polymer conjugates include those in which the substrate is a peptide, nucleotide, antigen, steroid, vitamin, drug, hapten, metabolite, toxin, environmental pollutant, amino acid, protein, nucleic acid, nucleic acid polymer, carbohydrate, lipid, ionic complex moiety, glass, or non-biological polymer. Alternatively, the substrate may be a cell, cellular system, cell fragment or subcellular particle (e.g., among others), viral particle, bacterial particle, viral component, biological cell (such as animal cell, plant cell, bacteria, yeast, or protist) or cellular component. Reactive polymers typically label functional groups on the cell surface, cell membrane, organelles, or cytoplasm.

[0312] Typical substrates include amino acids, peptides, proteins, tyramine, polysaccharides, ionic complexes, nucleosides, nucleotides, oligonucleotides, nucleic acids, haptens, psoralen, drugs, hormones, lipids, lipid complexes, polymers, polymer microparticles, biological cells, or viruses. More typically, substrates are peptides, proteins, nucleotides, oligonucleotides, or nucleic acids. When the polymer conjugates of the present invention are conjugated to such biopolymers, more polymer can be incorporated into each molecule to enhance the fluorescence signal. For polymer-antibody conjugates, it is preferred that one polymer is conjugated to the antibody.

[0313] In one embodiment, the substrate is an amino acid (including phosphonates, carbohydrates, or C1 to C2 amino acids). 25The conjugated protein (amino acid protected or substituted with a carboxylic acid) or a polymer of amino acids, such as a peptide or protein. Preferred peptide conjugations contain at least five amino acids, more preferably five to 36 amino acids. Preferred peptides include, but are not limited to, neuropeptides, cytokines, toxins, protease substrates, and protein kinase substrates. Preferred protein conjugations include enzymes, antibodies, lectins, glycoproteins, histones, albumins, lipoproteins, avidin, streptavidin, other avidin, protein A, protein G, phycobiliproteins and other fluorescent proteins, hormones, toxins, chemokines, and growth factors. In a preferred aspect, the conjugated protein is a polymeric antibody conjugation.

[0314] In one aspect of the invention, the substrate is a conjugated biological substrate, which is an antibody (including intact antibodies, antibody fragments, and antibody serum, etc.), amino acids, angiostatin or endostatin, avidin or streptavidin, biotin (e.g., aminobiotin, biocytin, dethiobiotin, etc.), blood component proteins (e.g., albumin, fibrinogen, plasminogen, etc.), dextran, enzymes, enzyme inhibitors, IgG binding proteins (e.g., protein A, protein G, protein A / G, etc.), fluorescent proteins (e.g., phycobiliproteins, jellyfish luminescent proteins, green fluorescent protein, etc.), growth factors, hormones, lectins (e.g., wheat germ lectin, concanavalin A, etc.), lipopolysaccharides, metal-binding proteins (e.g., calmodulin, etc.), microorganisms or parts thereof (e.g. Examples of biological agents include bacteria, viruses, yeast, etc.), neuropeptides and other bioactive factors (e.g., corticomerol, deltropin, endorphins, endorphins, tumor necrosis factor, etc.), non-biological particles (e.g., ferrofluids, gold, polystyrene, etc.), nucleotides, oligonucleotides, peptide toxins (e.g., apamin, bungalowtoxin, phalloidin, etc.), phospholipid-binding proteins (e.g., annexin, etc.), small molecule drugs (e.g., methotrexate, etc.), structural proteins (e.g., actin, fibronectin, laminin, tubuloassociated proteins, tubulin, etc.), or tyramine.

[0315] In another preferred embodiment, the biological substrate is a nucleic acid base, nucleoside, nucleotide, or nucleic acid polymer, including those modified to have additional connectors or spacer regions for attaching the polymer conjugate of the present invention, such as alkynyl links (US Patent No. 5,047,519), aminoallyl links (US Patent No. 4,711,955), or heteroatom-substituted connectors (US Patent No. 5,684,142) or other links. In another preferred embodiment, the conjugated biological substrate is a nucleoside or nucleotide analog that links a purine or pyrimidine base to a phosphate or polyphosphate moiety via a non-cyclic spacer region. In another preferred embodiment, the polymer conjugate is conjugated to the carbohydrate moiety of a nucleotide or nucleoside, typically via hydroxyl conjugation, but additionally via thiol or amino groups (US Patent Nos. 5,659,025, 5,668,268, 5,679,785). Typically, the conjugated nucleotides are nucleoside triphosphates, deoxynucleoside triphosphates, or dideoxynucleoside triphosphates. Incorporation of a methylene moiety or nitrogen or sulfur heteroatom into the phosphate or polyphosphate moiety is also useful. Nonpurine and nonpyrimidine bases, such as 7-deazopurine (US Patent No. 6,150,510), and nucleic acids containing such bases can also be coupled to the polymer conjugates of this invention. Nucleic acid adducts prepared by reacting depurinated nucleic acids with amines, hydrazides, or hydroxylamine derivatives provide additional means for labeling and detecting nucleic acids, for example, "A method for detecting abasic sites in living cells: age-dependent changes in baseexcision repair." Atamna H, Cheung I, Ames BN.PROC.NATL.ACAD.SCI.USA 97,686-691 (2000).

[0316] Preferred nucleic acid polymer conjugates are labeled, single-stranded or multi-stranded, natural or synthetic DNA or RNA, DNA or RNA oligonucleotides, or DNA / RNA hybrids, or incorporating rare linkers (such as morpholine-derived phosphate esters) or peptide nucleic acids (such as N-(2-aminoethyl)glycine units). When the nucleic acid is a synthetic oligonucleotide, it typically contains fewer than 50 nucleotides, more commonly fewer than 25 nucleotides. Peptide nucleic acid (PNA) conjugates (Nielsen et al., U.S. Patent No. 5,539,082) are preferred for certain applications because they typically have a faster hybridization rate.

[0317] In one embodiment, the conjugated oligonucleotide of the present invention is an aptamer for a specific target molecule, such as a metabolite, polymer conjugate, hapten, or protein. That is, the oligonucleotide has been selected to preferentially bind to the target molecule. Methods for preparing and screening aptamers for a given target molecule have been previously described and are known in the art [e.g., U.S. Patent No. 5,567,588 (1996) to Gold].

[0318] In another preferred embodiment, the substrate is a carbohydrate, which is typically a polysaccharide such as dextran, heparin, glycogen, amylopectin, mannan, inulin, starch, agarose, and cellulose. Alternatively, the carbohydrate is a polysaccharide, specifically a lipopolysaccharide. Preferred polysaccharide conjugates are dextran or lipopolysaccharide conjugates.

[0319] Conjugates with ionic complexes are used as indicators for calcium, sodium, magnesium, zinc, potassium, or other biologically important metal ions. Preferred ionic complexing moieties are crown ethers (US Patent No. 5,405,975); derivatives of 1,2-bis-(2-aminophenoxyethane)-N,N,N',N'-tetraacetic acid (BAPTA chelating agents; US Patent Nos. 5,453,517, 5,516,911, and 5,049,673); derivatives of 2-carboxymethoxyaniline-N,N-diacetic acid (APTRA chelating agents; AM.J. PHYSIOL., 256, C540 (1989)); or metal ion chelating agents based on pyridine and phenanthroline (US Patent No. 5,648,270); or derivatives of hypotriacetic acid, see, for example, "Single-step synthesis and characterization of biotinylated nitrilotriacetic acid, a unique reagent for the detection of histidine-tagged proteins immobilized on nitrocellulose", McMahan SA and Burgess RR. ANAL.BIOCHEM., 236, 101-106 (1996). Preferably, the ionic complex is a crown ether chelating agent, a BAPTA chelating agent, an APTRA chelating agent, or a derivative of hypozinotriacetic acid.

[0320] Other conjugates of non-biological materials include polymer conjugates of: organic or inorganic polymers, polymer films, polymer wafers, polymer membranes, polymer particles or polymer microparticles (magnetic and non-magnetic microspheres); iron, gold or silver particles; conductive and non-conductive metals and non-metals; and glass and plastic surfaces and particles. Conjugates are optionally prepared by copolymerization of polymer conjugates containing suitable functional groups during polymer preparation, or by chemical modification of polymers containing functional groups with suitable chemical reactivity. Other types of reactions that can be used to prepare polymer conjugates include catalytic polymerization or copolymerization of olefins and reactions of dienes with dienophiles, transesterification, or amino exchange. In another preferred embodiment, the conjugated biological substrate is glass or silica, which can be formed into optical fibers or other structures.

[0321] In one embodiment, conjugates of biopolymers such as peptides, proteins, oligonucleotides, and nucleic acid polymers are also labeled with at least a second fluorescent dye conjugate (optionally an additional polymer conjugate of the present invention) to form energy transfer pairs. In some aspects of the invention, the labeled conjugates act as enzyme substrates, and enzymatic hydrolysis disrupts the energy transfer. In another preferred embodiment of the invention, the energy transfer pairs incorporated into the polymer conjugates of the present invention are conjugated to oligonucleotides exhibiting effective fluorescence quenching in their hairpin conformation, such as the so-called "molecular beacons" of Tyagi et al., NATURE BIOTECHNOLOGY, 16, 49 (1998).

[0322] The preparation of polymer conjugates using reactive polymer conjugates is well documented, for example in U.S. Patents 8,158,444, 8,455,613, 8,354,239, 8,362,193, and 8,575,303 to Gaylord et al., and WO 2013 / 101902 to Chiu et al. Other biological applications of polyfluorene polymers have been well documented by Thomas III et al. (Chem. Rev. 2007, 107, 1339); Zhu et al. (Chem. Rev. 2012, 112, 4687); and Zhu et al. (Chem. Soc. Rev., 2011, 40, 3509). Conjugates are typically produced by mixing a suitable reactive polymer and the biological substrate to be conjugated in a suitable solvent in which both are soluble. The polymer conjugates of this invention are readily soluble in aqueous solutions, which facilitates conjugation reactions with most biological materials. For those photoactivated reactive polymer conjugates, conjugation requires irradiation of the reaction mixture to activate the reactive polymer conjugate.

[0323] Synthesis of reactive polymers

[0324] The synthesis of the reactive polymers of this invention depends on the initial preparation of certain key intermediates, as shown in Figure 1. For simplicity, all but a few possible substituents are shown as hydrogen. During or after synthesis, these basic structures may optionally be further substituted to obtain the corresponding polymer conjugate substituents as defined above. It is recognized that there are many possible variations that can produce equivalent results.

[0325] Methods for synthesizing polymers containing a variety of reactive functional groups (such as those described in Table 2) are well documented in the art. Particularly useful are amine-reactive polymer conjugates, such as “activated esters” of carboxylic acids, which are typically synthesized by coupling the carboxylic acid with a relatively acidic “leaving group.” Other preferred amine-reactive groups include: sulfonyl halides, prepared from sulfonic acids using halogenating agents such as PCl5 or POCl3; halotriazines, prepared by reacting cyanogen trihalides with amines; and isocyanates or isothiocyanates, prepared from amines and phosgene or thiophosgene, respectively. Polymers containing azides, alkynes, and tetraazines are particularly suitable for conjugation to substrates modified with click-reactive groups, such as antibodies modified from activated esters containing click-reactive groups.

[0326] Polymers containing amines and hydrazides are particularly suitable for conjugation to carboxylic acids, aldehydes, and ketones. Most commonly, these are synthesized via the reaction of activated esters of carboxylic acids or sulfonyl halides with diamines (such as cadaverine) or with hydrazine. Alternatively, aromatic amines are typically synthesized via the chemical reduction of nitro aromatic compounds. Amines and hydrazines are particularly suitable precursors for the synthesis of thiol-reactive haloacetamides or maleimides using standard methods.

[0327] Application and usage methods

[0328] In one aspect of the invention, the polymer conjugates of the invention are used for direct staining or labeling of samples so that the samples can be identified or quantified. For example, such polymer conjugates can be added as part of a biotarget analyte assay, as a detectable tracer element in biological or non-biological fluids; or for purposes such as photodynamic therapy of tumors, wherein the polymer-conjugated sample is irradiated to selectively destroy tumor cells and tissues; or generally for photoablation of arterial plaques or cells by photosensitizing to generate singlet oxygen. In a preferred embodiment, the polymer conjugates are used to stain samples containing a ligand for which the conjugated biological substrate is a complementary member of a specific binding pair (e.g., Table 3).

[0329] Typically, samples are obtained directly from a liquid source, or as a wash from a solid material (organic or inorganic) or a growth medium (in which cells have already been introduced for culture), or as a buffer solution in which cells have already been placed for evaluation. When the sample contains cells, the cells may optionally be single cells (including microorganisms) or multicellular cells associated with other cells in a two-dimensional or three-dimensional layer, including multicellular organisms, embryos, tissues, biopsy tissues, filaments, biofilms, etc.

[0330] Alternatively, the sample is a solid, optionally a smear or scraping, or a residue removed from a liquid or vapor by filtration. In one aspect of the invention, the sample is obtained from a biological fluid, including isolated or unfiltered biological fluids such as urine, cerebrospinal fluid, blood, lymph, tissue homogenate, interstitial fluid, cell extract, mucus, saliva, sputum, feces, physiological secretions, or other similar fluids. Alternatively, the sample is obtained from an environmental source, such as soil, water, or air; or from an industrial source, such as from waste streams, water sources, supply lines, or production batches.

[0331] Table 3. Representative specific binding pairs

[0332]

[0333] IgG is an immunoglobulin; aDNA and aRNA are antisense (complementary) strands used for hybridization.

[0334] In yet another embodiment, the sample is situated on or within a solid or semi-solid matrix. In one aspect of the invention, the matrix is ​​a membrane. In another aspect, the matrix is ​​an electrophoretic gel, such as those used for the separation and characterization of nucleic acids or proteins, or a blot prepared by transfer from an electrophoretic gel onto a membrane. In yet another aspect, the matrix is ​​a silicon wafer or glass slide, and the analyte of interest has been immobilized on a chip or glass slide in an array (e.g., the sample comprises a protein or nucleic acid polymer in a microarray). In yet another aspect, the matrix is ​​a microplate or microfluidic chip, and the sample is analyzed by automated methods, typically through various high-throughput screening methods, such as drug screening methods.

[0335] The polymer conjugates of the present invention are typically utilized by combining the polymer conjugates of the present invention, as described above, with a sample of interest under conditions selected to produce a detectable optical response. The term "polymer conjugate" is used herein to refer to all aspects of the claimed polymer conjugates. The polymer conjugates typically form a covalent association or complex with elements of the sample, or simply exist within the boundaries of the sample or a portion thereof. The sample is then irradiated with a wavelength selected to elicit an optical response. Typically, the sample is stained to determine specific characteristics of the sample by further comparing the optical response with a standard or expected response.

[0336] Detectable optical response refers to a change or occurrence of an optical signal that can be observed or detected by an instrument. Typically, a detectable response is a change in fluorescence, such as changes in fluorescence intensity, excitation or emission wavelength distribution, fluorescence lifetime, fluorescence polarization, or combinations thereof. The degree and / or location of staining, compared to a standard or expected response, indicates whether the sample possesses a given characteristic and to what extent it possesses that characteristic.

[0337] For biological applications, the polymer conjugates of this invention are typically used in the form of aqueous solutions (primarily aqueous or water-miscible solutions) prepared according to methods well known in the art. The exact concentration of the polymer conjugate depends on the experimental conditions and desired results. Optimal concentrations are determined through systematic variation until satisfactory results with minimal background fluorescence are obtained.

[0338] Polymer conjugates are most advantageous for staining samples containing biological components. Samples may contain heterogeneous mixtures of components (including intact cells, cell extracts, bacteria, viruses, organelles, and mixtures thereof), or single components or a homogeneous set of components (e.g., natural or synthetic amino acid, nucleic acid, or carbohydrate polymers, or lipid membrane complexes). These polymer conjugates are generally non-toxic to living cells and other biological components within the range of concentrations used.

[0339] Polymer conjugates are incorporated into the sample in any manner that facilitates contact between the polymer conjugate and the sample component of interest. Typically, only the polymer conjugate or a solution containing the polymer conjugate is added to the sample. Various protocols for cell staining with polymer conjugates can be employed, such as antibody-polymer conjugate staining for flow cytometry. Such protocols include intracellular staining of the sample for intracellular target antigens of interest using polymer conjugates. Membrane permeabilization treatments, such as electroporation, shock treatment, or high extracellular ATP, can be used to introduce selected polymer conjugates into cells. Alternatively, selected polymer conjugates can be physically inserted into cells, for example, via pressure microinjection, scratch loading, patch-clamp methods, or phagocytosis.

[0340] Polymer conjugates incorporating fatty amine or hydrazine residues can be microinjected into cells, where they can be immobilized in place using aldehyde immobilizers such as formaldehyde or glutaraldehyde. This immobilization capability allows such polymer conjugates to be used for intracellular applications, such as neuronal tracing.

[0341] Polymer conjugates with lipophilic substituents, such as phospholipids, are non-covalently incorporated into lipid assemblies, for example, as probes for membrane structures; or incorporated into liposomes, lipoproteins, membranes, plastics, lipophilic microspheres, or similar materials; or used for tracer purposes. Lipophilic polymer conjugates can also be used as fluorescent probes for membrane structures.

[0342] Using polymer conjugates to label active sites on cell surfaces, in cell membranes, or in intracellular compartments (such as organelles) or in the cytoplasm of cells allows for the determination of their presence or quantity in a sample, their accessibility, or their spatial and temporal distribution. Photoreactive polymer conjugates can similarly be used for the photolabeling of components of the outer membrane of biological cells, or as photofixable polar tracers for cells.

[0343] Optionally, the sample is washed after staining to remove residual, excess, or unbound polymer conjugates. During staining, the sample is optionally combined with one or more other solutions, including a washing solution, a permeation and / or fixation solution, and a solution containing an additional detection reagent. According to methods well known in the art, the additional detection reagent typically produces a detectable response due to the presence of specific cellular components, intracellular biological substrates, or cellular conditions. Multicolor application is possible when the additional detection reagent has or produces a product with spectral properties different from the subject polymer conjugate. This is particularly useful when the additional detection reagent is a polymer conjugate or polymer conjugate-conjugate of the present invention, which has spectral properties detectably distinct from the stained polymer conjugate.

[0344] The polymer conjugates of the present invention are used according to methods widely known in the art; for example, antibody conjugates are used in microscopy and immunofluorescence assays; and nucleotide or oligonucleotide conjugates are used in nucleic acid hybridization assays and nucleic acid sequencing (e.g., U.S. Patent No. 5,332,666 (1994) to Prober et al.; U.S. Patent No. 5,171,534 (1992) to Smith et al.; U.S. Patent No. 4,997,928 (1991) to Hobbs et al.; and WO Appl. 94 / 05688 to Menchen et al.). The polymer conjugate-conjugate combinations of the various independent polymer conjugates of the present invention have the utility of multicolor applications.

[0345] At any time after or during staining, the sample is irradiated with light at a selected wavelength to provide a detectable optical response, and observed using a tool for detecting the optical response. Devices that can be used to irradiate the polymer conjugates of the present invention include, but are not limited to, handheld UV lamps, mercury arc lamps, xenon lamps, lasers, and laser diodes. These irradiation sources are optionally integrated into laser scanners, fluorescence microplate readers, standard or miniature fluorometers, or chromatographic detectors. A preferred embodiment of the invention uses polymer conjugates excitable at or near a wavelength of 405 nm.

[0346] Optical responses may be detected optionally by visual inspection or by using any of the following devices: CCD camera, video camera, photographic film, laser scanning device, fluorometer, photodiode, quantum counter, epifluorescence microscope, scanning microscope, flow cytometer, fluorescence microplate reader, or by means of amplifying the signal, such as a photomultiplier tube. When examining samples using flow cytometry, the examination may optionally include sorting portions of the sample based on their fluorescence responses.

[0347] One aspect of the invention is the formulation of a kit that facilitates various assays using any polymer conjugate of the invention as described above. The kits of the invention typically contain the fluorescent polymer conjugate of the invention, wherein the conjugated biological substrate is a specific binding pair member, or a nucleoside, nucleotide, oligonucleotide, nucleic acid polymer, peptide, or protein. The kit optionally also contains one or more buffers, typically in aqueous solution. The kits of the invention optionally also contain additional detection reagents, purification media for purifying the resulting labeled biological substrate, luminescent standards, enzymes, enzyme inhibitors, organic solvents, or instructions for performing the assays of the invention.

[0348] Example

[0349] Examples of synthetic strategies for selected polymer conjugates of the present invention are provided in the following examples, along with their characterization, synthetic precursors, conjugates, and methods of use. Further modifications and variations will be apparent to those skilled in the art. The following examples are given to illustrate the practice of the invention. They are not intended to limit or define the entire scope of the invention. It should be understood that the invention is not limited to the specific aspects described, as these aspects can vary. It should also be understood that the terminology used herein is for the purpose of describing specific aspects only and is not intended to be restrictive, as the scope of the invention will be defined only by the appended claims. When providing ranges of values, it should be understood that, unless the context clearly indicates otherwise, every intermediary value (to one-tenth of the lower limit unit) between the upper and lower limits of the range, as well as any other specified value or intermediary value within the specified range, is covered within the scope of the invention. The upper and lower limits of these smaller ranges may be independently included within the smaller range and also covered within the invention, conditional on any explicitly excluded limit value within the specified range. When a specified range includes one or two limits, the range excluding any one or both of those included limits is also included in the invention.

[0350] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar to or equivalent to those described herein may also be used in the practice or experimentation of this invention, representative illustrative methods and materials are described hereafter.

[0351] Example 1. Preparation of Compound 2

[0352] 1 2

[0354] Compound 1 (36.6 g, Tianjin Biolite) was suspended in 400 mL MeCN. Allylamine (30 mL, 4 equivalents) was rapidly added. The mixture was stirred at room temperature for 40 minutes and concentrated. 10 g NaHCO3 and brine were added to the residue, and the mixture was extracted three times with DCM. The organic layer was collected, dried over Na2SO4, concentrated, and purified by rapid silica gel chromatography to give product 2 (21.75 g) as a yellow solid.

[0355] Example 2. Preparation of Compound 3

[0356] twenty three

[0358] Compound 2 (4.7 g) was added to a solution of KOH (3 g) in diethylene glycol (60 mL). The mixture was heated at 180 °C for 1 hour, then cooled to room temperature and diluted with water. The precipitated solid was collected by filtration and purified by silica gel chromatography to give product 3 (3.75 g) as a pale yellow solid.

[0359] Preparation of Compound 4 in Example 3

[0360] 3 4

[0362] In an ice bath, toluene (50 mL) and 50% NaOH (28 mL) were added to a mixture of compound 3 (4.94 g) and TBAB (0.644 g, 0.1 equivalent). The mixture was stirred at room temperature under argon for 16 hours, then diluted with ice water. The mixture was extracted twice with DCM. The organic phases were combined and washed with water. The collected solution was dried over Na2SO4, concentrated, and purified by rapid silica gel chromatography to give product 4 (9.36 g) as a yellow-orange solid.

[0363] Example 4. Preparation of Compound 5

[0364] 4 5

[0366] Compound 4 (9.2 g) was mixed with Pd(OAc)₂ (0.205 g, 0.05 equivalents), PPh₃ (1.10 g, 0.23 equivalents), and 1,3-dimethylbarbituric acid (8.56 g, 3 equivalents) in 250 mL of DCM. The reaction mixture was stirred at 35 °C for 2 hours. The reaction mixture was washed with NaHCO₃ solution and brine, dried over Na₂SO₄, concentrated, and purified by silica gel chromatography to give product 5 (9.44 g) as a yellow solid.

[0367] Example 5. Preparation of Compound 6

[0368] 5 6

[0370] Compound 5 (9.44 g) was dissolved in 400 mL of DCM, and iPr2NEt (14 mL, 4 equivalents) was added. The mixture was cooled in an ice bath, and succinate monoethyl ester chloride (5.05 g, 1.5 equivalents) was added. The reaction was stirred at room temperature for 20 min, then quenched with brine. The organic phase was separated, dried over Na2SO4, concentrated, and purified by silica gel chromatography to give product 6 (9.83 g) as a yellowish-brown solid.

[0371] Example 6. Preparation of Compound 7

[0372] 6 7

[0374] 15 mL of TFA was added to compound 6 (9.8 g) in 30 mL of DCM. The reaction was stirred at room temperature for 1.5 hours. The mixture was concentrated and azeotropically reacted with DCM, toluene, and finally hexane and DCM to give product 7 (7.32 g) as a yellow solid.

[0375] Example 7. Preparation of Compound 8

[0376] 7 8

[0378] Compound 7 (2.93 g) was dissolved in 20 mL of AcOH. Iodine (0.6 g, 0.4 equivalent), KIO3 (0.32 g, 0.24 equivalent), and 2.5 mL of 33% H2SO4 were added. The reaction was stirred at 50 °C for 4.5 hours, and then diluted with water. 25 mL of saturated NaHCO3 was added to obtain a solution, which was purified by reversed-phase HPLC using a C18 column and lyophilized to give product 8 (1.95 g).

[0379] Example 8. Preparation of Compound 9

[0380] 8 9

[0382] Compound 8 (1.95 g) was dissolved in 20 mL of DMF, and 0.15 mL of bromine (2 equivalents) was added. The mixture was stirred at room temperature for 30 hours. The reaction was quenched with Na₂SO₃ solution. NaHCO₃ (saturated, 25 mL) was added to obtain a solution, which was purified by reversed-phase HPLC on a C18 column and lyophilized to give product 9 (1.8 g).

[0383] Example 9. Preparation of Compound 10

[0384] 9 10

[0386] At 0 °C, Et3N (1.4 mL, 6 equivalents) was added to a mixture of compound 9 (1.8 g) and THF (20 mL), followed by the dropwise addition of ethyl chloroformate (0.63 mL, 4 equivalents). The mixture was stirred at room temperature for 30 minutes. The white precipitate was filtered off, and the filtrate was concentrated. After drying under high vacuum at 0 °C for 1 hour, THF (20 mL) was added, followed by the dropwise addition of sodium borohydride (0.37 g, 6 equivalents) in water (2 mL). The mixture was stirred at 0 °C for 30 minutes. The reaction was quenched with NH4Cl solution. More H2O was added to dissolve all the precipitate. The mixture was concentrated to remove THF and extracted with EtOAc (3 x 50 mL) until no product was found in the aqueous layer. The organic phase was dried over Na2SO4, concentrated, and purified by rapid silica gel chromatography to give product 10 (1 g) as a white solid.

[0387] Example 10. Preparation of Compound 11

[0388] 10 11

[0390] Compound 10 (1.0 g) was dissolved in 10 mL of DMF and bis(pinacol)diboron (0.413 g, 1.4 equivalents) was added, followed by Pd(dppf)Cl2 (0.04 g, 0.05 equivalents) and potassium acetate (0.47 g, 4 equivalents). The mixture was degassed with argon for 10 minutes. The reaction was stirred at 80 °C for 2 hours. At room temperature, EtOAc (100 mL) was added and quenched with brine. The organic phase was separated, dried over Na2SO4, concentrated, and purified by silica gel chromatography to give product 11 (0.9 g) as a light brown solid.

[0391] Example 11. Preparation of Compound 12

[0392] 11 12

[0394] Compound 11 (0.9 g) was dissolved in THF (10 mL), followed by the addition of Et3N (1.22 mL, 6 equivalents) and then TsCl (1.12 g, 4 equivalents). The reaction was stirred at room temperature for 18 hours. The mixture was concentrated and purified by silica gel chromatography to give product 12 (1.5 g) as a light brown solid.

[0395] Example 12. Preparation of Compound 13

[0396] 12 13

[0398] PEG was added to a mixture of NaH (0.33 g, 6 equivalents) and anhydrous THF (40 mL) at 0 °C. 11 -OH (4.34 g, 6 equivalents). After 10 minutes at 0 °C, a solution of compound 12 (1.5 g) in anhydrous THF (20 mL) was added dropwise. The reaction was stirred at room temperature for 18 hours. The mixture was concentrated and chloroform (100 mL) was added. The organic phase was washed with H2O (100 mL), separated, dried over Na2SO4, concentrated, and purified by silica gel chromatography to give product 13 (3 g) as a colorless liquid.

[0399] Example 13. Preparation of Compound 14

[0400] 13 14

[0402] Under argon atmosphere, an aqueous solution of K₂CO₃ (2 M, 4 mL) was added to a Schlenk flask containing a solution of compound 13 (0.8 g) and compound TJ4003 (0.029 g, Tianjin Biolite) in DMF (6 mL), followed by the addition of tetrakis(triphenylphosphine)palladium (0.015 g, 0.03 equivalents). The mixture was degassed by three freeze-pump-thaw cycles and heated to 80 °C for 4 hours. At room temperature, pinacol phenylborate (0.043 g, 0.1 equivalents) was added to the reaction mixture under argon atmosphere, and the mixture was heated to 80 °C for 2 hours. At room temperature, EDTA (0.1 g) in 20% EtOH / H₂O (20 mL) was added to the reaction mixture and stirred at room temperature for 2 hours. The resulting mixture was filtered through a 0.45 µm cup filter. The filtered solution was diluted to a concentration of 2 mg / mL using 20% ​​EtOH / H₂O. The resulting dilution was dialyzed into 20% EtOH / H2O using a tangential flow filtration system with 30 kD and 750 kD molecular weight cutoff membranes until the polymer in the eluent was less than 0.1 mg / mL. The solution was concentrated and lyophilized to give compound 14 (0.5 g) as a yellow semi-solid.

[0403] Example 14. Preparation of Compound 15

[0404] 14 15

[0406] At room temperature, trifluoroacetic acid (7 mL) was added to a solution of compound 14 (500 mg) in dichloromethane (13 mL), followed by anisole (0.08 mL). The reaction mixture was stirred at room temperature for 2–3 hours. The solvent was removed and the mixture was dried overnight under high vacuum to give compound 15 (400 mg) as a pale yellow oil.

[0407] Example 15. Preparation of Compound 16

[0408]

[0409] 15 TJ4100 16

[0410] At room temperature, triethylamine (0.033 mL) was added to a solution of compound 15 (120 mg) in DMF (3 mL), followed by the addition of TJ4100 (2.1 mg, Tianjin Biolite). The mixture was stirred at room temperature for 1–2 hours. Ice water (3–6 g) was added to quench the reaction. The mixture was purified by P6DG column chromatography (water as solvent) to give a yellow solution. The final product was aliquoted into 16 equal parts according to the extinction coefficient.

[0411] Example 16. Preparation of fluorene polymer succinimide ester (compound 17)

[0412] 15 17

[0414] To a solution of compound 15 (100 mg) in DMF (10 ml), add 0.1 ml of a DMF solution of di(N-succinimide) glutarate (1 mg, AAT Bioquest) and 10 µl of triethylamine. Stir the reaction mixture at room temperature for 2 hours and concentrate under high vacuum to remove DMF. Wash the residue several times with diethyl ether until most of the unreacted di(N-succinimide) glutarate is removed. Rapidly dissolve the residue in cold acidic water (pH = 5) and extract three times with ether. Freeze-dry the aqueous solution to give the desired fluorene polymer succinimide ester as compound 17.

[0415] Example 17. Preparation of fluorene polymer maleimide (compound 18)

[0416] To a solution of compound 15 (100 mg) in DMF (10 ml), add 0.1 ml of a DMF solution of N-hydroxysuccinimide 3-maleiminopropionic acid (1 mg, AAT Bioquest) and 10 µl of triethylamine. The reaction mixture was stirred at room temperature for 2 hours and concentrated under high vacuum to remove DMF. The residue was dissolved in acidic water (pH = 5) and extracted three times with ethyl acetate. The aqueous solution was freeze-dried to give the desired fluorene polymer maleimide as compound 18.

[0417] The above examples of synthetic strategies for selected polymers of the present invention, along with their characterization, synthetic precursors, conjugates, and methods of use, are provided for illustrative purposes. Further modifications and variations will be apparent to those skilled in the art. For example, the second fluorophore conjugated to the polymers of the present invention (such as Cy3.5, Cy5, TAMRA, and Texas Red in the examples above) can be readily replaced with commercially available dyes listed in Table 1 to give the polymers different desired spectral properties. Furthermore, the polymers of the present invention can be further functionalized with different reactive functional group pairs as listed in Table 2. Known click reactive groups can also be added to the polymers of the present invention for bioorthogonal chemistry-based conjugation (see P. Agarwal and R. Bertozzi, Bioconjugate Chem., 2015, 26, 176-192; K. Lang and J. Chin, Chem. Reviews, 2014, 114, 4764-4806; MD Best, Biochemistry, 2009, 48, 6571-6584). Several other alternative polymer functionalization methods are well described in the literature (see U.S. Patent Nos. 8,158,444, 8,455,613, 8,354,239, 8,362,193, and 8,575,303; and WO2013 / 101902, granted to Chiu et al.).

[0418] Example 18. Preparation of goat anti-mouse IgG-fluorene polymer dye conjugate

[0419] Goat anti-mouse IgG (GAM) was dissolved in 10 mM NaHCO3 (pH 8.2) buffer to prepare a 5 mg / mL solution. A DMF solution of compound 17 (20 equivalents) was added to the GAM protein aqueous solution. The solution was incubated at room temperature for 3 hours, and the reaction mixture was transferred to an Amicon ultrafilter (MWCO = 10 kDa) to remove DMF. The protein was restored to its initial volume with PBS buffer.

[0420] Free polymers were removed using cation exchange chromatography. The conjugated mixture was loaded onto UNOsphere™ S resin (Bio-Rad) in low-salt buffer [50 mM MES buffer (pH=5.0)] and incubated at room temperature for 10 min. Sample loading was repeated three times to obtain maximum binding. After loading, the medium was washed with low-salt buffer to baseline (until absorbance at 414 nm was below 0.01) to remove all free polymers. The fluorene polymer dye-GAM conjugates retained on the cation exchange resin were released by increasing the pH and ionic strength with high-salt phosphate buffer [10 mM phosphate buffer (pH=7.4) + 1.0 M NaCl buffer / methanol, 90 / 10]. Protein A and Protein G affinity resins can also be used to remove free polymers with comparable results. A HiTrap Protein G HP 1 mL column (GE Lifesciences) was pre-equilibrated with 10 mM phosphate-buffered saline (pH 7.4). The SEC-purified product was slowly injected at <1 mg / mL, allowing incubation for 30 minutes to allow binding. The column was washed with >10 column volumes of 10 mM phosphate-buffered saline to remove unbound polymer material, while monitoring the eluent uptake at 280 nm and 414 nm to ensure removal of all excess material. The conjugate was eluted by washing the column with 4 column volumes of 0.1 M glycine (pH 2.3). The eluent fractions were combined, and the pH was adjusted back to neutral using 1 M Tris pH 8. After removing the free polymer, the conjugate solution was concentrated using an Amicon ultrafilter (MWCO = 30 kD) and loaded onto a size exclusion column (Superdex 200, GE Lifesciences) to separate conjugated and unconjugated antibodies. The column was equilibrated with PBS buffer, and the fluorene polymer-antibody conjugate was eluted before the free antibody.

[0421] For effective labeling, the degree of substitution should be between 1-3 moles of fluorene polymer dye to 1 mole of antibody for most antibodies. As is well known in the art, the optimal polymer dye / protein ratio depends on the properties of the antibody to be labeled. The optimal polymer dye / protein labeling ratio is empirically determined by preparing a series of dye conjugates within the range of polymer dye / protein ratios and comparing the desired signal / background. In some cases, a higher polymer dye / protein ratio can provide a bright signal, while in others, a higher ratio can reduce the affinity of the antibody to be labeled.

[0422] Example 19. Fluorene polymer conjugates for flow cytometry

[0423] Analyte-specific antibodies (i.e., labeled antibodies) conjugated to the fluorene polymer dyes of this invention can be used to analyze blood cells (e.g., in whole blood samples) by flow cytometry. The labeled antibodies are used to stain cellular proteins, and the labeled cells are detected by flow cytometry. The fluorene polymer bioconjugate is evaluated by a staining index as defined by BD Biosciences on a flow cytometer. See, for example, H. Maeker and J. Trotter, BD Biosciences Application Note: "Selecting Reagents for Multicolour Flow Cytometry", September 2009. The staining index reports a measure of polymer brightness (non-specific binding). Flow cytometry provides a method for measuring cells with a specific phenotype or an analyte of interest on specific microspheres. This can be achieved by directly labeling a primary antibody, or, if signal amplification is desired, by a secondary antibody or a combination of avidin-biotin and an avidin-polymer conjugate. Take a sufficient amount of cells of interest, centrifuge, wash in DPBS + 0.2% BSA and 0.05% NaN3, and then resuspend in staining buffer of fluorene polymer conjugate.

[0424] For a single incubation, cells are incubated together with a single conjugate specific to the antigen of interest, with negative cells used as a negative nonspecific binding reference. A control population or a commercially available conjugate is used as a positive control. The primary antibody-polymer conjugate is incubated for 30 minutes at a concentration diluted 10–500 nM.

[0425] For secondary antibody labeling, incubate the unlabeled primary antibody against the antigen of interest at a titration of 1-50 µg / ml or other amounts. After the first incubation, wash the cells with 5 volumes of staining buffer and centrifuge for 3-5 minutes. Species-reactive secondary fluorene polymer conjugates are incubated at 10-500 nM volumetric dilutions for 30-60 minutes. After the second incubation, wash the cells with 3-5 volumes of staining buffer and centrifuge for 3-5 minutes. Resuspend the cells in DPBS + 0.2% BSA, 0.05% sodium azide for testing.

[0426] For streptavidin-polymer conjugate labeling, incubate cells with a biotinylated primary antibody targeting the biomarker of interest, as detailed above for secondary antibody labeling, instead of an unlabeled primary antibody. After one wash, resuspend the cells and incubate for 30 minutes with a 1–100 nM volume diluted streptavidin-polymer conjugate. After a second incubation, wash the cells with 5 volumes of staining buffer and centrifuge for 3–5 minutes. Resuspend the cells for testing. If further signal amplification is required, cells can be incubated with an unlabeled primary antibody, followed by incubation with a species-reactive biotinylated secondary antibody, and then with the streptavidin conjugate.

[0427] It should be understood that the specific antibody conjugate used, as well as the specific reaction components and reaction conditions, can affect the results. Routine experiments should be performed to determine the preferred reaction components, such as buffers or lysis buffers, and reaction conditions, including staining time and temperature. Routine optimization of such assay conditions is based on standard practice in the field of immunostaining assays.

[0428] Example 20. MTA-modified (amine)

[0429] The antibody was dissolved in 0.1 M sodium bicarbonate (pH 8.5) at 10 mg / mL, and 1 mg of the antibody solution was transferred to a microcentrifuge vial. Tetraazine succinimide was dissolved in anhydrous DMSO at 10 mg / mL, and 1.08 μL was added to the antibody solution at room temperature. The reaction mixture was briefly vortexed and then reacted on a low-speed vortex mixer for 1 hour. At the end of 1 hour, the modified antibody was purified from the free tetraazine succinimide by desalting on a PD-10 column to 3 mM phosphate buffer, 35 mM NaCl, pH 7.4, and collected in an Amicon Ultra-4 30k molecular weight concentrator and concentrated to >5 mg / mL. The modified antibody was stored at 4°C until conjugation.

[0430] Example 21. MTA modification (thiol group)

[0431] The antibody was dissolved at 10 mg / mL in 10 mM phosphate buffer (pH 7.4), and 1 mg of the antibody solution was transferred to a microcentrifuge vial. The antibody was reduced by adding 1 mL of 1 M dithiothreitol (DTT) solution, briefly vortexing, and allowing to stand at room temperature for 30 minutes. The solution was then desalted on a PD-10 column (GE Lifesciences) to remove DTT to 3 mM phosphate buffer (pH 7.4). The desalted reduced antibody was then concentrated to 10 mg / mL on a 30 kDa concentrator (Millipore Amicon Ultra). 2.3 mL of 10 mg / mL maleimide-MTA in DMSO was added to the reduced antibody solution. The solution was briefly vortexed and incubated on a vortex mixer at room temperature (RT) for 90 minutes. At 90 minutes, the modified antibody was purified from free maleimide-MTA by desalting on a PD-10 column to 3 mM phosphate buffer, 35 mM NaCl, pH 7.4, and collected in an Amicon Ultra-4 30k molecular weight concentrator and concentrated to >5 mg / mL. The modified antibody was stored at 4°C until conjugation.

[0432] Example 22. Bioorthogonal polymer-antibody conjugation

[0433] The cyclooctene reactive polymer was stored at 5 mg / mL in DI H2O at -20°C and thawed immediately before use. The MTA-modified antibody (1 mg, 0.167 mL) was transferred to a microcentrifuge vial containing 0.104 mL of 3 mM phosphate buffer (pH 7.4). The polymer solution (0.133 mL, 0.67 mg polymer) was added, followed by 0.1 mL of ethanol. The solution was rapidly mixed with a pipette. The reaction mixture was vortexed at room temperature in the dark for 2 hours. After 2 hours, the reaction mixture was quenched by adding 6.7 mL of 10 mM cyclooctene quencher, and the mixture was allowed to vortex for another 10 minutes before purification.

[0434] Example 23. Size Exclusion (SEC) Purification

[0435] Antibody-polymer conjugates were purified from free antibodies using a Bio-Rad NGC FPLC system on a Superdex 200 Increase size exclusion column (24 mL). The conjugates were first centrifuged at 20,238 rcf for 5 min to agglomerate any precipitated or cross-linked conjugates. The supernatant was then injected at <1.5% v / v onto a 24 mL Superdex200 Increase 10 / 300 GL column (GE Lifesciences) and eluted with 10 mM phosphate-buffered saline (pH 7.4) at 1.0 mL / min. The first elution peak at 280 nm, containing the antibody-polymer conjugate, was collected, and any free polymer and any subsequent peaks were discarded. (See Figure 2)

[0436] The optical properties of the polymer attached to the connector are shown in Figures 3-8.

[0437] Example 24. Cell stimulation

[0438] The detection of some antigens requires stimulation of cells to upregulate their expression. For CD25, normal human peripheral blood cells were cultured in RPMI-1640 containing 10% fetal bovine serum, 1:100 penicillin / streptomycin, 1:100 sodium pyruvate, and 1:1000 2-mercaptoethanol (cRPMI) and incubated for 3 days with phytohemagglutinin at a 1:500 dilution. For TNF-α, normal human peripheral blood cells were cultured for 4–6 hours in cRPMI containing a 1:500 dilution of phorbol myristate acetate, iomycin, brefidobacterium A, and monensin. For Ki-67, normal human peripheral blood cells were cultured for 3 days in cRPMI containing plate-bound anti-human CD3 and soluble anti-human CD28 monoclonal antibodies. After the specified stimulation duration, cells were harvested, washed in flow cytometry staining buffer, and then stained with antibodies.

[0439] Example 25. Surface staining of mouse CD4 (clone RM4-5), human CD20 (clone 2H7), and human CD25 (clone BC96).

[0440] To stain for antigens found on cell surfaces, single-cell suspensions of mouse spleen cells or normal human peripheral blood cells were resuspended at 10⁷ cells / mL in phosphate-buffered saline (flow cytometry staining buffer) containing 3% fetal bovine serum and 0.09% sodium azide. One hundred μL of cell suspension was aliquoted into 12 x 75 mm tubes, and allophycocyanin (APC) conjugated antibody was added at the manufacturer's recommended optimal concentration. The polymer-conjugated antibody was added to the cells at a final concentration of 0.0006–0.01 mg / mL. The cell and antibody mixture was incubated at 4°C for 30 minutes, and then 3 mL of flow cytometry staining buffer was added to the cells. The cells were centrifuged at 600 x g for 5 minutes, and the supernatant was discarded. Cell clumps were resuspended in up to 0.4 mL of flow cytometry staining buffer and analyzed on an LSR Tortessa® SORP flow cytometer (Becton, Dickinson, and Company) equipped with UV (355 nm) and red (640 nm) laser lines and bandpass filters suitable for detecting APC (670 / 14) and basal and tandem polymer dyes (379 / 28, 585 / 15, and 820 / 60, respectively). A minimum of 10,000 lymphocytes were collected. Data were analyzed using FlowJo software (Becton, Dickinson, and Company).

[0441] Example 26. Intracellular staining of human TNFα (clone MAb11)

[0442] For intracellular staining and detection of TNF-α, a single-cell suspension of stimulated normal human peripheral blood cells was resuspended at 10⁷ cells / mL in flow cytometry staining buffer. One hundred μL of cell suspension was aliquoted into 12 x 75 mm tubes, and 0.1 mL of formaldehyde-based fixation buffer was added. Cells were incubated at room temperature for 20–30 minutes, then 3 mL of wash-based permeation buffer was added. Cells were centrifuged at 600 x g for 5 minutes, and the supernatant was discarded. Additional washing was performed with 3 mL of permeation buffer. Cells were resuspended in 0.1 mL of permeation buffer, and then APC or FITC-conjugated antibody was added at the manufacturer's recommended optimal concentration. The polymeric conjugated antibody was added to the cells at a final concentration of 0.0006–0.01 mg / mL, and the sample was incubated at room temperature for 30 minutes. The sample was washed twice with 3 mL of permeation buffer. After the final wash, the cell clumps were resuspended in up to 0.4 mL of flow cytometry staining buffer, and the samples were analyzed on an LSR Tortessa® SORP flow cytometer (Becton, Dickinson and Company) equipped with UV (355 nm), red (640 nm), and blue (488 nm) laser lines and bandpass filters suitable for detecting APC (670 / 14), FITC (530 / 30), and basic and tandem polymer dyes (379 / 28 and 820 / 60, respectively). A minimum of 10,000 lymphocytes were collected. Data were analyzed using FlowJo software (Becton, Dickinson and Company).

[0443] Example 27. Intracellular staining of human Ki-67 (clone 20Raj1)

[0444] For intracellular staining and detection of Ki-67, a single-cell suspension of stimulated normal human peripheral blood cells was resuspended at 10⁷ cells / mL in flow cytometry staining buffer. One hundred μL of cell suspension was aliquoted into 12 x 75 mm tubes, and 1 mL of formaldehyde-based and detergent-based fixation / permeabilization buffer was added. Cells were incubated at room temperature for 20–30 minutes, then 3 mL of detergent-based permeabilization buffer was added. Cells were centrifuged at 600 x g for 5 minutes, and the supernatant was discarded. Additional washing was performed with 3 mL of permeabilization buffer. Cells were resuspended in 0.1 mL of permeabilization buffer, and then APC conjugated antibody was added at the manufacturer's recommended optimal concentration. The polymeric conjugated antibody was added to the cells at a final concentration of 0.0006–0.01 mg / mL, and the sample was incubated at room temperature for 30 minutes. The sample was washed twice with 3 mL of permeabilization buffer. After the final wash, the cell clumps were resuspended in up to 0.4 mL of flow cytometry staining buffer, and the samples were analyzed on an LSR Tortessa® SORP flow cytometer (Becton, Dickinson and Company) equipped with UV (355 nm) and red (640 nm) laser lines and bandpass filters suitable for detecting APC (670 / 14) and tandem polymer dyes (820 / 60). A minimum of 10,000 lymphocytes were collected. Data were analyzed using FlowJo software (Becton, Dickinson and Company).

[0445] Figures 9-11 illustrate the performance of polymer conjugated antibodies based on fluorenzaheptatriene, fluorenzazeptatriene, and fluorenzaheptanane in flow cytometry analysis.

[0446] Table 4. Representative fluorene-based polymers

[0447]

[0448] Linker 1 is methylene, and linker 2 is PEG4.

[0449] The structures of the polymers in the table above are as follows:

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

Claims

1. A polymer comprising monomer units of formula A. A Where X is the number of monomer units of formula A in the polymer, wherein the monomer units of formula A are continuous or discontinuous, and where X is from 10 to 200. and one or more monomer units of formula B B Where Y is the number of monomer units of formula B in the polymer, wherein the monomer units of formula B are continuous or discontinuous, and where Y is from 0 to 100. and one or more monomer units of formula C C Where Z is the number of monomer units of formula C in the polymer, wherein the monomer units of formula C are continuous or discontinuous and where Z is from 0 to 100. Where A is O, S, N, or C; SG1, SG2, SG5, and SG6 are each independently polyethylene glycol, alkyl, carboxylalkyl, sulfonylalkyl, phosphonylalkyl, aminoalkyl, or biosubstrate conjugated via a connector; R1 is absent or is a biological substrate that is hydrogen, alkyl, amino, sulfonyl, polyethylene glycol, acceptor, linker and / or conjugated via a linker; R2 is hydrogen, alkyl, amino, sulfonyl, polyethylene glycol, acceptor, linker and / or a biological substrate conjugated via a linker; SG3, SG4, R3, and R4 are independently hydrogen, halogen, polyethylene glycol, or connectors; The connector is an alkyl chain or a polyethylene glycol chain; The biological substrates mentioned therein are specific binding pairs of members, nucleosides, nucleotides, nucleic acid polymers, amino acid polymers, nucleic acids, or carbohydrates; The polymer ends are independently hydrogen, alkyl, halogen, boron carbonyl, aryl, heteroaryl groups or bio-substrate connected via a linker; The ratio of X / (Y+Z) is 0.3-1.0, and The sum of X+Y+Z is between 15 and 50. Y is present in the polymer at a concentration of at least 40%. The receptors mentioned therein include fluorophores or fluorescent dyes.

2. The polymer of claim 1, wherein the polymer of the amino acids is a peptide having 5-36 amino acids.

3. The polymer of claim 1, wherein the polymer of the amino acids is a protein selected from enzymes, antibodies, lectins, glycoproteins, histones, albumins, lipoproteins, avidin, protein A, protein G, phycobiliproteins, fluorescent proteins, hormones, toxins, chemokines, and growth factors.

4. The polymer of claim 1, wherein the biological substrate is an oligonucleotide.

5. The polymer of claim 1, wherein the monomer units of formulas A, B and C are directly connected to each other.

6. The polymer of claim 1, wherein the ratio of the receptor to the polymer is 0.01-0.

2.

7. The polymer of claim 1, wherein the ratio of X / (Y+Z) is 0.4-1.

0.

8. The polymer of claim 1, wherein SG1, SG2, SG5 and SG6 are independently polyethylene glycol 3 to polyethylene glycol 30.

9. The polymer of claim 1, wherein the monomer unit of formula B comprises B Where Y is the number of monomer units of formula B in the polymer, wherein the monomer units of formula B are continuous or discontinuous and where Y is from 0 to 100; and SG3, SG4, R3, and R4 are independently fluorine, hydrogen, polyethylene glycol, or connectors.

10. The polymer of claim 1, wherein A is C; and wherein R1 and R2 are each polyethylene glycol.

11. The polymer of claim 1, wherein A is N; and wherein R1 is absent and R2 is or .

12. The polymer of claim 1, wherein the receptor further comprises fluorescein, rhodamine, p-methaminophenol, anthocyanin, BODIPY, squaric acid cyanide, coumarin, perylene diimide, diketopyrrolopyrrole, porphyrin, or phthalocyanine.

13. The polymer of claim 1, wherein formula A comprises 、 、 、 or ， Where m and n range from 5 to 20.

14. The polymer of claim 1, wherein formula B comprises 、 、 or Where m ranges from 5 to 20.

15. The polymer of claim 1, wherein the receptor comprises: or 。 16. A polymer-antibody conjugate, said polymer-antibody conjugate comprising The polymer and antibody of claim 1, wherein the polymer is conjugated to the antibody.

17. The polymer-antibody conjugate of claim 16, wherein the antibody is a goat anti-mouse IgG antibody, a goat anti-rabbit IgG antibody, a goat anti-human IgG antibody, a donkey anti-mouse IgG antibody, a donkey anti-rabbit IgG antibody, a donkey anti-human IgG antibody, a chicken anti-mouse IgG antibody, a chicken anti-rabbit IgG antibody, or a chicken anti-human IgG antibody.

18. The polymer-antibody conjugate of claim 16, wherein monomer A of the polymer comprises fluorenzaxepin, fluorenzazepicycloheptatriene, or fluorenzacycloheptatriene.

19. A method for detecting an analyte in a sample, the method comprising: a) Combining the sample with the detection reagent, provided that the detection reagent containing the polymer as described in claim 1 binds to the analyte; as well as b) Detect the analyte bound to the detection reagent by fluorescence detection.

20. The method of claim 19, wherein the biological substrate is an antibody.

21. The method of claim 19, wherein the biological substrate is an anti-digoxin antibody.

22. The method of claim 19, wherein the biological substrate is a goat anti-mouse IgG antibody, a goat anti-rabbit IgG antibody, a goat anti-human IgG antibody, a donkey anti-mouse IgG antibody, a donkey anti-rabbit IgG antibody, a donkey anti-human IgG antibody, a chicken anti-mouse IgG antibody, a chicken anti-rabbit IgG antibody, or a chicken anti-human IgG antibody.

23. The method of claim 19, wherein the biological substrate is avidin, streptavidin, neutral avidin, avidin DN, or avidin D.

24. The method of claim 19, wherein the analyte is a target protein expressed on the cell surface.

25. The method of claim 19, wherein the analyte is a target protein expressed intracellularly.

26. A method for detecting an analyte in a sample, the method comprising: a) Combining the sample with the detection reagent, provided that the detection reagent comprising the polymer-antibody conjugate as described in claim 16 binds the analyte; as well as b) Detect the analyte bound to the detection reagent by fluorescence detection.

27. The method of claim 26, wherein the antibody is a goat anti-mouse IgG antibody, a goat anti-rabbit IgG antibody, a goat anti-human IgG antibody, a donkey anti-mouse IgG antibody, a donkey anti-rabbit IgG antibody, a donkey anti-human IgG antibody, a chicken anti-mouse IgG antibody, a chicken anti-rabbit IgG antibody, or a chicken anti-human IgG antibody.

28. The method of claim 26, wherein the analyte is a target protein expressed on the cell surface.

29. The method of claim 26, wherein the analyte is a target protein expressed intracellularly.

30. The method of claim 26, wherein the monomer A of the polymer comprises fluorenzaoxetine, fluorenzaazaheptatriene, or fluorenzacycloheptane.

31. A system for detecting an analyte in a sample, the system comprising: Detection reagents comprising the polymer-antibody conjugate as described in claim 16 One or more cells, and an optical response device.

32. The system of claim 31, wherein the antibody is a goat anti-mouse IgG antibody, a goat anti-rabbit IgG antibody, a goat anti-human IgG antibody, a donkey anti-mouse IgG antibody, a donkey anti-rabbit IgG antibody, a donkey anti-human IgG antibody, a chicken anti-mouse IgG antibody, a chicken anti-rabbit IgG antibody, or a chicken anti-human IgG antibody.

33. The system of claim 31, wherein the monomer A of the polymer comprises fluorenzaoxetine, fluorenzaazaheptatriene, or fluorenzacycloheptane.

34. The system of claim 31, wherein the analyte is a target protein expressed on the cell surface.

35. The system of claim 31, wherein the analyte is a target protein expressed intracellularly.

36. The system of claim 31, wherein the optical response device is a CCD camera, a video camera, photographic film, a laser scanning device, a fluorometer, a photodiode, a quantum counter, an epifluorescence microscope, a scanning microscope, a flow cytometer, or a fluorescence microplate reader.

37. A kit comprising... The polymer of claim 1, wherein the polymer comprises a conjugated biosubstrate selected from polymers that specifically bind to members, nucleosides, nucleotides, nucleic acid polymers, nucleic acids, carbohydrates, and amino acids to produce a labeled biosubstrate, and One or more buffers.

38. The kit of claim 37, wherein the polymer of said amino acids is a peptide having 5-36 amino acids.

39. The kit of claim 37, wherein the polymer of the amino acids is a protein selected from enzymes, antibodies, lectins, glycoproteins, histones, albumins, lipoproteins, avidin, protein A, protein G, phycobiliproteins, fluorescent proteins, hormones, toxins, chemokines, and growth factors.

40. The kit of claim 37, wherein the conjugated biological substrate is an oligonucleotide.

41. The kit of claim 37, further comprising one or more of the following: a detection reagent, a purification medium for purifying the obtained labeled biological substrate, a luminescent standard, an enzyme, an enzyme inhibitor, an organic solvent, or instructions for performing the assay.

42. A kit comprising... The polymer-antibody conjugate as described in claim 16, and One or more buffers.

43. The kit of claim 41, further comprising one or more of the following: a detection reagent, a purification medium for purifying the obtained labeled biological substrate, a luminescent standard, an enzyme, an enzyme inhibitor, an organic solvent, or instructions for performing the assay.

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