Polymer Dots

Abstract

A polymer dot is provided that includes a functionalized fluorescent polymer and an amphiphilic molecule.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 884,706, filed August 9, 2019, the contents of which are incorporated by reference. [Background technology]

[0002] Due to their greater brightness and photostability than traditional organic dyes, nanoparticle-based fluorescent probes, such as quantum dots and polymer-based dots (or polymer dots), have been used in various fluorescence-based techniques to study biological systems. However, inorganic quantum dots can be degraded by reactive oxygen species, releasing toxic heavy metals. Therefore, the use of quantum dots is limited to ex vivo applications. Fluorescent polymer dots do not have heavy metals that leach into solution and can therefore be used for in vivo applications. For use in biological systems, fluorescent probes are conjugated to proteins (e.g., antibodies) via functional groups on the probe's surface. For polymer-based nanoparticles, the polymers used to produce the nanoparticles are hydrophobic and do not always have functional groups on the particle's surface to which proteins can be attached. One approach used to functionalize the surface of polymer dots is to apply a hydrophilic coating to the surface of the polymer dots. The hydrophilic coating has hydrophilic functional groups to which proteins can be conjugated. Summary of the Invention [Means for solving the problem]

[0003] Polymer dots, methods for producing polymer dots, and bioconjugates of such polymer dots are provided.

[0004] In one embodiment, the polymer dot comprises a fluorescent polymer having a hydrophilic region with a hydrophobic region and a hydrophilic functional group, and an amphiphilic molecule having a hydrophobic region and a hydrophilic region, the hydrophilic functional group being utilized for conjugation. In some embodiments, the hydrophobic regions of the fluorescent polymer and the amphiphilic molecule are embedded in the hydrophobic core of the polymer dot, the hydrophilic regions of the fluorescent polymer and the amphiphilic molecule form a hydrophilic outer layer, and the hydrophilic functional group in the fluorescent polymer is located in the hydrophilic outer layer on the surface of the polymer dot.

[0005] In some embodiments, the fluorescent polymer is a heteropolymer. In some embodiments, the heteropolymer comprises at least two different monomers. In certain embodiments, the monomers are BODIPY, BODIPY derivatives, fluorene, fluorene derivatives, benzothiadiazole, benzothiadiazole derivatives, benzoxadiol, and / or benzoxadiol derivatives. In some embodiments, the monomers are [ka] (dibromobenzoxadiol), [ka] (4,7 bis(2-bromo-5-thienyl)-2,1,3-benzothiadiazole), [ka] (9,9-dioctyl-2,7-dibromofluorene), [ka] (9,9-dioctylfluorene-2,7-diboronic acid bis(1,3-propanediol) ester), [ka] and / or [ka] (n=10-30). In one embodiment, n=22. In some embodiments, the fluorescent polymer is a homopolymer.

[0006] In some embodiments, the hydrophilic functional group is a carboxyl, amino, mercapto, azide, alkyne, aldehyde, hydroxyl, carbonyl, sulfate, sulfonate, phosphate, cyanate, succinimidyl ester, strained alkyne, azide, diene, alkene, tetrazine, strained alkene, cyclooctyne, phosphine group, or derivatives thereof. In certain embodiments, the hydrophilic functional group in the polymer is conjugated to a biomolecule. In some embodiments, the biomolecule is a synthetic or naturally occurring protein, glycoprotein, polypeptide, amino acid, nucleic acid, carbohydrate, lipid, or fatty acid. In some embodiments, the biomolecule is an antibody. In some embodiments, the hydrophilic region of the amphiphilic molecule comprises a polyalkylene glycol. In some embodiments, the polyalkylene glycol is polyethylene glycol. In some embodiments, the size of the polymer dot is about 5 to 20 nanometers. In some embodiments, the weight ratio of the amphiphilic molecule to the fluorescent polymer is about 10% to about 200%. In other embodiments, the weight ratio is expressed as a range of amphiphilic molecule to fluorescent polymer from about 0.1 to about 2 (i.e., about 0.1 to 2.0:1). Some of these embodiments result in weight ratios of about 0.25:1, about 0.5:1, about 0.75:1, about 1:1, about 1.25:1, about 1.5:1, about 1.75:1, or about 2:1. In some embodiments, the hydrophobic region of the amphiphilic molecule comprises a lipid moiety, including, but not limited to, a 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) moiety, a 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) moiety, a 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) moiety, and a (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) (DPPE) moiety. In some embodiments, the amphiphilic molecule is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DSPE-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-hydroxyl-1000 or -2000] (DSPE-PEG-OH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DSPE-PEG-OCH3), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DSPE-PEG-NH2), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DSPE-PEG-COOH) 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DSPE-PEG-maleimide) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DSPE-PEG-biotin), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DMPE-PEG), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)hydroxyl-1000 or -2000] (DMPE-PEG-OH), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DMPE-PEG-OCH3), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DMPE-PEG-NH2), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DMPE-PEG-COOH), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DMPE-PEG-maleimide), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DMPE-PEG-biotin), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DLPE-PEG), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxyl-1000 or -2000] (DLPE-PEG-OH), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DLPE-PEG-OCH3), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DLPE-PEG-NH2), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DLPE-PEG-COOH), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DLPE-PEG-maleimide), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DLPE-PEG-biotin), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DPPE-PEG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxyl-1000 or -2000] (DPPE-PEG-OH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DPPE-PEG-OCH3), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DPPE-PEG-NH2), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DPPE-PEG-COOH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DPPE-PEG-maleimide), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DPPE-PEG-biotin), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DSPE-PAA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DSPE-PAA-OH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DSPE-PAA-OCH3), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DSPE-PAA-NH2), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DSPE-PAA-COOH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DSPE-PAA-maleimide), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DSPE-PAA-biotin), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DMPE-PAA), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DMPE-PAA-OH), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DMPE-PAA-OCH3), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DMPE-PAA-NH2), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DMPE-PAA-COOH), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DMPE-PAA-maleimide), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DMPE-PAA-biotin), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DLPE-PAA), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DLPE-PAA-OH), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DLPE-PAA-OCH3), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DLPE-PAA-NH2), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DLPE-PAA-COOH), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DLPE-PAA-maleimide), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DLPE-PAA-biotin), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DPPE-PAA), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DPPE-PAA-OH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DPPE-PAA-OCH3) or 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DPPE-PAA-NH2), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DPPE-PAA-COOH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DPPE-PAA-maleimide), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DPPE-PAA-biotin).

[0007] In one embodiment, a method for producing polymer dots includes preparing a mixture of a fluorescent polymer and an amphiphilic molecule in an aprotic solvent and adding the mixture to a protic solvent to form the polymer dot, wherein the fluorescent polymer comprises a hydrophobic region and a hydrophilic region, the hydrophilic region having a hydrophilic functional group, and the amphiphilic molecule comprises a hydrophobic region and a hydrophilic region, and the hydrophilic functional group is utilized for conjugation. In some embodiments, the hydrophobic regions of the fluorescent polymer and the amphiphilic molecule are embedded in the hydrophobic core of the polymer dot, the hydrophilic regions of the fluorescent polymer and the amphiphilic molecule form a hydrophilic outer layer, and the hydrophilic functional group in the fluorescent polymer is located in the hydrophilic outer layer on the surface of the polymer dot. In some embodiments, the aprotic solvent is tetrahydrofuran. In certain embodiments, the protic solvent is water. In some embodiments, the method further includes conjugating a biomolecule to the polymer dot via the hydrophilic functional group.

[0008] In one embodiment, a method for detecting a target molecule in a biological sample comprises contacting the biological sample with a polymer dot as described above and elsewhere herein. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a bar graph of the staining index for polymer dot anti-CD4 antibody conjugates and control conjugates. [Figure 2] FIG. 2 is a histogram plot of positive and negative cell populations for the polymer dot anti-CD4 antibody conjugates and the control conjugate. [Figure 3]FIG. 3 shows fluorescent polymers such as polyfluorenes (such as PDHF and PFO), poly(phenyleneethynylenes) (such as PPE), poly(phenylenevinylenes) (such as MEH-PPV and CN-PPV), fluorene-based copolymers (such as PFPV, PFBT, and PFDBT5), and related derivatives disclosed in Wu and Chiu, Angew. Chem Int. Ed. 2013, 52:3086-3109, which is incorporated herein by reference in its entirety. [Figure 4A] Figures 4A-4B show the particle size distribution of polymer dots produced by the disclosed method (Pdots 488 / 700 (Figure 4A) and Pdots 405 / 610 (Figure 4B)). As shown in the figures, more than 50% of the particles have an average effective diameter of about 25 nm or less. [Figure 4B] Figures 4A-4B show the particle size distribution of polymer dots produced by the disclosed method (Pdots 488 / 700 (Figure 4A) and Pdots 405 / 610 (Figure 4B)). As shown in the figures, more than 50% of the particles have an average effective diameter of about 25 nm or less. DETAILED DESCRIPTION OF THE INVENTION

[0010] Described herein are polymer dots, methods for their fabrication, and biomolecular conjugates thereof. Surface functionalization of the polymer dots is achieved by blending functionalized fluorescent polymers with amphiphilic molecules during fabrication. The polymer dots are stable (e.g., do not aggregate or precipitate from solution) and can be conjugated to biomolecules due to their surface reactive groups. The conjugates are used in a number of different applications, including, but not limited to, flow cytometry, fluorescence-activated sorting, immunofluorescence, immunohistochemistry, fluorescence multiplexing, single-molecule imaging, single-particle tracking, protein folding, protein rotational dynamics, DNA and genetic analysis, protein analysis, metabolite analysis, lipid analysis, FRET-based sensors, high-throughput screening, cellular imaging, in vivo imaging, fluorescence-based biological assays such as immunoassays and enzyme-based assays, and various fluorescence techniques in biological assays and measurements.

[0011] definition Unless otherwise indicated, the following terms used in this application, including the specification and claims, have the definitions set forth below. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Definitions of standard chemical terms can be found in reference texts including Carey and Sundberg (2007) "Advanced Organic Chemistry, 5th Ed." Vols. A and B, Springer Science+Business Media LLC, New York. The practice of the present invention employs, unless otherwise indicated, conventional methods of synthetic organic chemistry, mass spectrometry, chromatographic preparation and analytical methods, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology.

[0012] The term "about" or "approximately" refers to within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement method. The terms "about" and "approximately" are intended to encompass ranges of ±25%, ±20%, ±10%, or ±5% of a given value. With respect to polymer dot size, the term "about" or "approximately" indicates that the polymer dot size is of the stated size with a 0-10% variation around the value (X ±10%). Thus, a polymer dot having a diameter of approximately 20 nm includes a polymer dot having a diameter of 18-22 nm. When the term "about" is used in reference to a ratio (e.g., about 0.1:1), the term "about" applies to both values; specifically, the term is considered to apply to both a value of 0.1 and a value of 1 in the exemplified ratio.

[0013] In this disclosure, ranges are written succinctly to avoid lengthy listings of all values ​​within the range. Any appropriate value within the range may be selected as the upper, lower, or terminus of the range, where appropriate. For example, a range of 0.1 to 1.0 represents the end values ​​of 0.1 and 1.0, intermediate values ​​of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9, and all intermediate ranges encompassed within 0.1 to 1.0, such as 0.2 to 0.5, 0.2 to 0.8, and 0.7 to 1.0.

[0014] As used herein, the term "polymer dot" refers to a structure comprising one or more polymers that have been broken down into stable submicron-sized particles. The polymer dots provided herein may be formed by any method known in the art for breaking down polymers, including, but not limited to, methods that rely on precipitation, methods that rely on the formation of emulsions (e.g., miniemulsions or microemulsions), and methods that rely on condensation. In some embodiments, the polymer dots are formed by nanoprecipitation.

[0015] As used herein, a "polymer" is a molecule composed of at least two repeating structural units, typically linked by a covalent chemical bond. The repeating structural unit may be a type of monomer, and the resulting polymer is a homopolymer. In some embodiments, a polymer can contain two different types of monomers, or three different types of monomers, or more types of monomers. The different types of monomers can be distributed along the polymer chain in various ways. For example, three different types of monomers can be randomly distributed along the polymer. The distribution of monomers along the polymer can be expressed in different ways. The number of repeating structural units (e.g., monomers) along the length of a polymer can be represented by "n." In some embodiments, n can be, for example, at least 2, at least 100, at least 500, at least 1000, at least 5000, at least 10,000, or at least 100,000, or more. In certain embodiments, n ranges from 2 to 10,000, 20 to 10,000, 20 to 500, 50 to 300, 100 to 1000, or 500 to 10,000. Polymers generally have an extended molecular structure comprising a backbone optionally containing pendant side groups. Polymers provided herein can include, but are not limited to, linear and branched polymers.

[0016] As used herein, the term "nanoparticle" refers to a particle having a dimension less than about 1000 nm.

[0017] As used herein, the term "aprotic solvent" refers to a polar solvent that does not contain acidic hydrogen and does not act as a hydrogen bond donor.

[0018] As used herein, the term "protic solvent" refers to a solvent that contains dissociable H+ ions.

[0019] As used herein, the term "amphiphilic molecule" or "amphiphilic matrix" refers to a molecule that contains both hydrophobic and hydrophilic segments within the molecule. The term "hydrophilic" in this context refers to the segment of an amphiphilic molecule that has a high affinity for aqueous solutions, such as water. The term "hydrophobic" in this context refers to the segment of an amphiphilic molecule that repels aqueous solutions, such as water.

[0020] The term "lipid moiety" refers to a moiety that contains at least one lipid. As used herein, the term "lipid" refers to a small molecule having hydrophobic or amphiphilic properties, including, but not limited to, fats, waxes, fatty acids, cholesterol, sterols, phospholipids, monoglycerides, diglycerides, and triglycerides. Fatty acids may be saturated, monounsaturated, or polyunsaturated. Examples of fatty acids include, but are not limited to, butyric acid (C4), caproic acid (C6), caprylic acid (C8), capric acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid (C16), palmitoleic acid (C16), stearic acid (C18), isostearic acid (C18), oleic acid (C18), vaccenic acid (C18), These include linoleic acid (C18), alpha-linoleic acid (C18), gamma-dioleic acid (C18), arachidic acid (C20), gadoleic acid (C20), arachidonic acid (C20), eicosapentaenoic acid (C20), behenic acid (C22), erucic acid (C22), docosahexaenoic acid (C22), lignoceric acid (C24), and hexacosanoic acid (C26). The lipid portion contains several fatty acid groups that use branching groups such as lysine and other branched amines.

[0021] The term "functional group," as used herein, refers to any chemical unit that can be attached to a fluorescent polymer by any stable physical or chemical association, thereby altering the surface of the polymer dot, e.g., allowing the surface to be conjugated to a biomolecule (e.g., bioconjugation). The functional group is covalently attached to the backbone, side chain, or one of the terminal units of the fluorescent polymer. Functional groups include, but are not limited to, aldehyde, alkene, alkyl, alkyne, strained alkyne, amino, azide, carbonyl, carboxyl, cyano, cyclooctyne, dieno, ester, succinimidyl ester, haloalkyl, hydroxyl, imide, ketone, maleimide, mercapto, phosphate, phosphine, sulfate, sulfonate, substituted derivatives thereof, or combinations thereof. In general, any functional group suitable for bioconjugation can be used. Such functional groups are described, for example, in Bioconjugate Techniques (Academic Press, New York, 2013), which is incorporated herein by reference in its entirety for all purposes.

[0022] As used herein, the term "hydrophilic functional group" refers to a functional group that is hydrophilic in nature.

[0023] As used herein, the term "derivative" refers to a chemical substance or compound obtained or derived from another. For example, a BODIPY derivative is derived from BODIP.

[0024] The term "aliphatic," as used herein, refers to organic compounds or radicals characterized by a straight- or branched-chain structure, or a closed ring structure, any of which may contain saturated carbon bonds and, optionally, one or more non-conjugated carbon-carbon unsaturated bonds, such as carbon-carbon double bonds. Aliphatic groups have 1 to 24 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms.

[0025] As used herein, the term "biomolecule" refers to a synthetic or naturally occurring protein (e.g., an antibody), glycoprotein, peptide, amino acid, metabolite, drug, toxin, nucleic acid, nucleotide, carbohydrate, sugar, lipid, or fatty acid.

[0026] "Antibody" refers to immunoglobulin, composite (e.g., fusion), or fragmentary forms thereof. This term includes polyclonal or monoclonal antibodies of the IgA, IgD, IgE, IgG, and IgM classes derived from human or other mammalian cell lines, including natural or genetically modified forms such as, but not limited to, humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro-generated antibodies. "Antibody" also includes composite forms, including, but not limited to, fusion proteins with an immunoglobulin portion. "Antibody" also includes antibody fragments, such as Fab, F(ab'), Fv, scFv, Fd, dAb, and Fc, regardless of whether they retain antigen-binding function.

[0027] Polymer Dots In one embodiment, the polymer dot comprises a fluorescent polymer having a hydrophobic region and a hydrophilic region, and the hydrophilic region comprises a hydrophilic functional group. The polymer dot further comprises an amphiphilic molecule having a hydrophobic region and a hydrophilic region. The hydrophobic region of the fluorescent polymer and the amphiphilic molecule is embedded in the hydrophobic core of the polymer dot, the hydrophilic region of the fluorescent polymer and the amphiphilic molecule forms a hydrophilic outer layer, and the hydrophilic functional group in the fluorescent polymer is located in the hydrophilic outer layer on the surface of the polymer dot. By being localized on the surface of the polymer dot, the hydrophilic functional group is available for attachment to a biomolecule. In certain embodiments, the fluorescent polymer is conjugated and comprises a π-electron delocalized backbone. The fluorescent polymer can be partially or fully conjugated. As used herein, the term "fluorescent polymer" refers to a polymer that exhibits fluorescent properties. Non-limiting examples of fluorescent polymers include polyfluorenes (such as PDHF and PFO), poly(phenyleneethynylenes) (such as PPE), poly(phenylenevinylenes) (such as MEH-PPV and CN-PPV), fluorene-based copolymers (such as PFPV, PFBT, and PFDBT5), and related derivatives (see Figure 3 in Wu and Chiu, Angew. Chem Int. Ed. 2013, 52:3086-3109, which is incorporated herein by reference in its entirety). Other non-limiting examples of fluorescent polymers include BODIPY-containing polymers and BODIPY monomers forming polymers such as those disclosed in Rong et al., ACS Nano, 2013, 7(1):376-384, which is incorporated herein by reference in its entirety.

[0028] In one embodiment, the fluorescent polymer is a heteropolymer comprising at least two different monomers. In one embodiment, at least one monomer is fluorescent, and at least one monomer is "functionalized" by being attached or linked (e.g., covalently attached) to a hydrophilic functional group. In one embodiment, the hydrophilic functional group is attached to a side chain of a monomer within the polymer chain of the fluorescent polymer. In some embodiments, the hydrophilic functional group is attached to a terminal unit of the fluorescent polymer. In certain embodiments, the fluorescent polymer comprises from about 6 to less than about 10% of the monomers linked to hydrophilic functional groups.

[0029] Examples of monomers for forming fluorescent polymers include, but are not limited to, BODIPY, BODIPY derivatives, fluorene, fluorene derivatives, benzothiadiazole, benzothiadiazole derivatives, benzoxadiazole, and benzoxadiol derivatives. In some embodiments, the heteropolymer comprises at least two different monomers. In some embodiments, the monomers are selected from the group consisting of dibromobenzoxadiol, 4,7-bis(2-bromo-5-thienyl)-2,1,3-benzothiadiazole, 9,9-dioctyl-2,7-dibromofluorene, 9,9-dioctylfluorene-2,7-diboronic acid bis(1,3-propanediol) ester, and the like. [ka] and / or [ka] where n=10-30. Having polymer side chains with n=10-30 creates "arms" where the hydrophilic functional groups in the fluorescent polymer are located in the hydrophilic outer layer on the surface of the polymer dot, making the hydrophilic functional groups available for bioconjugation. In one embodiment, n=22.

[0030] The amphiphilic molecules help maintain the water solubility and stability of the polymer dots in solution without causing aggregation for at least about 1 week, 1 month, 3 months, 6 months, 1 year, 3 years, or 5 years or more. The hydrophobic region of the amphiphilic molecule comprises a saturated or unsaturated aliphatic chain moiety having 1 to 24 carbon atoms, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms. In embodiments, the aliphatic chain moiety forms part of the lipid moiety. The lipid moiety includes, but is not limited to, a 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) moiety, a 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) moiety, a 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) moiety, or a 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) moiety.

[0031] The hydrophilic region of the amphiphilic molecule comprises a hydrophilic polymer, including, but not limited to, polyoxyalkylene, polyalkylene glycol, and polycarboxyalkylene. Examples of hydrophilic polymers include, but are not limited to, polyethylene glycol (PEG), polypropylene glycol, polybutylene glycol, polycarboxymethylene, polycarboxyethylene (or polyacrylic acid (PAA)), polycarboxypropylene, and polycarboxybutylene.

[0032] In some embodiments, the molecular weight of PEG is about 800 to about 5000, about 800 to about 4800, about 800 to about 4600, about 800 to about 4400, about 800 to about 4200, about 800 to about 4000, about 800 to about 3800, about 800 to about 3600, about 800 to about 3400, about 800 to about 3200, about 800 to about 3000, about 800 to about 2800, about 800 to about 2600, about 800 to about 2400, about 800 to about 2200, about 800 to about 2000, about 800 to about 1800, about 800 to about 16 ... 00, about 800 to about 1400, about 800 to about 1200, about 800 to about 1000, about 1000 to about 2000, about 1000 to about 3000, about 1000 to about 4000, about 1000 to about 5000, about 800, about 1000, about 1200, about 1400, about 1600, about 1800, about 2000, about 2200, about 2400, about 2600, about 2800, about 3000, about 3200, about 3400, about 3600, about 3800, about 4000, about 4200, about 4400, about 4600, about 4800, and about 5000.

[0033] Amphiphilic molecules include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DSPE-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-hydroxyl-1000 or -2000] (DSPE-PEG-OH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DSPE-PEG-OCH3), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DSPE-PEG-NH2), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DSPE-PEG-COOH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DSPE-PEG-maleimide), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DSPE-PEG-biotin), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DMPE-PEG), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)hydroxyl-1000 or -2000] (DMPE-PEG-OH), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DMPE-PEG-OCH3), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DMPE-PEG-NH2), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DMPE-PEG-COOH), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DMPE-PEG-maleimide), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DMPE-PEG-biotin), 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DLPE-PEG) 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxyl-1000 or -2000] (DLPE-PEG-OH) 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DLPE-PEG-OCH3), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DLPE-PEG-NH2), 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DLPE-PEG-COOH) 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DLPE-PEG-maleimide), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DLPE-PEG-biotin), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DPPE-PEG) 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxyl-1000 or -2000] (DPPE-PEG-OH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DPPE-PEG-OCH3), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DPPE-PEG-NH2), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DPPE-PEG-COOH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DPPE-PEG-maleimide), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DPPE-PEG-biotin), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DSPE-PAA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DSPE-PAA-OH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DSPE-PAA-OCH3), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DSPE-PAA-NH2), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DSPE-PAA-COOH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DSPE-PAA-maleimide), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DSPE-PAA-biotin), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DMPE-PAA), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DMPE-PAA-OH), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DMPE-PAA-OCH3), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DMPE-PAA-NH2), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DMPE-PAA-COOH), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DMPE-PAA-maleimide), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DMPE-PAA-biotin), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DLPE-PAA), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DLPE-PAA-OH), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DLPE-PAA-OCH3), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DLPE-PAA-NH2), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DLPE-PAA-COOH), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DLPE-PAA-maleimide), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DLPE-PAA-biotin), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DPPE-PAA), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DPPE-PAA-OH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DPPE-PAA-OCH3), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DPPE-PAA-NH2), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DPPE-PAA-COOH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DPPE-PAA-maleimide) and An example is 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DPPE-PAA-biotin).

[0034] In some embodiments, the weight ratio of amphiphilic molecule to fluorescent polymer (expressed as w / w%) ranges from about 10% to about 200%, from about 10% to about 175%, from about 10% to about 150%, from about 10% to about 100%, from about 10% to about 75%, or from about 100% to about 200%. In some embodiments, the weight ratio of amphiphilic molecule to fluorescent polymer is about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, or about 200%. In various other embodiments, the weight ratio of amphiphilic molecule to fluorescent polymer ranges from about 40% to about 100% (or about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%). These values ​​can also be expressed in decimal form rather than as percentages (e.g., 0.1, 0.25, etc., as shown in Table 1). In other embodiments, the weight ratio (w / w) is expressed as a range of amphiphilic molecule to fluorescent polymer that is about 0.1 to about 2 (i.e., about 0.1 to 2.0:1). Some of these embodiments result in weight ratios of about 0.25:1, about 0.5:1, about 0.75:1, about 1:1, about 1.25:1, about 1.5:1, about 1.75:1, or about 2:1. Too low a weight ratio results in unstable polymer dots that aggregate, while too high a weight ratio results in modest particle size growth (see Table 1). In some instances, using a higher weight ratio causes problems in downstream processes (e.g., low conjugation yield, see Table 6). Table 1 also shows various weight ratios of amphiphilic molecule to polymer (e.g., 0.25 mg amphiphilic molecule per mg fluorescent polymer).

[0035] In embodiments, the size (average effective diameter) of the polymer dots ranges from about 5 nm to about 25 nm, from about 10 nm to about 25 nm, from about 10 nm to about 25 nm, or from about 15 nm to about 25 nm. In alternative embodiments, the size of the polymer dots ranges from about 10 nm to about 20 nm, from about 10 nm to about 15 nm, or from about 15 nm to about 20 nm. In some embodiments, the size of the polymer dots is about 10 nm, about 15 nm, about 20 nm, or about 25 nm. Still other embodiments provide polymer dots with an average effective diameter of about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, about 20 nm, about 21 nm, about 22 nm, about 23 nm, about 24 nm, or about 25 nm.

[0036] In other embodiments, populations of polymer dots formed by the disclosed methods have a size (average effective diameter) of about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, about 20 nm, about 21 nm, about 22 nm, about 23 nm, about 24 nm, or about 25 nm, wherein at least 50% of the polymer dots in the population have an average effective diameter that is equal to or less than a specified value. Still other embodiments provide populations of polymer dots where at least 50% have an average effective diameter equal to or less than about 25 nm and at least greater than (<) about 5 nm (e.g., at least 50% of the polymer dots in the population have an average effective diameter in the range of about 5 nm to about 25 nm). Yet other embodiments provide a population of polymer dots, at least 50% of which have an average effective diameter of about 20 nm or less and at least greater than (<) about 5 nm (e.g., at least 50% of the polymer dots in the population have an average effective diameter in the range of about 5 nm to about 20 nm).

[0037] Bioconjugation of polymer dots Also provided is a method for conjugating polymer dots to biomolecules. The biomolecules are conjugated to the surface of the polymer dots via hydrophilic functional groups in the fluorescent polymer used to generate the polymer dots. In one embodiment, the biomolecules are monoclonal antibodies that bind to antigens on the surface of cells.

[0038] The method used to attach or conjugate biomolecules to the surface of the polymer dots varies depending on the type of functional group present on the surface. For example, the attachment of proteins to polymer dot-NH or polymer dot-COOH may be via a carboimide-mediated coupling reaction. In one embodiment, the properties of the biomolecule-conjugate-functionalized polymer dots are not altered upon bioconjugation.

[0039] In another embodiment, a method for detecting a target molecule in a biological sample comprises contacting the biological sample with the disclosed polymer dots.

[0040] Polymer dot manufacturing method Also provided is a method for fabricating polymer dots, the method comprising preparing a mixture of a fluorescent polymer and an amphiphilic molecule in an aprotic solvent, the fluorescent polymer comprising a hydrophobic region and a hydrophilic region, the hydrophilic region having a hydrophilic functional group, and the amphiphilic molecule comprising a hydrophobic region and a hydrophilic region.

[0041] The next step of the method involves adding the mixture to a protic solvent to form polymer dots, in which the hydrophobic regions of the fluorescent polymer and amphiphilic molecules are embedded in the hydrophobic core of the polymer dot, the hydrophilic regions of the fluorescent polymer and amphiphilic molecules form a hydrophilic outer layer, and the hydrophilic functional groups in the fluorescent polymer are located in the hydrophilic outer layer on the surface of the polymer dot.

[0042] When a mixture of fluorescent polymers and amphiphilic molecules in an aprotic solvent is added to a protic solvent, spherical polymer dots are formed. The reduction in hydrophobicity leads to the collapse of the hydrophobic regions of the fluorescent polymer and amphiphilic molecules. The polymer dots thus formed have hydrophobic regions embedded in the core and hydrophilic regions of the fluorescent polymer, and amphiphilic molecules on the outer surface so that functional groups are available for bioconjugation.

[0043] Examples of aprotic solvents include, but are not limited to, tetrahydrofuran, ether, dichloromethane, acetone, acetonitrile, and dimethylformamide. Exemplary protic solvents include, but are not limited to, water, methanol, ethanol, propanol, and butanol.

[0044] In some embodiments, the method further comprises controlling the size of the polymer dots by adjusting the initial concentration of the fluorescent polymer in the aprotic solvent. Controlling the size of the polymer dots allows for the use of the polymer dots in specific applications. For example, polymer dots with a size of about 10 nm to about 20 nm can be used in flow cytometry. Polymer dots of other appropriate sizes are useful in different applications.

[0045] In some embodiments, the initial concentration of the fluorescent polymer in the aprotic solvent ranges from about 0.05 mg / mL to about 5.0 mg / mL, from about 0.1 to about 1.0 mg / mL, from about 0.2 mg / mL to about 1.0 mg / mL, from about 0.3 mg / mL to about 1.0 mg / mL, from about 0.4 mg / mL to about 1.0 mg / mL, from about 0.5 mg / mL to about 1.0 mg / mL, from about 0.6 mg / mL to about 1.0 mg / mL, from about 0.7 mg / mL to about 1.0 mg / mL, from about 0.8 mg / mL to about 1.0 mg / mL, or from about 0.9 mg / mL to about 1.0 mg / mL. In some embodiments, the initial concentration of the fluorescent polymer in the aprotic solvent is about 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or about 1.0 mg / mL.

[0046] In certain embodiments, the method further comprises controlling the stability of the polymer dots by controlling the initial concentration of the amphiphilic molecules in the aprotic solvent.

[0047] Various non-limiting embodiments include the following. 1. A fluorescent polymer having a hydrophobic region and a hydrophilic region having a hydrophilic functional group; an amphiphilic molecule having a hydrophobic region and a hydrophilic region; wherein the hydrophilic functional groups are utilized for conjugation. 2. The polymer dot of embodiment 1, wherein the hydrophobic regions of the fluorescent polymer and the amphiphilic molecule are embedded in a hydrophobic core of the polymer dot, the hydrophilic regions of the fluorescent polymer and the amphiphilic molecule form a hydrophilic outer layer, and the hydrophilic functional groups in the fluorescent polymer are located in the hydrophilic outer layer on the surface of the polymer dot. 3. The polymer dot of embodiment 1 or 2, wherein the fluorescent polymer is a heteropolymer. 4. The polymer dot of embodiment 3, wherein the heteropolymer comprises at least two different monomers. 5. The polymer dot of embodiment 4, wherein the monomers are each independently selected from the group consisting of BODIPY, BODIPY derivatives, fluorene, fluorene derivatives, benzothiadiazole, benzothiadiazole derivatives, benzoxadiazole, and benzoxadiol derivatives. 6. The monomers each independently represent: [ka] (dibromobenzoxadiol), [ka] (4,7 bis(2-bromo-5-thienyl)-2,1,3-benzothiadiazole), [ka] (9,9-dioctyl-2,7-dibromofluorene), [ka] (9,9-dioctylfluorene-2,7-diboronic acid bis(1,3-propanediol) ester), [ka] and [ka] (n=10-30). 7. The polymer dot of embodiment 6, wherein n is 22. 8. The polymer dot of any one of embodiments 1 to 7, wherein the hydrophilic functional group is selected from the group consisting of carboxyl, amino, mercapto, azide, alkyne, aldehyde, hydroxyl, carbonyl, sulfate, sulfonate, phosphate, cyanate, succinimidyl ester, strained alkyne, azide, diene, alkene, tetrazine, strained alkene, cyclooctyne, phosphine group, and derivatives thereof. 9. The polymer dot according to any one of embodiments 1 to 8, wherein the hydrophilic functional group in the polymer is conjugated to a biomolecule. 10. The polymer dot of embodiment 9, wherein the biomolecule is selected from the group consisting of synthetic or naturally occurring proteins, glycoproteins, polypeptides, amino acids, nucleic acids, carbohydrates, lipids, and fatty acids. 11. The polymer dot according to embodiment 9 or 10, wherein the biomolecule is an antibody. 12. The polymer dot of embodiment 1 or 2, wherein the hydrophilic region of the amphiphilic molecule comprises a polyalkylene glycol. 13. The polymer dot of embodiment 12, wherein the polyalkylene glycol is polyethylene glycol. 14. The polymer dot of any one of embodiments 1 to 13, wherein the size of the polymer dot is from about 5 to about 25 nanometers or from about 5 to about 20 nm. 15. The polymer dot according to any one of embodiments 1 to 14, wherein the weight ratio (w / w%) of the amphiphilic molecule to the fluorescent polymer is from about 10% to about 200%. 16. The polymer dot of embodiment 1 or 2, wherein the hydrophobic region of the amphiphilic molecule comprises a lipid moiety selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphoethanolamino (DSPE) moieties, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) moieties, 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) moieties, and (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) (DPPE) moieties. 17. The amphiphilic molecule is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DSPE-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-hydroxyl-1000 or -2000] (DSPE-PEG-OH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DSPE-PEG-OCH3), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DSPE-PEG-NH2), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DSPE-PEG-COOH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DSPE-PEG-maleimide), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DSPE-PEG-biotin), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DMPE-PEG), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)hydroxyl-1000 or -2000] (DMPE-PEG-OH), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DMPE-PEG-OCH3), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DMPE-PEG-NH2), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DMPE-PEG-COOH), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DMPE-PEG-maleimide), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DMPE-PEG-biotin), 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DLPE-PEG) 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxyl-1000 or -2000] (DLPE-PEG-OH) 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DLPE-PEG-OCH3), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DLPE-PEG-NH2), 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DLPE-PEG-COOH) 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DLPE-PEG-maleimide), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DLPE-PEG-biotin), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DPPE-PEG) 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxyl-1000 or -2000] (DPPE-PEG-OH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DPPE-PEG-OCH3), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DPPE-PEG-NH2), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DPPE-PEG-COOH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DPPE-PEG-maleimide), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DPPE-PEG-biotin), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DSPE-PAA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DSPE-PAA-OH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DSPE-PAA-OCH3), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DSPE-PAA-NH2), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DSPE-PAA-COOH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DSPE-PAA-maleimide), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DSPE-PAA-biotin), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DMPE-PAA), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DMPE-PAA-OH), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DMPE-PAA-OCH3), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DMPE-PAA-NH2), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DMPE-PAA-COOH), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DMPE-PAA-maleimide), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DMPE-PAA-biotin), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DLPE-PAA), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DLPE-PAA-OH), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DLPE-PAA-OCH3), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DLPE-PAA-NH2), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DLPE-PAA-COOH), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DLPE-PAA-maleimide), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DLPE-PAA-biotin), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DPPE-PAA), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DPPE-PAA-OH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DPPE-PAA-OCH3), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DPPE-PAA-NH2), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DPPE-PAA-COOH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DPPE-PAA-maleimide), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DPPE-PAA-biotin) 17. The polymer dot according to any one of embodiments 1 to 16, selected from the group consisting of: 18. A method for producing polymer dots, comprising: preparing a mixture of an amphiphilic molecule and a fluorescent polymer in an aprotic solvent; adding the mixture to a protic solvent to form polymer dots; The fluorescent polymer comprises a hydrophobic region and a hydrophilic region, the hydrophilic region having a hydrophilic functional group, the amphiphilic molecule comprises a hydrophobic region and a hydrophilic region, the weight ratio (w / w) of the amphiphilic molecule to the fluorescent polymer (amphiphilic molecule:fluorescent polymer) is 0.1 to 2:1 or about 10% to about 200%, and the hydrophilic functional group is utilized for conjugation. 19. The method of embodiment 18, wherein the hydrophobic regions of the fluorescent polymer and the amphiphilic molecule are embedded in the hydrophobic core of the polymer dot, the hydrophilic regions of the fluorescent polymer and the amphiphilic molecule form a hydrophilic outer layer, and the hydrophilic functional groups in the fluorescent polymer are located in the hydrophilic outer layer on the surface of the polymer dot. 20. The method of embodiment 18 or 19, wherein the aprotic solvent is tetrahydrofuran. 21. The method of embodiment 18 or 19, wherein the protic solvent is water. 22. The method of any one of embodiments 18 to 21, further comprising conjugating a biomolecule to the polymer dot via the hydrophilic functional group. 23. The amphiphilic molecule is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DSPE-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-hydroxyl-1000 or -2000] (DSPE-PEG-OH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DSPE-PEG-OCH3), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DSPE-PEG-NH2), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DSPE-PEG-COOH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DSPE-PEG-maleimide), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DSPE-PEG-biotin), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DMPE-PEG), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)hydroxyl-1000 or -2000] (DMPE-PEG-OH), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DMPE-PEG-OCH3), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DMPE-PEG-NH2), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DMPE-PEG-COOH), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DMPE-PEG-maleimide), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DMPE-PEG-biotin), 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DLPE-PEG) 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxyl-1000 or -2000] (DLPE-PEG-OH) 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DLPE-PEG-OCH3), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DLPE-PEG-NH2), 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DLPE-PEG-COOH) 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DLPE-PEG-maleimide), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DLPE-PEG-biotin), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DPPE-PEG) 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxyl-1000 or -2000] (DPPE-PEG-OH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DPPE-PEG-OCH3), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DPPE-PEG-NH2), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DPPE-PEG-COOH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DPPE-PEG-maleimide), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DPPE-PEG-biotin), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DSPE-PAA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DSPE-PAA-OH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DSPE-PAA-OCH3), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DSPE-PAA-NH2), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DSPE-PAA-COOH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DSPE-PAA-maleimide), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DSPE-PAA-biotin), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DMPE-PAA), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DMPE-PAA-OH), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DMPE-PAA-OCH3), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DMPE-PAA-NH2), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DMPE-PAA-COOH), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DMPE-PAA-maleimide), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DMPE-PAA-biotin), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DLPE-PAA), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DLPE-PAA-OH), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DLPE-PAA-OCH3), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DLPE-PAA-NH2), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DLPE-PAA-COOH), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DLPE-PAA-maleimide), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DLPE-PAA-biotin), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DPPE-PAA), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DPPE-PAA-OH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DPPE-PAA-OCH3), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DPPE-PAA-NH2), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DPPE-PAA-COOH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DPPE-PAA-maleimide), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DPPE-PAA-biotin) 23. The method of any one of embodiments 18 to 22, selected from the group consisting of: 24. The weight ratio (expressed as a percentage) of amphiphilic molecule to fluorescent polymer is about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105% , about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, or about 200%. 25. The method of any one of embodiments 18-23, wherein the weight ratio of amphiphilic molecule to fluorescent polymer is about 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1.0:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2.0:1. 26. A population of polymer dots produced by the method of any one of embodiments 18 to 25, wherein the population of polymer dots has an average effective diameter of about 5 nm to about 25 nm, with the proviso that at least 50% of the polymer dots in the population have an average effective diameter of less than about 25 nm. 27. A method for detecting a target molecule in a biological sample, comprising contacting the biological sample with a polymer dot of any one of embodiments 1 to 17 or a population of polymer dots described in embodiment 26, and detecting the target molecule. 28. The polymer dot of any one of embodiments 1 to 17, wherein the polymer dots have an average effective diameter of about 5 nm to about 25 nm, provided that at least 50% of the polymer dots in the population have an average effective diameter of less than about 25 nm. 29. The polymer dot according to embodiment 1 or 2, wherein the fluorescent polymer is a homopolymer.

[0048] Example The following examples are illustrative only, and not limiting. Those of ordinary skill in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield substantially the same or similar results.

[0049] Example 1 - Determination of the optimal ratio of DSPE-PEG-OH to Polymer 700 Polymer 700 was dissolved in 50 mL of destabilized tetrahydrofuran (THF) to produce a 200 ppm polymer solution. Using a 25 mg / mL stock solution of DSPE-PEG-OH nanosoft polymer in anhydrous dimethyl sulfoxide, six different mixtures of polymer and PEG-lipid were produced by adding 0.25, 0.5, 0.75, 1.25, 1.5, or 1.75 mass equivalents of DSPE-PEG-OH to the polymer solution. For each mixture, 100 mL of nanopure water was cooled in an ice bath (temperature = 5-6 °C) for 30 min while the polymer and PEG-lipid mixture in THF was cooled for 15 min (temperature = 2-3 °C).

[0050] To form polymer dots, each mixture of polymer and PEG-lipid in THF was pumped (speed = 30 mL / min) into 100 mL of cold nanopure water while stirring (speed = 11.0-11.2 K rpm). The THF in the polymer dot mixture was evaporated for 3 h using a cetriVap vacuum concentrator. This process also removed some water, resulting in a polymer dot solution of approximately 200 ppm.

[0051] Batches of polymer dots were further concentrated to 5000 ppm using Corning Spin-X UF Concentrators or Sartorius Vivacell 250 (100k MWCO PES).

[0052] The size of each lot of polymer dots was measured by dynamic light scattering (DLS). Quantum yield (QY) was determined by photoluminescence QY. The percent yield of the polymer dot lot was also calculated. The results are shown in Table 1. The results indicate that the ratio of DSPE-PEG-OH to polymer affects the size of the polymer dots. Polymer dots with less than 1.25 mg of DSPE-PEG-OH per mg of polymer yielded p-dots of 6–7 nm (by DLS). However, after concentration to 5000 ppm, the size of these same polymer dots increased to 17–20 nm (by DLS). The increase in size was due to aggregate formation, resulting in low yields (44–69%). The quantum yield also decreased for these lots. Adding 1.5 or 1.75 mg of DSPE-PEG-OH per mg of polymer slightly increased the DLS size, increasing the cost of polymer dot production.

[0053] [Table 1]

[0054] [Table 2]

[0055] Example 2 - Formation of polymer dots using polymer 700 and DSPE-PEG-OH Polymer 700 was dissolved in 50 mL of destabilized tetrahydrofuran (THF) to produce a 200 ppm polymer solution. Using a 25 mg / mL stock solution of DSPE-PEG-OH (Nano-soft Polymers) in anhydrous dimethyl sulfoxide, 1.25 mass equivalents of DSPE-PEG-OH were added to the polymer solution to produce a polymer-PEG-lipid mixture. The polymer-PEG-lipid mixture in THF was cooled for 15 minutes (temperature = 2-3 °C) while 100 mL of nanopure water was cooled in an ice bath (temperature = 5-6 °C) for 30 minutes.

[0056] To form polymer dots, the mixture of polymer and PEG-lipid in THF was pumped (rate = 30 mL / min) into cold nanopure water while stirring (rate = 11.0-11.2 K rpm). The THF in the polymer dot mixture was evaporated for 3 h using a cetriVap vacuum concentrator. This process also removed some water, resulting in a polymer dot solution of approximately 200 ppm.

[0057] Some batches of polymer dots were further concentrated to 5000 ppm using Corning Spin-X UF concentrators (100k MWCO PES). Alternatively, multiple batches of polymer dots were pooled together and concentrated to 5000 ppm using a Sartorius Vivacell 250 (100k MWCO PES) in preparation for protein conjugation.

[0058] The size of the polymer dots, as measured by dynamic light scattering (DLS), along with the % yield for four different lots of polymer dots, is shown in Table 2. The results show consistent size from lot to lot. The results also show that concentration to approximately 5000 ppm resulted in a slight decrease in yield for three of the four lots.

[0059] [Table 3]

[0060] Example 3 - Formation of polymer dots using polymer 460 and DSPE-PEG-OH In this example, the same method as in Example 2 was used to generate polymer dots with a different polymer (polymer 460).

[0061] The size of each lot of polymer dots was measured by dynamic light scattering (DLS). The quantum yield (QY) was determined by photoluminescence. The percent yield of the polymer dot lots was also calculated. The results are shown in Table 3. The results show consistent size from lot to lot. The percent yield varied from 70% to 91% for the 200 ppm polymer dots. The results also show a slight decrease in yield for all three lots at concentrations up to approximately 5000 ppm. The quantum yield did not change at concentrations up to approximately 5000 ppm.

[0062] [Table 4]

[0063] Example 4 - Formation of polymer dots using polymer 700 and DSPE-PEG-OCH3 Different types of PEG-lipids were used to generate polymer dots. The same method as in Example 2 was used, except that DSPE-PEG-OCH3 was used instead of DSPE-PEG-OH. Two different ratios of PEG-lipid to polymer were also tested: 1.25 mg or 0.75 mg lipid per mg polymer.

[0064] The size of each batch of polymer dots was measured by dynamic light scattering (DLS). The percent yield of the polymer dot batch was also calculated. The results are shown in Tables 4 and 5 below. The results show similar results in size and yield for both PEG-lipid to polymer ratios tested, indicating that less DSPE-PEG-OCH3 is required to stabilize the polymer dots.

[0065] [Table 5]

[0066] [Table 6]

[0067] Example 5 - Binding of IgG antibodies to polymer dots 250 μl (1.25 mg) of the polymer dots from Example 2 were first activated by incubating them in a 1.5 mL Eppendorf tube with 6.3 μl of 250 mM sulfo-NHS (Thermo Fisher) and 3.2 μl of 200 mM EDC (Thermo Fisher) for 30 minutes at room temperature. Next, 3.2 μl of 3 M triethanolamine (Sigma-Aldrich) and 3.2 μl of 1 M aminoethylmaleimide (Sigma-Aldrich) were added to the tube, mixed, and the tube was incubated in the dark at room temperature for 3 hours.

[0068] Anti-CD4 IgG (Bio-Rad) was activated by adding 2.0 μl of 0.5 M EDTA (Sigma-Aldrich) and 2.0 μl of 10 mg / mL iminothiolane (Thermo Fisher Scientific) to 200 μl (1 mg) of anti-CD4 IgG in a 1.5 mL Eppendorf tube. The solution was mixed and incubated at room temperature for 1 hour.

[0069] The activated polymer dots were quenched by adding 20 μl of 0.5 M taurine (Sigma-Aldrich) to the polymer dot solution, which was mixed and incubated in the dark at room temperature for 15 minutes.

[0070] Both the activated polymer dots and anti-CD4 IgG were desalted using a 40K MWCO spin column (Thermo Fisher Scientific), and the total amount of each desalted product was determined using absorbance. A 3:1 ratio of polymer dots to IgG was used in the conjugation reaction, which was incubated overnight at 4°C in the dark. The conjugation was quenched by adding 1 / 200 volume of 25% N-ethylmaleimide (Sigma-Aldrich) and incubating at room temperature in the dark for 30 minutes.

[0071] Free anti-CD4 antibody was removed from the polymer dot-IgG conjugates by size exclusion chromatography.

[0072] Example 6 - Characterization of polymer dots conjugated to IgG versus control conjugates by flow cytometry Flow cytometry was used to determine how the polymer dot anti-CD4 IgG conjugates from Example 5 (or test conjugates) compared to a control conjugate (PerCPCy5.5, BioLegend). A ZE5 Sapphire flow cytometer (Bio-Rad) was used for the experiments.

[0073] The staining index was determined for the test and control conjugates. The staining index was calculated using the formula: (MFIpos-MFIneg) / (2*rSDneg), where MFIpos is the median fluorescence intensity of the positive population, MFIneg is the median fluorescence intensity of the negative population, and rSDneg is the relative standard deviation of the negative population. Referring to Figure 1, the test conjugate gives a significantly higher staining index (>>2x) than the control conjugate.

[0074] Histogram plots of positive and negative cell populations were obtained for the test (6452-66-1) and control (PerCPCy5.5) conjugates. The results are shown in Figure 2. For the test conjugates, SEC refers to size exclusion chromatography. The positive peak shape and height of the positive and negative peaks for the test conjugates were comparable to those of the control conjugate.

[0075] Example 7 - Effect of PEG-lipids on P-dot stability experiment: For each polymer, two sets of small P-dots were generated: one containing DSPE-PEG1000-OH (PEG-lipid) and the other without PEG-lipid. The amount of PEG-lipid added was different for each polymer and had been previously determined in separate experiments. Both sets of small P-dots were concentrated to ∼5000 ppm. Particle size and yield recovery were evaluated to determine P-dot stability. result:

[0076] [Table 7]

[0077] The instability of small P-dots produced without PEG-lipids was already evident even at concentrations up to 5000 ppm. For polymers 405 / 610, 405 / 790, and 405 / 515, aggregation was observed early after removal of THF. This resulted in low yields (~60%) compared to small P-dots produced with PEG-lipids (80–97%).

[0078] It was not possible to concentrate the small P-dots produced without PEG-lipids to ~5000 ppm. Aggregates formed and adhered to the concentrator membrane, resulting in low yields (12–24%) that were still diluted (1000–2000 ppm). In contrast, the small P-dots produced with PEG-lipids retained their small particle size with little or no process loss.

[0079] Example 8: Accelerated Stability Study Experimental Method: Accelerated shelf-life testing was performed at 37°C. Based on the Arrhenius model, assuming an activation energy of 15 kcal / mol for the reaction, 20 days at 37°C is theoretically equivalent to one year at 4°C (the recommended storage temperature).

[0080] The Pdots were divided into seven Eppendorf tubes. Six samples were stored at 37°C and removed at different time points (see Table 7 below). One sample was stored at 4°C and served as a control.

[0081] [Table 8]

[0082] After removal from the incubation chamber, the samples were examined for aggregates and particle size was also measured. To pass the stability test, the sample must be free of aggregates and its particle size must be within 15% of the particle size of the reference sample.

[0083] [Table 9] All patents, patent applications, and other published references cited herein are hereby incorporated by reference in their entirety.

Claims

1. a fluorescent polymer having a hydrophobic region and a hydrophilic region having a hydrophilic functional group; an amphiphilic molecule having a hydrophobic region and a hydrophilic region; Including, the hydrophilic functional group is a carboxyl group, an amino group, a mercapto group, an azide group, an aldehyde group, a hydroxyl group, a carbonyl group, a sulfate group, a sulfonate group, a phosphate group, a cyanate group, a succinimidyl ester group, or a combination thereof; the hydrophobic region of the amphiphilic molecule comprises a lipid moiety selected from the group consisting of a 1,2-distearoyl-sn-glycero-3-phosphoethanolamino (DSPE) moiety, a 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) moiety, a 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) moiety, and a (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) (DPPE) moiety; the fluorescent polymer comprises fluorescent monomers, less than 10% of which are bound to hydrophilic functional groups; the fluorescent monomer is selected from the group consisting of BODIPY, a BODIPY derivative, fluorene, a fluorene derivative, benzothiadiazole, a benzothiadiazole derivative, benzoxadiazole, and a benzoxadiazole derivative; The weight ratio (expressed as w / w %) of said amphiphilic molecule to said fluorescent polymer is between 7.5% and 250%.

2. 2. The polymer dot of claim 1, wherein the hydrophobic regions of the fluorescent polymer and the amphiphilic molecule are embedded in a hydrophobic core of the polymer dot, the hydrophilic regions of the fluorescent polymer and the amphiphilic molecule form a hydrophilic outer layer, and the hydrophilic functional groups in the fluorescent polymer are located in the hydrophilic outer layer on the surface of the polymer dot.

3. The polymer dot according to claim 1 or 2, wherein the fluorescent polymer is a heteropolymer.

4. The polymer dot of claim 3 , wherein the heteropolymer comprises at least two different monomers.

5. The monomers each independently represent: 【Chemistry 1】 (dibromobenzoxadiazole), 【Chemistry 2】 (4,7 bis(2-bromo-5-thienyl)-2,1,3-benzothiadiazole), 【Transformation 3】 (9,9-dioctyl-2,7-dibromofluorene), 【Chemistry 4】 (9,9-dioctylfluorene-2,7-diboronic acid bis(1,3-propanediol) ester), 【Transformation 5】 and 【Transformation 6】 (n=10-30), The polymer dot according to claim 1 .

6. The polymer dot of claim 5 , wherein n is 22.

7. The polymer dot according to any one of claims 1 to 6, further comprising a biomolecule bound to the polymer dot via the hydrophilic functional group.

8. The polymer dot of claim 7 , wherein the biomolecule is selected from the group consisting of synthetic or naturally occurring proteins, glycoproteins, polypeptides, amino acids, nucleic acids, carbohydrates, lipids, and fatty acids.

9. The polymer dot according to claim 7 or 8, wherein the biomolecule is an antibody.

10. The polymer dot of claim 1 or 2, wherein the hydrophilic region of the amphiphilic molecule comprises a polyalkylene glycol.

11. The polymer dot of claim 10 , wherein the polyalkylene glycol is polyethylene glycol.

12. The polymer dot according to any one of claims 1 to 11, wherein the size of the polymer dot is 4.5 to 27.5 nanometers.

13. The amphiphilic molecule is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DSPE-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-hydroxyl-1000 or -2000] (DSPE-PEG-OH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DSPE-PEG-OCH3), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DSPE-PEG-NH2), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DSPE-PEG-COOH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DSPE-PEG-maleimide), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DSPE-PEG-biotin), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DMPE-PEG), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)hydroxyl-1000 or -2000] (DMPE-PEG-OH), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DMPE-PEG-OCH3), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DMPE-PEG-NH2), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DMPE-PEG-COOH), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DMPE-PEG-maleimide), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DMPE-PEG-biotin), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DLPE-PEG) 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxyl-1000 or -2000] (DLPE-PEG-OH) 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DLPE-PEG-OCH3), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DLPE-PEG-NH2), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DLPE-PEG-COOH) 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DLPE-PEG-maleimide), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DLPE-PEG-biotin), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DPPE-PEG) 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxyl-1000 or -2000] (DPPE-PEG-OH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DPPE-PEG-OCH3), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DPPE-PEG-NH2), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DPPE-PEG-COOH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DPPE-PEG-maleimide), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DPPE-PEG-biotin), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DSPE-PAA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DSPE-PAA-OH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DSPE-PAA-OCH3), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DSPE-PAA-NH2), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DSPE-PAA-COOH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DSPE-PAA-maleimide), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DSPE-PAA-biotin), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DMPE-PAA), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DMPE-PAA-OH), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DMPE-PAA-OCH3), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DMPE-PAA-NH), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DMPE-PAA-COOH), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DMPE-PAA-maleimide), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DMPE-PAA-biotin), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DLPE-PAA), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DLPE-PAA-OH), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DLPE-PAA-OCH3), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DLPE-PAA-NH2), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DLPE-PAA-COOH), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DLPE-PAA-maleimide), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DLPE-PAA-biotin), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DPPE-PAA), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DPPE-PAA-OH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DPPE-PAA-OCH3), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DPPE-PAA-NH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DPPE-PAA-COOH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DPPE-PAA-maleimide), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DPPE-PAA-biotin) The polymer dot according to any one of claims 1 to 12, selected from the group consisting of:

14. 1. A method for producing polymer dots, comprising: preparing a mixture of an amphiphilic molecule and a fluorescent polymer in an aprotic solvent; adding the mixture to a protic solvent to form polymer dots; the fluorescent polymer comprises a hydrophobic region and a hydrophilic region, the hydrophilic region having a hydrophilic functional group, the amphiphilic molecule comprises a hydrophobic region and a hydrophilic region, the weight ratio (w / w) of the amphiphilic molecule to the fluorescent polymer (amphiphilic molecule:fluorescent polymer) is 7.5% to 250%, and the hydrophilic functional group is a carboxyl group, an amino group, a mercapto group, an azide group, an aldehyde group, a hydroxyl group, a carbonyl group, a sulfate group, a sulfonate group, a phosphate group, a cyanate group, a succinimidyl ester group, or a combination thereof; the hydrophobic region of the amphiphilic molecule comprises a lipid moiety selected from the group consisting of a 1,2-distearoyl-sn-glycero-3-phosphoethanolamino (DSPE) moiety, a 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) moiety, a 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) moiety, and a (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) (DPPE) moiety; the fluorescent polymer comprises fluorescent monomers, less than 10% of which are bound to hydrophilic functional groups; The method, wherein the fluorescent monomer is selected from the group consisting of BODIPY, BODIPY derivatives, fluorene, fluorene derivatives, benzothiadiazole, benzothiadiazole derivatives, benzoxadiazole, and benzoxadiazole derivatives.

15. 15. The method of claim 14, wherein the hydrophobic regions of the fluorescent polymer and the amphiphilic molecules are embedded in a hydrophobic core of the polymer dot, the hydrophilic regions of the fluorescent polymer and the amphiphilic molecules form a hydrophilic outer layer, and the hydrophilic functional groups in the fluorescent polymer are located in the hydrophilic outer layer on the surface of the polymer dot.

16. 16. The method of claim 14 or 15, wherein the aprotic solvent is tetrahydrofuran.

17. 16. The method of claim 14 or 15, wherein the protic solvent is water.

18. The method of any one of claims 14 to 17, further comprising conjugating a biomolecule to the polymer dot via the hydrophilic functional group.

19. The amphiphilic molecule is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DSPE-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-hydroxyl-1000 or -2000] (DSPE-PEG-OH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DSPE-PEG-OCH3), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DSPE-PEG-NH2), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DSPE-PEG-COOH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DSPE-PEG-maleimide), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DSPE-PEG-biotin), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DMPE-PEG), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)hydroxyl-1000 or -2000] (DMPE-PEG-OH), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DMPE-PEG-OCH3), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DMPE-PEG-NH2), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DMPE-PEG-COOH), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DMPE-PEG-maleimide), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DMPE-PEG-biotin), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DLPE-PEG) 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxyl-1000 or -2000] (DLPE-PEG-OH) 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DLPE-PEG-OCH3), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DLPE-PEG-NH2), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DLPE-PEG-COOH) 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DLPE-PEG-maleimide), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DLPE-PEG-biotin), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DPPE-PEG) 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxyl-1000 or -2000] (DPPE-PEG-OH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DPPE-PEG-OCH3), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DPPE-PEG-NH2), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DPPE-PEG-COOH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DPPE-PEG-maleimide), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DPPE-PEG-biotin), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DSPE-PAA), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DSPE-PAA-OH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DSPE-PAA-OCH3), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DSPE-PAA-NH2), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DSPE-PAA-COOH), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DSPE-PAA-maleimide), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DSPE-PAA-biotin), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DMPE-PAA), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DMPE-PAA-OH), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DMPE-PAA-OCH3), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DMPE-PAA-NH), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DMPE-PAA-COOH), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DMPE-PAA-maleimide), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DMPE-PAA-biotin), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DLPE-PAA), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DLPE-PAA-OH), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DLPE-PAA-OCH3), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DLPE-PAA-NH2), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DLPE-PAA-COOH), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DLPE-PAA-maleimide), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DLPE-PAA-biotin), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DPPE-PAA), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DPPE-PAA-OH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DPPE-PAA-OCH3), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DPPE-PAA-NH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DPPE-PAA-COOH), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DPPE-PAA-maleimide), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DPPE-PAA-biotin) The method of any one of claims 14 to 18, selected from the group consisting of:

20. A method for detecting a target molecule in a biological sample, comprising contacting the biological sample with a polymer dot according to any one of claims 1 to 13 and detecting the target molecule.

21. 10. The polymer dots of claim 1, wherein the polymer dots have an average effective diameter between 4.5 nm and 27.5 nm, with the proviso that at least 50% of the polymer dots in the population have an average effective diameter less than 27.5 nm.

22. The polymer dot according to claim 1 or 2, wherein the fluorescent polymer is a homopolymer.

23. A method for detecting a target molecule in a biological sample, comprising contacting the biological sample with a population of polymer dots produced by the method of any one of claims 14 to 19 and detecting the target molecule; The population of polymer dots has an average effective diameter of 4.5 nm to 27.5 nm, with the proviso that at least 50% of the polymer dots in the population have an average effective diameter of less than 27.5 nm.

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