Polymer Dots

Abstract

Polymer dots comprising a functionalized fluorescent polymer and an amphiphilic molecule are provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 884,706, filed on August 9, 2019, which is incorporated herein by reference in its entirety. Background Art

[0003] Nanoparticle-based fluorescent probes, such as quantum dots and polymer-based dots (or polymer dots), are used in various fluorescence-based techniques to study biological systems due to their high brightness and photostability, which are superior to traditional organic dyes. However, inorganic quantum dots can be degraded by reactive oxygen species, releasing toxic heavy metals. Therefore, the use of quantum dots is limited to in vitro applications. Fluorescent polymer dots do not contain heavy metals that can be leached into the solution and can therefore be used for in vivo applications. In order to be used in biological systems, fluorescent probes are coupled to proteins (e.g., antibodies) via functional groups on the probe surface. For polymer-based nanoparticles, the polymer used to make the nanoparticles is hydrophobic and may not always have functional groups on the particle surface to which proteins can be attached. One method for functionalizing the surface of polymer dots is to apply a hydrophilic coating to the surface of the polymer dots. The hydrophilic coating has one or more hydrophilic functional groups to which proteins can be coupled. Summary of the invention

[0004] Polymer dots, methods for preparing polymer dots, and bioconjugates of such polymer dots are provided.

[0005] In one embodiment, the polymer dot comprises a fluorescent polymer having a hydrophobic region and a hydrophilic region, wherein the hydrophilic region has a hydrophilic functional group; and an amphiphilic molecule having a hydrophobic region and a hydrophilic region, wherein the hydrophilic functional group is available for coupling. In some embodiments, 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.

[0006] In some embodiments, the fluorescent polymer is a heteropolymer. In some embodiments, the heteropolymer comprises at least two different monomers. In certain embodiments, the monomer is BODIPY, a BODIPY derivative, fluorene, a fluorene derivative, benzothiadiazole, a benzothiadiazole derivative, a benzoxadiole and / or a benzoxadiazole derivative. In some embodiments, the monomer is:

[0007] (dibromobenzoxadiazole),

[0008] (4,7-bis(2-bromo-5-thienyl)-2,1,3-benzothiadiazole),

[0009] (9,9-dioctyl-2,7-dibromofluorene),

[0010] (9,9-dioctylfluorene-2,7-diboric acid bis(1,3-propylene glycol) ester),

[0011] and / or

[0012]

[0013] wherein n = 10-30. In one embodiment, n = 22. In some embodiments, the fluorescent polymer is a homopolymer.

[0014] In some embodiments, the hydrophilic functional group is a carboxyl, amino, sulfhydryl, azido, alkyne, aldehyde, hydroxyl, carbonyl, sulfate, sulfonate, phosphate, cyanate, succinimidyl ester, strained alkyne, azide, diene, olefin, tetrazine, strained alkene, cyclooctyne, phosphine group or a derivative thereof. In certain embodiments, the hydrophilic functional group in the polymer is coupled 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 dots is about 5-20 nanometers. In some embodiments, the weight ratio of the amphiphilic molecule to the fluorescent polymer is about 10% to about 200%. In another embodiment, the weight ratio is expressed as a range of about 0.1 to about 2 amphiphilic molecules to fluorescent polymers (i.e., about 0.1-2.0: 1). Some of these embodiments provide a weight ratio 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 portion, including but not limited to, a 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) portion, a 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) portion, a 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) portion, and a (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) (DPPE) portion. In some embodiments, the amphiphilic molecule is:

[0015] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DSPE-PEG);

[0016] 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -poly(ethylene glycol)-hydroxy-1000 or -2000] (DSPE-PEG-OH);

[0017] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000](DSPE-PEG-OCH3),

[0018] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DSPE-PEG-NH2);

[0019] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DSPE-PEG-COOH);

[0020] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DSPE-PEG-maleimide);

[0021] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine N-[biotinyl(polyethylene glycol)-1000 or -2000] (DSPE-PEG-biotin);

[0022] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DMPE-PEG);

[0023] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxy-1000 or -2000] (DMPE-PEG-OH);

[0024] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DMPE-PEG-OCH3);

[0025] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DMPE-PEG-NH2);

[0026] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DMPE-PEG-COOH);

[0027] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DMPE-PEG-maleimide);

[0028] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DMPE-PEG-biotin);

[0029] 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DLPE-PEG);

[0030] 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxy-1000 or -2000] (DLPE-PEG-OH);

[0031] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DLPE-PEG-OCH3);

[0032] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DLPE-PEG-NH2);

[0033] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DLPE-PEG-COOH);

[0034] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DLPE-PEG-maleimide);

[0035] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DLPE-PEG-biotin);

[0036] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DPPE-PEG);

[0037] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxy-1000 or -2000] (DPPE-PEG-OH);

[0038] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DPPE-PEG-OCH3);

[0039] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DPPE-PEG-NH2);

[0040] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DPPE-PEG-COOH);

[0041] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DPPE-PEG-maleimide);

[0042] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DPPE-PEG-biotin);

[0043] 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -[(polyacrylic acid)] (DSPE-PAA);

[0044] 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DSPE-PAA-OH);

[0045] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)](DSPE-PAA-OCH3),

[0046] 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DSPE-PAA-NH2);

[0047] 1,2-distearoyl- sn -glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DSPE-PAA-COOH);

[0048] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DSPE-PAA-maleimide);

[0049] 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DSPE-PAA-biotin);

[0050] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DMPE-PAA);

[0051] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DMPE-PAA-OH);

[0052] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DMPE-PAA-OCH3);

[0053] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DMPE-PAA-NH2);

[0054] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DMPE-PAA-COOH);

[0055] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DMPE-PAA-maleimide);

[0056] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DMPE-PAA-biotin);

[0057] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DLPE-PAA);

[0058] 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DLPE-PAA-OH);

[0059] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DLPE-PAA-OCH3);

[0060] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DLPE-PAA-NH2);

[0061] 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DLPE-PAA-COOH);

[0062] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DLPE-PAA-maleimide);

[0063] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DLPE-PAA-biotin);

[0064] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DPPE-PAA);

[0065] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DPPE-PAA-OH);

[0066] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DPPE-PAA-OCH3); or

[0067] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DPPE-PAA-NH2);

[0068] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DPPE-PAA-COOH);

[0069] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DPPE-PAA-maleimide);

[0070] 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DPPE-PAA-biotin).

[0071] In one embodiment, a method for preparing a polymer dot comprises: preparing a mixture of a fluorescent polymer and an amphiphilic molecule in an aprotic solvent, 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; adding the mixture to a protic solvent to form a polymer dot, wherein the hydrophilic functional group is available for coupling. 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 groups in the fluorescent polymer are 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 comprises coupling a biomolecule to the polymer dot via the hydrophilic functional group.

[0072] In one embodiment, a method of detecting a target molecule in a biological sample comprises contacting the biological sample with a polymer dot as described above and elsewhere herein.

[0073] BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 is a bar graph of the staining index of the polymer dot-anti-CD4 antibody conjugate and the control conjugate.

[0075] Figure 2 are histograms of positive and negative cell populations for control conjugate and polymer dot-anti-CD4 antibody conjugate.

[0076] Figure 3 Fluorescent polymers are depicted, such as polyfluorenes (e.g., PDHF and PFO), poly(styrene ethene) (e.g., PPE), poly(styrene vinyl) (e.g., MEH-PPV and CN-PPV), fluorene-based copolymers (e.g., as PFPV, PFBT, and PFDBT5), and related derivatives, which are described in Wu and Chiu, Angew. Chem. Int. Ed. 2013, 52:3086–3109, incorporated herein by reference in its entirety).

[0077] Figures 4A-4B The size distribution of the polymer dots produced by the disclosed method (P-dots 488 / 700 ( Figure 4A ) and P-point 405 / 610( Figure 4B )). As shown, more than 50% of the particles have an average effective diameter less than or equal to about 25 nm. DETAILED DESCRIPTION

[0078] Polymer dots, methods for their manufacture, and their biomolecule conjugates are described herein. Surface functionalization of polymer dots is achieved by mixing fluorescent polymers with functional groups with amphiphilic molecules during the manufacturing process. Polymer dots are stable (e.g., do not aggregate and precipitate from solution) and can be conjugated to biomolecules due to the surface reactive groups. The conjugates can be used in a large 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 gene analysis, protein analysis, metabolite analysis, lipid analysis, FRET-based sensors, high throughput screening, cell imaging, in vivo imaging, fluorescence-based bioassays such as immunoassays and enzyme-based assays, and various fluorescence techniques and measurements in bioassays.

[0079] definition

[0080] Unless otherwise indicated, the following terms used in this application (including the specification and claims) have the following definitions. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Definitions of standard chemical terms can be found in reference works, including Carey and Sundberg (2007) "Advanced Organic Chemistry 5th Edition". th Ed.)" Volumes A and B, Springer Science+Business Media LLC, New York. Unless otherwise indicated, the practice of the present invention will employ conventional methods of synthetic organic chemistry, mass spectroscopy, chromatography, protein chemistry, biochemistry, recombinant DNA techniques and pharmaceutical preparative and analytical methods.

[0081] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend on how the value is measured or determined, i.e., the limits of the measurement system. 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" may mean that the polymer dot size has the stated size with a 0-10% variation (X ± 10%) around that value. Thus, a polymer dot having a diameter of about 20 nm includes polymer dots having a diameter between 18 nm and 22 nm. When the term "about" is used in a ratio (e.g., about 0.1:1), it should be understood that the term "about" applies to both values, specifically, in this exemplary ratio, the term applies to the 0.1 value and the 1 value.

[0082] In the present disclosure, ranges are shorthand to avoid unnecessary detailed listing and description of each value in the range. Where appropriate, any suitable value in the range may be selected as the upper limit, lower limit or end point of the range. For example, the range 0.1-1.0 represents the final values ​​0.1 and 1.0, and the intermediate values ​​0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges are included in 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc.

[0083] As used herein, the term "polymer dots" refers to structures comprising one or more polymers that have been collapsed into stable submicron-sized particles. The polymer dots provided herein can be formed by any method known in the art for collapsing polymers, including but not limited to methods that rely on precipitation, methods that rely on the formation of emulsions (e.g., microemulsions or microemulsions), and methods that rely on condensation. In some embodiments, polymer dots are formed by nanoprecipitation.

[0084] As used herein, "polymer" is a molecule consisting of at least two repeating structural units, usually connected by covalent chemical bonds. The repeating structural unit may be a type of monomer, and the resulting polymer is a homopolymer. In some embodiments, the polymer may include two different types of monomers, or three different types of monomers, or more types of monomers. Different types of monomers can be distributed along the polymer chain in a variety of ways. For example, three different types of monomers can be randomly distributed along the polymer. The distribution of monomers along the polymer can be represented in different ways. The number of repeating structural units (such as monomers) along the length of the 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, or at least 10000, or at least 100000 or more. In certain embodiments, n can range from 2 to 10000, 20 to 10000, 20 to 500, 50 to 300, 100 to 1000 or 500 to 10000. Polymers generally have an extended molecular structure, including a main chain that optionally contains side groups. The polymers provided herein can include, but are not limited to, linear polymers and branched polymers.

[0085] As used herein, the term "nanoparticle" refers to a particle having a size less than about 1000 nm.

[0086] As used herein, the term "aprotic solvent" refers to a polar solvent that does not contain acidic hydrogens and does not act as a hydrogen bond donor.

[0087] As used herein, the term "protic solvent" refers to a solvent containing dissociable H+ ions.

[0088] As used herein, the term "amphiphile" or "amphiphilic matrix" refers to a molecule that contains hydrophobic and hydrophilic segments within the molecule. In this context, the term "hydrophilic" refers to a segment of an amphiphile that has a high affinity for aqueous solutions, such as water. In this context, the term "hydrophobic" refers to a segment of an amphiphile that repels aqueous solutions, such as water.

[0089] The term "lipid moiety" refers to a moiety comprising at least one lipid. As used herein, the term "lipid" refers to a small molecule having a hydrophobic or amphipathic nature, including but not limited to fats, waxes, fatty acids, cholesterol, sterols, phospholipids, monoglycerides, diglycerides, and triglycerides. Fatty acids can 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), linoleic acid (C18), α-linoleic acid (C18), γ-linolenic acid (C18), arachidic acid (C20), gadolinic 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 moiety may include several fatty acid groups using branching groups such as lysine and other branching amines.

[0090] As used herein, the term "functional group" refers to any chemical unit that can be attached to a fluorescent polymer, for example, by any stable physical or chemical association, thereby changing the surface of the polymer point, for example, making the surface available for coupling with a biomolecule (e.g., bioconjugation). The functional group can be covalently linked to one of the main chain, side chain, or termination units of the fluorescent polymer. The functional group can be, but is not limited to, any of the following: aldehydes, olefins, alkyls, alkynes, strained alkynes, amino groups, azido groups, carbonyl groups, carboxyl groups, cyano groups, cyclooctynes, dienes, esters, succinimidyl esters, haloalkyl groups, hydroxyls, imino groups, ketones, maleimides, sulfhydryl groups, phosphates, phosphines, sulfates, sulfonates, substituted derivatives thereof, or combinations thereof. In general, any functional group suitable for bioconjugation can be used. Such functional groups are described in, for example, Bioconjugate Techniques, (Academic Press, New York, 2013), which is incorporated herein by reference in its entirety for all purposes.

[0091] The term "hydrophilic functional group" as used herein refers to a functional group that is hydrophilic in nature.

[0092] As used herein, the term "derivative" refers to a chemical substance or compound that is obtained from or is considered to be derived from another chemical substance or compound. For example, a BODIPY derivative is derived from BODIPY.

[0093] The term "aliphatic" as used herein refers to an organic compound or group characterized by a linear or branched 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 may have 1 to 24 carbon atoms, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 carbon atoms.

[0094] As used herein, the term "biomolecule" refers to a synthetic or naturally occurring protein (eg, an antibody), glycoprotein, peptide, amino acid, metabolite, drug, toxin, nucleic acid, nucleotide, carbohydrate, sugar, lipid, or fatty acid.

[0095] "Antibody" refers to an immunoglobulin, its complex (such as a fusion) or its fragment form. The term includes, but is not limited to: polyclonal or monoclonal antibodies of the IgA, IgD, IgE, IgG and IgM classes derived from human or other mammalian cell lines, including natural forms or genetically modified forms, such as humanized, human, single chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted and in vitro generated antibodies. "Antibody" also includes complex forms, including but not limited to fusion proteins with immunoglobulin portions. "Antibody" also includes antibody fragments, such as Fab, F(ab')2, Fv, scFv, Fd, dAb, Fc, whether or not they retain antigen binding function.

[0096] Polymer Dots

[0097] In one embodiment, the polymer dot includes a fluorescent polymer having a hydrophobic region and a hydrophilic region, the hydrophilic region comprising a hydrophilic functional group. The polymer dot also includes 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 groups in the fluorescent polymer are located in the hydrophilic outer layer on the surface of the polymer dot. By being positioned on the surface of the polymer dot, the hydrophilic functional groups can be used for attachment to biomolecules. In certain embodiments, the fluorescent polymer is conjugated and comprises a π-electron delocalized backbone. The fluorescent polymer can be partially or completely conjugated. As used herein, the term "fluorescent polymer" refers to a polymer that exhibits fluorescent properties. Non-limiting examples of fluorescent polymers include polyfluorenes (e.g., PDHF and PFO), poly(phenylene vinylene) (e.g., PPE), poly(phenylene vinylene) (e.g., MEH-PPV and CN-PPV), fluorene-based copolymers (e.g., PFPV, PFBT, and PFDBT5), and related derivatives (see Figure 3, from 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 that form such polymers, which are described in Rong et al., ACS Nano, 2013, 7(1):376-384, which is incorporated herein by reference in its entirety.

[0098] 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 bonded) to a hydrophilic functional group. In one embodiment, the hydrophilic functional group is attached to the side chain of the monomer within the polymer chain of the fluorescent polymer. In some embodiments, the hydrophilic functional group is attached to the terminal unit of the fluorescent polymer. In certain embodiments, the fluorescent polymer comprises less than about 6% to about 10% of the monomer connected to the hydrophilic functional group.

[0099] Exemplary monomers for forming fluorescent polymers include, but are not limited to, BODIPY, BODIPY derivatives, fluorene, fluorene derivatives, benzothiadiazole, benzothiadiazole derivatives, benzoxadiazole, and benzoxadiazole derivatives. In some embodiments, the heteropolymer comprises at least two different monomers. In some embodiments, the monomers are dibromobenzoxadiazole; 4,7-bis(2-bromo-5-thienyl)-2,1,3-benzothiadiazole; 9,9-dioctyl-2,7-dibromofluorene; 9,9-dioctylfluorene-2,7-diboric acid bis(1,3-propylene glycol) ester;

[0100] and / or

[0101] Where n=10-30. The polymer side chains with where n=10-30 create an "arm" that allows the hydrophilic functional groups in the fluorescent polymer to be located in the hydrophilic outer layer of the polymer dot surface, thereby making the hydrophilic functional groups available for bioconjugation. In one embodiment, n=22.

[0102] The amphiphilic molecule helps maintain the water solubility and stability of the polymer dots in solution without causing aggregation for at least about a week, 1 month, 3 months, 6 months, 1 year, 3 years, or 5 years or longer. The hydrophobic region of the amphiphilic molecule comprises a saturated or unsaturated fatty chain portion having 1-24 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 an embodiment, the aliphatic chain portion forms part of the lipid portion. 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.

[0103] The hydrophilic region of the amphiphilic molecule comprises a hydrophilic polymer. Hydrophilic polymers include, but are not limited to, polyoxyalkylenes, polyalkylene glycols, and polycarboxyalkylenes. Exemplary hydrophilic polymers include, but are not limited to, polyethylene glycol (PEG), polypropylene glycol, polybutylene glycol, polycarboxymethylene, polycarboxyvinyl (or polyacrylic acid (PAA)), polycarboxypropylene, and polycarboxybutylene.

[0104] In some embodiments, the molecular weight of PEG ranges from about 800 to about 5000, or from about 800 to about 4800, or from about 800 to about 4600, or from about 800 to about 4400, or from about 800 to about 4200, or from about 800 to about 4000, or from about 800 to about 3800, or from about 800 to about 3600, or from about 800 to about 3400, or from about 800 to about 3200, or from about 800 to about 3000, or from about 800 to about 2800, or from about 800 to about 2600, or from about 800 to about 2400, or from about 800 to about 2200, or from about 800 to about 2000, or from about 800 to about 1800, or from about 800 to about 1800. To about 1600, or about 800 to about 1400, or about 800 to about 1200, or about 800 to about 1000, or about 1000 to about 2000, or about 1000 to about 3000, or about 1000 to about 4000, or about 1000 to about 5000, or about 800, about 1000, about 1200, about 1400, about 1600, or 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, about 5000.

[0105] Exemplary amphiphilic molecules include, but are not limited to, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DSPE-PEG);

[0106] 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -poly(ethylene glycol)-hydroxy-1000 or -2000] (DSPE-PEG-OH);

[0107] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DSPE-PEG-OCH3);

[0108] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DSPE-PEG-NH2);

[0109] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DSPE-PEG-COOH);

[0110] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DSPE-PEG-maleimide);

[0111] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine N-[biotinyl(polyethylene glycol)-1000 or -2000] (DSPE-PEG-biotin);

[0112] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DMPE-PEG);

[0113] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxy-1000 or -2000] (DMPE-PEG-OH);

[0114] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DMPE-PEG-OCH3);

[0115] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DMPE-PEG-NH2);

[0116] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DMPE-PEG-COOH);

[0117] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DMPE-PEG-maleimide);

[0118] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DMPE-PEG-biotin);

[0119] 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DLPE-PEG);

[0120] 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxy-1000 or -2000] (DLPE-PEG-OH);

[0121] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DLPE-PEG-OCH3);

[0122] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DLPE-PEG-NH2);

[0123] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DLPE-PEG-COOH);

[0124] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DLPE-PEG-maleimide);

[0125] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DLPE-PEG-biotin);

[0126] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DPPE-PEG);

[0127] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxy-1000 or -2000] (DPPE-PEG-OH);

[0128] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DPPE-PEG-OCH3);

[0129] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DPPE-PEG-NH2);

[0130] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DPPE-PEG-COOH);

[0131] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DPPE-PEG-maleimide);

[0132] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DPPE-PEG-biotin);

[0133] 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -[(polyacrylic acid)] (DSPE-PAA);

[0134] 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DSPE-PAA-OH);

[0135] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DSPE-PAA-OCH3);

[0136] 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DSPE-PAA-NH2);

[0137] 1,2-distearoyl- sn -glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DSPE-PAA-COOH);

[0138] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DSPE-PAA-maleimide);

[0139] 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DSPE-PAA-biotin);

[0140] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DMPE-PAA);

[0141] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DMPE-PAA-OH);

[0142] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DMPE-PAA-OCH3);

[0143] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DMPE-PAA-NH2);

[0144] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DMPE-PAA-COOH);

[0145] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DMPE-PAA-maleimide);

[0146] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DMPE-PAA-biotin);

[0147] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DLPE-PAA);

[0148] 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DLPE-PAA-OH);

[0149] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DLPE-PAA-OCH3);

[0150] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DLPE-PAA-NH2);

[0151] 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DLPE-PAA-COOH);

[0152] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DLPE-PAA-maleimide);

[0153] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DLPE-PAA-biotin);

[0154] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DPPE-PAA);

[0155] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DPPE-PAA-OH);

[0156] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DPPE-PAA-OCH3);

[0157] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DPPE-PAA-NH2);

[0158] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DPPE-PAA-COOH);

[0159] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DPPE-PAA-maleimide); and

[0160] 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DPPE-PAA-biotin).

[0161] In some embodiments, the weight ratio (expressed as w / w %) of the amphiphilic molecule to the fluorescent polymer 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 the amphiphilic molecule to the 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 the amphiphilic molecule to the 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 format instead of percentages (e.g., 0.1, 0.25, etc., as shown in Table 1). In another embodiment, the weight ratio (w / w) is expressed as a range of about 0.1 to about 2 amphiphilic molecules to fluorescent polymers (i.e., about 0.1-2.0:1). Some of these embodiments provide a weight ratio 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 can result in unstable polymer point aggregation, while too high a weight ratio can moderately increase particle size (see Table 1). In some cases, using higher weight ratios can lead to problems in downstream processes (eg, low coupling yields, see Table 6). Table 1 also illustrates various weight ratios of amphiphile to polymer (eg, 0.25 mg amphiphile / mg fluorescent polymer).

[0162] 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. Other embodiments provide polymer dots having 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.

[0163] In other embodiments, the polymer dot population formed by the disclosed methods has 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 polymer dot population have an average effective diameter less than or equal to the value. Other embodiments provide a polymer dot population wherein at least 50% of the average effective diameter is less than or equal to (≤) about 25 nm and greater than at least about 5 nm (e.g., at least 50% of the polymer dots in the population have an average effective diameter ranging from about 5 nm to about 25 nm). Other embodiments provide a population of Pdots wherein at least 50% have an average effective diameter less than or equal to (≤) about 20 nm and greater than at least about 5 nm (eg, at least 50% of the Pdots in the population have an average effective diameter ranging from about 5 nm to about 20 nm).

[0164] Bioconjugation of polymer dots

[0165] Also provided is a method for coupling polymer dots to biomolecules. The biomolecules are coupled to the surface of the polymer dots through the hydrophilic functional groups in the fluorescent polymer used to make the polymer dots. In one embodiment, the biomolecule is an antibody that can bind to a cell surface antigen.

[0166] The method used to attach or couple a biomolecule to the surface of a polymer dot will depend on the type of functional groups located on the surface. For example, attachment of a protein to a polymer dot-NH2 or polymer dot-COOH can be performed via a carboximide-mediated coupling reaction. In one embodiment, the properties of the functionalized polymer dot to which the biomolecule is coupled are not altered upon bioconjugation.

[0167] In another aspect, a method of detecting a target molecule in a biological sample comprises contacting the biological sample with the disclosed polymer dots.

[0168] Methods for making polymer dots

[0169] A method for making polymer dots is also provided, which includes preparing a mixture of a fluorescent polymer and an amphiphilic molecule in an aprotic solvent, wherein the fluorescent polymer includes a hydrophobic region and a hydrophilic region, the hydrophilic region having a hydrophilic functional group, and the amphiphilic molecule includes a hydrophobic region and a hydrophilic region.

[0170] The next step of the method includes adding the mixture to a protic solvent to form polymer dots. In the resulting polymer dots, the hydrophobic regions of the fluorescent polymer and the amphiphilic molecule are embedded in the hydrophobic core of the polymer dots, 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 dots.

[0171] When a mixture of fluorescent polymer and amphiphilic molecules in an aprotic solvent is added to a protic solvent, spherical polymer dots are formed. The reduction in hydrophobicity causes the hydrophobic regions of the fluorescent polymer and amphiphilic molecules to collapse. 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, thereby making functional groups available for bioconjugation.

[0172] Exemplary aprotic solvents include, but are not limited to, tetrahydrofuran, diethyl ether, dichloromethane, acetone, acetonitrile, and dimethylformamide. Exemplary protic solvents include, but are not limited to, water, methanol, ethanol, propanol, and butanol.

[0173] 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 the use of polymer dots in certain applications. For example, polymer dots having a size of about 10 nm to about 20 nm can be used in flow cytometry. Polymer dots having other suitable sizes can be used for different applications.

[0174] 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, about 0.1 to about 1.0 mg / mL, about 0.2 mg / mL to about 1.0 mg / mL, about 0.3 mg / mL to about 1.0 mg / mL, about 0.4 mg / mL to about 1.0 mg / mL, about 0.5 mg / mL to about 1.0 mg / mL, about 0.6 mg / mL to about 1.0 mg / mL, about 0.7 mg / mL to about 1.0 mg / mL, about 0.8 mg / mL to about 1.0 mg / mL, or 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.

[0175] In certain embodiments, the method further comprises controlling the stability of the Pdots by controlling the initial concentration of the amphiphilic molecule in the aprotic solvent.

[0176] Various non-limiting embodiments include:

[0177] 1. A polymer dot comprising:

[0178] a fluorescent polymer having a hydrophobic region and a hydrophilic region, wherein the hydrophilic region has a hydrophilic functional group; and

[0179] Amphiphilic molecules with hydrophobic and hydrophilic regions,

[0180] The hydrophilic functional groups can be used for coupling.

[0181] 2. The polymer dots as described in embodiment 1, wherein the hydrophobic regions of the fluorescent polymer and the amphiphilic molecules are embedded in the hydrophobic core of the polymer dots, 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 dots.

[0182] 3. The polymer dot of embodiment 1 or 2, wherein the fluorescent polymer is a heteropolymer.

[0183] 4. The polymer dot of embodiment 3, wherein the heteropolymer comprises at least two different monomers.

[0184] 5. The polymer dot according to 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 benzoxadiazole derivatives.

[0185] 6. The polymer dot according to embodiment 5, wherein the monomers are each independently selected from the group consisting of:

[0186] (dibromobenzoxadiazole),

[0187] (4,7-bis(2-bromo-5-thienyl)-2,1,3-benzothiadiazole),

[0188] (9,9-dioctyl-2,7-dibromofluorene),

[0189] (9,9-dioctylfluorene-2,7-diboric acid bis(1,3-propylene glycol) ester),

[0190] and

[0191]

[0192] Where n=10-30.

[0193] 7. The polymer dot of embodiment 6, wherein n is 22.

[0194] 8. The polymer dot of any one of embodiments 1-7, wherein the hydrophilic functional group is selected from the group consisting of carboxyl, amino, thiol, azido, alkyne, aldehyde, hydroxyl, carbonyl, sulfate, sulfonate, phosphate, cyanate, succinimide ester, strained alkyne, azide, diene, olefin, tetrazine, strained olefin, cyclooctyne, phosphine group, and derivatives thereof.

[0195] 9. The polymer dot of any one of embodiments 1-8, wherein the hydrophilic functional group in the polymer is coupled to a biomolecule.

[0196] 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.

[0197] 11. The polymer dot of embodiment 9 or 10, wherein the biomolecule is an antibody.

[0198] 12. The polymer dot of embodiment 1 or 2, wherein the hydrophilic region of the amphiphilic molecule comprises polyalkylene glycol.

[0199] 13. The polymer dot of embodiment 12, wherein the polyalkylene glycol is polyethylene glycol.

[0200] 14. The polymer dot of any one of embodiments 1-13, wherein the size of the polymer dot is from about 5 to about 25 nm or from about 5 to about 20 nm.

[0201] 15. The polymer dot of any one of embodiments 1-14, wherein the weight ratio (w / w %) of the amphiphilic molecule to the fluorescent polymer is from about 10% to about 200%.

[0202] 16. The polymer dot of embodiment 1 or 2, wherein the hydrophobic region of the amphiphilic molecule comprises a lipid portion selected from the group consisting of a 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) portion, a 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) portion, a 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) portion, and a (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) (DPPE) portion.

[0203] 17. The polymer dot of any one of embodiments 1-16, wherein the amphiphilic molecule is selected from the group consisting of:

[0204] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DSPE-PEG);

[0205] 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -poly(ethylene glycol)-hydroxy-1000 or -2000] (DSPE-PEG-OH);

[0206] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DSPE-PEG-OCH3);

[0207] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DSPE-PEG-NH2);

[0208] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DSPE-PEG-COOH);

[0209] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DSPE-PEG-maleimide);

[0210] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine N-[biotinyl(polyethylene glycol)-1000 or -2000] (DSPE-PEG-biotin);

[0211] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DMPE-PEG);

[0212] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxy-1000 or -2000] (DMPE-PEG-OH);

[0213] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DMPE-PEG-OCH3);

[0214] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DMPE-PEG-NH2);

[0215] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DMPE-PEG-COOH);

[0216] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DMPE-PEG-maleimide);

[0217] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DMPE-PEG-biotin);

[0218] 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DLPE-PEG);

[0219] 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxy-1000 or -2000] (DLPE-PEG-OH);

[0220] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DLPE-PEG-OCH3);

[0221] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DLPE-PEG-NH2);

[0222] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DLPE-PEG-COOH);

[0223] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DLPE-PEG-maleimide);

[0224] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DLPE-PEG-biotin);

[0225] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DPPE-PEG);

[0226] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxy-1000 or -2000] (DPPE-PEG-OH);

[0227] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DPPE-PEG-OCH3);

[0228] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DPPE-PEG-NH2);

[0229] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DPPE-PEG-COOH);

[0230] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DPPE-PEG-maleimide);

[0231] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DPPE-PEG-biotin);

[0232] 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -[(polyacrylic acid)] (DSPE-PAA);

[0233] 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DSPE-PAA-OH);

[0234] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)](DSPE-PAA-OCH3),

[0235] 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DSPE-PAA-NH2);

[0236] 1,2-distearoyl- sn -glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DSPE-PAA-COOH);

[0237] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DSPE-PAA-maleimide);

[0238] 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DSPE-PAA-biotin);

[0239] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DMPE-PAA);

[0240] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DMPE-PAA-OH);

[0241] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DMPE-PAA-OCH3);

[0242] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DMPE-PAA-NH2);

[0243] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DMPE-PAA-COOH);

[0244] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DMPE-PAA-maleimide);

[0245] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DMPE-PAA-biotin);

[0246] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DLPE-PAA);

[0247] 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DLPE-PAA-OH);

[0248] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DLPE-PAA-OCH3);

[0249] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DLPE-PAA-NH2);

[0250] 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DLPE-PAA-COOH);

[0251] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DLPE-PAA-maleimide);

[0252] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DLPE-PAA-biotin);

[0253] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DPPE-PAA);

[0254] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DPPE-PAA-OH);

[0255] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DPPE-PAA-OCH3); and

[0256] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DPPE-PAA-NH2);

[0257] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DPPE-PAA-COOH);

[0258] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DPPE-PAA-maleimide);

[0259] 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DPPE-PAA-biotin).

[0260] 18. A method for preparing a polymer dot, the method comprising:

[0261] preparing a mixture of an amphiphilic molecule and a fluorescent polymer in an aprotic solvent, wherein the fluorescent polymer comprises a hydrophobic region and a hydrophilic region, the hydrophilic region has a hydrophilic functional group, the amphiphilic molecule comprises a hydrophobic region and a hydrophilic region, and 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%;

[0262] adding the mixture to a protic solvent to form polymer dots,

[0263] The hydrophilic functional groups are available for coupling.

[0264] 19. A method as described in 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.

[0265] 20. The method of embodiment 18 or 19, wherein the aprotic solvent is tetrahydrofuran.

[0266] 21. The method of embodiment 18 or 19, wherein the protic solvent is water.

[0267] 22. The method of any one of embodiments 18-21, further comprising coupling the biomolecule to the polymer point via a hydrophilic functional group.

[0268] 23. The method of any one of embodiments 18-22, wherein the amphiphilic molecule is selected from the group consisting of:

[0269] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DSPE-PEG);

[0270] 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -poly(ethylene glycol)-hydroxy-1000 or -2000] (DSPE-PEG-OH);

[0271] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DSPE-PEG-OCH3);

[0272] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DSPE-PEG-NH2);

[0273] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DSPE-PEG-COOH);

[0274] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DSPE-PEG-maleimide);

[0275] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine N-[biotinyl(polyethylene glycol)-1000 or -2000] (DSPE-PEG-biotin);

[0276] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DMPE-PEG);

[0277] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxy-1000 or -2000] (DMPE-PEG-OH);

[0278] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DMPE-PEG-OCH3);

[0279] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DMPE-PEG-NH2);

[0280] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DMPE-PEG-COOH);

[0281] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DMPE-PEG-maleimide);

[0282] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DMPE-PEG-biotin);

[0283] 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DLPE-PEG);

[0284] 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxy-1000 or -2000] (DLPE-PEG-OH);

[0285] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DLPE-PEG-OCH3);

[0286] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DLPE-PEG-NH2);

[0287] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DLPE-PEG-COOH);

[0288] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DLPE-PEG-maleimide);

[0289] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DLPE-PEG-biotin);

[0290] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000] (DPPE-PEG);

[0291] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxy-1000 or -2000] (DPPE-PEG-OH);

[0292] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000] (DPPE-PEG-OCH3);

[0293] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000] (DPPE-PEG-NH2);

[0294] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000] (DPPE-PEG-COOH);

[0295] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000] (DPPE-PEG-maleimide);

[0296] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000] (DPPE-PEG-biotin);

[0297] 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -[(polyacrylic acid)] (DSPE-PAA);

[0298] 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DSPE-PAA-OH);

[0299] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)](DSPE-PAA-OCH3),

[0300] 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DSPE-PAA-NH2);

[0301] 1,2-distearoyl- sn -glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DSPE-PAA-COOH);

[0302] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DSPE-PAA-maleimide);

[0303] 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DSPE-PAA-biotin);

[0304] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DMPE-PAA);

[0305] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DMPE-PAA-OH);

[0306] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DMPE-PAA-OCH3);

[0307] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DMPE-PAA-NH2);

[0308] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DMPE-PAA-COOH);

[0309] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DMPE-PAA-maleimide);

[0310] 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DMPE-PAA-biotin);

[0311] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DLPE-PAA);

[0312] 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DLPE-PAA-OH);

[0313] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DLPE-PAA-OCH3);

[0314] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DLPE-PAA-NH2);

[0315] 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DLPE-PAA-COOH);

[0316] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DLPE-PAA-maleimide);

[0317] 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DLPE-PAA-biotin);

[0318] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)] (DPPE-PAA);

[0319] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)] (DPPE-PAA-OH);

[0320] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)] (DPPE-PAA-OCH3); and

[0321] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)] (DPPE-PAA-NH2);

[0322] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)] (DPPE-PAA-COOH);

[0323] 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)] (DPPE-PAA-maleimide);

[0324] 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)] (DPPE-PAA-biotin).

[0325] 24. A method as described in any of embodiments 18-23, wherein the weight ratio (expressed as a percentage) of the amphiphilic molecule to the 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%.

[0326] 25. A method as described in any of embodiments 18-23, wherein the weight ratio of the amphiphilic molecule to the 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.

[0327] 26. A population of polymer dots produced by the method of any one of embodiments 18-25, wherein the population of polymer dots has 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.

[0328] 27. A method for detecting a target molecule in a biological sample, the method comprising: contacting the biological sample with the polymer dot described in any one of embodiments 1-17 or the polymer dot group described in embodiment 26, and detecting the target molecule.

[0329] 28. The polymer dots of any of embodiments 1-17, wherein the average effective diameter of the polymer dots is from 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 less than about 25 nm.

[0330] 29. The polymer dot of embodiment 1 or 2, wherein the fluorescent polymer is a homopolymer.

[0331] Example

[0332] The following examples are provided for illustration only and are not intended to be limiting. It will be readily apparent to those skilled in the art that there are a number of non-critical parameters that can be varied or modified and yield substantially the same or similar results.

[0333] Example 1 - Determination of the Optimal Ratio of DSPE-PEG-OH to Polymer 700

[0334] Polymer 700 was dissolved in 50 mL of unstable tetrahydrofuran (THF) to prepare a 200 ppm polymer solution. Using a 25 mg / mL stock solution of DSPE-PEG-OH Nanosoft polymer in anhydrous dimethyl sulfoxide, 0.25, 0.5, 0.75, 1.25, 1.5 or 1.75 mass equivalents of DSPE-PEG-OH were added to the polymer solution to prepare six different mixtures of polymer and PEG-lipid. For each mixture, 100 mL of nanopure water was cooled in an ice bath for 30 minutes (temperature = 5-6 ° C), while the mixture of polymer and PEG-lipid in THF was cooled for 15 minutes (temperature = 2-3 ° C).

[0335] To form the polymer dots, each mixture of polymer and PEG-lipid in THF was injected into 100 mL of cold nanopure water by a pump (rate = 30 mL / min) under stirring (rate = 11.0-11.2K rpm). The THF in the polymer dot mixture was evaporated using a cetriVap vacuum concentrator for 3 hours. This step also removed some water, resulting in a polymer dot solution of approximately 200 ppm.

[0336] Batches of Pdots were further concentrated to 5000 ppm using a Corning Spin-XUF concentrator or a Sartorius Vivacell 250 (100k MWCO PES).

[0337] The size of each batch of polymer dots was measured by dynamic light scattering (DLS). The quantum yield (QY) was determined by photoluminescence QY. The percentage yield of a large number of polymer dots was also calculated. The results are shown in Table 1. The results show that the ratio of DSPE-PEG-OH to polymer affects the size of the polymer dots. Polymer dots containing less than 1.25 mg DSPE-PEG-OH per mg of polymer produced 6-7 nm p dots (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 is attributed to the formation of aggregates, resulting in low yields (44-69%). The quantum yield of these batches also decreased. Adding 1.5 or 1.75 mg DSPE-PEG-OH per mg of polymer resulted in a slight increase in DLS size and increased the production cost of the polymer dots.

[0338] Table 1 - Results of different ratios of DSPE-PEG-OH to polymer 700

[0339]

[0340] Table 6 - Coupling yield as a function of lipid / polymer ratio (DSPE-PEG-OH to polymer 610)

[0341] mg lipid / mg polymer % Coupling Yield Loss 0.75 33％ 1.0 25％ 1.25 50％

[0342] Example 2 - Preparation of Polymer Dots Using Polymer 700 and DSPE-PEG-OH

[0343] Polymer 700 was dissolved in 50 mL of unstable tetrahydrofuran (THF) to prepare 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 prepare a mixture of polymer and PEG-lipid. 100 mL of nanopure water was cooled in an ice bath for 30 minutes (temperature = 5-6 ° C), and the mixture of polymer and PEG-lipid in THF was cooled for 15 minutes (temperature = 2-3 ° C).

[0344] To form the polymer dots, a mixture of polymer and PEG-lipid in THF was injected into cold nanopure water by a pump (rate = 30 mL / min) under stirring (rate = 11.0-11.2K rpm). The THF in the polymer dot mixture was evaporated using a cetriVap vacuum concentrator for 3 hours. This step also removed some water, resulting in a polymer dot solution of approximately 200 ppm.

[0345] Some batches of Pdots were further concentrated to 5000 ppm using a Corning Spin-X UF concentrator (100k MWCO PES). Alternatively, multiple batches of Pdots were pooled together and concentrated to 5000 ppm using a Sartorius Vivacell 250 (100k MWCO PES) in preparation for coupling with proteins.

[0346] The size of the polymer dots measured by dynamic light scattering (DLS) and the percent yield of the polymer dots of four different batches are given in Table 2. The results show that the size is consistent between batches. The results also show that there is some loss in yield in three of the four batches when concentrated to about 5000 ppm.

[0347] Table 2 – Polymer dot batch investigation using Polymer 700 and DSPE-PEG-OH

[0348]

[0349] Example 3 - Preparation of Polymer Dots Using Polymer 460 and DSPE-PEG-OH

[0350] In this example, the same method as in Example 2 was used to prepare Pdots with a different polymer (Polymer 460).

[0351] The size of each batch of polymer dots was measured by dynamic light scattering (DLS). The quantum yield (QY) was determined by photoluminescence. The percentage yield of a large number of polymer dots was also calculated. The results are shown in Table 3. The results show that the size is consistent between batches. For 200ppm polymer dots, the yield percentage ranged from 70% to 91%. The results also show that when concentrated to about 5000ppm, there is some loss in yield for all three batches. The quantum yield does not change when the concentration reaches about 5000ppm.

[0352] Table 3 – Polymer dot batch investigation using polymer 460 and DSPE-PEG-OH

[0353]

[0354] Example 4 - Preparation of Polymer Dots Using Polymer 700 and DSPE-PEG-OCH3

[0355] Polymer dots were prepared using different types of PEG-lipids. The same method as in Example 2 was used, except that DSPE-PEG-OCH3 was used instead of DSPE-PEG-OH. In addition, two different ratios of PEG-lipid to polymer were tested: 1.25 mg or 0.75 mg lipid per mg polymer.

[0356] The size of each batch of polymer dots was measured by dynamic light scattering (DLS). The percent yield of a large number of polymer dots was also calculated. The results are given in Tables 4 and 5 below. The results show that for the two ratios of PEG-lipid to polymer tested, the results in terms of size and yield are similar, indicating that less DSPE-PEG-OCH3 may be needed to stabilize the polymer dots.

[0357] Table 4 - Polydot batches containing 1.25 mg DSPE-PEG-OCH3 per mg polymer 700

[0358]

[0359] Table 5 - Polymer dot batches containing 0.75 mg DSPE-PEG-OCH3 per mg polymer 700

[0360]

[0361]

[0362] Example 5 - Coupling of IgG Antibodies to Polymer Dots

[0363] The polymer dots from Example 2 were first activated by incubating 250 μl (1.25 mg) of the polymer dots with 6.3 μl of 250 mM sulfo-NHS (ThermoFisher) and 3.2 μl of 200 mM EDC (ThermoFisher) in a 1.5 mL Eppendorf tube at room temperature for 30 minutes. Then 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 at room temperature for 3 hours in the dark.

[0364] 2.0 μl 0.5M EDTA (Sigma-Aldrich) and 2.0 μl 10 mg / mL iminothiolane (Thermo Fisher) were added to 200 μl (1 mg) anti-CD4 IgG in a 1.5 mL Eppendorf tube to activate anti-CD4 IgG (Bio-Rad). The solution was mixed and incubated at room temperature for 1 hour.

[0365] The activated Pdots were quenched by adding 20 μl of 0.5 M taurine (Sigma-Aldrich) to the Pdot solution. The Pdot solution was mixed and incubated at room temperature in the dark for 15 minutes.

[0366] Both activated Pdots and anti-CD4 IgG were desalted using 40K MWCO spin columns (Thermo Fisher Scientific), and the total amount of each desalted product was determined by absorbance. The ratio of Pdots to IgG used in the coupling reaction was 3:1, and the reaction was incubated overnight at 4°C in the dark. The coupling was quenched by adding 1 / 200 volume of 25% N-ethylmaleimide (Sigma-Aldrich) and incubated at room temperature in the dark for 30 minutes.

[0367] Free anti-CD4 antibodies were removed from the polymer dot-IgG conjugates by size exclusion chromatography.

[0368] Example 6 - Characterization of Polymer Dots Conjugated to IgG vs. Control Conjugates by Flow Cytometry

[0369] Flow cytometry was used to determine how the polymer dot-anti-CD4 IgG conjugates (or test conjugates) from Example 5 compared to a control conjugate (PerCPCy5.5; from BioLegend). A ZE5 Sapphire flow cytometer (BioLegend Laboratories, Inc.) was used in the experiments.

[0370] Determine the staining index of the test and control conjugates. The staining index is calculated as follows: (MFI 阳 -MFI 阴) / (2*rSD 阴 ), where MFI 阳 is the median fluorescence intensity of the positive population, MFI 阴 is the median fluorescence intensity of the negative population, rSD 阴 is the relative standard deviation of the negative population. Figure 1 As shown, the staining index of the test conjugate was significantly higher (>>2x) than that of the control conjugate.

[0371] Histograms of positive and negative cell populations were obtained for the test (6452-66-1) and control (PerCPCy5.5) conjugates. Figure 2 As shown. For the test conjugate, SEC refers to size exclusion chromatography. The positive peak shape and the heights of the positive and negative peaks of the test conjugate were comparable to those of the control conjugate.

[0372] Example 7 - Effect of PEG-lipid on Pdot Stability

[0373] experiment:

[0374] For each polymer, two sets of small P dots were made: 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 was previously determined in different experiments. Both sets of small P dots were concentrated to about 5000 ppm. Particle size and yield recovery were evaluated to determine P dot stability.

[0375] result:

[0376]

[0377]

[0378]

[0379]

[0380]

[0381] Even before concentration to 5000 ppm, the instability of small Pdots prepared without PEG-lipids was already evident. For polymers 405 / 610, 405 / 790, and 405 / 515, aggregation was observed as early as after removal of THF. This resulted in a lower yield (about 60%) compared to small Pdots made with PEG-lipids (80-97%).

[0382] It was not possible to concentrate small Pdots without PEG-lipids to about 5000 ppm. Aggregates formed and stuck to the membrane of the concentrator, resulting in low yields (12-24%), but they were still diluted (1000-2000 ppm). In contrast, small Pdots made with PEG-lipids retained their small particle size with little or no step loss.

[0383] Example 8 - Accelerated Stability Study

[0384] Experimental methods:

[0385] The accelerated shelf life test was conducted at 37°C. Based on the Arrhenius model, assuming a reaction activation energy of 15 kcal / mol, 20 days at 37°C is theoretically equivalent to 1 year at 4°C (the recommended storage temperature).

[0386] The P spots were aliquoted into 7 Eppendorf tubes. Six (6) samples were stored at 37°C and removed at different time points. (See Table 7 below.) One (1) sample was stored at 4°C for reference.

[0387]

[0388] After removal from the incubation chamber, the samples are inspected for aggregates and the particle size is measured. To pass the stability test, the samples must be free of aggregates and their particle size must be within 15% of the reference sample particle size.

[0389] Pdot 488 / 700 / DSPE-PEG1000-OH - Real-time stability data:

[0390] time Particle size from DLS t＝0 16nm t = 1.5 years 16nm

[0391] Pdot 488 / 700 / DSPE-PEG1000-OH-Accelerated stability data:

[0392]

[0393]

[0394] Pdot 405 / 610 / DSPE-PEG1000-OH-Accelerated stability data:

[0395]

[0396] All patents, patent applications, and other published references cited in this specification are hereby incorporated by reference in their entirety.

Claims

1. A polymer dot comprising: A fluorescent polymer having a hydrophobic region and a hydrophilic region, the hydrophilic region having a hydrophilic functional group, the fluorescent polymer comprising a heteropolymer comprising at least two different monomers; and Amphiphilic molecules with hydrophobic and hydrophilic regions, The hydrophilic functional groups are available for coupling. One of the monomers is wherein n=10-30, and the other monomers are each independently selected from the group consisting of BODIPY, BODIPY derivatives, fluorene, fluorene derivatives, benzothiadiazole, benzothiadiazole derivatives, benzoxadiazole and benzoxadiazole derivatives, and The hydrophobic region of the amphiphilic molecule comprises a lipid portion selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine and (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine).

2. The polymer dot according to claim 1, wherein The hydrophobic regions of the fluorescent polymer and the amphiphilic molecules are embedded in the hydrophobic core of the Pdots, 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 Pdots.

3. The polymer dot of claim 1, wherein the monomers are each independently selected from the group consisting of: (2,5-dibromobenzoxadiazole), (4,7-bis(2-bromo-5-thienyl)-2,1,3-benzothiadiazole), (9,9-dioctyl-2,7-dibromofluorene), (9,9-dioctylfluorene-2,7-diboric acid bis(1,3-propylene glycol) ester), and The polymer dot of claim 1 , wherein n is 22.

5. The polymer dot of claim 1 or 2, wherein the hydrophilic functional group is selected from the group consisting of carboxyl, amino, thiol, azido, alkyne, aldehyde, hydroxyl, carbonyl, sulfate, sulfonate, phosphate, cyanate, succinimide ester, strained alkyne, azide, diene, olefin, tetrazine, strained olefin, cyclooctyne, phosphine group, and derivatives thereof.

6. The polymer dot of claim 5, wherein The carboxylic acid groups are coupled to biomolecules.

7. The polymer dot of claim 6, wherein the biomolecule is selected from the group consisting of synthetic or naturally occurring proteins, polypeptides, amino acids, nucleic acids, carbohydrates, and lipids.

8. The polymer dot of claim 6 or 7, wherein the biomolecule is an antibody.

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

10. The polymer dot of claim 9, wherein the polyalkylene glycol is polyethylene glycol.

11. The polymer dot of claim 1 or 2, wherein the size of the polymer dot is 5 to 25 nm or 5 to 20 nm. 12 . The polymer dot of claim 1 , wherein a weight ratio (w / w %) of the amphiphilic molecule to the fluorescent polymer is 10% to 200%.

13. The polymer dot of claim 1 or 2, wherein the amphiphilic molecule is selected from the group consisting of: 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000]; 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -poly(ethylene glycol)-hydroxy-1000 or -2000]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine N-[biotinyl(polyethylene glycol)-1000 or -2000]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxy-1000 or -2000]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000]; 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxy-1000 or -2000]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxy-1000 or -2000]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)]; 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)]; 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)]; 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)]; 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)]; 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)]; and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)]; and 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)].

14. A method for preparing a polymer dot, the method comprising: preparing a mixture of an amphiphilic molecule and a fluorescent polymer in an aprotic solvent, wherein the fluorescent polymer comprises a hydrophobic region and a hydrophilic region, the hydrophilic region has a hydrophilic functional group, the amphiphilic molecule comprises a hydrophobic region and a hydrophilic region, and the weight ratio (w / w) of the amphiphilic molecule to the fluorescent polymer (amphiphilic molecule:fluorescent polymer) is 0.1 to 2:1, or 10% to 200%; adding the mixture to a protic solvent to form polymer dots, The hydrophilic functional groups are available for coupling, and wherein the polymer comprises at least two different monomers and one monomer is wherein n=10-30, and the other monomers are independently selected from the group consisting of BODIPY, BODIPY derivatives, fluorene, fluorene derivatives, benzothiadiazole, benzothiadiazole derivatives, benzoxadiazole and benzoxadiazole derivatives, and The hydrophobic region of the amphiphilic molecule comprises a lipid portion selected from the group consisting of a 1,2-distearoyl-sn-glycero-3-phosphoethanolamine portion, a 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine portion, a 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine portion, and a (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) portion.

15. The method of claim 14, wherein: The hydrophobic regions of the fluorescent polymer and the amphiphilic molecules are embedded in the hydrophobic core of the Pdots, 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 Pdots.

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

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

18. The method of claim 14 or 15, further comprising: The carboxylic acid groups in the monomers point-couple the biomolecules to the polymer.

19. The method of claim 14 or 15, wherein the amphiphilic molecule is selected from the group consisting of: 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000]; 1,2-distearoyl- sn -glycero-3-phosphoethanolamine- N -poly(ethylene glycol)-hydroxy-1000 or -2000]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine N-[biotinyl(polyethylene glycol)-1000 or -2000]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxy-1000 or -2000]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000]; 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxy-1000 or -2000]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-1000 or -2000]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyethylene glycol)-hydroxy-1000 or -2000]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000 or -2000]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-1000 or -2000]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000 or -2000]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000 or -2000]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-1000 or -2000]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)], 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)]; 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)]; 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)]; 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)]; 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)]; 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)]; 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)]; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)]; 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[(polyacrylic acid)]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxy(polyacrylic acid)]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyacrylic acid)]; and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyacrylic acid)]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyacrylic acid)]; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyacrylic acid)]; 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyacrylic acid)].

20. The method of claim 14 or 15, wherein the weight ratio of the amphiphilic molecule to the fluorescent polymer is 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.

21. A population of polymer dots produced by the method of claim 14 or 15, wherein the population of polymer dots has an average effective diameter of 5 nm to 25 nm, provided that at least 50% of the polymer dots in the population have an average effective diameter of less than 25 nm.

22. A method for detecting a target molecule in a biological sample, the method comprising contacting the biological sample with the polymer dots of claim 1 or 2 and detecting the target molecule.

23. The population of polymer dots of claim 1, wherein the average effective diameter of the polymer dots is 5 nm to 25 nm, with the proviso that at least 50% of the polymer dots in the population have an average effective diameter less than 25 nm.

24. A method for detecting a target molecule in a biological sample, the method comprising contacting the biological sample with the polymer dot population of claim 21 and detecting the target molecule.

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