Preparation of ultra-small nano probe with efficient biomarker performance and super-resolution imaging application of ultra-small nano probe

Abstract

The invention provides preparation of an ultra-small nano probe with efficient biomarker performance and super-resolution imaging application of the ultra-small nano probe. The nanodot is of a core-shell structure, and the core-shell structure comprises a fluorescent core, a fluorescent layer and a fluorescent layer, wherein the fluorescent core is composed of a single-chain semiconductor polymer; the shell layer is composed of an amphiphilic compound, a hydrophobic part of the amphiphilic compound is combined with the semiconductor polymer, a hydrophilic group is located on the surface of the shell layer to serve as an active group, the average particle size of the nanodot is smaller than 5 nm, and the nanodot is of a glassy structure. The nanodot can realize high-density specific marking of various subcellular organelles and nano-scale biological fluorescence imaging.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, and particularly relates to a super-small nanoprobe with high-efficiency biomarker performance and a preparation method and super-resolution imaging application thereof. BACKGROUND

[0002] In recent years, fluorescent nanoparticles have attracted widespread attention in the field of fluorescence imaging due to their high brightness and excellent light stability. Currently, fluorescent nanoprobes face two major challenges in biological targeting labeling: ① The complex particle composition can lead to uncontrollable surface functional groups, which in turn affect the functional modification with targeted biomolecules. ② The large particle size (> 10 nm) often causes large steric hindrance or particle aggregation problems, thereby reducing the labeling density. Therefore, when applied in biological labeling imaging, the large size and complex surface chemistry of current fluorescent nanoparticles not only limit the specific labeling density of biological target samples, but also affect the imaging resolution at the nanoscale.

[0003] Wu Yayun et al. developed a polymer nanoparticle fluorescent probe with a particle size of about 20 nm, which successfully realized the long-term visualization of endocytic vesicle interaction (Anal Chem 2020, 92, 12088-12096). However, the large particle size limits the labeling ability of the probe for the fine structure of subcellular organelles, resulting in discontinuous distribution of the label, which makes it difficult to accurately analyze the subtle structural features of subcellular targets. Quantum dots (QDs) are a classic type of fluorescent nanoprobes. In order to achieve excellent fluorescence properties in aqueous solution, nuclear shell coating and ligand modification treatment are usually required, but this inevitably leads to the problems of large particle size (> 10 nm) and complex surface chemistry in biological system applications (Chem Soc Rev, 2015, 44, 4792).

[0004] Therefore, it is urgent to develop a new type of fluorescent nanoprobe, which is different from the existing large size (> 10 nm) or complex composition nanoprobes. It needs to have clear composition, ultra-small size, easy-to-functionalize surface properties, and multi-color adjustable fluorescence, so as to realize high-density specific labeling of multiple targets in biological systems and subcellular organelle fine structure imaging at the nanoscale. SUMMARY

[0005] The present application realizes high-density specific labeling of various subcellular targets, such as microtubulin (Tubulin), mitochondrial outer membrane protein (Tom 20) and clathrin-coated vesicles (CCPs) and the like, by preparing super-small nanodots (<5nm, suPdots) composed of a single polymer chain and modifying the surface functional groups (-COOH) of the suPdots with antibodies, and clearly visualizing the characteristic structures of subcellular organelles. In addition, the multicolor fluorescence characteristics of suPdots enable us to simultaneously visualize the characteristic structures of various subcellular organelles. Under stimulated emission depletion super-resolution imaging (STED), the fine hollow ring structure of suPdots-labeled CCPs can be resolved, thereby realizing the best biological imaging performance among all current STED nanoprobes, and solving the problem that current fluorescent nanoprobes are difficult to realize high-density specific biological labeling.

[0006] In one aspect of the present application, a nanodot is provided, wherein the nanodot is a core-shell structure, and the core-shell structure comprises:

[0007] a fluorescent core composed of a single-chain semiconductor polymer;

[0008] a shell layer composed of an amphiphilic compound, wherein the hydrophobic group of the amphiphilic compound is combined with the semiconductor polymer, and the hydrophilic group is located on the surface of the shell layer as an active group, the average particle size of the nanodot is <5nm, and the nanodot has improved light stability and anti-photobleaching property.

[0009] In one embodiment of the present application, the semiconductor polymer is selected from conjugated polymers, polythiophene, polyfluorene, polystyrene, polyphenylacetylene and derivatives thereof. In one embodiment of the present application, the semiconductor polymer is selected from CNPPV, PDFDP or a combination thereof.

[0010] In one embodiment of the present application, the amphiphilic compound comprises a hydrophobic group and a hydrophilic group, and the hydrophilic group is selected from carboxyl, amino or hydroxyl. In one embodiment of the present application, the amphiphilic compound is selected from polystyrene maleic anhydride.

[0011] In one embodiment of the present application, the nanodot is a glassy structure. In one embodiment of the present application, the nanodot is prepared by a glass freezing method. In one embodiment of the present application, the semiconductor polymer and the amphiphilic compound are dissolved and placed in a low-temperature environment for rapid cooling, so that they are converted into a glassy state to obtain the nanodot.

[0012] In one embodiment of the present application, the cooling rate of the rapid cooling is greater than 1000℃ / min.

[0013] In one embodiment of the present application, the nanodot further comprises a modification.

[0014] In one embodiment of the present application, the modification group is covalently coupled to the nanodot via a reactive group. In one embodiment of the present application, the modification group comprises an antibody, an antibody fragment, a ligand, a receptor, a peptide, a protein, a nucleic acid, an aptamer, a saccharide, a drug molecule, a small molecule compound, a fluorescent probe, a radioactive label, an enzyme, a signaling molecule, a polymeric modification group, or a magnetic / functional nanoparticle. In one embodiment of the present application, the modification group is an antibody or an antibody fragment, and exemplary modification groups include, but are not limited to, an anti-tubulin antibody or an antibody fragment, an anti-mitochondrial outer membrane protein antibody or an antibody fragment, and an anti-clathrin-coated vesicle antibody or an antibody fragment.

[0015] In one embodiment of the present application, the nanodot can be used for bioimaging. In one embodiment of the present application, the nanodot can be used for high-density specific labeling of various subcellular organelles; including resolving the fine structure of various subcellular organelles, and visualizing the relative distribution of various subcellular organelles at the nanoscale.

[0016] In a second aspect of the present application, a method for preparing a nanodot is provided, which is prepared by a glass freezing method.

[0017] In one embodiment of the present application, the method comprises the following steps:

[0018] (a) dissolving a semiconductor polymer and an amphiphilic compound in a solvent to form a mixed solution;

[0019] (b) rapidly cooling the mixed solution in a low-temperature environment to convert it into a glass state;

[0020] (c) removing the solvent to obtain a glassy nanodot product.

[0021] In one embodiment of the present application, the solvent is tetrahydrofuran, dioxane, dichloromethane, or a combination thereof. In one embodiment of the present application, the solvent is a mixed solvent of tetrahydrofuran and dioxane, and the volume ratio of tetrahydrofuran to dioxane is 2:8.

[0022] In one embodiment of the present application, the concentration of the semiconductive polymer is 0.1-2 mg / mL, such as 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL; the concentration of the amphiphilic compound is 10-100 mg / mL, such as 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL. The volume ratio of the semiconductive polymer and the amphiphilic compound is 1:1-1:10, such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10. In one embodiment of the present application, the concentration of the semiconductive polymer is 1 mg / mL; the concentration of the amphiphilic compound is 50 mg / mL; and the volume ratio of the semiconductive polymer and the amphiphilic compound is 1:1.

[0023] In one embodiment of the present application, the cooling rate of the rapid cooling is greater than 1000 ℃ / min.

[0024] In one embodiment of the present application, the low temperature is liquid nitrogen temperature.

[0025] In one embodiment of the present application, the mixed solution is slowly dropped from a certain height to a freezing platform at liquid nitrogen temperature, so that it is rapidly solidified at low temperature to form a glass structure. In one embodiment of the present application, the mixed solution is slowly dropped from a height of 0.5 m to a freezing platform at liquid nitrogen temperature.

[0026] In one embodiment of the present application, the freezing platform is a glass freezing platform.

[0027] In one embodiment of the present application, the solvent is removed by freeze-drying.

[0028] In one embodiment of the present application, the obtained dry powder is hydrolyzed and / or dispersed in an aqueous solution (such as an alkaline solution) to obtain single-stranded ultra-small polymer nanodots stably dispersed in the aqueous solution. In the aqueous solution, the single-stranded polymer is wound intramolecularly to form stably dispersed single-stranded ultra-small polymer nanodots.

[0029] In one embodiment of the present application, the method further comprises the steps of centrifugation, filtration and ultrafiltration purification.

[0030] In one embodiment of the present application, the method further comprises modifying the nanodots.

[0031] In one embodiment of the present application, the modification group is covalently coupled to the nanodot via an active group. In one embodiment of the present application, the modification group is covalently coupled to the carboxyl group of the nanodot under the action of an activating agent. In one embodiment of the present application, the activating agent is 5 μL (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) (EDC, 5 mg / mL).

[0032] In one embodiment of the present application, the modification group comprises an antibody, an antibody fragment, a ligand, a receptor, a peptide, a protein, a nucleic acid, an aptamer, a saccharide, a drug molecule, a small molecule compound, a fluorescent probe, a radioactive label, an enzyme, a signal molecule, a polymer modification group, or a magnetic / functional nanoparticle. In one embodiment of the present application, the modification group is an antibody or an antibody fragment, including but not limited to an anti-tubulin antibody or an antibody fragment, an anti-mitochondrial outer membrane protein antibody or an antibody fragment, and an anti-clathrin-coated vesicle antibody or an antibody fragment.

[0033] In a third aspect of the present application, a nanoprobe, a kit, or a composition is provided, comprising the nanodot described above.

[0034] In a fourth aspect of the present application, the nanodot, the nanoprobe, the kit, or the composition described above is used in biological imaging.

[0035] In one embodiment of the present application, the nanodot has a slow decay rate (>140 seconds) and has stronger light stability in long-time imaging.

[0036] In one embodiment of the present application, the nanodot can be used for high-density specific labeling of various subcellular organelles; including resolving the fine structure of various subcellular organelles, and visualizing the relative distribution position of various subcellular organelles at the nanoscale. In one embodiment of the present application, the organelle comprises microtubules, mitochondria, vesicles, etc.

[0037] In a fifth aspect of the present application, a detection method is provided, comprising using the nanodot, the nanoprobe, the kit, or the composition described above for detection.

[0038] In one embodiment of the present application, the detection method comprises: 1) preparing a nanodot-labeled sample to be detected; and 2) using fluorescence imaging for detection.

[0039] In an embodiment of the present application, step 1) comprises fixing, permeabilizing and blocking treatment after the cells are inoculated and cultured to an appropriate density, followed by incubation with specific antibodies and biotin-coupled ultrasmall polymer dots in turn, and preparation for fluorescence imaging through washing steps. Alternatively, step 1) comprises fixing, permeabilizing and blocking treatment after the cells are inoculated and cultured to an appropriate density, followed by incubation labeling with specific antibodies and two different biotin-coupled ultrasmall polymer dots of different species, and preparation for fluorescence imaging through washing steps.

[0040] In an embodiment of the present application, step 2) comprises using STED super-resolution imaging detection, using a STED microscope to image the sample, and achieving high-resolution fluorescence imaging by optimizing the excitation wavelength and loss laser intensity. Alternatively, using a single excitation light and loss light combined with different collection wavelengths, based on the characteristics of the ultrasmall polymer nanodots, high-resolution fluorescence imaging of multiple targets is achieved.

[0041] Compared with the prior art, the present application has the following beneficial effects:

[0042] The present application solves the key problems of insufficient labeling density and limited imaging resolution in biological target labeling of existing nanoprobes by developing a new type of fluorescent nanoprobe with clear composition and ultrasmall size, which significantly expands the application scope of fluorescent nanoprobes in biological imaging.

[0043] Through efficient coupling with biological antibodies, the nanodot can achieve high-density specific labeling of multiple subcellular organelles, exhibiting the best biological imaging effect in the current STED nanoprobes. It not only can clearly analyze the fine structure of various subcellular organelles, but also can visualize the relative distribution position of multiple subcellular organelles at the nanoscale under the premise of simplifying the STED imaging light path. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 Schematic diagram of synthesis strategy of ultrasmall nanoprobes.

[0045] Figure 2 Size and morphology characterization of ultrasmall polymer dots.

[0046] Figure 3 Light stability characterization of ultrasmall polymer dots and traditional dyes.

[0047] Figure 4 Single-particle STED super-resolution imaging of ultrasmall polymer dots.

[0048] Figure 5 Immunofluorescence biological labeling imaging of ultrasmall polymer dots. From left to right, tubulin; clathrin-coated pits (CCPs) and mitochondrial outer membrane protein Tom 20.

[0049] Figure 6 This image shows a two-color immunofluorescence biolabeling technique for ultrasmall polymer dots. Green represents CNPPV suPdots, and burgundy represents PDFDP suPdots.

[0050] Figure 7 Super-resolution imaging of STED, an immunofluorescence biolabel for ultrasmall polymer dots. (a) Fluorescence confocal and STED super-resolution imaging of biolabeled clathrin endosomes; inset: histogram of the statistical distribution of clathrin endosome size. (bc) Fluorescence confocal and STED super-resolution imaging of biolabeled tubulin. (d) Fluorescence intensity profile distribution curves at the white and purple arrows.

[0051] Figure 8 Super-resolution imaging of STED, a two-color immunofluorescence biolabel for ultrasmall polymer dots. Two types of nanoparticles share a single excitation beam (Ex = 488 nm) and a loss beam (Em = 775 nm). Green represents CNPPV suPdots (tubulin), and burgundy represents PDFDP suPdots (clathrin invaginates). Detailed Implementation

[0052] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0053] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. CNPPV (poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-pheny-lene)], ADS 110 RE), PDFDP(poly[{9,9-dihexyl-2,7-bis(1-cyanovinylene)fluorene}-alt-co-{2,5-bis(N,N'-diphenylamino)-1,4-phenylene}], ADS 111 RE)

[0054] The single-chain ultrasmall polymer nanodot of the present application refers to an ultrasmall-sized nanodot formed by coiling a single semiconductor fluorescent polymer chain (or single-chain semiconductor fluorescent polymer). The polymer chain exists in the form of a single molecule, rather than a complex structure formed by aggregation or multi-chain entanglement of multiple polymer chains, and has a clearer internal composition and higher operability of nanodot surface chemistry.

[0055] Preparation of single-chain ultrasmall polymer nanodots in Example 1

[0056] The glassy freezing technique is used to prepare nanodots with ultrasmall size and ultrahigh brightness. The specific steps are as follows:

[0057] Take 200 μL of CNPPV or PDFDP polymer chain solution (1 mg / mL) and 200 μL (50 mg / mL) of styrene maleic anhydride into a mixed solvent (tetrahydrofuran / dioxane = 2:8), and mix the solution in an ultrasonic instrument. Slowly drop the mixed solution from a height of 0.5 m onto a clean glass plate pre-cooled by liquid nitrogen, so that it is rapidly solidified by low temperature. Further remove the solvent by freeze-drying, hydrolyze and disperse the obtained dry powder in an alkaline solution, and finally purify by centrifugation, filtration and ultrafiltration to obtain single-chain ultrasmall polymer nanodots CNPPVsuPdots and PDFDP suPdots stably dispersed in aqueous solution.

[0058] The schematic diagram of the principle of preparing nanodots with ultrasmall size and ultrahigh brightness by glassy freezing technique is shown in Figure 1 The size of suPdot is characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The results are shown in Figure 2 The average diameter of CNPPVsuPdot and PDFDP suPdot is distributed around 5 nm.

[0059] The light stability of ultrasmall polymer nanodots is detected as shown in Figure 3 Compared with traditional dyes, after continuous scanning for 150 frames under a confocal fluorescence microscope, the ultrasmall polymer nanodots can show better light stability, which is suitable for long-time imaging applications such as single-molecule tracking and super-resolution imaging such as STED requiring high laser power. Among them, the stability of PDFDP suPdot is the best.

[0060] Single-particle STED super-resolution imaging of ultrasmall polymer dots is shown in Figure 4The ultra-small polymer dots were shown to perform well in STED super-resolution imaging under Leica SP 8 STED 3X commercial instrument imaging, with resolution reduced from the diffraction limit (>200 nm) to the range of 20-50 nm, which is about 2-3 times higher than the previous large size polymer nanoparticles in imaging resolution, thus very suitable for single molecule or subcellular level of ultra-high resolution studies.

[0061] Example 2 Ultra-small polymer dot functionalization tag small molecule modification

[0062] Take 150 μL of ultra-small polymer dot solution into a 10 mL glass bottle container containing 850 μL of ultrapure water (MilliQ). 20 μL of PEG (5%), 20 μL of HEPES (1M), IgG (2.2 mg / mL, 5 μL, respectively anti-tubulin IgG, anti-mitochondrial outer membrane protein IgG and anti-clathrin coated vesicle IgG) and EDC (5 mg / mL, 8 μL) were added to the above 10 mL glass container in turn, and mixed thoroughly. After stirring at room temperature for 2 hours, then add BSA (0.025 mg / mL, 400 μL) solution to the above solution system and mix for 1 hour, after the reaction is completed, add 600 μL of MilliQ water, 40 μL of PEG (5%), 40 μL of HEPES (1M) and 4 μL of Trition X-100, then purify with 100KDa ultrafiltration centrifuge tube to remove excess antibody. Collect the concentrated ultra-small polymer dot bioantibody conjugate at 4°C for standby.

[0063] Example 3 Ultra-small polymer dot bioantibody conjugate for subcellular organelle labeling

[0064] Cells were seeded in advance into confocal imaging dishes and cultured overnight until the cell density reached about 70% for immunofluorescence labeling. Cells were fixed with a mixed solution of PFA (4%) and GA (0.1%) for 15 min. After fixation, cells were washed with PBS, and then permeabilized with Triton X-100 (0.5%) for 5 min, followed by incubation in blocking buffer containing 5% BSA and 0.1% Triton X-100 in PBS for 30 min. To specifically label tubulin, cells were then incubated with rabbit anti-a tubulin (Abeam, 1 pg / mL) in blocking buffer for 1 h, and after washing with PBS, further incubated with super small polymer nanoparticle bioconjugate in blocking buffer for another 1 h. For mitochondria and clathrin staining, the steps of fixation, permeabilization and blocking were the same as before. Then cells were incubated with rabbit anti-Tom20 (Thermofisher, 1 :50) or rabbit anti-Clathrin (Abeam, 1 :2000) in blocking buffer for 1 h, and after washing with PBS for 5 min for three times, stored at 4 °C for fluorescence confocal imaging. For dual-color labeling imaging, two different species of antibodies were used for labeling, and further incubation with CNPPV and PDFDP super small polymer nanoparticle bioconjugates, and the remaining steps were the same as single-color labeling.

[0065] Example 4 STED super-resolution microscopy imaging

[0066] Super-resolution imaging was performed using a commercial STED instrument (Leica SP 8 STED 3X) fluorescence microscope, and a 100x oil objective was used. The excitation wavelength was selected as 488 nm, and the corresponding collection wavelength was selected according to different polymer dot samples. Confocal fluorescence imaging of the sample was performed. The specific parameters of the imaging were adjusted according to the state of the sample and the fluorescence intensity to achieve the best imaging effect. Under the best confocal imaging conditions, the STED imaging mode was turned on, and the loss laser was selected as 775 nm. By adjusting the intensity of the loss laser, the best STED super-resolution imaging effect was achieved.

[0067] Immunofluorescence bio-labeling imaging of super small polymer dots Figure 5The fluorescence signals of microtubule (Tubulin), coated pits (CCPs) and mitochondrial outer membrane protein Tom20 are shown from left to right in the image, indicating that different fluorescently labeled antibodies (such as anti-tubulin IgG, anti-clathrin IgG and anti-Tom20 IgG) can be successfully and specifically labeled with various target proteins. The experimental results show that the use of functionalized ultra-small polymer nanodots can successfully achieve immunofluorescence labeling of Tubulin, CCPs and Tom20 targets, showing high specificity and high density labeling capability, and is suitable for dynamic imaging of multiple proteins in cells and relative distribution position research under nanoscale.

[0068] Figure 6 In the above, the effectiveness of CNPPV suPdots and PDFDP suPdots in dual-color immunofluorescence imaging is due to the multicolor adjustable fluorescence characteristics of suPdots, which can simultaneously label and image multiple biological targets such as Tubulin, CCPs, and Tubulin, TOM20, etc. under fluorescence confocal microscopic imaging, demonstrating its potential in multi-target imaging of cytoskeleton, endocytosis and mitochondria, etc. with high biological and diagnostic value. Figure 7 In the above, the high resolution (50-125 nm) and light stability of CNPPV suPdots and PDFDP suPdots in STED imaging successfully realize the visualization of the fine subcellular structure of CCP and microtubule, achieving the best results of current nanoprobes in subcellular structure labeling imaging, and are suitable for studying the endocytosis process or microtubule-CCP interaction in cells.

[0069] Example 5 Multi-target STED super-resolution imaging

[0070] Based on the large Stokes shift characteristics of ultra-small polymer dots, multi-target STED super-resolution imaging can be achieved using the same excitation light and loss light with a STED commercial instrument (Leica SP8STED 3x), overcoming the need for complex optical paths for multi-color STED imaging. Specifically, the excitation wavelength is 488 nm, the collection wavelengths are "500-600" and "630-700" respectively, and the loss wavelength is 775 nm. By adjusting the intensity of the loss laser, the best STED dual-color super-resolution imaging effect is achieved.

[0071] The results are as follows Figure 8As shown, the results show that the ultra-small polymer dots have excellent super-resolution performance in two-color immunofluorescence STED super-resolution imaging, the resolution is improved from the diffraction limit to the sub-100 nm level, and the fine labeling and relative position distribution of microtubules and other target proteins (such as CCPs or Tom20) at the nanoscale are successfully realized, which is suitable for multi-dimensional and multi-target relative position distribution analysis of subcellular structures at the nanoscale in cells.

[0072] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although modifications or equivalent replacements are made with reference to the preferred embodiments, they should be covered in the scope of the claims of the present application without departing from the purpose and scope of the technical solutions of the present application.

Claims

1. A nanodot, characterized in that, The nanodot is a core-shell structure, the core-shell structure comprising: a fluorescent core consisting of a single-stranded semiconductor polymer; a shell layer consisting of an amphiphilic compound, the hydrophobic group of the amphiphilic compound binding with the semiconductor polymer, the hydrophilic group on the surface of the shell layer as an active group, the average particle size of the nanodot < 5 nm, the nanodot having improved light stability and anti-photobleaching.

2. A nanodot according to claim 1, wherein, The semiconductor polymer is selected from conjugated polymers, polythiophene, polyfluorene, polystyrene, polyphenylacetylene and derivatives thereof; alternatively, the semiconductor polymer is selected from CNPPV, PDFDP or a combination thereof.

3. The nanodot of claim 1, wherein the nanodot is a semiconductor nanodot. The amphiphilic compound comprises a hydrophobic group and a hydrophilic group, the hydrophilic group selected from carboxyl, amino or hydroxyl; alternatively, the amphiphilic compound is selected from polystyrene maleic anhydride.

4. The nanodot of claim 1, wherein, The nanodot is a glassy structure; Alternatively, the nanodot is prepared by a glass freezing method; Alternatively, the semiconductor polymer and the amphiphilic compound are dissolved and placed in a low temperature environment for rapid cooling to convert them into a glassy state to obtain the nanodot; alternatively, the cooling rate of the rapid cooling is greater than 1000℃ / min.

5. The nanodot of claim 1, wherein The nanodot further comprises a modification; Alternatively, the modification group comprises an antibody, an antibody fragment, a ligand, a receptor, a peptide, a protein, a nucleic acid, an aptamer, a sugar, a drug molecule, a small molecule compound, a fluorescent probe, a radioactive label, an enzyme, a signal molecule, a polymer modification group or a magnetic / functional nanoparticle; alternatively, the modification group is an antibody or an antibody fragment.

6. A method of preparing nanodots, comprising: Prepared by a glass freezing method; Alternatively, the method comprises the following steps: (a) dissolving a semiconductor polymer and an amphiphilic compound in a solvent to form a mixed solution; (b) placing the mixed solution in a low temperature environment for rapid cooling to convert them into a glassy state; (c) removing the solvent to obtain a glassy nanodot product.

7. The method for preparing nanodots as described in claim 6, characterized in that, The solvent is tetrahydrofuran, dioxane, dichloromethane or a combination thereof; Alternatively, the concentration of the semiconductor polymer is 0.1-2 mg / mL; alternatively, the concentration of the amphiphilic compound is 10-100 mg / mL; alternatively, the volume ratio of the semiconductor polymer to the amphiphilic compound is 1:1-1:10; Alternatively, the cooling rate of the rapid cooling is greater than 1000℃ / min; alternatively, the low temperature is liquid nitrogen temperature; alternatively, the mixed solution is slowly dropped from a certain height onto a freezing table at liquid nitrogen temperature to rapidly solidify it at low temperature to form a glassy structure; alternatively, the solvent is removed by freeze-drying; alternatively, the obtained dry powder is hydrolyzed and / or dispersed in an aqueous solution to obtain a single-stranded ultra-small polymer nanodot; alternatively, the single-stranded polymer is wound intramolecularly to form a single-stranded ultra-small polymer nanodot in the aqueous solution; Alternatively, the method further comprises the steps of centrifugation, filtration and ultrafiltration purification; alternatively, the method further comprises modifying the nanodot.

8. A nanoprobe, kit or composition comprising the nanodot of any one of claims 1-5.

9. Use of the nanodots of any one of claims 1-5, the nanoprobes, kits or compositions of claim 8 in bioimaging. Optionally, the nanodots can be used for high-density specific labeling of various subcellular organelles. Optionally, the nanodots can be used for resolving the fine structure of various subcellular organelles, and visualizing the relative distribution of various subcellular organelles at nanoscale; optionally, the organelles include microtubules, peroxisomes, vesicles.

10. A detection method comprising using the nanodots of any one of claims 1-5, the nanoprobes, kits or compositions of claim 8 for detection. Optionally, the detection method comprises: 1) preparing nanodot-labeled samples to be detected; 2) using fluorescence imaging for detection.

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