Semiconductor polymer dot, preparation method thereof and fluorescent probe

Abstract

The invention provides a semiconductor polymer dot, a preparation method thereof and a fluorescent probe. The semiconductor polymer point comprises a fluorescent polymer and an amphiphilic organic polymer, the surface of the fluorescent polymer is coated with the amphiphilic organic polymer, and the average particle size of the semiconductor polymer point is 1-10.5 nm; the fluorescent polymer comprises a semiconductor polymer with a trans-olefin structure and poly [(BODIPY)-co-(9, 9-dioctyl fluorenyl-2, 7-diyl)] or the semiconductor polymer with the trans-olefin structure. The semiconductor polymer point comprises a fluorescent polymer and an amphiphilic organic polymer coating the fluorescent polymer, and by limiting the selection of the fluorescent polymer, the semiconductor polymer point with a specific average particle size has a good scintillation characteristic, and can realize high-order SOFI imaging of a single particle and a cellular level.

Description

Technical Field

[0001] The present application relates to the field of fluorescence imaging technology, and in particular to a semiconductor polymer dot and a preparation method thereof, as well as a fluorescent probe. Background Art

[0002] With the introduction of the famous "Abbe limit" theory, the potential of optical microscopes has been fully developed. When the distance between two point light sources is less than 200 nm, the two light spots cannot be distinguished due to mutual overlap. The resolution of ordinary optical microscopes has reached the limit of 0.2 μm and cannot be improved. In order to deeply study the fine structure within the cell and the synergy between different organelles, it is necessary to break the limitation of light diffraction on resolution, and super-resolution microscopy technology came into being. Compared with traditional fluorescence microscopy, super-resolution fluorescence microscopy breaks through the optical diffraction limit and cleverly applies the principles of structural optics, single-molecule imaging and resonance energy transfer, providing biological researchers with powerful tools to achieve detailed observation and in-depth exploration of the submicroscopic structure, function and kinematics of biological macromolecules of organelles. Among them, super-resolution microscopy mainly includes stimulated emission depletion microscopy (STED), structured illumination microscopy (SIM), photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STROM), super-resolution optical fluctuation imaging (SOFI), and super-resolution imaging technology based on expansion of biological samples (Expansion Microscopy, ExM).

[0003] Compared with other imaging methods, SOFI has attracted more and more attention due to its simple optical equipment, no background noise, short sampling time, and less photobleaching and phototoxicity to samples. SOFI can reconstruct higher-resolution images by collecting the fluorescence fluctuations of particles and performing statistical analysis and calculation of the cross-cumulants. Since the images collected are the intensity fluctuations of fluorescent molecules, the noise during shooting can be easily removed, and the higher the order of the cumulants, the higher the resolution. The results of SOFI high-order reconstruction are highly dependent on the blinking characteristics of the fluorescent probe.

[0004] Fluorescent probes used for SOFI mainly include semiconductor polymer dots, inorganic quantum dots, organic small molecule dyes, fluorescent proteins, and organic semiconductor polymers. Compared with other fluorescent probes, semiconductor polymer dots (Pdots) have the advantages of high brightness, good stability, easy optical regulation, and good biocompatibility, and are more suitable for SOFI imaging. However, the size and blinking characteristics of traditional semiconductor polymer dots cannot meet the needs of high-order SOFI imaging. Summary of the invention

[0005] Based on this, it is necessary to provide a semiconductor polymer dot and a preparation method thereof and a fluorescent probe whose size and scintillation characteristics can meet the requirements of high-order super-resolution imaging.

[0006] The present application discloses a semiconductor polymer dot, wherein the semiconductor polymer dot comprises a fluorescent polymer and an amphiphilic organic polymer, wherein the amphiphilic organic polymer is coated on the surface of the fluorescent polymer, and the average particle size of the semiconductor polymer dot is 1 nm to 10.5 nm;

[0007] Wherein, the fluorescent polymer includes a semiconductor polymer having a trans-olefin structure and poly[(fluoroborane dipyrrole)-co-(9,9-dioctylfluorenyl-2,7-diyl)] or the semiconductor polymer having a trans-olefin structure.

[0008] In one embodiment, the semiconductor polymer having a trans-olefin structure includes one or more of poly[{9,9-dioctyl-2,7-dienylidene-fluorenylene}-alternating-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}], poly[dibenzothiophene-4,4'-dimethoxy-2,2'-divinyl], poly(p-phenylene vinylene) and poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene vinylene].

[0009] In one embodiment, the mass of the amphiphilic organic polymer is 15% to 20% of the mass of the fluorescent polymer.

[0010] In one embodiment, the fluorescent polymer is poly[{9,9-dioctyl-2,7-diethenylidene-fluorenylene}-alternate-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}], having the structural formula , wherein the molecular weight is 20000~100000, and the average particle size of the semiconductor polymer dots is 7nm~9nm.

[0011] In one embodiment, the fluorescent polymer is poly[dibenzothiophene-4,4'-dimethoxy-2,2'-divinyl] having the structural formula , wherein the molecular weight is 20000~100000, and the average particle size of the semiconductor polymer dots is 8nm~10.5nm.

[0012] In one embodiment, the fluorescent polymer is the poly[{9,9-dioctyl-2,7-diethenylidene-fluorenylene}-alternating-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}] and the poly[(fluoroborane dipyrrole)-co-(9,9-dioctylfluorenyl-2,7-diyl)] in a mass ratio of (1-5):1, and the structural formula of the poly[(fluoroborane dipyrrole)-co-(9,9-dioctylfluorenyl-2,7-diyl)] is , wherein the molecular weight is 5000~50000, and the average particle size of the semiconductor polymer dots is 8nm~10nm.

[0013] In one embodiment, the amphiphilic organic polymer includes one or both of polystyrene-polyethylene glycol-carboxylic acid and poly(styrene-co-maleic anhydride).

[0014] Furthermore, the present application provides a method for preparing the semiconductor polymer dots as described above, comprising the following steps:

[0015] Providing a mixed raw material, the mixed raw material comprising the fluorescent polymer and the amphiphilic organic polymer;

[0016] The mixed raw materials are co-precipitated to coat the surface of the fluorescent polymer with the amphiphilic organic polymer to prepare semiconductor polymer dots with an average particle size of 1 nm to 10.5 nm;

[0017] The fluorescent polymer includes a semiconductor polymer having a trans-olefin structure and poly[(fluoroborane dipyrrole)-co-(9,9-dioctylfluorenyl-2,7-diyl)] or a semiconductor polymer having a trans-olefin structure.

[0018] In one embodiment, the step of preparing the semiconductor polymer dots further includes: the mixed raw material also includes a good solvent, and the mixed raw material is added into a poor solvent to allow the fluorescent polymer and the amphiphilic organic polymer to co-precipitate in the poor solvent.

[0019] The present application also provides a fluorescent probe, comprising the semiconductor polymer dots as described above.

[0020] In one embodiment, the fluorescent probe is used for high-order super-resolution imaging.

[0021] The semiconductor polymer dots provided in the present application include fluorescent polymers and amphiphilic organic polymers encapsulating the fluorescent polymers. By limiting the selection of fluorescent polymers, the semiconductor polymer dots with an average particle size of 1 nm to 10.5 nm have good scintillation properties, and high-order SOFI imaging at the single particle and cellular levels can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] FIG1 is a dynamic light scattering test size distribution diagram of Example 1 to Comparative Example 1, (1) dynamic light scattering result diagram of Example 1; (2) dynamic light scattering result diagram of Example 2; (3) dynamic light scattering result diagram of Example 3; (4) dynamic light scattering result diagram of Comparative Example 1;

[0024] Figure 2 Spectra of Examples 1 to 3; (1) Spectrum of Example 1; (2) Spectrum of Example 2; (3) Spectrum of Example 3;

[0025] Figure 3 Comparison of the flickering characteristics and fluorescence characteristics of Example 1 and Example 3; (1) Fluorescence intensity flickering characteristic diagrams of Example 1 and Example 3; (2) Fluorescence dark state distribution statistics of Example 3 and its power law distribution fitting; (3) Typical fluorescence flickering on / off time ratio of Example 1; (4) Typical fluorescence flickering on / off time ratio of Example 3; (5) Fluorescence intensity flickering characteristic diagram of Comparative Example 1;

[0026] Figure 4 The figures are single-particle SOFI wide-field imaging and high-order reconstruction images; (1) the wide-field image and the eighth-order reconstruction image of Example 1; (2) the wide-field resolution of the single particle shown by the arrow in Figure (1) and the resolution after the eighth-order reconstruction; (3) the wide-field image of Example 2 and its eighth-order reconstruction image; (4) the wide-field resolution of the single particle shown by the arrow in Figure (3) and the resolution after the eighth-order reconstruction; (5) the wide-field image of Example 3 and its eighth-order reconstruction image; (6) the wide-field resolution of the single particle shown by the arrow in Figure (5) and the resolution after the eighth-order reconstruction; (7) the wide-field image of Comparative Example 1 and its eighth-order reconstruction image; (8) the wide-field resolution of the single particle shown by the arrow in Figure (7) and the resolution after the eighth-order reconstruction;

[0027] Figure 5The following are SOFI images of specifically labeled cell microtubules; (1) wide-field images of specifically labeled cell microtubules in Example 3; (2) an enlarged image of the cell microtubule structure in the yellow frame in Figure (1) and the 4th, 6th, and 8th order SOFI reconstructed images of the region; (3) a wide-field image of specifically labeled cell microtubules in Example 1 and an 8th order SOFI reconstructed image; (4) a quantitative analysis of the resolution of the region indicated by the arrow in Figure (2); (5) a quantitative analysis of the resolution of the region indicated by the arrow in Figure (3); (6) a wide-field image of specifically labeled cell microtubules in Comparative Example 1 and an 8th order SOFI reconstructed image; (7) a quantitative analysis of the resolution of the region in Figure (6). DETAILED DESCRIPTION

[0028] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0030] The term "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, wherein the any and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. For example, "A and / or B" includes three parallel solutions: A, B, and "a combination of A and B".

[0031] In this article, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and other expressions that mean "one or more" are also understood in the same way unless otherwise specified.

[0032] In this document, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent technical solutions are related in terms of the content covered, but should not be understood as limiting the previous technical solution, nor can they be understood as limiting the scope of protection of this document. In this document, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0033] Herein, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel schemes of "yes" or "no". If multiple "optional" items appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" item is independent of each other. In this application, descriptions such as "optionally contain", "optionally include", etc. mean "contain or not contain". "Optional component X" means that component X exists or does not exist, or means that component X is contained or not contained.

[0034] In this document, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0035] In this article, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0036] Herein, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​within the numerical interval is considered to be continuous and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval only refers to integers within the numerical interval, including the two endpoint integers of the numerical range, and each integer between the two endpoints, is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0037] Herein, the term "room temperature" or "normal temperature" generally refers to 4°C to 35°C, for example, 20°C ± 5°C. In some embodiments herein, "room temperature" or "normal temperature" refers to 10°C to 30°C. In some embodiments herein, "room temperature" or "normal temperature" refers to 20°C to 30°C.

[0038] In this article, if there are multiple steps involved in the method flow, unless there is a clear different description in this article, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0039] The present application discloses a semiconductor polymer dot, wherein the semiconductor polymer dot comprises a fluorescent polymer and an amphiphilic organic polymer, wherein the amphiphilic organic polymer is coated on the surface of the fluorescent polymer, and the average particle size of the semiconductor polymer dot is 1 nm to 10.5 nm;

[0040] The fluorescent polymer includes a semiconductor polymer having a trans-olefin structure and poly[(fluoroborane dipyrrole)-co-(9,9-dioctylfluorenyl-2,7-diyl)] or a semiconductor polymer having a trans-olefin structure.

[0041] It can be understood that the fluorescent polymer includes a mixture of a semiconductor polymer having a trans-olefin structure and poly[(fluoroboranedipyrrole)-co-(9,9-dioctylfluorenyl-2,7-diyl)], or a semiconductor polymer having a trans-olefin structure.

[0042] The semiconductor polymer dots provided in this application include fluorescent polymers and amphiphilic organic polymers coated with fluorescent polymers. By limiting the selection of fluorescent polymers, semiconductor polymer dots with an average particle size of 1nm to 10.5nm have good scintillation characteristics and can achieve high-order SOFI imaging at the single particle and cell level. Specifically, after performing eighth-order reconstruction of single particles and cell microtubules, it can be found that the resolution of semiconductor polymer dots has reached about 60 nm.

[0043] In a specific example, the trans-olefin structured semiconductor polymer includes one or more of poly[{9,9-dioctyl-2,7-dienylidene-fluorenylene}-alternating-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}], poly[dibenzothiophene-4,4'-dimethoxy-2,2'-divinyl], poly(p-phenylene vinylene) and poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylethynylene].

[0044] Furthermore, the molecular weight of the semiconductor polymer with a trans-olefin structure is 10,000-150,000.

[0045] Specifically, the fluorescent polymer can be poly[{9,9-dioctyl-2,7-divinylene-fluorenylene}-alternate-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}] (PFPV), or poly[dibenzothiophene-4,4'-dimethoxy-2,2'-divinylene] (PFVA), or a mixture of poly[{9,9-dioctyl-2,7-dienylidene-fluorenylene}-alternate-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}] (PFPV) and poly[dibenzothiophene-4,4'-dimethoxy-2,2'-divinyl] (PFVA), the fluorescent polymer may also be a mixture of poly[{9,9-dioctyl-2,7-dienylidene-fluorenylene}-alternate-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}] (PFPV) and poly[(fluoroboranedipyrrole)-co-(9,9-dioctylfluorenyl-2,7-diyl)] (PF5BODIPY565), poly[dibenzothiophene-4,4'-dimethoxy-2,2'-divinyl] (PFVA) and poly[(fluoroboranedipyrrole)-co-(9,9-dioctylfluorenyl-2,7-diyl)] (PF5BODIPY565).

[0046] In a specific example, the mass of the amphiphilic organic polymer is 15% to 20% of the mass of the fluorescent polymer. Specifically, the mass of the amphiphilic organic polymer can be, but is not limited to, 15%, 16%, 17%, 18%, 19% or 20% of the mass of the fluorescent polymer.

[0047] In a specific example, the fluorescent polymer is poly[{9,9-dioctyl-2,7-diethenylidene-fluorenylene}-alternate-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}], having the structural formula , wherein the molecular weight is 20000-100000, and the average particle size of the semiconductor polymer dots is 7nm-9nm. Specifically, the average particle size of the semiconductor polymer dots can be, but is not limited to, 7nm, 7.5nm, 8nm, 8.5nm or 9nm.

[0048] In a specific example, the fluorescent polymer is poly[dibenzothiophene-4,4'-dimethoxy-2,2'-divinyl] having the structural formula , wherein the molecular weight is 20000-100000, and the average particle size of the semiconductor polymer dots is 8nm-10.5nm. Preferably, the average particle size of the semiconductor polymer dots can be, but is not limited to, 8nm, 8.5nm, 9nm, 9.5nm, 10nm, 10.36nm or 10.5nm.

[0049] In a specific example, the fluorescent polymer is a poly[{9,9-dioctyl-2,7-diethenylidene-fluorenyl}-alternating-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}] and a poly[(fluoroborane dipyrrole)-co-(9,9-dioctylfluorenyl-2,7-diyl)] in a mass ratio of (1-5):1, and the structural formula of the poly[(fluoroborane dipyrrole)-co-(9,9-dioctylfluorenyl-2,7-diyl)] is , wherein the molecular weight is 5000-50000, and the average particle size of the semiconductor polymer dots is 8nm-10nm. Further, the mass ratio of the two components of the fluorescent polymer can be, but not limited to, 1:1, 2:1, 3:1, 4:1 or 5:1, and the average particle size of the semiconductor polymer dots can be, but not limited to, 8nm, 8.5 nm, 9 nm, 9.5 nm or 10 nm.

[0050] In a specific example, the amphiphilic organic polymer includes one or both of polystyrene-polyethylene glycol-carboxylic acid and poly(styrene-co-maleic anhydride).

[0051] Preferably, the amphiphilic organic polymer is poly(styrene-co-maleic anhydride) (PSMA) having a CAS number of 26762-29-8 and a structural formula of , wherein the molecular weight is 500 to 3000. Specifically, the molecular weight of poly(styrene-co-maleic anhydride) can be, but is not limited to, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900 or 3000.

[0052] In a specific example, the structural formula of polyparaphenylene vinylene (PPV) is The structural formula of poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylethynylene] (MEH-PPV) is .

[0053] Furthermore, the present application provides a method for preparing the semiconductor polymer dots as described above, comprising the following steps:

[0054] Providing a mixed raw material, the mixed raw material comprising a fluorescent polymer and an amphiphilic organic polymer;

[0055] The mixed raw materials are co-precipitated to allow the amphiphilic organic polymer to cover the fluorescent polymer, thereby preparing semiconductor polymer dots with an average particle size of 1 nm to 10.5 nm;

[0056] The fluorescent polymer includes a semiconductor polymer having a trans-olefin structure and poly[(fluoroborane dipyrrole)-co-(9,9-dioctylfluorenyl-2,7-diyl)] or a semiconductor polymer having a trans-olefin structure.

[0057] It can be understood that the method for preparing semiconductor polymer dots includes the following steps:

[0058] A mixed raw material is provided, the mixed raw material includes a fluorescent polymer and an amphiphilic organic polymer, the fluorescent polymer includes a semiconductor polymer with a trans-olefin structure and poly[(fluoroborane dipyrrole)-co-(9,9-dioctylfluorenyl-2,7-diyl)] or a semiconductor polymer with a trans-olefin structure; the mixed raw material is co-precipitated to make the amphiphilic organic polymer cover the fluorescent polymer, and prepare semiconductor polymer dots with an average particle size of 1nm to 10.5nm.

[0059] In a specific example, the step of preparing semiconductor polymer dots further includes: the mixed raw material also includes a good solvent, and the mixed raw material is added into a poor solvent to co-precipitate the fluorescent polymer and the amphiphilic organic polymer in the poor solvent.

[0060] Specifically, the good solvent may be, but is not limited to, one or two of tetrahydrofuran (THF) and ethanol, and the poor solvent may be, but is not limited to, one or more of water.

[0061] During the reprecipitation process, the mixed raw materials including the good solvent are rapidly mixed with the poor solvent (ultrasonic state). After mixing, the solubility of the fluorescent polymer and the amphiphilic organic polymer suddenly decreases. Under the influence of the hydrophobic effect between polymer chains or single chain segments, a suspension of semiconductor polymer dots is finally formed.

[0062] The present application also provides a fluorescent probe, comprising the semiconductor polymer dots as described above.

[0063] Furthermore, the fluorescent probe of the present application is used for high-order super-resolution imaging.

[0064] It can be understood that high order specifically refers to a reconstruction order of 8.

[0065] The present application is further described in detail below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manuals or conventional conditions in the art, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0066] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed. "Normal temperature" refers to 25°C; "normal pressure" refers to 100KPa or 101KPa.

[0067] The poly[{9,9-dioctyl-2,7-dienylidene-fluorenylene}-alternate-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}] (PFPV) used in the following examples was purchased from Luminescence Technology Crop. Product No. LT-A1013; poly[dibenzothiophene-4,4'-dimethoxy-2,2'-divinyl] (PFVA) was purchased from Luminescence Technology Crop. Product No. LT-A1011; poly(styrene-co-maleic anhydride) (PSMA) was purchased from SIGMA-ALDRICH; PFBT was purchased from Ruixun Company, CAS No. 210347-52-7; poly[(fluoroboron dipyrrole)-co-(9,9-dioctylfluorenyl-2,7-diyl)] (PF5BODIPY565) It is self-made, with a molecular weight of Mn of 11418 and a PDI of 1.554562. The specific preparation steps of PF5BODIPY565 are as follows:

[0068] Step 1:

[0069] Powdered I2 (3.04 g, 12 mmol) and NaIO4 (0.86 g, 4 mmol) were slowly added to stirred 98% H2SO4 (50 ml). Stirring was continued at room temperature for 30 minutes to obtain a dark brown iodide solution. p-Tolualdehyde (1.5 g, 14 mmol) was added to the iodide solution in one portion and the resulting solution was stirred at room temperature for 5 hours. The reaction mixture was then slowly poured into stirred ice water. The crude solid product was collected by filtration, washed with water until the filtrate was neutral, and dried in vacuo in the dark to obtain a light brown powder, which was recrystallized from ethyl acetate to obtain a light yellow solid 4-methyl-3,5-diiodobenzaldehyde.

[0070] The specific reactions are as follows The yield of 4-methyl-3,5-diiodobenzaldehyde was 2.13 g, 40.9%.

[0071] 1 H NMR (CDCl3, 500MHz): δ=9.823 (s, 1H), 8.306 (d, 2H), 2.842 (s, 3H). 13C NMR (CDCl3, 125MHz): δ=188.63, 149.91, 140.42, 136.62, 99.54, 35.54.

[0072] Step 2:

[0073] To a solution of 4-methyl-3,5-diiodobenzaldehyde (1.5 g, 4.2 mmol) and 2,4-dimethyl-1H-pyrrole (1 g, 10.5 mmol) in dry CH2Cl2 (120 mL) at room temperature, 110 μL of trifluoroacetic acid in dry CH2Cl2 (5 mL) was slowly added. After stirring for 3 h under ice-bath cooling, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (0.95 g, 4.2 mmol) was added and stirred for 10 min. The solution was stirred for another 1 h at room temperature. NEt3 (10 mL, 72 mmol) was added, followed by the slow addition of BF3•Et2O (12 mL, 81 mmol). After stirring for 10 h at room temperature, the reaction mixture was washed with saturated aqueous Na2CO3 (2×100 mL), dried over Na2SO4, and concentrated on a rotary evaporator. The brown oily residue was purified by silica gel column chromatography, hexane / CH2Cl2 = 3:1. The green fluorescent product fraction was dried to obtain a red solid as monomer 1.

[0074] The specific reactions are as follows ,

[0075] Yield of monomer 1: 0.48 g, 19.5%. 1 H NMR (CDCl3, 500 MHz): δ = 7.94 (d, 2 H), 6.40 (s, 2 H), 3.07 (t, 4 H), 2.83 (t, 3 H), 2.54 (t, 4 H), 1.86 (m, 4 H), 1.76 (m, 4 H). 13C NMR (CDCl3, 125 MHz): δ = 158.3, 144.7, 140.7, 136.4, 135.5, 133.6, 129.9, 126.3, 98.7, 35.3, 24.9, 23.4, 23.0, 22.5. HRMS (ESI). (M+, C24H23BF2I2N2 ) Calculated 643.0090; Found 643.0095.

[0076] Step 3:

[0077] In a 100 mL round-bottom flask were added the above monomer 1 (0.1 mmol, 64.2 mg), monomer 2 (1.02 mmol, 0.045 mmol, 569.16 mg), monomer 3 (0.9 mmol, 577 mg), toluene (20 mL), Na2CO3 (2 M, 10 mL) and ethanol (5.7 mL). The mixture was stirred at room temperature, degassed and re-injected with N2, and this was repeated 4 times before and after the addition of Pd(PPh3)4 (0.02 mmol, 23 mg). The reactants were stirred at 83 °C for 30 h, and then phenylboronic acid (100 mg) dissolved in THF (1 mL) was added. After two hours, bromobenzene (1 mL) was added and stirred for further 2 hours, and the mixture was poured into methanol (100 mg). The precipitate was filtered off and washed with methanol, water and acetone to remove monomers, small molecular oligomers and inorganic salts. The crude product was dissolved in DCM (15 mL), filtered through a 0.2 mm membrane, and reprecipitated in methanol (150 mL). The powder was then stirred in acetone (200 mL) overnight, then collected by filtration and dried in vacuo.

[0078] The specific reactions are as follows:

[0079]

[0080] Example 1

[0081] This embodiment provides a method for preparing semiconductor polymer dots with an average particle size of 7 nm, wherein the semiconductor polymer dots include a fluorescent polymer and an amphiphilic organic polymer coating the fluorescent polymer, the fluorescent polymer is PFPV, the amphiphilic organic polymer is PSMA, and PSMA accounts for 20% of the semiconductor polymer dots:

[0082] Use an electronic balance to weigh the PFPV polymer and PSMA polymer respectively. After weighing, use THF to dissolve them into a 1000 mg / L PFPV solution, and ultrasonicate for 1 min to completely dissolve them. Use the same method to prepare a 1000 mg / L PSMA solution. Turn on the temperature control table in the fume hood and preheat it at 95 °C.

[0083] Prepare a mixed raw material solution, use THF to dilute 1000 mg / L PFPV solution to 100 mg / L, where the functional polymer PSMA accounts for 20% of the luminescent polymer. Specifically, add 0.5 mL of 1000 mg / L PFPV solution to a glass bottle, then add 0.1 mL of 1000 mg / L PSMA solution, and finally add 4.4 mL THF to dilute the 1000 mg / L PFPV solution to 100 mg / L, and ultrasonicate for 30 s to make it evenly distributed.

[0084] Add 10 g of deionized water to a new glass bottle, place the glass bottle with deionized water in the sonicator and ensure that the liquid level of the deionized water in the glass bottle is below the liquid level of the water in the sonicator. Turn on the ultrasound, use a pipette to take 1 mL of the mixed raw material solution and vertically and quickly inject it into the glass bottle, so that the mixed polymer stretched in THF is quickly injected into the poor solvent and folded into nanoparticles. Each time the raw material solution is added, an interval of about 30 seconds is allowed. After all the raw material solutions are added, sonicate for about 3 minutes to make them evenly distributed. Then take out the glass bottle and put it on the preheated heating table. Pass nitrogen into the glass bottle for about half an hour to discharge the THF in the system, so that the polymer is evenly distributed in the aqueous solution. Decide whether to continue passing nitrogen according to the concentration of the required semiconductor polymer dots. After the THF in the glass bottle is completely removed, turn off the heating table to cool the solution. When the solution temperature drops to room temperature, use a syringe to remove the solution and pass it through a water filter with a pore size of 0.22 μm to remove aggregates. Seal the bottle with sealing film and wrap the bottle with tin foil, then place it in a refrigerator at 4 ℃ for later use.

[0085] Example 2

[0086] This embodiment provides a method for preparing semiconductor polymer dots with an average particle size of 10 nm, wherein the semiconductor polymer dots include a fluorescent polymer and an amphiphilic organic polymer coating the fluorescent polymer, the fluorescent polymer is PFVA, the amphiphilic organic polymer is PSMA, and PSMA accounts for 20% of the semiconductor polymer dots:

[0087] The preparation method is different from that of Example 1 only in that PFPV is replaced by PFVA.

[0088] Example 3

[0089] This embodiment provides a method for preparing semiconductor polymer dots with an average particle size of 9 nm, wherein the semiconductor polymer dots include a fluorescent polymer and an amphiphilic organic polymer coating the fluorescent polymer, the fluorescent polymer is PFPV doped with PF5BODIPY565, the amphiphilic organic polymer is PSMA, and PSMA accounts for 20% of the semiconductor polymer dots:

[0090] The preparation method is different from that of Example 1 only in that PFPV is replaced by 1000 ppm of PFPV and 1000 ppm of PF5BODIPY565 mixed in equal volumes.

[0091] Comparative Example 1

[0092] This comparative example provides a semiconductor polymer dot with an average particle size of 16 nm. The difference from Example 1 is that PFPV is replaced by PFBT. The structural formula of PFBT is .

[0093] Detection methods and test results

[0094] As shown in FIG1 , the size distribution is tested using dynamic light scattering. It can be seen that the average particle size of the semiconductor polymer dots containing semiconductor polymers having a trans-olefin structure in Examples 1, 2 and 3 is all below 10 nm, while the average particle size of the semiconductor polymer dots in Comparative Example 1 is 16 nm.

[0095] like Figure 2 The figures show the spectral tests performed on Examples 1 to 3, respectively, where Abs is absorbance and EM is excitation wavelength.

[0096] Analysis of single particle scintillation and fluorescence characteristics

[0097] In order to further determine whether the semiconductor polymer dots prepared in each embodiment are suitable for SOFI imaging, single particles of the semiconductor polymer dots prepared in each embodiment are analyzed. Before single particle SOFI imaging is performed, a single particle sample is prepared first.

[0098] The preparation of single particle samples specifically includes: using a 22×22 mm microscope cover glass for the single particle sample, placing the cover glass in an isopropanol solution of sodium hydroxide for 30 min, and then ultrasonicating it in deionized water for 30 min. After taking it out, blow dry with N2, and drop 60 μL of 5×10 -4 The cover glass was chemically modified with a 3-aminopropyltriethoxysilane ethanol solution of 10 mol / L, and then rinsed with deionized water and dried with nitrogen after standing for 5 min. 600 μL of 0.01 mg / L semiconductor polymer dots prepared in each embodiment and comparative example were added dropwise, and then rinsed with deionized water after standing for 30 min and dried for later use.

[0099] After preparing the single particle sample, SOFI imaging can be performed and the imaging device can be debugged. Turn on the EMCCD detector, computer, and laser controller in turn, select 100X objective lens, add lens oil, place the prepared sample flat on the stage of the microscope, turn on the computer operating program and laser, use 488 nm excitation light for excitation, and filter the fluorescence signal through the bandpass ZET488 filter. The attenuator uses OD×3, the excitation light intensity is 5%, and the gain is 80%. Debug the focus to find single particles. After finding a suitable field of view, collect images to test the scintillation characteristics and fluorescence characteristics of single particles. The image size is 256×256 pixels, and 1000 frames are shot. It takes 20 seconds. The test results are as follows: Figure 3 shown.

[0100] Single-particle SOFI imaging and eighth-order reconstruction

[0101] The fluorescence flickering characteristics of the PFPV semiconductor polymer dots (Pdots) of Example 1, the doped semiconductor polymer dots (Pdots) of Example 3, and the PFBT semiconductor polymer dots (Pdots) of Comparative Example 1 were quantitatively analyzed, mainly including the change of fluorescence intensity over time, the fluorescence flickering on / off time ratio, the fluorescence dark state distribution statistics and its power law distribution. It can be found that Figure 3 As shown in (1), the fluorescence intensity of the PFPV Pdots of Example 1 and the doped Pdots of Example 3 changes significantly over time, and both have relatively excellent blinking characteristics. In order to verify that Pdots meet the requirements of SOFI imaging for fluorescent probes and that the fluorescent probes need to blink randomly, the dark state distribution of Pdots is analyzed. The results are as follows Figure 3 (2) shows that its dark state distribution conforms to the power law distribution, which is similar to the fluorescence flashing of inorganic quantum dots, that is, it can be considered that Pdots flash randomly. Figure 3 The typical fluorescence blinking on / off ratios of (3) and (4) further indicate that the prepared small-sized Pdots have good blinking properties and are suitable for use as fluorescent probes for high-order SOFI. Figure 3 As shown in (5), the fluorescence intensity of PFBTPdots in comparative example 1 does not change significantly over time, and its blinking characteristics are poor, making it unsuitable as a fluorescent probe for high-order SOFI.

[0102] The single-particle wide-field image was analyzed after eight-order reconstruction, such as Figure 4 As shown in the figure, it can be found that each particle is evenly distributed without aggregation under wide-field conditions, the signal-to-noise ratio and brightness are both high. By statistically accumulating the mutual accumulation, the collected single-particle data are reconstructed in a high-order manner, the Airy disk generated by light diffraction is reduced, and the semiconductor polymer particles are becoming clearer and clearer. The results of wide-field and eighth-order SOFI reconstruction of PFPV Pdots doped in Example 3 are analyzed. Figure 4It can be seen from (5) and (6) that under wide field conditions, the two particles in the area indicated by the white arrow cannot be distinguished due to the presence of the Airy disk. However, after the eighth-order reconstruction of SOFI, the Airy disk is reduced, the resolution is improved, and the two particles in the white area can be easily distinguished. In order to further explore the resolution improvement effect, the resolution of the area indicated by the white arrow is quantitatively analyzed, and the intensity distribution is Gaussian fitted. After the eighth-order reconstruction, the resolution of the optical system (the full width at half maximum of the curve FWHM) is improved from 403nm in the wide field to 72nm, which is 5.6 times higher than that in the wide field. After the eighth-order reconstruction, the resolution of the doped Pdots is improved to 66nm, which is 6.5 times higher. After the eighth-order reconstruction, the resolution of the semiconductor polymer quantum dots provided in Comparative Example 1 is only improved from 386nm to 111nm, which is only 3.48 times higher. The resolution improvement effect is not as good as that of the semiconductor polymer dots in the embodiment. The above results further demonstrate the potential of small-sized Pdots as high-order SOFI fluorescent probes.

[0103] Pdots specifically label microtubules for SOFI imaging and high-order reconstruction

[0104] Bioconjugation and enrichment of polymer dots

[0105] In order to specifically label cell microtubules, the Pdots sample must be coupled to biological streptavidin (SA), so that the Pdots can be combined with antibodies that can specifically target microtubules to achieve specific labeling of cell microtubules. The following are the steps for coupling the Pdots sample with SA:

[0106] (1) Prepare a 4 mg / L standard solution. The concentration of the Pdots sample is obtained based on the fact that the fluorescence intensity is proportional to the concentration. Add 10 mL of deionized water to a glass bottle and place it in an ultrasonicator for sonication. Keep the water level in the glass bottle below the level of the ultrasonic water. Take 40 μL of a 1000 mg / L PFPV / PFBT tetrahydrofuran solution and inject it vertically and quickly into the glass bottle. After sonication for 2-3 min, take it out to obtain a standard solution with a concentration of 4 mg / L.

[0107] (2) Calibrate the concentration of Pdots samples by absorption spectrum. Turn on the UV-visible spectrophotometer, use a four-way glass cuvette, add 2 mL of deionized water to scan the baseline, adjust to zero, take out the cuvette, pour out the water, wash and blow dry the cuvette, then add 2 mL of 4 mg / L standard sample to scan its UV absorption spectrum and record the peak value of the spectrum. Take out the cuvette, pour out the standard sample, wash and blow dry the cuvette, then add 2 mL of Pdots sample, scan its UV absorption spectrum and record the peak value of the spectrum. The concentration of the Pdots sample can be obtained by comparing the peak values ​​of the two.

[0108] (3) Pdots coupled with biological streptavidin. Take a 1.5 mL EP tube and add 1 mL of 50 mg / L Pdots sample, 20 μL of 1M HEPES buffer solution, 20 μL of 5% PEG aqueous solution, 20 μL of 5 mg / mL EDC aqueous solution, and 60 μL of 1 mg / mL SA in sequence. Wrap the EP tube with tin foil and place it on a mixer at room temperature at 20 r / min for 4 hours to obtain Pdots-SA.

[0109] (4) Concentrate Pdots-SA by ultrafiltration centrifugation. This step is mainly to remove excess SA in the coupling biological streptavidin reaction. First, prepare the eluent with 1 mL HEPES, 1 mL of 5% PEG aqueous solution and 48 mL water. Select a 100 KDa ultrafiltration tube, add 4 mL of water, select a 2# rotor, and run at 3500 r for 3 min 30 s to check whether the ultrafiltration tube is leaking. After checking, add Pdots-SA and eluent. Add 10 μL of 10% TritonX-100 to every 2 mL of eluent to prevent nanoparticle aggregation (except the last time). Use a 2# rotor and run at 3000 r / min for 3 min. After each ultrafiltration centrifugation, pour out the lower clear liquid, add the eluent and a certain amount of TritonX-100, and repeat this five times. The Pdots-SA solution is concentrated to about 200 μL for the last time, and its average particle size and concentration are tested for subsequent experiments.

[0110] Polymer dots specifically label cell microtubules

[0111] When the cells in the culture medium grow to about 60%, the cell microtubule-specific labeling experiment can be performed. The experimental steps are as follows:

[0112] (1) Prepare the solution.

[0113] Extraction solution: Mix 0.1 mM PIPES solution, 0.2% TritonX-100 aqueous solution and 1 mM EGTA solution evenly, gradually add diluted magnesium chloride aqueous solution, stir and detect the pH value of the solution while adding, until the solution is just clear; Fixation solution: Mix 4% paraformaldehyde (PFA) and 0.1% glutaraldehyde (GA) evenly (diluted with extraction solution); Blocking solution: Dissolve 5% bovine serum albumin (BSA) and 0.1% TritonX-100 in PBS and mix by ultrasonic.

[0114] (2) Extraction.

[0115] Aspirate the original culture medium in the culture dish, add 2 mL PBS to rinse twice, aspirate the PBS and add 100 μL extraction solution for 30 s (no more than 40 s).

[0116] (3) Fixed.

[0117] Aspirate the extract, add 100 μL of the prepared fixative, place in a 4 ℃ refrigerator for 15 min, take out, aspirate the fixative, add PBS to rinse, add 2 mL each time, shake on a shaker for 1 min, aspirate PBS, and repeat 3 times. The following steps are all performed on a shaker at a speed of 40 r / min.

[0118] (4) Punching.

[0119] Add 100 μL of 0.5% TritonX-100 and treat for 5 min before aspirating.

[0120] (5) Closed.

[0121] Add 100 μL of the prepared blocking solution and treat for 30 min before aspirating.

[0122] (6) Primary antibody.

[0123] 1 μL of primary antibody was diluted 100-fold with blocking solution, incubated on a shaker for 60 min, then aspirated and washed 3 times with PBS, each time for 5 min.

[0124] (7) Secondary antibody.

[0125] Dilute 1 μL of secondary antibody 100-fold with blocking solution, incubate on a shaker for 60 min, then aspirate and wash three times with PBS, 5 min each time.

[0126] (8) Dyes.

[0127] Dilute Pdots-SA to 15 mg / L with blocking solution, add it to the culture dish and incubate for 1 h. After aspirating the Pdots-SA solution, wash it with PBS for 3 times. Finally, add about 2 mL of PBS, seal it with sealing film and store it in a 4 ℃ refrigerator (up to one week).

[0128] Cellular SOFI imaging experiment

[0129] The cell microtubule SOFI imaging experiment is similar to the single-particle SOFI experiment.

[0130] In order to minimize the impact of photobleaching on the experiment, a deoxygenation system was added to all samples used in the experiment, that is, 0.3 mL of 20 mM glucose and 4 mL of 100 nM glucose oxidase were added to the culture dish.

[0131] Next, a cell microtubule SOFI experiment was conducted. The prepared cell sample was placed on the stage, the EMCCD camera was turned on, the objective lens was adjusted to 100X and immersion oil was added, the computer and the laser controller were turned on, and the laser switch was turned on when the light on the laser controller stopped flashing. The cell samples labeled with PFPV Pdots in Example 1, doped PFPV Pdots in Example 3, and PFBTPdots in Comparative Example 1 were all excited with a 488 nm laser, and the fluorescence signal was filtered through a bandpass ZET488 filter. The attenuation film was selected as OD×3, the excitation light intensity was selected as 5%, the gain was 80%, and an additional 1.6x zoom was introduced. The focal length was adjusted to find a suitable cell microtubule structure. After finding a suitable field of view, images were collected, and 1500 frames were collected each time, which took 30 s.

[0132] The cell microtubule results of the PFPV Pdots of Example 1, the doped PFPV Pdots of Example 3, and the PFBT Pdots of Comparative Example 1 were analyzed. Figure 5 As shown in (1), due to the high brightness of the semiconductor polymer itself, the signal-to-noise ratio of the wide-field image is relatively high. However, due to the influence of light diffraction, many structures are not clear and the imaging of cell microtubules is coarse. Figure 5 (2) There are many cell microtubules overlapping in the wide-field image. As the SOFI reconstruction order increases, the cell microtubule structure becomes clearer and thinner. The microtubule structure originally overlapping in the wide field can be clearly distinguished. The triangular area generated by the overlap of three microtubules can be clearly seen, and the signal is basically not lost. The microtubule structure is still very continuous.

[0133] In order to further analyze the resolution improvement effect of SOFI high-order reconstruction of the two materials, the resolution was quantitatively analyzed. The results are as follows: Figure 5 (4) and (5). Figure 5 By analyzing the resolution of the area indicated by the white arrow in (2), it can be seen that the resolution of a single cell microtubule marked by doped PFPV Pdots in Example 3 is increased from 360 nm in wide field to 65 nm after SOFI eighth-order reconstruction, and the resolution is increased by about 5.6 times. By analyzing the resolution of the cell microtubule marked by PFPV Pdots in Example 1, it can be seen that Figure 5 The resolution of the area indicated by the white arrow in (3) is also improved to 64 nm after the eighth-order SOFI reconstruction. Figure 5 The resolution of the larger-sized semiconductor polymer dots provided in the comparative example (7) was improved to 139 nm after the eighth-order SOFI reconstruction. From the above results, it can be concluded that the small-sized Pdots with trans-olefin structure have excellent blinking properties and are very suitable for use as fluorescent probes for high-order SOFI.

[0134] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0135] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the specification and drawings may be used to interpret the scope of the claims.

Claims

1. A semiconductor polymer dot, characterized in that: The semiconductor polymer dots include a fluorescent polymer and an amphiphilic organic polymer, the amphiphilic organic polymer is coated on the surface of the fluorescent polymer, and the average particle size of the semiconductor polymer dots is 1 nm to 10.5 nm; Wherein, the fluorescent polymer includes a semiconductor polymer having a trans-olefin structure and poly[(fluoroborane dipyrrole)-co-(9,9-dioctylfluorenyl-2,7-diyl)] or the semiconductor polymer having a trans-olefin structure.

2. The semiconductor polymer dot according to claim 1, wherein The semiconductor polymer having a trans-olefin structure includes one or more of poly[{9,9-dioctyl-2,7-dienylidene-fluorenylene}-alternating-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}], poly[dibenzothiophene-4,4'-dimethoxy-2,2'-divinyl], poly(p-phenylene vinylene) and poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylethynylene].

3. The semiconductor polymer dot according to claim 1 or 2, characterized in that The mass of the amphiphilic organic polymer is 15% to 20% of the mass of the fluorescent polymer.

4. The semiconductor polymer dot according to claim 2, wherein The fluorescent polymer is poly[{9,9-dioctyl-2,7-diethenylidene-fluorenylene}-alternate-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}], and has the structural formula: , wherein the molecular weight is 20000~100000, and the average particle size of the semiconductor polymer dots is 7nm~9nm.

5. The semiconductor polymer dot according to claim 2, wherein: The fluorescent polymer is poly[dibenzothiophene-4,4'-dimethoxy-2,2'-divinyl], and the structural formula is , wherein the molecular weight is 20000~100000, and the average particle size of the semiconductor polymer dots is 8nm~10.5nm.

6. The semiconductor polymer dot according to claim 2, wherein: The fluorescent polymer is the poly[{9,9-dioctyl-2,7-diethenylidene-fluorenylene}-alternating-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}] and the poly[(fluoroborane dipyrrole)-co-(9,9-dioctylfluorenyl-2,7-diyl)] in a mass ratio of (1-5):

1. The structural formula of the poly[(fluoroborane dipyrrole)-co-(9,9-dioctylfluorenyl-2,7-diyl)] is , wherein the molecular weight is 5000~50000, and the average particle size of the semiconductor polymer dots is 8nm~10nm.

7. The semiconductor polymer dot according to any one of claims 1, 2, and 4 to 6, wherein: The amphiphilic organic polymer includes one or both of polystyrene-polyethylene glycol-carboxylic acid and poly(styrene-co-maleic anhydride).

8. A method for preparing a semiconductor polymer dot according to any one of claims 1 to 7, characterized in that: The following steps are involved: Providing a mixed raw material, the mixed raw material comprising the fluorescent polymer and the amphiphilic organic polymer; The mixed raw materials are co-precipitated to coat the surface of the fluorescent polymer with the amphiphilic organic polymer to prepare semiconductor polymer dots with an average particle size of 1 nm to 10.5 nm; Wherein, the fluorescent polymer includes a semiconductor polymer having a trans-olefin structure and poly[(fluoroborane dipyrrole)-co-(9,9-dioctylfluorenyl-2,7-diyl)] or the semiconductor polymer having a trans-olefin structure.

9. A fluorescent probe, characterized in that Comprising the semiconductor polymer dots as described in any one of claims 1 to 7.

10. The fluorescent probe according to claim 9, characterized in that Used for high-order super-resolution imaging.

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