Synthesis method of rhodamine polymer super-resolution nanoprobe

Abstract

The invention discloses a synthesis method of a rhodamine polymer super-resolution nanoprobe, and belongs to the technical field of fluorescent probes. The synthesis method of the rhodamine polymer super-resolution nanoprobe comprises the following steps: synthesis of a compound 2-(4-vinyl-phenoxy)-ethanol, synthesis of a multi-component copolymer, synthesis of a rhodamine-based polymer fluorescent dye and preparation of polymer nanoparticles. The prepared rhodamine polymer super-resolution nanoprobe is high in fluorescence quantum efficiency, high in light stability, capable of effectively avoiding aggregation-induced emission quenching, small in size and suitable for application and popularization, and images with higher resolution can be observed when the rhodamine polymer super-resolution nanoprobe is used for super-resolution imaging.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a method for synthesizing a rhodamine polymer super-resolution nanoprobe. Background Art

[0002] As a non-destructive labeling technology, fluorescent labeling technology provides important technical support for cell visualization. Through targeted fluorescent labeling technology, proteins, nucleic acids or organelles in cells are fluorescently labeled, providing a powerful tool for the relevant properties and functions of single cells. With the improvement of science and technology, people can break through the resolution limitations of traditional optical microscopes and observe and study cells, subcellular structures and molecular dynamic behaviors with higher resolution. However, super-resolution imaging requires probes to have high fluorescence brightness, excellent photostability and other conditions. Therefore, people have a considerable interest in developing brighter and more photostable fluorescent probes. In the prior art, a Chinese invention patent with publication number CN117447699A discloses a method for preparing and using a nano-dendritic polymer fluorescent probe, wherein Arg is grafted onto the surface of a polyamidoamine (PAMAM) dendritic macromolecule to obtain a PAMAM-Arg polymer (PR), and then 1,6,7,12-tetrachloro-3,4,9,10-perylenetetracarboxylic dianhydride (tetrachloroperylene anhydride) (TCPBA (TA)) is encapsulated in the hydrophobic inner cavity of PAMAM to obtain nanoparticles TA@PR, which are then coupled to double-stranded DNA (dsDNA) with smaller nanoparticles (112 nm) to synthesize TA@PR-dsDNA. The Chinese invention patent with publication number CN111592630A discloses a polymer fluorescent nanoprobe for liver-targeted visualized ratiometric detection of hypochlorous acid and its preparation and application. The fluorescent sensor is a new type of ratiometric fluorescent nanoprobe prepared from 1-propynyl-2-(((dodecylthio)carbonylthio)thio)-2-methylpropionate, styrene (St), polyethylene glycol methyl ether (PEGMA), 1-pyrenecarboxaldehyde and 1-(2-hydroxyethyl)-2,3,3-trimethyl-3H-indole-1-ammonium bromide, acryloyl chloride, tetraacetyl-α-D-bromogalactose, and sodium azide as raw materials. Summary of the invention

[0003] The purpose of the present invention is to provide a method for synthesizing a rhodamine polymer super-resolution nanoprobe.

[0004] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: A method for synthesizing a rhodamine polymer super-resolution nanoprobe comprises: S1, preparing a multi-polymer copolymer by reacting an enol reagent with a comonomer, wherein the enol reagent includes 2-(4-vinyl-phenoxy)-ethanol, the comonomer includes at least styrene, and the amount ratio of 2-(4-vinyl-phenoxy)-ethanol to styrene is 0.1-0.5 g: 1-3 mL; S2, a multi-polymer copolymer is reacted with a rhodamine-based fluorescent dye to prepare a rhodamine-based polymer fluorescent dye, wherein the rhodamine-based fluorescent dye includes at least one of fluorescein, rhodamine B and 6-HMSiR-COOH; S3, a rhodamine-based polymer fluorescent dye and a functional agent are prepared in a solvent to obtain polymer nanoparticles, namely, a rhodamine polymer super-resolution nanoprobe, and the functional agent includes PSMA.

[0005] The rhodamine-based fluorescent dye selected by the present invention has excellent properties such as high fluorescence quantum yield, large molar extinction coefficient, good photostability, good biocompatibility, etc., and can be well applied to super-resolution imaging. At the same time, under the initiation of AIBN, the comonomer styrene and the enol reagent form a polymer chain through polymerization reaction, and finally combine with the rhodamine-based fluorescent dye through a dehydration condensation reaction, that is, the fluorescent group is covalently modified on the side chain of the polymer chain, thereby introducing steric hindrance between molecules, preventing approach and aggregation, reducing the occurrence of π-π stacking and aggregation-induced quenching between molecules, and the rhodamine polymer super-resolution nanoprobe prepared by the nanoprecipitation method often has good dispersibility and long-term stability.

[0006] Preferably, the comonomer also includes vinyl phenyl acetate and 2-vinyl nicotinate methyl ester. The low polarity phenyl ester and methyl ester groups in vinyl phenyl acetate and 2-vinyl nicotinate are introduced to undergo free radical polymerization with styrene to form polymer chains with low polarity, which are then easily formed into uniform and stable agglomerated particles in the solution, thereby improving the fluorescence intensity and photostability of the rhodamine polymer super-resolution nanoprobe.

[0007] Preferably, the ratio of vinyl phenyl acetate to styrene is 0.05-0.3 g: 1-3 mL, and the ratio of 2-vinyl nicotinate to styrene is 0.05-0.3 g: 1-3 mL.

[0008] Preferably, in the preparation of the multi-polymer copolymer, toluene is used as the solvent and AIBN is used as the initiator.

[0009] Preferably, in the preparation of the rhodamine-based polymer fluorescent dye, DCM is used as a solvent, and EDCl and DMAP are used as reaction aids.

[0010] Preferably, tetrahydrofuran is used as a solvent in the preparation of the polymer nanoparticles.

[0011] Preferably, the mass ratio of the rhodamine-based polymer fluorescent dye to PSMA is 4-10:1-4.

[0012] Preferably, nanofiltration membrane is also used for filtration during the preparation of polymer nanoparticles.

[0013] The invention discloses a rhodamine polymer super-resolution nanoprobe prepared by the synthesis method.

[0014] The invention discloses the use of the above-mentioned rhodamine polymer super-resolution nanoprobe in a targeted fluorescent labeling reagent and / or a cell labeling reagent.

[0015] The invention discloses a method for synthesizing a rhodamine polymer super-resolution nanoprobe, comprising the synthesis of a compound 2-(4-vinyl-phenoxy)-ethanol, the synthesis of a multi-polymer copolymer, the synthesis of a rhodamine-based polymer fluorescent dye and the preparation of polymer nanoparticles, and specifically comprising the following steps: 1) Synthesis of compound 2-(4-vinyl-phenoxy)-ethanol: Ethylene glycol, 4-vinylbenzyl chloride, NaOH and H2O were mixed, stirred in a constant temperature oil bath at 50-80°C for 18-36 h, cooled, washed with an appropriate amount of DCM, dried over anhydrous Na2SO4, filtered, the organic solvent was removed in vacuo, and purified by column chromatography to obtain an oily substance, namely 2-(4-vinyl-phenoxy)-ethanol.

[0016] 2) Synthesis of multi-component copolymer: dissolve the enol reagent and the comonomer in toluene, add AIBN, and then heat at 50-90 °C under nitrogen for 12-36 h. After cooling, add hexane to precipitate, filter, dissolve the precipitate in DCM, and then stir and precipitate in MeOH for 1-3 times, and vacuum dry at 60-90 °C for 12-36 h to obtain the multi-component copolymer.

[0017] 3) Synthesis of rhodamine-based polymer fluorescent dye: Dissolve the multi-polymer copolymer, EDCl and DMAP in DCM, add rhodamine-based fluorescent dye dropwise at 0-5°C, react at room temperature for 12-48 h, extract with water, wash with brine, dry with Na2SO4, remove the solvent under reduced pressure, add dropwise to MeOH, filter, and vacuum dry at 60-90°C for 12-48 h to obtain the rhodamine-based polymer fluorescent dye.

[0018] 4) Preparation of polymer nanoparticles: The rhodamine-based polymer fluorescent dye and the functional reagent were dissolved in tetrahydrofuran, injected into water in an ultrasonic water bath and ultrasonicated for 1-5 min. Nitrogen was blown at 50-80 °C for 1-2 h, and the polymer nanoparticles were filtered with a 220 nm filter membrane while hot, i.e., the rhodamine polymer super-resolution nanoprobe.

[0019] Preferably, in the synthesis of 2-(4-vinyl-phenoxy)-ethanol, the ratio of ethylene glycol to H2O is 10-15 g:0.1-0.3 mL, the ratio of 4-vinylbenzyl chloride to H2O is 1 g:0.1-0.3 mL, and the ratio of NaOH to H2O is 0.1-0.4 g:0.1-0.3 mL.

[0020] Preferably, in the synthesis of 2-(4-vinyl-phenoxy)-ethanol, the volume ratio of PE and EA in the mobile phase in column chromatography purification is 5-10:1-5.

[0021] Preferably, in the synthesis of the multi-polymer, the enol reagent comprises 2-(4-vinyl-phenoxy)-ethanol.

[0022] Preferably, in the synthesis of the multi-component copolymer, the comonomer includes at least styrene.

[0023] Preferably, in the synthesis of the multi-polymer copolymer, the dosage ratio of 2-(4-vinyl-phenoxy)-ethanol and toluene is 0.1-0.5 g:5-10 mL, the volume ratio of styrene and toluene is 1-3:5-10, the dosage ratio of AIBN and toluene is 5-15 mg:5-10 mL, the volume ratio of hexane and toluene is 100-200:5-10, and the volume ratio of DCM and toluene is 1-2:5-10.

[0024] The synthesis reaction formula of the multi-polymer copolymer is shown in formula (1).

[0025]

[0026] Preferably, in the synthesis of rhodamine-based polymer fluorescent dye, the ratio of the multipolymer to DCM is 150-300 mg: 20-50 mL, the ratio of EDCl to DCM is 45-60 mg: 20-50 mL, and the ratio of DMAP to DCM is 1-5 mg: 20-50 mL.

[0027] Preferably, in the synthesis of the rhodamine-based polymer fluorescent dye, the rhodamine-based fluorescent dye is one of fluorescein, rhodamine B and 6-HMSiR-COOH, and the amount ratio of the rhodamine-based fluorescent dye to DCM is 1-100 mg: 20-50 mL. The rhodamine-based polymer fluorescent dye formed with fluorescein as the rhodamine-based fluorescent dye is named P1, and the reaction formula is shown in formula (2); the rhodamine-based polymer fluorescent dye formed with rhodamine B as the rhodamine-based fluorescent dye is named P2, and the reaction formula is shown in formula (3); the rhodamine-based polymer fluorescent dye formed with 6-HMSiR-COOH as the rhodamine-based fluorescent dye is named P3, and the reaction formula is shown in formula (4).

[0028]

[0029]

[0030]

[0031] Preferably, in the synthesis of the rhodamine-based polymer fluorescent dye, the saline solution is saturated saline solution.

[0032] Preferably, in the preparation of polymer nanoparticles, the functional reagent includes PSMA, the rhodamine-based polymer fluorescent dye and tetrahydrofuran are used in a ratio of 4-10 mg: 50-100 mL, the functional reagent and tetrahydrofuran are used in a ratio of 1-4 mg: 50-100 mL, and the volume ratio of water and tetrahydrofuran is 250-500: 50-100.

[0033] More preferably, in the synthesis of the multi-polymer copolymer, the comonomers further include vinyl phenyl acetate and 2-vinyl nicotinate methyl ester, the amount ratio of vinyl phenyl acetate to styrene is 0.05-0.3 g: 1-3 mL, and the amount ratio of 2-vinyl nicotinate to styrene is 0.05-0.3 g: 1-3 mL.

[0034] More preferably, in the synthesis of the multi-copolymer, the comonomer also includes acryloxypropyl trimethoxysilane, and the amount ratio of acryloxypropyl trimethoxysilane to styrene is 0.1-0.6 g: 1-3 mL. Acryloxypropyl trimethoxysilane can undergo free radical random polymerization with other comonomers and introduce new groups, which work together with the phenyl ester and methyl ester groups introduced by vinyl phenyl acetate and 2-vinyl nicotinate methyl ester, thereby improving the fluorescence intensity and photostability of the nanoprobe.

[0035] The present invention adds vinyl phenyl acetate and 2-vinyl nicotinate methyl ester in the synthesis of multi-polymer copolymers to generate polymer chains containing multiple functional groups, and finally synthesizes rhodamine polymer super-resolution nanoprobes, which has the following beneficial effects: high fluorescence quantum efficiency, strong photostability, can effectively avoid aggregation-induced luminescence quenching, and is small in size and suitable for application and promotion. Therefore, the purpose of the present invention is to provide a method for synthesizing rhodamine polymer super-resolution nanoprobes. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of 2-[(4-vinylphenyl)methoxy]ethanol.

[0037] Figure 2 This is the carbon NMR spectrum of 2-[(4-vinylphenyl)methoxy]ethanol.

[0038] Figure 3 This is a graph showing the DLS characterization test results of the rhodamine polymer super-resolution nanoprobe of Examples 1-3.

[0039] Figure 4 The graphs show the test results of ultraviolet absorption spectrum and fluorescence spectrum of rhodamine polymer super-resolution nanoprobe of Examples 1-3.

[0040] Figure 5 This is the confocal imaging result of cell microtubules labeled by the rhodamine polymer super-resolution nanoprobe in Example 1-3.

[0041] Figure 6 This is the STED imaging result of cell microtubules labeled with the rhodamine polymer super-resolution nanoprobe in Example 1-3. DETAILED DESCRIPTION

[0042] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] The following first describes the concepts involved in the present application in conjunction with the accompanying drawings. It should be noted that the following description of each concept is only to make the content of the present application easier to understand, and does not limit the scope of protection of the present application; at the same time, the embodiments and features in the embodiments of the present application can be combined with each other in the absence of conflict. The present application will be described in detail with reference to the accompanying drawings and in conjunction with the embodiments.

[0044] Example 1 A method for synthesizing a rhodamine polymer super-resolution nanoprobe comprises the following steps: synthesizing a compound 2-(4-vinyl-phenoxy)-ethanol, synthesizing a multi-polymer copolymer, synthesizing a rhodamine-based polymer fluorescent dye, and preparing polymer nanoparticles. 1) Synthesis of compound 2-(4-vinyl-phenoxy)-ethanol: Ethylene glycol, 4-vinylbenzyl chloride, NaOH and H2O were mixed, stirred in a constant temperature oil bath at 70 ℃ for 24 h, cooled, washed with an appropriate amount of DCM, dried over anhydrous Na2SO4, filtered, and the organic solvent was removed in vacuo. The oily substance, i.e., 2-(4-vinyl-phenoxy)-ethanol, was purified by column chromatography. The ratio of ethylene glycol to H2O was 11.03 g:0.21 mL, the ratio of 4-vinylbenzyl chloride to H2O was 1 g:0.21 mL, and the ratio of NaOH to H2O was 0.27 g:0.21 mL. The volume ratio of mobile phase PE to EA in column chromatography purification was 7:3.

[0045] 2) Synthesis of multi-polymer copolymer: The enol reagent and the comonomer were dissolved in toluene, AIBN was added, and then heated at 70 °C under nitrogen for 24 h, cooled to room temperature, hexane was added for precipitation, filtered, the precipitate was dissolved in DCM, and then stirred and precipitated in MeOH twice, and vacuum dried at 80 °C for 24 h to obtain a multi-polymer copolymer. The enol reagent was 2-(4-vinyl-phenoxy)-ethanol, the comonomer was styrene, the amount ratio of 2-(4-vinyl-phenoxy)-ethanol and toluene was 0.172 g:5 mL, the volume ratio of styrene and toluene was 1:5, the amount ratio of AIBN initiator and toluene was 6 mg:5 mL, the volume ratio of hexane and toluene was 100:5, and the volume ratio of DCM and toluene was 1:5.

[0046] 3) Synthesis of rhodamine-based polymer fluorescent dye: The multi-polymer copolymer, EDCl and DMAP were dissolved in DCM, and the rhodamine-based fluorescent dye was added dropwise at 0 ℃, reacted at room temperature for 24 h, extracted with water, washed with brine, dried with Na2SO4, and the solvent was removed under reduced pressure. The mixture was added dropwise to MeOH, filtered, and dried under vacuum at 80 ℃ for 24 h to obtain the rhodamine-based polymer fluorescent dye. The amount ratio of the multi-polymer copolymer to DCM was 200 mg:30 mL, the amount ratio of EDCl to DCM was 48 mg:30 mL, and the amount ratio of DMAP to DCM was 4 mg:30 mL. The rhodamine-based fluorescent dye added dropwise was fluorescein, and the amount ratio of the rhodamine-based fluorescent dye to DCM was 100 mg:30 mL. In the extraction, the volume ratio of water to DCM was 1:5. The brine was saturated saline.

[0047] 4) Preparation of polymer nanoparticles: The rhodamine-based polymer fluorescent dye and the functional reagent were dissolved in tetrahydrofuran, and quickly injected into water in an ultrasonic water bath, and ultrasonicated for 2 min. Under the condition of heating at 65 °C, the ultrasonicated solution was blown with nitrogen for 1 h, and then filtered with a 220 nm filter membrane while hot to obtain polymer nanoparticles, i.e., rhodamine polymer super-resolution nanoprobes. The functional reagent was PSMA, the dosage ratio of rhodamine-based polymer fluorescent dye and tetrahydrofuran was 4 mg:50 mL, the dosage ratio of functional reagent and tetrahydrofuran was 1 mg:50 mL, and the volume ratio of water and tetrahydrofuran was 250:50.

[0048] Example 2 A method for synthesizing a rhodamine polymer super-resolution nanoprobe, the difference between this embodiment and embodiment 1 is that in the synthesis of the rhodamine-based polymer fluorescent dye, the rhodamine-based fluorescent dye added dropwise is rhodamine B, and the amount ratio of the rhodamine-based fluorescent dye to DCM is 100 mg: 30 mL. Others are the same as embodiment 1.

[0049] Example 3 A method for synthesizing a rhodamine polymer super-resolution nanoprobe, the difference between this embodiment and embodiment 1 is that in the synthesis of the rhodamine-based polymer fluorescent dye, the rhodamine-based fluorescent dye added dropwise is 6-HMSiR-COOH, and the amount ratio of the rhodamine-based fluorescent dye to DCM is 2 mg:30 mL. Others are the same as embodiment 1.

[0050] Example 4 A method for synthesizing a rhodamine polymer super-resolution nanoprobe. The difference between this embodiment and embodiment 1 is that in the synthesis of the multi-polymer copolymer, the comonomers are styrene, vinyl phenyl acetate and 2-vinyl nicotinate methyl ester, wherein the amount ratio of vinyl phenyl acetate to styrene is 0.172 g:1 mL, the amount ratio of 2-vinyl nicotinate methyl ester to styrene is 0.171 g:1 mL, and the rest is the same as embodiment 1.

[0051] Example 5 A method for synthesizing a rhodamine polymer super-resolution nanoprobe, the difference between this embodiment and embodiment 4 is that in the synthesis of the rhodamine-based polymer fluorescent dye, the rhodamine-based fluorescent dye added dropwise is rhodamine B, and the amount ratio of the rhodamine-based fluorescent dye to DCM is 100 mg: 30 mL. Others are the same as embodiment 4.

[0052] Example 6 A method for synthesizing a rhodamine polymer super-resolution nanoprobe, the difference between this embodiment and embodiment 4 is that in the synthesis of the rhodamine-based polymer fluorescent dye, the rhodamine-based fluorescent dye added dropwise is 6-HMSiR-COOH, and the amount ratio of the rhodamine-based fluorescent dye to DCM is 2 mg:30 mL. Others are the same as embodiment 4.

[0053] Example 7 A method for synthesizing a rhodamine polymer super-resolution nanoprobe. The difference between this embodiment and embodiment 4 is that in the synthesis of the multi-polymer copolymer, the amount ratio of vinyl acetate phenyl ester to styrene in the comonomer is 0.085 g:1 mL, the amount ratio of 2-vinylnicotinate methyl ester to styrene is 0.085 g:1 mL, and the rest is the same as embodiment 4.

[0054] Example 8 A method for synthesizing a rhodamine polymer super-resolution nanoprobe, the difference between this embodiment and embodiment 7 is that in the synthesis of the rhodamine-based polymer fluorescent dye, the rhodamine-based fluorescent dye added dropwise is rhodamine B, and the amount ratio of the rhodamine-based fluorescent dye to DCM is 100 mg: 30 mL. Others are the same as embodiment 7.

[0055] Example 9 A method for synthesizing a rhodamine polymer super-resolution nanoprobe, the difference between this embodiment and embodiment 7 is that in the synthesis of the rhodamine-based polymer fluorescent dye, the rhodamine-based fluorescent dye added dropwise is 6-HMSiR-COOH, and the amount ratio of the rhodamine-based fluorescent dye to DCM is 2 mg:30 mL. Others are the same as embodiment 7.

[0056] Example 10 A method for synthesizing a rhodamine polymer super-resolution nanoprobe. The difference between this embodiment and embodiment 7 is that in the synthesis of the multi-polymer copolymer, the comonomers are styrene, vinyl phenyl acetate, 2-vinyl nicotinate methyl ester and acryloxypropyl trimethoxy silane, wherein the usage ratio of acryloxypropyl trimethoxy silane to styrene is 0.245 g:1 mL, and the rest is the same as embodiment 7.

[0057] Embodiment 11 A method for synthesizing a rhodamine polymer super-resolution nanoprobe, the difference between this embodiment and embodiment 10 is that in the synthesis of the rhodamine-based polymer fluorescent dye, the rhodamine-based fluorescent dye added dropwise is rhodamine B, and the amount ratio of the rhodamine-based fluorescent dye to DCM is 100 mg: 30 mL. Others are the same as embodiment 10.

[0058] Example 12 A method for synthesizing a rhodamine polymer super-resolution nanoprobe, the difference between this embodiment and embodiment 10 is that in the synthesis of the rhodamine-based polymer fluorescent dye, the rhodamine-based fluorescent dye added dropwise is 6-HMSiR-COOH, and the amount ratio of the rhodamine-based fluorescent dye to DCM is 2 mg:30 mL. Others are the same as embodiment 10.

[0059] Embodiment 13 A method for synthesizing a rhodamine polymer super-resolution nanoprobe. The difference between this embodiment and embodiment 10 is that in the synthesis of the multi-polymer copolymer, the usage ratio of acryloxypropyltrimethoxysilane and styrene in the copolymer monomer is 0.122 g:1 mL, and the rest is the same as embodiment 10.

[0060] Embodiment 14 A method for synthesizing a rhodamine polymer super-resolution nanoprobe, the difference between this embodiment and embodiment 13 is that in the synthesis of the rhodamine-based polymer fluorescent dye, the rhodamine-based fluorescent dye added dropwise is rhodamine B, and the amount ratio of the rhodamine-based fluorescent dye to DCM is 100 mg: 30 mL. Others are the same as embodiment 13.

[0061] Embodiment 15 A method for synthesizing a rhodamine polymer super-resolution nanoprobe, the difference between this embodiment and embodiment 13 is that in the synthesis of the rhodamine-based polymer fluorescent dye, the rhodamine-based fluorescent dye added dropwise is 6-HMSiR-COOH, and the amount ratio of the rhodamine-based fluorescent dye to DCM is 2 mg:30 mL. Others are the same as embodiment 13.

[0062] Comparative Example 1 The difference between this comparative example and Example 4 is that in the synthesis of the multi-component copolymer, the comonomer does not contain vinyl phenyl acetate, and the rest is the same as Example 4.

[0063] Comparative Example 2 The difference between this comparative example and Example 4 is that in the synthesis of the multi-component copolymer, the comonomer does not contain 2-vinylnicotinate methyl ester, and the rest is the same as Example 4.

[0064] Comparative Example 3 The difference between this comparative example and Example 10 is that in the synthesis of the multi-component copolymer, the comonomer does not contain vinyl phenyl acetate and 2-vinyl nicotinate methyl ester, and the rest is the same as Example 10.

[0065] H NMR spectroscopy test The results of the H NMR spectrum test of 2-[(4-vinylphenyl)methoxy]ethanol are as follows Figure 1 shown.

[0066] 1 H NMR (400 MHz, CDCl3): δ 7.45 – 7.25 (m, 4H), 6.71 (dd, J = 17.6,10.9 Hz, 1H), 5.74 (d, J = 17.6 Hz, 1H), 5.24 (d, J = 10.9 Hz, 1H), 4.54 (s, 2H), 3.80 – 3.68 (m, 2H), 3.63 – 3.52 (m, 2H), 2.34 (bs, 1H). Carbon NMR Spectroscopy Test The results of the carbon NMR spectrum test of 2-[(4-vinylphenyl)methoxy]ethanol are as follows Figure 2 shown.

[0067] 13 C NMR (101 MHz, CDCl3): δ 137.53, 137.19, 136.48, 128.06, 126.33, 113.99, 73.02, 71.40, 61.88. DLS Characterization Test The rhodamine polymer super-resolution nanoprobes of Examples 1-3 were subjected to DLS characterization.

[0068] DLS characterization test results are as follows Figure 3 As shown, Figure 3 Figure a is the particle size distribution diagram. Figure 3 Figure b is a potential diagram. It can be seen that the particle size of the prepared rhodamine polymer super-resolution nanoprobe is basically distributed around 30 nm, which is relatively small; it can also be seen that the potential peak of the rhodamine polymer super-resolution nanoprobe is around -30 mV, which has good stability.

[0069] UV absorption spectrum and fluorescence spectrum test The rhodamine polymer super-resolution nanoprobes of Examples 1-3 were subjected to ultraviolet absorption spectroscopy and fluorescence spectroscopy tests, wherein the excitation wavelength when measuring the fluorescence spectrum of the rhodamine polymer super-resolution nanoprobe of Example 1 was 484 nm, the excitation wavelength when measuring the fluorescence spectrum of the rhodamine polymer super-resolution nanoprobe of Example 2 was 569 nm, and the excitation wavelength when measuring the fluorescence spectrum of the rhodamine polymer super-resolution nanoprobe of Example 3 was 658 nm.

[0070] The UV absorption spectrum and fluorescence spectrum test results are as follows Figure 4 As shown, Figure 4 Figure a in the figure is a graph showing the test results of ultraviolet absorption spectrum and fluorescence spectrum of rhodamine polymer super-resolution nanoprobe in Example 1; Figure 4 Figure b is a graph showing the test results of ultraviolet absorption spectrum and fluorescence spectrum of rhodamine polymer super-resolution nanoprobe in Example 2; Figure 4 Figure c in the figure is the test result of ultraviolet absorption spectrum and fluorescence spectrum of rhodamine polymer super-resolution nanoprobe in Example 3. It can be seen that the prepared rhodamine polymer super-resolution nanoprobe has a regular absorption peak in the ultraviolet absorption spectrum, and can efficiently emit light of a specific wavelength after being excited in the fluorescence spectrum, showing good stability and selectivity, and has excellent optical properties.

[0071] Confocal imaging and STED imaging application tests The rhodamine polymer super-resolution nanoprobes of Examples 1-3 were used to label cell microtubules and imaged on confocal and STED. Specifically: (1) African green monkey kidney cells BS-C-1 cells (commercially purchased) were selected and seeded in confocal microplates, with approximately 40,000 cells per well. The cells were cultured at 37°C and 5% CO2 for 24 h. The culture medium of the BS-C-1 cells in the culture dish was aspirated and washed three times with PBS buffer solution. (2) Add 0.5% Triton X-100 at a volume of 200 μL / well and shake for 30 s; (3) Quickly aspirate Triton X-100, add fixative at a volume of 200 μL / well, shake in the dark for 15 min, then aspirate the fixative and rinse three times with DPBS buffer solution; the fixative is 3.2% paraformaldehyde by volume; (4) Add sodium borohydride solution at a volume of 200 μL / well, shake in the dark for 10 min, and rinse three times with DPBS buffer solution; the concentration of sodium borohydride solution is 1 mg / mL.

[0072] (5) Add 200 μL of blocking solution, shake for 45 min, then aspirate and wash with DPBS buffer solution; the blocking solution is PBS solution, which contains BSA and Triton X-100, with a volume ratio of BSA of 5% and a volume ratio of Triton X-100 of 0.5%; (6) Dilute the primary antibody Anti-beta Tubulin (Abcam, cat. no. Ab179513) solution in blocking solution at a volume ratio of 1:200, add 150 μL of the primary antibody dilution solution to the culture dish and gently shake on a shaker for 45 min, and wash three times with DPBS buffer solution; (7) Dilute the secondary antibody Goat Polyclonal Antibody to Rabbit IgG (H&L) - Biotin (Yuantai Biotechnology, cat. no. P50075) solution in the blocking solution at a volume ratio of 1:200. Add 150 μL of the secondary antibody dilution solution to the culture dish and shake gently on a shaker for 45 min. Wash three times with DPBS buffer solution. (8) 4 mL of rhodamine polymer super-resolution nanoprobe, 240 μL of streptavidin, 80 μL of polyethylene glycol solution and 80 μL of 4-hydroxyethylpiperazineethanesulfonic acid solution were mixed and stirred evenly, and then 60 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added. After thorough mixing, the mixture was reacted for 4 h. Free streptavidin was removed by ultrafiltration at 100K to obtain modified rhodamine polymer super-resolution nanoprobe particles. The concentration of streptavidin was 1 mg / mL. The volume fraction of the polyethylene glycol solution was 5%, and the solvent was H2O. The concentration of 4-hydroxyethylpiperazineethanesulfonic acid was 1 mol / L, and the pH was 7.3. The concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was 5 mg / mL.

[0073] (9) Pour the modified rhodamine polymer super-resolution nanoprobe particles into the culture dish in step (7) and shake gently for 30 min; wash thoroughly with DPBS buffer solution and store in a refrigerator at 4 °C.

[0074] (10) Confocal imaging: CSU-W1-SoRa was used. Since the laser used could not be adjusted over the entire wavelength band, 488, 561, and 640 nm were selected as the excitation wavelengths for confocal imaging of the cells. The excitation wavelength when the rhodamine polymer super-resolution nanoprobe of Example 1 was used was 488 nm, the excitation wavelength when the rhodamine polymer super-resolution nanoprobe of Example 2 was used was 561 nm, and the excitation wavelength when the rhodamine polymer super-resolution nanoprobe of Example 3 was used was 640 nm.

[0075] (11) Super-resolution imaging: STED was used for super-resolution imaging. When the rhodamine polymer super-resolution nanoprobe of Example 1 was used, the excitation light was 484 nm and the depletion light was 660 nm. When the rhodamine polymer super-resolution nanoprobe of Example 2 was used, the excitation light was 569 nm and the depletion light was 660 nm. After the operation, two images with different characteristics were obtained. One was a CLSM image generated only after the excitation process was completed, and the other was a STED image obtained after the excitation process and further depletion light processing. The full width at half maximum (FWHM) is the result obtained by Gaussian fitting.

[0076] Confocal imaging test results are as follows Figure 5 As shown, Figure 5 Figure a in the figure is the confocal imaging result of the rhodamine polymer super-resolution nanoprobe labeling cell microtubules in Example 1, Figure 5 Figure b is the confocal imaging result of the rhodamine polymer super-resolution nanoprobe labeling cell microtubules in Example 2. Figure 5 Figure c in the figure is the confocal imaging result of the rhodamine polymer super-resolution nanoprobe labeling cell microtubules in Example 3. The STED imaging test results are shown in Figure 3. Figure 6 As shown, Figure 6 Figure a in the figure is the CLSM image and STED image of the cell microtubules labeled by the rhodamine polymer super-resolution nanoprobe in Example 1. Figure 6 Figure b shows the fluorescence signal intensity value of cell microtubules marked by the rhodamine polymer super-resolution nanoprobe in Example 1. Figure 6 Figure c in the figure is the CLSM image and STED image of the cell microtubules labeled by the rhodamine polymer super-resolution nanoprobe in Example 2. Figure 6Figure d in the figure is the fluorescence signal intensity value of the cell microtubules marked by the rhodamine polymer super-resolution nanoprobe in Example 2. It can be seen that the rhodamine polymer super-resolution nanoprobes in Examples 1-3 have a relatively continuous imaging effect on cell microtubules. It can also be seen that the STED image resolution after imaging with the rhodamine polymer super-resolution nanoprobes in Examples 1 and 2 is significantly improved compared to the CLSM image resolution. The rhodamine polymer super-resolution nanoprobe in Example 1 is improved from the original 316.56 nm to 137.7 and 78.7 nm, and the resolution of STED after imaging with the rhodamine polymer super-resolution nanoprobe in Example 2 is improved from the original 264 nm to 155 nm compared to confocal. However, since the rhodamine polymer super-resolution nanoprobe in Example 3 uses a light scintillation dye, the fluorescence brightness after labeling is relatively low, and the STED imaging effect is not good.

[0077] Fluorescence intensity test A rhodamine polymer super-resolution nanoprobe with rhodamine-based fluorescent dye as fluorescein was selected, and the fluorescence spectrum of the nanoprobe was measured using a fluorescence spectrophotometer. The normalized intensity was calculated as follows: N = E / E0 × 100%, where E is the fluorescence intensity of the nanoprobe, and E0 is the fluorescence intensity of the nanoprobe with the strongest fluorescence intensity. The test results are shown in Table 1.

[0078] Table 1 Fluorescence intensity test results

[0079] It can be seen from Table 1 that the fluorescence intensity of the rhodamine polymer super-resolution nanoprobe of the embodiment is relatively high, which indicates that the polymer chain formed by the polymerization reaction of two monomers, styrene and 2-(4-vinyl-phenoxy)-ethanol, is combined with the rhodamine-based dye, which can introduce steric hindrance, prevent the molecules from approaching and aggregating, reduce the π-π stacking between molecules, and improve the fluorescence intensity; it can also be seen that the fluorescence intensity of the rhodamine polymer super-resolution nanoprobe of Examples 4 and 7 is higher than that of Example 1, which indicates that the polymer chain formed by the introduction of vinyl acetate phenyl ester and 2-vinyl nicotinate methyl ester and other monomers contains phenyl ester and methyl ester with low polarity, and is easy to show uniform and stable agglomerated particles in the solution, thereby improving the fluorescence intensity; it can also be seen that the fluorescence intensity of the rhodamine polymer super-resolution nanoprobe of Example 4 is higher than that of Example 7, which indicates that the higher the amount of vinyl acetate phenyl ester and 2-vinyl nicotinate methyl ester used, the better the fluorescence intensity; it can also be seen that the fluorescence intensity of the rhodamine polymer super-resolution nanoprobe of Examples 10 and 13 is higher than that of Example 4, which indicates that the introduction of acryloyloxy The new group in propyltrimethoxysilane can react with other monomers to form a polymer chain, thereby improving the fluorescence intensity of the rhodamine polymer super-resolution nanoprobe; it can also be seen that the fluorescence intensity of the rhodamine polymer super-resolution nanoprobe in Example 10 is higher than that in Example 13, which indicates that adding an appropriate amount of acryloxypropyltrimethoxysilane can improve the fluorescence intensity of the rhodamine polymer super-resolution nanoprobe; it can also be seen that the fluorescence intensity of the rhodamine polymer super-resolution nanoprobe in Example 4 is higher than that in Comparative Examples 1 and 2, which indicates that the rhodamine polymer super-resolution nanoprobe introduces a polymer chain formed by vinyl acetate phenyl ester and 2-vinyl nicotinate methyl ester and other monomers, and the fluorescence intensity is higher than that of a single substance introduced; it can also be seen that the fluorescence intensity of the rhodamine polymer super-resolution nanoprobe in Example 10 is higher than that in Comparative Example 3, which indicates that the effect of introducing acryloxypropyltrimethoxysilane into the rhodamine polymer super-resolution nanoprobe on improving the fluorescence intensity is lower than the effect of introducing a composite addition of vinyl acetate phenyl ester, 2-vinyl nicotinate methyl ester and acryloxypropyltrimethoxysilane.

[0080] Fluorescence stability test The rhodamine polymer super-resolution nanoprobe in the embodiment of the rhodamine-based fluorescent dye was selected as fluorescein for testing. The rhodamine polymer super-resolution nanoprobe was continuously irradiated with 484 nm excitation light for 1 h, and the irradiated solution was taken to measure its fluorescence intensity, and the fluorescence stability was evaluated by the fluorescence intensity retention rate. The calculation formula of the fluorescence intensity retention rate is: R=E / E0×100%, where E is the fluorescence intensity after 1 h, and E0 is the original fluorescence intensity. The test results are shown in Table 2.

[0081] Table 2 Fluorescence stability test results

[0082] It can be seen from Table 1 that the fluorescence intensity retention rates of the rhodamine polymer super-resolution nanoprobes in the embodiments are all relatively high, which indicates that the polymer chain formed by the polymerization reaction of the two monomers of styrene and 2-(4-vinyl-phenoxy)-ethanol is combined with the rhodamine-based fluorescent dye, which can introduce steric hindrance, prevent the molecules from approaching and aggregating, reduce the occurrence of π-π stacking and aggregation-induced quenching between molecules, and improve the fluorescence intensity retention rate; it can also be seen that the fluorescence intensity retention rates of the rhodamine polymer super-resolution nanoprobes in Examples 4 and 7 are higher than those in Example 1, which indicates that the polymer chains formed by the introduction of vinyl phenyl acetate and 2-vinyl nicotinate methyl ester and other monomers contain phenyl esters and methyl esters with low polarity, which are easy to show uniform and stable agglomerated particles in the solution, so they show better photostability and improve the fluorescence intensity retention rate; it can also be seen that the fluorescence intensity retention rate of the rhodamine polymer super-resolution nanoprobe in Example 4 is higher than that in Example 7, which indicates that the higher the usage of vinyl phenyl acetate and 2-vinyl nicotinate methyl ester in the multi-component mixture, the better the fluorescence intensity retention rate; it can also be seen that the fluorescence intensity retention rates of the rhodamine polymer super-resolution nanoprobes in Examples 10 and 13 are higher than Example 4, which shows that the new group introduced into acryloxypropyltrimethoxysilane can work together with other monomers to form a polymer chain, which can improve the fluorescence intensity retention rate of the rhodamine polymer super-resolution nanoprobe; it can also be seen that the fluorescence intensity retention rate of the rhodamine polymer super-resolution nanoprobe in Example 10 is higher than that in Example 13, which shows that adding an appropriate amount of acryloxypropyltrimethoxysilane can improve the fluorescence intensity retention rate of the rhodamine polymer super-resolution nanoprobe; it can also be seen that the fluorescence intensity retention rate of the rhodamine polymer super-resolution nanoprobe in Example 4 is higher than that in Comparative Examples 1 and 2. Ratio 2, which shows that the fluorescence intensity retention rate of the rhodamine polymer super-resolution nanoprobe introduced by the polymer chain formed by vinyl phenyl acetate and 2-vinyl nicotinate methyl ester and other monomers is higher than that of the single introduction of one substance; it can also be seen that the fluorescence intensity retention rate of the rhodamine polymer super-resolution nanoprobe in Example 10 is higher than that in Comparative Example 3, which shows that the effect of introducing acryloxypropyltrimethoxysilane into the rhodamine polymer super-resolution nanoprobe on improving the fluorescence intensity retention rate is lower than the effect of introducing the composite addition of vinyl phenyl acetate, 2-vinyl nicotinate and acryloxypropyltrimethoxysilane.

[0083] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any form. Any technical personnel in this field may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0084] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and its core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the invention to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A method for synthesizing a rhodamine polymer super-resolution nanoprobe, comprising: S1, preparing a multi-polymer copolymer by reacting an enol reagent with a comonomer, wherein the enol reagent includes 2-(4-vinyl-phenoxy)-ethanol, the comonomer includes at least styrene, and the amount ratio of 2-(4-vinyl-phenoxy)-ethanol to styrene is 0.1-0.5 g: 1-3 mL; S2, a multi-polymer copolymer is reacted with a rhodamine-based fluorescent dye to prepare a rhodamine-based polymer fluorescent dye, wherein the rhodamine-based fluorescent dye includes at least one of fluorescein, rhodamine B and 6-HMSiR-COOH; S3, preparing polymer nanoparticles, namely, rhodamine polymer super-resolution nanoprobes, by combining rhodamine-based polymer fluorescent dye and functional reagent in a solvent, wherein the functional reagent includes PSMA.

2. The method for synthesizing a rhodamine polymer super-resolution nanoprobe according to claim 1, characterized in that: The comonomers also include vinyl phenyl acetate and 2-vinyl nicotinate methyl ester.

3. The method for synthesizing a rhodamine polymer super-resolution nanoprobe according to claim 2, characterized in that: The usage ratio of vinyl phenyl acetate to styrene is 0.05-0.3 g:1-3 mL, and the usage ratio of 2-vinyl nicotinate methyl ester to styrene is 0.05-0.3 g:1-3 mL.

4. The method for synthesizing a rhodamine polymer super-resolution nanoprobe according to claim 1, characterized in that: In the preparation of the multi-polymer, toluene is used as a solvent and AIBN is used as an initiator.

5. The method for synthesizing a rhodamine polymer super-resolution nanoprobe according to claim 1, characterized in that: In the preparation of the rhodamine-based polymer fluorescent dye, DCM is used as a solvent, and EDCl and DMAP are used as reaction aids.

6. The method for synthesizing a rhodamine polymer super-resolution nanoprobe according to claim 1, characterized in that: In the preparation of the polymer nanoparticles, tetrahydrofuran is used as a solvent.

7. The method for synthesizing a rhodamine polymer super-resolution nanoprobe according to claim 1, characterized in that: The mass ratio of the rhodamine-based polymer fluorescent dye to PSMA is 4-10:1-4.

8. The method for synthesizing a rhodamine polymer super-resolution nanoprobe according to claim 1, characterized in that: In the preparation of the polymer nanoparticles, a nanofiltration membrane is also used for filtration.

9. The rhodamine polymer super-resolution nanoprobe prepared by the synthesis method according to any one of claims 1 to 8.

10. Use of the rhodamine polymer super-resolution nanoprobe according to claim 9 in a targeted fluorescent labeling reagent and / or a cell labeling reagent.

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