Synthesis method of a rhodamine polymer super-resolution nanoprobe

Abstract

The present invention discloses a method for synthesizing a rhodamine polymer super-resolution nanoprobe, belonging to the technical field of fluorescent probes. The method for synthesizing the rhodamine polymer super-resolution nanoprobe includes: the synthesis of compound 2-(4-vinyl-phenoxy)-ethanol, the synthesis of a multi-component copolymer polymer, the synthesis of a rhodamine-based polymer fluorescent dye, and the preparation of polymer nanoparticles. The rhodamine polymer super-resolution nanoprobe prepared by the present invention has a relatively high fluorescence quantum efficiency, strong photostability, can effectively avoid aggregation-induced emission quenching, has a small size and is suitable for application and popularization, and can observe higher-resolution images when used for super-resolution imaging.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes, and particularly relates to a synthesis method of a rhodamine polymer super-resolution nanoprobe. Background Art

[0002] As a non-destructive labeling technology, the fluorescent labeling technology provides important technical support for cell visualization. By using the targeted fluorescent labeling technology to fluorescently label proteins, nucleic acids or organelles in cells, it provides a powerful tool for studying the related properties and functions of single cells. With the improvement of technology, people can break through the resolution limit 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 conditions such as high fluorescence brightness and excellent photostability. Therefore, there is a considerable interest in developing brighter and more photostable fluorescent probes. In the prior art, the Chinese invention patent with the publication number CN117447699A discloses a preparation method and application of a nano-dendritic polymer fluorescent probe. In this fluorescent probe, Arg is grafted onto the surface of polyamidoamine (PAMAM) dendrimers to obtain a PAMAM-Arg polymer (PR), and then 1,6,7,12-tetrachloro-3,4,9,10-perylene tetracarboxylic dianhydride (tetrachloroperylene dianhydride) (TCPBA(TA)) is encapsulated in the hydrophobic inner cavity of PAMAM to obtain nanoparticles TA@PR, and double-stranded DNA (dsDNA) is coupled with smaller nanoparticles (112 nm) to synthesize TA@PR-dsDNA. The Chinese invention patent with the publication number CN111592630A discloses a liver-targeted visual ratio detection polymer fluorescent nanoprobe for hypochlorous acid and its preparation and application. This fluorescent sensor is a novel ratio fluorescent nanoprobe prepared from 1-propynyl-2-(((dodecylthio)thioxomethyl)thio)-2-methylpropionate, styrene (St), methoxypolyethylene glycol (PEGMA), 1-pyrenecarboxaldehyde and 1-(2-hydroxyethyl)-2,3,3-trimethyl-3H-indolium-1-bromide, acryloyl chloride, tetraacetyl-α-D-galactosyl bromide, and sodium azide as raw materials. Summary of the Invention

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

[0004] The technical solution adopted by the present invention to achieve the above purpose is as follows:

[0005] A synthesis method of a rhodamine polymer super-resolution nanoprobe, comprising:

[0006] S1. The enol reagent reacts with the comonomer to prepare a multi-component copolymerized polymer. The enol reagent includes 2-(4-vinyl-phenoxy)-ethanol, and the comonomer includes at least styrene. The dosage ratio of 2-(4-vinyl-phenoxy)-ethanol to styrene is 0.1-0.5 g: 1-3 mL.

[0007] S2. The multi-component copolymerized polymer reacts with the rhodamine-based fluorescent dye to prepare a rhodamine-based polymer fluorescent dye. The rhodamine-based fluorescent dye includes at least one of fluorescein, rhodamine B, and 6-HMSiR-COOH.

[0008] S3. The rhodamine-based polymer fluorescent dye and the functional reagent are used in a solvent to prepare polymer nanoparticles, namely rhodamine polymer super-resolution nanoprobes. The functional reagent includes PSMA.

[0009] The selected rhodamine-based fluorescent dye in the present invention has excellent properties such as high fluorescence quantum yield, large molar extinction coefficient, good photostability, and good biocompatibility, and can be well applied to super-resolution imaging. Meanwhile, under the initiation of AIBN, the comonomer styrene and the enol reagent form a polymer chain through a polymerization reaction, and finally combine with the rhodamine-based fluorescent dye through a dehydration condensation reaction, that is, a fluorescent group is covalently modified on the side chain of the polymer chain, thereby introducing steric hindrance between molecules to prevent approaching and aggregation, reducing the occurrence of Π-Π stacking and aggregation-induced quenching between molecules, and the rhodamine polymer super-resolution nanoprobes prepared by the nanoprecipitation method often have good dispersibility and long-term stability.

[0010] Preferably, the comonomer further includes phenyl vinyl acetate and methyl 2-vinylnicotinate. The introduction of low-polarity phenyl ester and methyl ester groups in phenyl vinyl acetate and methyl 2-vinylnicotinate together with styrene undergoes a free radical polymerization reaction to form a polymer chain with lower polarity, and then uniform and stable aggregated particles are easily formed in the solution, improving the fluorescence intensity and photostability of the rhodamine polymer super-resolution nanoprobes.

[0011] Preferably, the dosage ratio of phenyl vinyl acetate to styrene is 0.05-0.3 g: 1-3 mL, and the dosage ratio of methyl 2-vinylnicotinate to styrene is 0.05-0.3 g: 1-3 mL.

[0012] Preferably, in the preparation of the multi-component copolymerized polymer, toluene is used as the solvent and AIBN is used as the initiator.

[0013] Preferably, in the preparation of the rhodamine-based polymer fluorescent dye, DCM is used as the solvent, and EDCl and DMAP are used as reaction aids.

[0014] Preferably, in the preparation of the polymer nanoparticles, tetrahydrofuran is used as the solvent.

[0015] Preferably, the mass ratio of the rhodamine-based polymer fluorescent dye to PSMA is 4-10:1-4.

[0016] Preferably, in the preparation of the polymer nanoparticles, a nanofiltration membrane is also used for filtration.

[0017] The present invention discloses a rhodamine polymer super-resolution nanoprobe prepared by the above synthesis method.

[0018] The present invention discloses the use of the above rhodamine polymer super-resolution nanoprobe in a targeted fluorescent labeling reagent and / or a cell labeling reagent.

[0019] The present invention discloses a synthesis method of a rhodamine polymer super-resolution nanoprobe, including the synthesis of compound 2-(4-vinyl-phenoxy)-ethanol, the synthesis of a multi-component copolymeric polymer, the synthesis of a rhodamine-based polymer fluorescent dye, and the preparation of polymer nanoparticles, which specifically includes the following steps:

[0020] 1) Synthesis of compound 2-(4-vinyl-phenoxy)-ethanol: Mix ethylene glycol, 4-vinylbenzyl chloride, NaOH, and H2O, stir in a constant temperature oil bath at 50-80 °C for 18-36 h, cool, wash with an appropriate amount of DCM, dry with anhydrous Na2SO4, filter, remove the organic solvent under vacuum, and purify by column chromatography to obtain an oily substance, namely 2-(4-vinyl-phenoxy)-ethanol.

[0021] 2) Synthesis of the multi-component copolymeric polymer: Dissolve the enol reagent and the comonomer in toluene, add AIBN, then heat under nitrogen at 50-90 °C for 12-36 h, cool, add hexane to precipitate, filter, dissolve the precipitate in DCM, and then stir and precipitate in MeOH 1-3 times, dry under vacuum at 60-90 °C for 12-36 h to obtain the multi-component copolymeric polymer.

[0022] 3) Synthesis of the rhodamine-based polymer fluorescent dye: Dissolve the multi-component copolymeric polymer, EDCl, and DMAP in DCM, dropwise add the rhodamine-based fluorescent dye at 0-5 °C, react at room temperature for 12-48 h, add water for extraction, wash with brine, dry with Na2SO4, remove the solvent under reduced pressure, dropwise add to MeOH, filter, and dry under vacuum at 60-90 °C for 12-48 h to obtain the rhodamine-based polymer fluorescent dye.

[0023] 4) Preparation of polymer nanoparticles: Dissolve the rhodamine-based polymer fluorescent dye and the functional reagent in tetrahydrofuran, inject into water in an ultrasonic water bath and sonicate for 1-5 min. Blow nitrogen at 50-80 °C for 1-2 h, filter while it is hot with a 220 nm filter membrane to obtain polymer nanoparticles, namely the rhodamine polymer super-resolution nanoprobe.

[0024] Preferably, in the synthesis of 2-(4-vinyl-phenoxy)-ethanol, the dosage ratio of ethylene glycol to H2O is 10-15 g: 0.1-0.3 mL, the dosage ratio of 4-vinylbenzyl chloride to H2O is 1 g: 0.1-0.3 mL, and the dosage ratio of NaOH to H2O is 0.1-0.4 g: 0.1-0.3 mL.

[0025] Preferably, in the synthesis of 2-(4-vinyl-phenoxy)-ethanol, the volume ratio of mobile phase PE to EA in column chromatography purification is 5-10: 1-5.

[0026] Preferably, in the synthesis of the multi-component copolymerized polymer, the enol reagent includes 2-(4-vinyl-phenoxy)-ethanol.

[0027] Preferably, in the synthesis of the multi-component copolymerized polymer, the comonomer includes at least styrene.

[0028] Preferably, in the synthesis of the multi-component copolymerized polymer, the dosage ratio of 2-(4-vinyl-phenoxy)-ethanol to toluene is 0.1-0.5 g: 5-10 mL, the volume ratio of styrene to toluene is 1-3: 5-10, the dosage ratio of AIBN to toluene is 5-15 mg: 5-10 mL, the volume ratio of hexane to toluene is 100-200: 5-10, and the volume ratio of DCM to toluene is 1-2: 5-10.

[0029] The synthesis reaction formula of the multi-component copolymerized polymer is shown in Formula (1).

[0030]

[0031] Preferably, in the synthesis of the rhodamine-based polymer fluorescent dye, the dosage ratio of the multi-component copolymerized polymer to DCM is 150-300 mg: 20-50 mL, the dosage ratio of EDCl to DCM is 45-60 mg: 20-50 mL, and the dosage ratio of DMAP to DCM is 1-5 mg: 20-50 mL.

[0032] Preferably, in the synthesis of rhodamine-based polymeric fluorescent dyes, the rhodamine-based fluorescent dye is one of fluorescein, rhodamine B, and 6-HMSiR-COOH, and the dosage ratio of the rhodamine-based fluorescent dye to DCM is 1-100 mg: 20-50 mL. The rhodamine-based polymeric 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 polymeric 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 polymeric 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).

[0033]

[0034]

[0035]

[0036] Preferably, in the synthesis of rhodamine-based polymeric fluorescent dyes, the brine is saturated brine.

[0037] Preferably, in the preparation of polymer nanoparticles, the functional reagent includes PSMA, and the dosage ratio of the rhodamine-based polymeric fluorescent dye to tetrahydrofuran is 4-10 mg: 50-100 mL, and the dosage ratio of the functional reagent to tetrahydrofuran is 1-4 mg: 50-100 mL, and the volume ratio of water to tetrahydrofuran is 250-500: 50-100.

[0038] More preferably, in the synthesis of the multi-component copolymerized polymer, the comonomer further includes phenyl vinyl acetate and methyl 2-vinylnicotinate, and the dosage ratio of phenyl vinyl acetate to styrene is 0.05-0.3 g: 1-3 mL, and the dosage ratio of methyl 2-vinylnicotinate to styrene is 0.05-0.3 g: 1-3 mL.

[0039] More preferably, in the synthesis of the multi-component copolymerized polymer, the comonomer further includes 3-(trimethoxysilyl)propyl acrylate, and the dosage ratio of 3-(trimethoxysilyl)propyl acrylate to styrene is 0.1-0.6 g: 1-3 mL. 3-(trimethoxysilyl)propyl acrylate can undergo a free radical random polymerization reaction with other comonomers and introduce a new group, which acts together with the phenyl ester and methyl ester groups introduced by phenyl vinyl acetate and methyl 2-vinylnicotinate, thereby improving the fluorescence intensity and photostability of the nanoprobe.

[0040] Since vinyl phenyl acetate and methyl 2-vinylnicotinate are added in the synthesis of the multi-component copolymer polymer, a polymer chain containing multiple functional groups is generated, and finally a rhodamine polymer super-resolution nanoprobe is synthesized. Therefore, the present invention has the following beneficial effects: high fluorescence quantum efficiency, strong photostability, can effectively avoid aggregation-induced emission quenching, and the size is small and suitable for application and promotion. Therefore, the object of the present invention is to provide a method for synthesizing a rhodamine polymer super-resolution nanoprobe. Description of the Drawings

[0041] Figure 1 1H NMR spectrum of 2-[(4-vinylphenyl)methoxy]ethanol.

[0042] Figure 2 13C NMR spectrum of 2-[(4-vinylphenyl)methoxy]ethanol.

[0043] Figure 3 DLS characterization test result graph of the rhodamine polymer super-resolution nanoprobe of Examples 1-3.

[0044] Figure 4 UV absorption spectrum and fluorescence spectrum test result graph of the rhodamine polymer super-resolution nanoprobe of Examples 1-3.

[0045] Figure 5 Confocal imaging result graph of the rhodamine polymer super-resolution nanoprobe of Examples 1-3 labeling cell microtubules.

[0046] Figure 6 STED imaging result graph of the rhodamine polymer super-resolution nanoprobe of Examples 1-3 labeling cell microtubules. Detailed Embodiments

[0047] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0048] The concepts involved in the present application will be described below with reference to the accompanying drawings first. It should be noted here that the following descriptions of each concept are only for making the content of the present application easier to understand, and do not represent a limitation on the protection scope of the present application; at the same time, without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The present application will be described in detail below with reference to the drawings and in combination with the embodiments.

[0049] Example 1

[0050] A method for synthesizing a rhodamine polymer super-resolution nanoprobe, including the synthesis of compound 2-(4-vinyl-phenoxy)-ethanol, the synthesis of a multi-component copolymer polymer, the synthesis of a rhodamine-based polymer fluorescent dye, and the preparation of polymer nanoparticles, specifically includes the following steps:

[0051] 1) Synthesis of compound 2-(4-vinyl-phenoxy)-ethanol: Mix ethylene glycol, 4-vinylbenzyl chloride, NaOH, and H2O, stir in an oil bath at 70 °C for 24 h, cool, wash with an appropriate amount of DCM, dry with anhydrous Na2SO4, filter, remove organic solvents under vacuum, and purify by column chromatography to obtain an oily substance, namely 2-(4-vinyl-phenoxy)-ethanol. The dosage ratio of ethylene glycol to H2O is 11.03 g:0.21 mL, the dosage ratio of 4-vinylbenzyl chloride to H2O is 1 g:0.21 mL, and the dosage ratio of NaOH to H2O is 0.27 g:0.21 mL. In column chromatography purification, the volume ratio of the mobile phase PE to EA is 7:3.

[0052] 2) Synthesis of a multi-component copolymer polymer: Dissolve the enol reagent and the comonomer in toluene, add AIBN, then heat at 70 °C under nitrogen for 24 h, cool to room temperature, precipitate with hexane, filter, dissolve the precipitate in DCM, and then stir and precipitate 2 times in MeOH, and dry under vacuum at 80 °C for 24 h to obtain a multi-component copolymer polymer. The enol reagent is 2-(4-vinyl-phenoxy)-ethanol, the comonomer is styrene, the dosage ratio of 2-(4-vinyl-phenoxy)-ethanol to toluene is 0.172 g:5 mL, the volume ratio of styrene to toluene is 1:5, the dosage ratio of the AIBN initiator to toluene is 6 mg:5 mL, the volume ratio of hexane to toluene is 100:5, and the volume ratio of DCM to toluene is 1:5.

[0053] 3) Synthesis of a rhodamine-based polymer fluorescent dye: Dissolve the multi-component copolymer polymer, EDCl, and DMAP in DCM, dropwise add the rhodamine-based fluorescent dye at 0 °C, react at room temperature for 24 h, extract with water, wash with brine, dry with Na2SO4, remove the solvent under reduced pressure, drop it into MeOH, filter, and dry under vacuum at 80 °C for 24 h to obtain a rhodamine-based polymer fluorescent dye. The dosage ratio of the multi-component copolymer polymer to DCM is 200 mg:30 mL, the dosage ratio of EDCl to DCM is 48 mg:30 mL, and the dosage ratio of DMAP to DCM is 4 mg:30 mL. The dropped rhodamine-based fluorescent dye is fluorescein, and the dosage ratio of the rhodamine-based fluorescent dye to DCM is 100 mg:30 mL. In extraction, the volume ratio of water to DCM is 1:5. The brine is saturated saline.

[0054] 4) Preparation of Polymer Nanoparticles: Dissolve the rhodamine-based polymer fluorescent dye and the functional reagent in tetrahydrofuran, quickly inject it into water in an ultrasonic water bath, and ultrasonicate for 2 min. Under the condition of heating at 65 °C, blow nitrogen into the sonicated solution for 1 h, then filter it while it is still hot through a 220 nm filter membrane to obtain polymer nanoparticles, namely rhodamine polymer super-resolution nanoprobes. The functional reagent is PSMA, the dosage ratio of the rhodamine-based polymer fluorescent dye to tetrahydrofuran is 4 mg:50 mL, the dosage ratio of the functional reagent to tetrahydrofuran is 1 mg:50 mL, and the volume ratio of water to tetrahydrofuran is 250:50.

[0055] Example 2

[0056] A method for synthesizing a rhodamine polymer super-resolution nanoprobe. The difference between this example and Example 1 is as follows: In the synthesis of the rhodamine-based polymer fluorescent dye, the dropped rhodamine-based fluorescent dye is Rhodamine B, and the dosage ratio of the rhodamine-based fluorescent dye to DCM is 100 mg:30 mL. The others are the same as in Example 1.

[0057] Example 3

[0058] A method for synthesizing a rhodamine polymer super-resolution nanoprobe. The difference between this example and Example 1 is as follows: In the synthesis of the rhodamine-based polymer fluorescent dye, the dropped rhodamine-based fluorescent dye is 6-HMSiR-COOH, and the dosage ratio of the rhodamine-based fluorescent dye to DCM is 2 mg:30 mL. The others are the same as in Example 1.

[0059] Example 4

[0060] A method for synthesizing a rhodamine polymer super-resolution nanoprobe. The difference between this example and Example 1 is as follows: In the synthesis of the multi-component copolymerized polymer, the copolymerization monomers are styrene, phenyl vinyl acetate, and methyl 2-vinylnicotinate. Among them, the dosage ratio of phenyl vinyl acetate to styrene is 0.172 g:1 mL, and the dosage ratio of methyl 2-vinylnicotinate to styrene is 0.171 g:1 mL. The others are the same as in Example 1.

[0061] Example 5

[0062] A method for synthesizing a rhodamine polymer super-resolution nanoprobe. The difference between this example and Example 4 is as follows: In the synthesis of the rhodamine-based polymer fluorescent dye, the dropped rhodamine-based fluorescent dye is Rhodamine B, and the dosage ratio of the rhodamine-based fluorescent dye to DCM is 100 mg:30 mL. The others are the same as in Example 4.

[0063] Example 6

[0064] A method for synthesizing a rhodamine polymer super-resolution nanoprobe. The difference between this example and Example 4: In the synthesis of the rhodamine-based polymer fluorescent dye, the added rhodamine-based fluorescent dye is 6-HMSiR-COOH, and the dosage ratio of the rhodamine-based fluorescent dye to DCM is 2 mg: 30 mL. The others are the same as in Example 4.

[0065] Example 7

[0066] A method for synthesizing a rhodamine polymer super-resolution nanoprobe. The difference between this example and Example 4: In the synthesis of the multi-component copolymerized polymer, the dosage ratio of phenyl vinyl acetate to styrene in the comonomers is 0.085 g: 1 mL, and the dosage ratio of methyl 2-vinylnicotinate to styrene is 0.085 g: 1 mL. The others are the same as in Example 4.

[0067] Example 8

[0068] A method for synthesizing a rhodamine polymer super-resolution nanoprobe. The difference between this example and Example 7: In the synthesis of the rhodamine-based polymer fluorescent dye, the added rhodamine-based fluorescent dye is rhodamine B, and the dosage ratio of the rhodamine-based fluorescent dye to DCM is 100 mg: 30 mL. The others are the same as in Example 7.

[0069] Example 9

[0070] A method for synthesizing a rhodamine polymer super-resolution nanoprobe. The difference between this example and Example 7: In the synthesis of the rhodamine-based polymer fluorescent dye, the added rhodamine-based fluorescent dye is 6-HMSiR-COOH, and the dosage ratio of the rhodamine-based fluorescent dye to DCM is 2 mg: 30 mL. The others are the same as in Example 7.

[0071] Example 10

[0072] A method for synthesizing a rhodamine polymer super-resolution nanoprobe. The difference between this example and Example 7: In the synthesis of the multi-component copolymerized polymer, the comonomers are styrene, phenyl vinyl acetate, methyl 2-vinylnicotinate, and acryloxypropyltrimethoxysilane, and the dosage ratio of acryloxypropyltrimethoxysilane to styrene is 0.245 g: 1 mL. The others are the same as in Example 7.

[0073] Example 11

[0074] A method for synthesizing a rhodamine polymer super-resolution nanoprobe. The difference between this example and Example 10: In the synthesis of the rhodamine-based polymer fluorescent dye, the added rhodamine-based fluorescent dye is rhodamine B, and the dosage ratio of the rhodamine-based fluorescent dye to DCM is 100 mg: 30 mL. The others are the same as in Example 10.

[0075] Example 12

[0076] A synthesis method of a rhodamine polymer super-resolution nanoprobe. The difference between this example and Example 10: In the synthesis of the rhodamine-based polymer fluorescent dye, the added rhodamine-based fluorescent dye is 6-HMSiR-COOH, and the dosage ratio of the rhodamine-based fluorescent dye to DCM is 2 mg: 30 mL. Other conditions are the same as in Example 10.

[0077] Example 13

[0078] A synthesis method of a rhodamine polymer super-resolution nanoprobe. The difference between this example and Example 10: In the synthesis of the multi-component copolymerized polymer, the dosage ratio of acryloxypropyltrimethoxysilane to styrene in the comonomers is 0.122 g: 1 mL. Other conditions are the same as in Example 10.

[0079] Example 14

[0080] A synthesis method of a rhodamine polymer super-resolution nanoprobe. The difference between this example and Example 13: In the synthesis of the rhodamine-based polymer fluorescent dye, the added rhodamine-based fluorescent dye is Rhodamine B, and the dosage ratio of the rhodamine-based fluorescent dye to DCM is 100 mg: 30 mL. Other conditions are the same as in Example 13.

[0081] Example 15

[0082] A synthesis method of a rhodamine polymer super-resolution nanoprobe. The difference between this example and Example 13: In the synthesis of the rhodamine-based polymer fluorescent dye, the added rhodamine-based fluorescent dye is 6-HMSiR-COOH, and the dosage ratio of the rhodamine-based fluorescent dye to DCM is 2 mg: 30 mL. Other conditions are the same as in Example 13.

[0083] Comparative Example 1

[0084] The difference between this comparative example and Example 4: In the synthesis of the multi-component copolymerized polymer, the comonomers do not contain phenyl vinyl acetate. Other conditions are the same as in Example 4.

[0085] Comparative Example 2

[0086] The difference between this comparative example and Example 4: In the synthesis of the multi-component copolymerized polymer, the comonomers do not contain methyl 2-vinylnicotinate. Other conditions are the same as in Example 4.

[0087] Comparative Example 3

[0088] The difference between this comparative example and Example 10: In the synthesis of the multi-component copolymerized polymer, the comonomers do not contain phenyl vinyl acetate and methyl 2-vinylnicotinate. Other conditions are the same as in Example 10.

[0089] 1H NMR test

[0090] The 1H NMR test results of 2-[(4-vinylphenyl)methoxy]ethanol are as Figure 1 follows.

[0091] 1 H NMR (400 MHz, CDCl3) :δ 7.45 – 7.25 (m, 4H), 6.71 (dd, J J = 17.6,10.9 Hz, 1H), 5.74 (d, J J = 17.6 Hz, 1H), 5.24 (d, J 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).

[0092] 13C NMR test

[0093] The 13C NMR test results of 2-[(4-vinylphenyl)methoxy]ethanol are as Figure 2 follows.

[0094] 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.

[0095] DLS characterization test

[0096] The rhodamine polymer super-resolution nanoprobes of Examples 1-3 were characterized by DLS.

[0097] The DLS characterization test results are as Figure 3 follows. Figure 3 Figure a in Figure 3 is the particle size distribution diagram, and figure b in

[0098] is the potential diagram. It can be seen that the particle sizes of the prepared rhodamine polymer super-resolution nanoprobes are basically distributed around 30 nm, and the sizes are small; it can also be seen that the potential peak value of the rhodamine polymer super-resolution nanoprobes is around -30 mV, indicating good stability.

[0099] The rhodamine polymer super-resolution nanoprobes of Examples 1-3 were subjected to ultraviolet absorption spectroscopy and fluorescence spectroscopy tests. Among them, the excitation wavelength for measuring the fluorescence spectrum of the rhodamine polymer super-resolution nanoprobe in Example 1 was 484 nm, the excitation wavelength for measuring the fluorescence spectrum of the rhodamine polymer super-resolution nanoprobe in Example 2 was 569 nm, and the excitation wavelength for measuring the fluorescence spectrum of the rhodamine polymer super-resolution nanoprobe in Example 3 was 658 nm.

[0100] The test results of ultraviolet absorption spectroscopy and fluorescence spectroscopy are as Figure 4 shown. Figure 4 Figure a in Figure 4 is the test result diagram of ultraviolet absorption spectroscopy and fluorescence spectroscopy of the rhodamine polymer super-resolution nanoprobe in Example 1; Figure 4 Figure b in

[0101] Confocal imaging and STED imaging application tests

[0102] The rhodamine polymer super-resolution nanoprobes of Examples 1-3 were used for cell microtubule labeling and imaged on confocal and STED. Specifically:

[0103] (1) African green monkey kidney cells BS-C-1 cells (purchased commercially) were selected, and cells were inoculated in a confocal dish, about 40,000 cells per well, and the cells were cultured for 24 h under the conditions of 37 °C and 5% CO2; the culture medium of BS-C-1 cells in the culture dish was aspirated and washed 3 times with PBS buffer solution;

[0104] (2) Added Triton X-100 with a volume ratio of 0.5% at a volume of 200 μL / well and shaken for 30 s;

[0105] (3) Quickly aspirated Triton X-100, added the fixative at a volume of 200 μL / well and shaken in the dark for 15 min, then aspirated the fixative and rinsed three times with DPBS buffer solution; the fixative was paraformaldehyde with a volume ratio of 3.2%;

[0106] (4) Added sodium borohydride solution at a volume of 200 μL / well, shaken in the dark for 10 min, and rinsed three times with DPBS buffer solution; the concentration of the sodium borohydride solution was 1 mg / mL.

[0107] (5) Add 200 μL of blocking solution, shake for 45 min, then aspirate and wash thoroughly with DPBS buffer solution; the blocking solution is PBS solution containing BSA and Triton X-100, the volume ratio of BSA is 5%, and the volume ratio of Triton X-100 is 0.5%.

[0108] (6) Dilute the primary antibody Anti-beta Tubulin (Abcam, cat. no. Ab179513) solution at a volume ratio of 1:200 in the blocking solution, add 150 μL of the primary antibody dilution to the culture dish and gently shake on a shaker for 45 min, and wash 3 times with DPBS buffer solution.

[0109] (7) Dilute the secondary antibody Goat Polyclonal Antibody to Rabbit IgG (H&L) - Biotin (Yuantai Biotech, cat. no. P50075) solution at a volume ratio of 1:200 in the blocking solution, add 150 μL of the secondary antibody dilution to the culture dish and gently shake on a shaker for 45 min, and wash 3 times with DPBS buffer solution.

[0110] (8) Mix 4 mL of rhodamine polymer super-resolution nanoprobe, 240 μL of streptavidin, 80 μL of polyethylene glycol solution, and 80 μL of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid solution, stir evenly, add 60 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, mix well and react for 4 h, and ultrafilter with 100K to remove free streptavidin to obtain modified rhodamine polymer super-resolution nanoprobe particles; the concentration of streptavidin is 1 mg / mL; the volume fraction of the polyethylene glycol solution is 5%, and the solvent is H2O; the concentration of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid is 1 mol / L, pH = 7.3; the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 5 mg / mL.

[0111] (9) Pour the modified rhodamine polymer super-resolution nanoprobe particles into the culture dish in step (7), gently shake for 30 min; wash thoroughly with DPBS buffer solution, and store at 4 °C in the refrigerator.

[0112] (10) Confocal imaging: Using CSU-W1-SoRa, since the laser used cannot be adjusted across the entire wavelength range, 488, 561, and 640 nm were selected as the excitation wavelengths for confocal imaging of cells. The excitation wavelength for the rhodamine polymer super-resolution nanoprobe in Example 1 was 488 nm, the excitation wavelength for the rhodamine polymer super-resolution nanoprobe in Example 2 was 561 nm, and the excitation wavelength for the rhodamine polymer super-resolution nanoprobe in Example 3 was 640 nm.

[0113] (11) Super-resolution imaging: STED was used for super-resolution imaging. For the rhodamine polymer super-resolution nanoprobe in Example 1, a 484 nm excitation light was paired with a 660 nm depletion light, and for the rhodamine polymer super-resolution nanoprobe in Example 2, a 569 nm excitation light was paired with a 660 nm depletion light. After the operation, two images with different characteristics were obtained. One was the CLSM image generated only after the excitation process was completed, and the other was the STED image obtained after the excitation process and further depletion light treatment. The full width at half maximum (FWHM) was the result obtained by Gaussian fitting.

[0114] The test results of confocal imaging are as Figure 5 shown. Figure 5 In Figure a, it is the confocal imaging result diagram of the rhodamine polymer super-resolution nanoprobe in Example 1 labeling cell microtubules. Figure 5 In Figure b, it is the confocal imaging result diagram of the rhodamine polymer super-resolution nanoprobe in Example 2 labeling cell microtubules. Figure 5 In Figure c, it is the confocal imaging result diagram of the rhodamine polymer super-resolution nanoprobe in Example 3 labeling cell microtubules. The test results of STED imaging are as Figure 6 shown. Figure 6 In Figure a, it is the CLSM image and STED image of the rhodamine polymer super-resolution nanoprobe in Example 1 labeling cell microtubules. Figure 6 In Figure b, it is the fluorescence signal intensity value of the rhodamine polymer super-resolution nanoprobe in Example 1 labeling cell microtubules. Figure 6 In Figure c, it is the CLSM image and STED image of the rhodamine polymer super-resolution nanoprobe in Example 2 labeling cell microtubules. Figure 6Figure d in [Example 2] shows the fluorescence signal intensity values of the rhodamine polymer super-resolution nanoprobe labeling cell microtubules. It can be seen that the rhodamine polymer super-resolution nanoprobes in [Examples 1-3] all have relatively continuous imaging effects on cell microtubules. It can also be seen that the resolution of the STED images after imaging with the rhodamine polymer super-resolution nanoprobes in [Examples 1 and 2] is significantly improved compared to the resolution of the CLSM images. The resolution of the rhodamine polymer super-resolution nanoprobe in [Example 1] has increased from the original 316.56 nm to 137.7 and 78.7 nm, and the resolution of the STED image after imaging with the rhodamine polymer super-resolution nanoprobe in [Example 2] has increased from the original 264 nm to 155 nm compared to confocal microscopy. However, since the rhodamine polymer super-resolution nanoprobe in [Example 3] uses a photoswitching dye, the fluorescence brightness after labeling is relatively low, and the STED imaging effect is not good.

[0115] Fluorescence intensity test

[0116] Select a rhodamine polymer super-resolution nanoprobe with a rhodamine-based fluorescent dye as fluorescein, and use a fluorescence spectrophotometer to measure the fluorescence spectrum of the nanoprobe. The calculation formula for the normalized intensity is: 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.

[0117] Table 1 Test results of fluorescence intensity

[0118]

[0119] As can be seen from Table 1, the fluorescence intensities of the rhodamine polymer super-resolution nanoprobes in the examples are all relatively high. This indicates that the polymer chains formed by the polymerization reaction of styrene and 2-(4-vinyl-phenoxy)-ethanol monomers combined with the rhodamine-based dye can introduce steric hindrance, prevent molecules from approaching and aggregating, reduce intermolecular Π-Π stacking, and improve the fluorescence intensity. It can also be seen that the fluorescence intensities of the rhodamine polymer super-resolution nanoprobes in Example 4 and Example 7 are higher than those in Example 1. This shows that the polymer chains formed by introducing phenyl acetate vinyl ester and methyl 2-vinylnicotinate with other monomers contain low-polar phenyl esters and methyl esters, which are prone to show uniform and stable aggregated particles in solution, thus improving the fluorescence intensity. It can also be seen that the fluorescence intensity of the rhodamine polymer super-resolution nanoprobe in Example 4 is higher than that in Example 7. This indicates that the higher the usage amounts of phenyl acetate vinyl ester and methyl 2-vinylnicotinate, the better the fluorescence intensity. It can also be seen that the fluorescence intensities of the rhodamine polymer super-resolution nanoprobes in Example 10 and Example 13 are higher than those in Example 4. This shows that the new groups introduced in acryloxypropyltrimethoxysilane can act together with other monomers to form polymer chains, 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. This 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 those in Comparative Example 1 and Comparative Example 2. This shows that the fluorescence intensity of the rhodamine polymer super-resolution nanoprobe with the polymer chains formed by phenyl acetate vinyl ester and methyl 2-vinylnicotinate and other monomers is higher than that with 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. This indicates that the effect of improving the fluorescence intensity by introducing acryloxypropyltrimethoxysilane in the rhodamine polymer super-resolution nanoprobe is lower than the effect of the combined addition of phenyl acetate vinyl ester, methyl 2-vinylnicotinate, and acryloxypropyltrimethoxysilane.

[0120] Fluorescence stability test

[0121] The rhodamine polymer super-resolution nanoprobe of the example using a rhodamine-based fluorescent dye as fluorescein was selected for testing. The rhodamine polymer super-resolution nanoprobe was continuously irradiated with a 484 nm excitation light for 1 h, and the fluorescence intensity of the irradiated solution was measured. The fluorescence stability was evaluated by the fluorescence intensity retention rate. The calculation formula for 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.

[0122] Table 2 Fluorescence stability test results

[0123]

[0124] As can be seen from Table 1, the fluorescence intensity retention rates of the rhodamine polymer super-resolution nanoprobes in the examples are all relatively high. This indicates that the polymer chains formed by the polymerization reaction of two monomers, styrene and 2-(4-vinyl-phenoxy)-ethanol, combined with the rhodamine-based fluorescent dye can introduce steric hindrance, prevent molecules from approaching and aggregating, reduce intermolecular Π-Π stacking and aggregation-induced quenching, 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 Example 4 and Example 7 are higher than that in Example 1. This indicates that the polymer chains formed by introducing phenyl acetate vinyl ester and methyl 2-vinylnicotinate with other monomers contain low-polarity phenyl esters and methyl esters, which are prone to show uniform and stable aggregated particles in solution, so they exhibit 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. This indicates that the higher the usage amounts of phenyl acetate vinyl ester and methyl 2-vinylnicotinate 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 Example 10 and Example 13 are higher than that in Example 4. This indicates that the new groups introduced in 3-(trimethoxysilyl)propyl acrylate can act together with other monomers to form polymer chains, 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. This indicates that adding an appropriate amount of 3-(trimethoxysilyl)propyl acrylate 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 Example 1 and Comparative Example 2. This indicates that the fluorescence intensity retention rate of the rhodamine polymer super-resolution nanoprobe is higher when introducing the polymer chains formed by phenyl acetate vinyl ester and methyl 2-vinylnicotinate with other monomers than when introducing a single 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. This indicates that the improvement effect of introducing 3-(trimethoxysilyl)propyl acrylate on the fluorescence intensity retention rate of the rhodamine polymer super-resolution nanoprobe is lower than the effect of the combined addition of phenyl acetate vinyl ester, methyl 2-vinylnicotinate and 3-(trimethoxysilyl)propyl acrylate.

[0125] The above-described examples and / or embodiments are only used to illustrate the preferred examples and / or embodiments for implementing the technology of the present invention, and do not impose any formal restrictions on the implementation modes of the technology of the present invention. Any person skilled in the art, without departing from the scope of the technical means disclosed in the content of the present invention, may make some changes or modifications to other equivalent embodiments, but should still be regarded as the same technology or embodiment as the present invention in essence.

[0126] In this text, specific examples are used to illustrate the principles and implementation manners of the present application. The description of the above embodiments is only for helping to understand the method and its core idea of the present application. The above is only the preferred implementation manner of the present application. It should be noted that due to the limited nature of literal expression and objectively infinite specific structures, for those of ordinary skill in the art, without departing from the principle of the present application, several improvements, retouches or changes can be made, or the above technical features can be combined in an appropriate manner; these improvements, retouches, changes or combinations, or directly applying the inventive concept and technical solution to other occasions without improvement, shall all be regarded as the protection scope of the present application.

Claims

1. A method for synthesizing a rhodamine polymer super-resolution nanoprobe, comprising: S1, an enol reagent reacts with a comonomer to prepare a multi-polymer copolymer, wherein the enol reagent includes 2-[(4-vinylphenyl)methoxy]ethanol, and the comonomer includes at least styrene, vinyl acetate phenyl ester and 2-vinyl nicotinate methyl ester, the amount ratio of vinyl acetate to styrene is 0.05-0.3 g: 1-3 mL, the amount ratio of 2-vinyl nicotinate methyl ester to styrene is 0.05-0.3 g: 1-3 mL; the amount ratio of 2-[(4-vinylphenyl)methoxy]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 silicon-rhodamine labeled carboxylic acid 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 prostate-specific membrane antigen PSMA.

2. 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 azobisisobutyronitrile AIBN is used as an initiator.

3. 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, dichloromethane DCM is used as a solvent, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDCl and 4-dimethylaminopyridine DMAP are used as reaction aids.

4. 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.

5. 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 the prostate-specific membrane antigen PSMA is 4-10:1-4.

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, a nanofiltration membrane is also used for filtration.

7. The rhodamine polymer super-resolution nanoprobe prepared by the synthesis method according to any one of claims 1 to 6.

8. Use of the rhodamine polymer super-resolution nanoprobe according to claim 7 in the preparation of targeted fluorescent labeling agents and / or cell labeling agents.

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