Preparation method and application of scopoletin functionalized magnetic nanoprobe

Abstract

The present invention discloses a preparation method and application of a scopolamine functionalized magnetic nanoprobe, including the preparation of amino-terminally modified nanoparticles Fe3O4@SiO2‑NH2, the preparation of a photoaffinity linker-scopolamine coupling product, and the preparation of a scopolamine functionalized magnetic nanoprobe. The method of the present invention is simple and rapid to operate, and does not require prior clarification of the structure-activity relationship of the active compound, and does not affect its all-round contact with the target protein. The present invention uses the prepared scopolamine functionalized magnetic nanoprobe to carry out target fishing in cells and animal models of rheumatoid arthritis, separates the fished target protein, and identifies it by mass spectrometry. It successfully passes a protein immunoblotting experiment, verifies that the target protein of scopolamine on the HFLS‑RA cell membrane is vimentin, and provides a new therapeutic target for rheumatoid arthritis.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to a preparation method and application of a scopoletin functionalized magnetic nanoprobe. Background Art

[0002] Rheumatoid arthritis (RA) is a common autoimmune disease, which is mainly manifested by synovial hyperplastic inflammation and bone destruction, which can lead to a significant decline in the patient's physical function and quality of life, thereby causing a social burden. The global incidence of RA is about 1%, and it can occur in all age groups. The cause is complex, the course of the disease is long and recurrent, and it is easy to cause multiple complications. RA has not yet been cured. Existing drugs include glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), and disease-modifying antirheumatic drugs (DMARDs); these drugs can only relieve symptoms, cannot control disease progression, and have inevitable adverse reactions. In recent years, highly targeted biological agents (such as TNF inhibitors) have been gradually applied to the treatment of RA; the recently emerged small molecule targeted drugs (such as JAK inhibitors) have further overcome the defects of protein biological agents. Therefore, the development of small molecule targeted drugs has become a new direction for RA drug research and is also the key to achieving personalized precision medicine.

[0003] Currently, chemically synthesized targeted drugs (such as small molecule JAK inhibitors: Tofacitinib, Baricitinib) face serious clinical safety issues. So far, attempts to modify the chemical structure of existing RA targeted drugs to reduce toxic side effects have not been successful. Compared with chemical syntheses, natural product compounds have complex and diverse skeletons and rich functional groups, and have unique biological activities, making them suitable as the starting point for new drug screening.

[0004] Studies have shown that scopolamine can safely and effectively treat RA, and there are target proteins in the RA model. For animal models, oral administration of the compound to rats with adjuvant arthritis (AIA) can significantly alleviate clinical symptoms (redness and swelling of the hind feet, ulceration of the tail), regulate inflammatory factor responses (selectively inhibit COX-2 expression, regulate the levels of cytokines IL-10 and IL-6 in ankle synovial tissue), improve joint lesions (soft tissue swelling, narrowing of the joint cavity, cartilage erosion, cell infiltration, pannus formation), and have no obvious cardiovascular toxicity. For cell membrane type, the compound can effectively inhibit the migration and invasion of inflammatory synovial cells and the NF-KB signaling pathway in fibroblast-like synoviocytes (HFLS-RA) of RA patients.

[0005] Target identification of natural active compounds is a key step in new drug development. It not only helps to discover compound structures with development potential, but also facilitates the safety and efficacy evaluation of compounds, and can even discover new therapeutic targets for complex diseases outside of classical pharmacological signaling pathways. Target identification methods for active compounds can be divided into two categories: indirect and direct. Indirect methods include phenotypic screening, gene screening, and virtual screening, which are limited in that false negative results cannot be ruled out. In contrast, direct methods are widely used, and active compounds are chemically connected to solid carriers based on "drug-target" affinity to enrich and identify target proteins.

[0006] Due to the excellent properties of magnetic nanoparticles (MNPs), they are suitable for preparing compound-functionalized magnetic nanoprobes and used as solid carriers for target fishing. At present, the synthesis of this emerging probe is mostly through structural modification of compounds and MNPs and then connection. However, structural modification of compounds is not conducive to efficient target fishing because: 1) before the structure-activity relationship of active compounds is clarified, changing the structure is not conducive to its all-round contact with target proteins; 2) natural active compounds have diverse structures. If the connection with MNPs needs to be designed separately according to the characteristics of the molecular structure, it is difficult to make probes in a unified and convenient way. Summary of the invention

[0007] To solve the above problems, the primary purpose of the present invention is to provide a method for preparing scopoletin functionalized magnetic nanoprobes. This method uses photocrosslinking technology, is simple and rapid to operate, does not require prior clarification of the structure-activity relationship of the active compound, and does not affect its all-round contact with the target protein.

[0008] The present invention is achieved through the following technical solutions:

[0009] A method for preparing a scopoletin functionalized magnetic nanoprobe comprises the following steps:

[0010] (1) dispersing ferric chloride hexahydrate, polyvinyl pyrrolidone and sodium acetate in ethylene glycol to react and obtain Fe3O4 nanoparticles; ultrasonically reacting the Fe3O4 nanoparticles with tetraethyl orthosilicate to obtain Fe3O4@SiO2 nanoparticles;

[0011] (2) Using ethanol as solvent, aminopropyltriethoxysilane was added to Fe3O4@SiO2 nanoparticles, and refluxed to obtain amino-terminally modified nanoparticles Fe3O4@SiO2-NH2;

[0012] (3) mixing the photoaffinity linker and scopoletin and fully dissolving them in an organic solvent, irradiating them with 365 nm ultraviolet light at room temperature, and after the reaction is completed, rotating and drying to remove the solvent to obtain a photoaffinity linker-scopoletin coupling product;

[0013] (4) The photoaffinity linker-scopolamine coupling product was re-dissolved in an organic solvent, and triethylamine was added dropwise. The amino-terminally modified nanoparticles Fe3O4@SiO2-NH2 were added, mixed and shaken overnight in the dark. After the reaction was completed, the product was separated by a magnet and washed with methanol to obtain a scopolamine functionalized magnetic nanoprobe.

[0014] Preferably, in step (3), the photoaffinity linker is one or more of 4-[3-(trifluoromethyl)-3H-diaziridine-3-yl]benzoic acid (TDBA), 3-(4-bromophenyl)-3-(trifluoromethyl)-3H-diaziridine (SDAD) or 3-methyl-3H-diaziridine-3-propionic acid (MPDA), more preferably 4-[3-(trifluoromethyl)-3H-diaziridine-3-yl]benzoic acid.

[0015] Preferably, in step (3), the molar ratio of the photoaffinity linker to scopoletin is 1-4:1.

[0016] Preferably, in step (3), the organic solvent is one or more of anhydrous methanol, dichloromethane or carbon tetrachloride, more preferably anhydrous methanol.

[0017] Preferably, in step (3), the ultraviolet light irradiation time at room temperature is 15-60 minutes.

[0018] Preferably, in step (4), the organic solvent is a mixed solution of isopropanol and dimethylformamide in a volume ratio of 1:1, dimethylformamide or 2-(7-azobenzotriazole)-tetramethyluronium hexafluorophosphate, or more, preferably a mixed solution of isopropanol and dimethylformamide in a volume ratio of 1:1.

[0019] The present invention also provides a scopoletin functionalized magnetic nanoprobe, which is prepared by the above-mentioned preparation method.

[0020] The present invention also provides the use of the above-mentioned scopoletin functionalized magnetic nanoprobe in target protein fishing and / or identification, and the scopoletin functionalized magnetic nanoprobe is used to target fish for the rheumatoid arthritis model protein group. After the fished target protein is separated by gel electrophoresis and identified by liquid phase mass spectrometry, it is verified by protein immunoblotting that the target protein of scopoletin on the HFLS-RA cell membrane is vimentin, and the specific binding of vimentin to scopoletin includes covalent binding and hydrogen bonding.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention uses photo-crosslinking reaction technology, and the photoaffinity linker generates carbenes by ultraviolet light irradiation, and cross-links various active compounds on the solid surface in a non-selective manner. This method is suitable for various natural active compound structures, does not need to be individually designed according to the characteristics of the molecular structure, is simple and fast to operate, and does not require the structure-activity relationship of the active compound to be clarified in advance, and does not affect its all-round contact with the target protein.

[0023] The present invention uses the prepared scopoletin functionalized magnetic nanoprobe to carry out target fishing in cells and animal models of rheumatoid arthritis. After separation and mass spectrometry identification of the target protein, the protein immunoblotting experiment is successfully passed, verifying that the target protein of scopoletin on the HFLS-RA cell membrane is vimentin, providing a new therapeutic target for rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The flowchart for the preparation of amino-terminally modified nanoparticles Fe3O4@SiO2-NH2;

[0025] Figure 2 The flowchart is a preparation process of scopoletin functionalized magnetic nanoprobes;

[0026] Figure 3 Optimize the sequence of photocrosslinking reaction and dehydration condensation reaction;

[0027] Figure 4 Optimization results for photocrosslinking reaction (A: selection of photoaffinity linker; B: amount of photoaffinity linker; C: physical state of reaction system; D: selection of reaction system solvent; E: reaction time);

[0028] Figure 5 Optimization results for dehydration condensation reaction (A: physical state of reaction system; B: solvent selection of reaction system);

[0029] Figure 6 The validation results of the probe preparation method are as follows: functionalized magnetic nanoprobes with different compounds (A: Rhodamine B; B: Coumarin 6);

[0030] Figure 7 Results of fishing and verification of probe targets: Celecoxib-functionalized magnetic nanoprobes (Cel-MNPs) were used to fish and verify the target proteins (silver staining and protein immunoblotting results: 1, cell membrane protein solution; 2, blank control group; 3, normal fishing group; 4, competition control group. The arrow indicates the protein COX-2 that is specifically and covalently bound to Celecoxib);

[0031] Figure 8Silver staining results of Sco-MNPs fishing and separating target proteins from RA cell model protein group (1, cell membrane protein solution; 4, blank control group; 5, normal fishing group; 6, competition control group. Arrows indicate the main protein bands that are specifically and covalently bound to scopoletin);

[0032] Fig. 9 Silver staining results of Sco-MNPs fishing and isolating target proteins from the intestinal bacterial protein group of RA animal model (1, intestinal bacterial protein solution; 2, blank control group; 3, normal fishing group; 4, competitive control group. Arrows and boxes indicate the main protein bands that are specifically and covalently bound to scopoletin);

[0033] Fig.10 The results of immunoblotting experiments for the target proteins fished by Sco-MNPs from the RA cell model protein group: 1, cell membrane protein solution; 2, blank control group; 3, normal fishing group; 4, competition control group). DETAILED DESCRIPTION

[0034] The present invention is further described below by means of specific implementation methods. The following examples are preferred implementation methods of the present invention, but the implementation methods of the present invention are not limited to the following examples.

[0035] Example 1: Preparation of Scopoletin Functionalized Magnetic Nanoprobe

[0036] 20mmol of ferric chloride hexahydrate (FeCl3·6H2O), 0.05mmol of polyvinyl pyrrolidone (PVP) and 40mmol of sodium acetate (NaOAc) were dispersed in 20mL of ethylene glycol (EG), sealed in a polytetrafluoroethylene-lined stainless steel autoclave and reacted at 200℃ for 10h to obtain Fe3O4 nanoparticles (Fe3O4MNPs);

[0037] 50 mg of Fe3O4 MNPs were dispersed in 50 mL of ethanol / water / ammonia solution (45:5:1, v / v), 0.1 mmol of tetraethyl orthosilicate (TEOS) was added, and ultrasonic reaction was performed for 1 h to obtain Fe3O4@SiO2 nanoparticles (Fe3O4@SiO2 MNPs);

[0038] Using 45 mL of ethanol as solvent, 5 mL of aminopropyltriethoxysilane (APTES) was added to 50 mg of nanoparticles, and refluxed for 2 h. The product was separated by a magnet, washed with deionized water, and dried to obtain amino-terminally modified nanoparticles Fe3O4@Si02-NH2. The preparation process is as follows: Figure 1 As shown;

[0039] 1 μmol of 4-[3-(trifluoromethyl)-3H-diaziridin-3-yl]benzoic acid (TDBA) and 0.25 μmol of scopoletin were mixed and fully dissolved in 1 mL of anhydrous methanol, spread flat in a sealed glass container, and irradiated with 365 nm ultraviolet light at room temperature for 15 minutes at a distance of 1 cm. After the reaction was completed, the solvent was removed by rotary drying to obtain TDBA-scopoletin coupling product (TDBA-Sco);

[0040] TDBA-Sco was redissolved in 1 mL of isopropanol / dimethylformamide (1:1 volume ratio), and 1.6 μL of triethylamine was added, and 10 mg of NH2-MNPs was added. After mixing, the mixture was shaken overnight in the dark. After the reaction was completed, the product was separated by a magnetic magnet and washed with methanol to obtain scopoletin functionalized magnetic nanoprobes (Sco-MNPs). The preparation process is as follows: Figure 2 shown.

[0041] Example 2: Optimization of process conditions for preparing scopoletin functionalized magnetic nanoprobes

[0042] The preparation of scopolamine functionalized magnetic nanoprobes includes two main steps: coupling of photoaffinity linkers with scopolamine through photocrosslinking reaction, and coupling of amino groups at the end of MNPs with carboxyl groups of photoaffinity linkers through dehydration condensation reaction. Many factors will affect the coupling rate of scopolamine on the probe surface, thereby affecting the fishing effect of the final probe product. The main factors include: 1) the order of the two coupling reactions; 2) the selection and dosage of photoaffinity linkers, the physical state of the reaction system, the choice of solvent, and the reaction time during the photocrosslinking reaction; 3) the physical state of the reaction system and the choice of solvent during the dehydration condensation reaction.

[0043] The present invention optimizes and screens the above factors, and the specific process is as follows:

[0044] (1) Optimization of the sequence of two coupled reactions

[0045] Sample 1: First do the photocrosslinking reaction, then do the dehydration condensation reaction

[0046] 1 μmol 4-[3-(trifluoromethyl)-3H-bis(aziridin-3-yl)benzoic acid (TDBA) and 0.25 μmol scopoletin were mixed and fully dissolved in 1 mL of anhydrous methanol, spread flat in a sealed glass container, and irradiated with 365 nm ultraviolet light at room temperature for 15 minutes at a distance of 1 cm. After the reaction was completed, the solvent was removed by rotary drying to obtain TDBA-scopoletin coupling (TDBA-Sco). TDBA-Sco was re-dissolved in 1 mL of isopropanol / dimethylformamide (1:1 volume ratio), and 1.6 μL of triethylamine was dropped, and 10 mg of NH2-MNPs was added. After mixing, the mixture was shaken overnight in the dark. After the reaction was completed, the product was separated by magnet and washed with methanol to obtain scopoletin functionalized magnetic nanoprobes (Sco-MNPs).

[0047] Sample 2: Dehydration condensation reaction first, then photocrosslinking reaction

[0048] 4-[3-(Trifluoromethyl)-3H-diaziridin-3-yl]benzoic acid (TDBA) was dissolved in 1 mL of isopropanol / dimethylformamide (1:1 volume ratio), and 1.6 μL of triethylamine was added, and 10 mg of NH2-MNPs was added. After mixing, the product was separated by magnet and washed with methanol to obtain a blank probe (TDBA-MNPs). A blank probe containing 1 μmol of TDBA and 0.25 μmol of scopoletin were mixed and fully dissolved in 1 mL of anhydrous methanol, spread flat in a sealed glass container, and irradiated with 365 nm ultraviolet light at room temperature for 15 minutes at a distance of 1 cm. After the reaction was completed, the product was separated by magnet and washed with methanol to obtain scopoletin functionalized magnetic nanoprobes (Sco-MNPs).

[0049] For sample 1 and sample 2, after the second reaction was completed, the concentration of scopoletin in the supernatant was determined by LC-MS; this concentration was compared with the initial concentration of scopoletin to obtain the coupling rate of scopoletin on the probe surface. Figure 3 As shown, the scopoletin coupling rate of sample 1 is significantly higher than that of sample 2, so it is preferred to perform the photocrosslinking reaction first and then the dehydration condensation reaction.

[0050] (2) Optimization of photocrosslinking reaction

[0051] For each sample in this step, the ion peak area of ​​scopoletin in the supernatant before and after the reaction is determined by liquid chromatography-mass spectrometry; the two peak areas are subtracted and divided by the peak area before the reaction to obtain the coupling rate of scopoletin and the photoaffinity linker.

[0052] 2.1 Optimizing the selection of photoaffinity linkers:

[0053] Three 1 μmol photoaffinity linkers were mixed with 0.25 μmol scopoletin and fully dissolved in 1 mL of anhydrous methanol. The three photoaffinity linkers were 4-[3-(trifluoromethyl)-3H-diaziridin-3-yl]benzoic acid (TDBA), 3-(4-bromophenyl)-3-(trifluoromethyl)-3H-diaziridin (SDAD), and 3-methyl-3H-diaziridin-3-propionic acid (MPDA). The mixture was spread in a sealed glass container and irradiated with 365 nm ultraviolet light at room temperature for 15 minutes at a distance of 1 cm. The results are shown in Figure 2. Figure 4 As shown in A, three different photoaffinity linkers were used, and the coupling rates with scopoletin were different, from high to low, TDBA>SDAD>MPDA. Therefore, TDBA was the preferred optimal photoaffinity linker.

[0054] 2.2 Optimize the amount of photoaffinity linker:

[0055] 0.25, 1, 2.5 μmol TDBA and 0.25 μmol scopoletin were mixed and fully dissolved in 1 mL of anhydrous methanol, spread flat in a sealed glass container, and irradiated with 365 nm ultraviolet light at room temperature for 15 minutes at a distance of 1 cm. Figure 4 As shown in B, when the amount of the photoaffinity linker is less than or equal to 1 μmol, the coupling rate of scopoletin increases with the increase of the amount of the photoaffinity linker; but when the amount exceeds 1 μmol, the coupling rate of scopoletin decreases significantly with the increase of the amount of the photoaffinity linker. Therefore, the optimal amount of the photoaffinity linker is preferably 1 μmol.

[0056] 2.3 Optimize the physical state of the reaction system:

[0057] 1 μmol TDBA and 0.25 μmol scopoletin were mixed and fully dissolved in 1 mL of anhydrous methanol, spread flat in a sealed glass container, vacuum dried, nitrogen blow dried, and not dried in the solvent, and irradiated with 365 nm ultraviolet light at room temperature for 15 minutes at a distance of 1 cm. The results are shown in Figure 2. Figure 4 As shown in C, the coupling rates of the photoaffinity linker and scopoletin are different when using different drying methods, from high to low, no drying in the solvent > nitrogen blowing drying > vacuum drying. Therefore, the optimal physical state of the preferred reaction system is dissolved in the solution.

[0058] 2.4 Optimize the solvent selection of the reaction system:

[0059] 1 μmol TDBA and 0.25 μmol scopoletin were mixed and fully dissolved in 1 mL of anhydrous methanol, dichloromethane, and carbon tetrachloride, respectively, spread flat in a sealed glass container, and irradiated with 365 nm ultraviolet light at room temperature for 15 minutes at a distance of 1 cm. Figure 4 As shown in D, when using different solvents, the coupling rates of the photoaffinity linker and scopoletin are different, from high to low, anhydrous methanol > carbon tetrachloride > dichloromethane. Therefore, the best solvent for the preferred reaction system is anhydrous methanol.

[0060] 2.5 Optimize reaction time:

[0061] 1 μmol TDBA and 0.25 μmol scopoletin were mixed and fully dissolved in 1 mL of anhydrous methanol, spread flat in a sealed glass container, and irradiated with 365 nm ultraviolet light at room temperature for 15, 40, and 60 minutes at a distance of 1 cm. Figure 4 As shown in E, after 15 minutes, the coupling rate between the photoaffinity linker and scopoletin did not increase significantly with time. Therefore, the optimal reaction time was preferably 15 minutes.

[0062] (3) Optimization of dehydration condensation reaction

[0063] For each sample in this step, the ion peak area of ​​TDBA-scopoletin coupling product in the supernatant before and after the reaction was determined by liquid chromatography-mass spectrometry; the two peak areas were subtracted and divided by the peak area before the reaction to obtain the coupling rate between the coupling product and NH2-MNPs.

[0064] 3.1 Optimize the physical state of the reaction system:

[0065] 1 μmol TDBA and 0.25 μmol scopoletin were mixed and fully dissolved in 1 mL of anhydrous methanol, spread flat in a sealed glass container, and irradiated with 365 nm ultraviolet light at room temperature for 15 minutes at a distance of 1 cm. After the reaction was completed, the solvent was removed by rotary drying to obtain TDBA-scopoletin coupling product (TDBA-Sco). TDBA-Sco was re-dissolved with 1 mL of isopropanol / dimethylformamide (1:1 volume ratio) or vacuum dried, and then 1.6 μL of triethylamine was dropped, and 10 mg of NH2-MNPs was added. After mixing, it was shaken overnight in the dark. The results are as follows Figure 5 As shown in Figure A, the TDBA-Sco coupling rate of the sample in the solvent is significantly higher than that of the dry sample. Therefore, the optimal physical state of the preferred reaction system is dissolved in the solution.

[0066] 3.2 Optimize the solvent selection of the reaction system:

[0067] 1 μmol TDBA and 0.25 μmol scopoletin were mixed and fully dissolved in 1 mL of anhydrous methanol, spread flat in a sealed glass container, and irradiated with 365 nm ultraviolet light at room temperature for 15 minutes at a distance of 1 cm. After the reaction was completed, the solvent was removed by rotary drying to obtain TDBA-scopoletin coupling product (TDBA-Sco). TDBA-Sco was re-dissolved with 1 mL of isopropanol / dimethylformamide (IPA / DMF; 1:1 volume ratio), dimethylformamide (DMF), and 2-(7-azobenzotriazole)-tetramethyluronium hexafluorophosphate (HATU), and 1.6 μL of triethylamine was dropped, and 10 mg of NH2-MNPs was added. After mixing, it was shaken overnight in the dark. The results are as follows Figure 5 As shown in B, using different solvents, the coupling rates of TDBA-Sco and NH2-MNPs are different, from high to low, IPA / DMF>DMF>HATU. Therefore, the optimal solvent for the preferred reaction system is IPA / DMF (1:1 volume ratio).

[0068] Example 3: Validation of the probe preparation method

[0069] The preparation method was the same as that in Example 1, except that scopoletin was replaced with rhodamine B (red fluorescent dye) and coumarin 6 (green fluorescent dye) to prepare rhodamine B and coumarin 6 functionalized magnetic nanoprobes (Rho-MNPs and Cou-MNPs), respectively.

[0070] The microscopic observation results of Rho-MNPs and Cou-MNPs are shown in Figure 2. Figure 6 As shown. The product is quasi-circular, relatively uniform in size, and well dispersed, indicating that the physical properties of the probe product are satisfactory. The nanoparticles show uniform red light and green light under fluorescence, respectively, indicating that rhodamine B and coumarin 6 are uniformly coupled to the surface of the magnetic nanoprobe, and both compounds have a high coupling rate. Using a unified operating procedure, functionalized magnetic nanoprobes of different compounds were prepared, and obvious coupling effects were obtained, verifying that the probe preparation method of the present invention is feasible.

[0071] Example 4: Probe target fishing verification: Celecoxib functionalized magnetic nanoprobe fishing and verification of target protein

[0072] Prepare fishing buffer: add 50mM Tris (pH 7.6), 150mM NaCl, 2mM CaCl2, and 2mM MgCl2 to deionized water.

[0073] Prepare elution buffer: add 0.3% TritonX-100 to the fishing buffer.

[0074] Prepare electrophoresis buffer: 1 mL deionized water, 1 mL 0.5 M Tris-Cl (pH 6.8), 1 mL glycerol, 1 mL 10% SDS, 0.1 mL mercaptoethanol, and 0.2 mL 0.05% bromophenol blue.

[0075] (1) Preparation of celecoxib-functionalized magnetic nanoprobes

[0076] The preparation method is the same as that of Example 1, except that scopoletin is replaced with celecoxib (a COX-2 selective inhibitor) to prepare celecoxib functionalized magnetic nanoprobes (Cel-MNPs).

[0077] (2) Preparation of cell membrane protein solution:

[0078] HFLS-RA cells were taken, cell membrane proteins were extracted, and the concentration was adjusted to 3 mg / mL with fishing buffer.

[0079] (3) Prepare blank control group samples:

[0080] 0.5 μL DMSO was added to 250 μL cell membrane protein solution and incubated at 4°C for 1 h. Then 2.5 mg TDBA-MNPs (10 mg / mL, suspended in fishing buffer) was added and incubated overnight at 4°C with shaking. After incubation, the probes were separated and collected by magnet, washed three times with elution buffer, 100 μL electrophoresis buffer was added, boiled at 100°C for 5 minutes, and cooled to room temperature.

[0081] (4) Make a normal fishing group sample:

[0082] 0.5 μL DMSO was added to 250 μL cell membrane protein solution and incubated at 4°C for 1 h. Then 2.5 mg Cel-MNPs (10 mg / mL, suspended in fishing buffer) were added and incubated overnight at 4°C with shaking. After incubation, the probes were collected by magnetic separation, washed three times with elution buffer, 100 μL electrophoresis buffer was added, boiled at 100°C for 5 minutes, and cooled to room temperature.

[0083] (5) Preparation of competitive control group samples:

[0084] 0.5 μL of DMSO solution containing 10 mM celecoxib was added to 250 μL of cell membrane protein solution and incubated at 4°C for 1 h. Then 2.5 mg of Sco-MNPs (10 mg / mL, suspended in fishing buffer) was added and incubated overnight at 4°C with shaking. After incubation, the probes were collected by magnetic separation, washed three times with elution buffer, 100 μL of electrophoresis buffer was added, boiled at 100°C for 5 minutes, and cooled to room temperature.

[0085] (6) Target protein separation, identification and verification:

[0086] After boiling and cooling, the samples of each group were separated by gel electrophoresis to observe the difference of bands. Differential protein bands were cut, decolorized, dehydrated, alkylated, and enzymatically hydrolyzed to extract peptides; after peptides were purified, they were detected by liquid chromatography-mass spectrometry. The protein structure was inferred through database retrieval and comparison. The proteins in the bands were verified by western blotting.

[0087] Fishing and verification results are as follows Figure 7 As shown. There are many proteins in the cell membrane protein solution (lane 1), and the blank control group did not fish out obvious bands, indicating that the blank probe had no obvious non-specific binding with the protein group (lane 2). Comparing the normal fishing group with the blank control group, the normal fishing group had significantly more bands and darker colors, indicating that the coupled celecoxib on the probe had obvious specific binding with the protein, including hydrogen bonding and covalent bonding (lane 3). In the competitive control group, since part of the target protein had already undergone a relatively stable covalent bonding with the free celecoxib before fishing, the remaining part of the target protein could only be hydrogen bonded with the coupled celecoxib on the probe and fished out (lane 4). Comparing the normal fishing group with the competitive control group, it can be seen that the normal fishing group has a relatively obvious differential protein band at about 74kDa, indicating that the protein can be covalently bound to celecoxib. The results of protein immunoblotting proved that the protein was COX-2, which is the selective inhibition target of celecoxib. The above results show that the method of fishing for target proteins using compound functionalized probes of the present invention is feasible.

[0088] Example 5: Scopolamine functionalized magnetic nanoprobes (Sco-MNPs) fishing and isolating target proteins from the RA cell model proteome

[0089] (1) Prepare blank control group, normal fishing group, and competitive control group samples:

[0090] The method for preparing the cell membrane protein solution and each group of samples is the same as that in Example 4.

[0091] (2) Sample protein separation:

[0092] After boiling and cooling, the samples of each group were separated by gel electrophoresis and the bands were observed.

[0093] Observation results such as Figure 8As shown. There are many proteins in the cell membrane protein solution (lane 1), and the blank control group did not fish out obvious bands (lane 4). Comparing the normal fishing group with the blank control group, the bands in the normal fishing group are obvious, indicating that scopoletin has obvious specific binding with the protein group (lane 5). Comparing the normal fishing group with the competitive control group (lane 6), it can be seen that the normal fishing group has obvious differential protein bands at about 57, 50, and 40 kDa, among which the band at about 57 kDa is the most prominent, indicating that these proteins are the target proteins of scopoletin and can be covalently bound to scopoletin.

[0094] Example 6: Scopoletin functionalized magnetic nanoprobes (Sco-MNPs) fishing and isolating target proteins from the proteome of RA animal models

[0095] (1) Preparation of intestinal bacterial protein solution:

[0096] Male SD rats were subcutaneously injected with 0.1 mL of complete Freund's adjuvant at the base of the tail to induce adjuvant arthritis. Rats were killed 21 days after modeling and fresh feces were collected. 10 g of feces was added to 40 mL of PBS for thorough homogenization, centrifuged at 700 g for 1 min to obtain the supernatant, and then centrifuged at 6000 g for 3 min to obtain the precipitate, washed twice with 10 mL of PBS, added to 250 mL of anaerobic culture medium, and incubated in an anaerobic bag at 37 ° C for 12 h. After the incubation was completed, the precipitate was centrifuged at 4000 g for 15 min and suspended in 1 mL of PBS to obtain the intestinal bacteria culture solution. The intestinal bacteria culture solution was homogenized with HEPES lysis buffer, centrifuged at 4 ° C 700 g for 5 min to obtain the supernatant, ultrafiltered to obtain the upper filtrate, and washed with lysis buffer to obtain the intestinal bacteria protein solution.

[0097] (2) Prepare blank control group, normal fishing group, and competitive control group samples:

[0098] The method is the same as that of Example 4, except that the cell membrane protein solution is replaced with an intestinal bacteria protein solution.

[0099] (3) Sample protein separation:

[0100] After boiling and cooling, the samples of each group were separated by gel electrophoresis and the bands were observed.

[0101] Observation results such as Fig. 9As shown. There are many proteins in the cell membrane protein solution (lane 1), and the blank control group did not fish out obvious bands (lane 2). Comparing the normal fishing group with the blank control group, the bands in the normal fishing group are obvious, indicating that scopoletin has obvious specific binding with the protein group (lane 3). Comparing the normal fishing group with the competitive control group (lane 4), it can be seen that the normal fishing group has obvious differential protein bands at about 66, 50-20 kDa, among which the band at about 57 kDa is the most prominent, indicating that these proteins are the target proteins of scopoletin and can be covalently bound to scopoletin.

[0102] Example 7: Confirmation of the target proteins angling from the RA cell model proteome by Sco-MNPs

[0103] According to the results of Example 5, the differential protein band at about 57 kDa was cut. The cut protein band was decolorized, dehydrated, alkylated, and enzymatically hydrolyzed in sequence to extract peptides; after the peptides were purified, they were detected by liquid chromatography-mass spectrometry. After database retrieval and comparison, it was inferred that the protein was vimentin.

[0104] Western blotting results Fig.10 As shown, the band at about 57 kDa was confirmed to be vimentin. Vimentin was visible in the cell membrane protein solution (lane 1), the normal fishing group (lane 3), and the competitive control group (lane 4), while the blank fishing group (lane 2) did not fish out the protein. Among them, the band in the normal fishing group (lane 3) was significantly thicker and thicker than that in the competitive control group (lane 4), indicating that vimentin can specifically bind to scopoletin, including both hydrogen bonding and covalent bonding.

Claims

1. A method for preparing a scopoletin functionalized magnetic nanoprobe, characterized in that: The steps include: (1) Dispersing ferric chloride hexahydrate, polyvinyl pyrrolidone and sodium acetate in ethylene glycol to react, thereby obtaining Fe3O4 nanoparticles; and ultrasonically reacting the Fe3O4 nanoparticles with tetraethyl orthosilicate to obtain Fe3O4@SiO2 nanoparticles; (2) Using ethanol as solvent, aminopropyltriethoxysilane was added to Fe3O4@SiO2 nanoparticles, and refluxed to obtain amino-terminally modified nanoparticles Fe3O4@SiO2-NH2; (3) mixing a photoaffinity linker and scopolamine and fully dissolving them in an organic solvent, irradiating them with 365 nm ultraviolet light at room temperature, and after the reaction is completed, rotating and drying to remove the solvent to obtain a photoaffinity linker-scopolamine coupling product; the photoaffinity linker is 4-[3-(trifluoromethyl)-3H-diaziridin-3-yl]benzoic acid; the molar ratio of the photoaffinity linker to scopolamine is 1-4:1; and the organic solvent is anhydrous methanol; (4) The photoaffinity linker-scopolamine coupling product is re-dissolved in an organic solvent, and triethylamine is added dropwise, and the amino-terminally modified nanoparticles Fe3O4@SiO2-NH2 are added. After mixing, the mixture is shaken in the dark overnight. After the reaction is completed, the product is separated by a magnet and washed with methanol to obtain a scopolamine functionalized magnetic nanoprobe; the organic solvent is a mixed solution of isopropanol and dimethylformamide in a volume ratio of 1:

1.

2. The method for preparing the scopoletin functionalized magnetic nanoprobe according to claim 1, characterized in that: In step (3), the ultraviolet light irradiation time at room temperature is 15-60 minutes.

3. A scopoletin functionalized magnetic nanoprobe prepared by the preparation method according to any one of claims 1-2.

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