A C-reactive protein thin film solid-phase extraction column and its preparation method and application

Abstract

The present invention discloses a preparation method of a C-reactive protein thin film solid phase extraction column and its preparation method and application. The C-reactive protein thin film solid phase extraction column prepared by the present invention has many advantages compared with traditional affinity chromatography: it has higher non-specific adsorption, does not introduce impurities, has simple steps, and high recovery rate. The C-reactive protein thin film solid phase extraction column prepared by the present invention can also be applied to the purification of CRP in actual samples such as human serum and mouse plasma to obtain high-purity CRP with complete structure and function.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and particularly relates to a C-reactive protein thin film solid-phase extraction column and its preparation method and application. Background Art

[0002] CRP (C-reactive protein) is a homologous pentameric protein belonging to the pentraxin family. It is produced in liver hepatocytes, smooth muscle cells, macrophages, and adipocytes. When various infectious diseases occur, tissue damage substances and inflammatory substances enter the blood circulation in large quantities, and through the mediation of corresponding cytokines, they stimulate the liver to synthesize CRP, resulting in an increase in CRP in the body's response. Therefore, CRP protein is used as a biomolecule for inflammatory biomarkers [Hirschfield G M, Smith M D, Ley S V, et al. Clinical Science, 2003, 104(s49):65P - 66P]. The content of CRP protein in a normal body is relatively low and can be found in blood (103–104 ng / mL) [Okamura J M, Miyagi J M, Terada K, et al. Journal of clinical laboratory analysis, 1990, 4(3):231 - 235] and saliva, but the content is much lower (102 to 103 times less) [Floriano P N, Christodoulides N, Miller C S, et al. Clinical chemistry, 2009, 55(8):1530 - 1538. Ouellet-Morin I, Danese A, Williams B, et al. Brain, Behavior, and Immunity, 2011, 25(4):640 - 646.]. So far, CRP is closely related to the pathological processes of various diseases such as cardiovascular diseases, neonatal sepsis in children, and Alzheimer's disease.

[0003] Under physiological conditions, the average serum concentration of CRP is 0.8 mg / L. In normal, healthy individuals, the baseline level of CRP ranges from 1 to 5 mg / L [Pepys M B, Hirschfield G M. The Journal of clinical investigation, 2003, 111(12):1805 - 1812.]. However, in the case of inflammation, CRP levels can increase sharply up to 10,000 - fold within 24 - 72 hours [Clyne B, Olshaker J S. The Journal of emergency medicine, 1999, 17(6):1019 - 1025.]. Once secreted, the half - life of CRP is approximately 19 hours. Given that the clearance of CRP follows first - order kinetics, the serum level of CRP is mainly due to the result of any ongoing hepatic synthesis caused by inflammation. These properties make CRP an ideal marker for tissue inflammation and infection and have led to the widespread adoption of CRP testing in clinical medicine. [McFadyen J D, Zeller J, Potempa L A, et al. Vertebrate and invertebrate respiratory proteins, lipoproteins and other body fluid proteins, 2020:499 - 520.].

[0004] The ability of CRP to trigger an inflammatory response is structurally related to each protomer containing a binding face and an effector face. The binding face contains a phosphocholine binding site that facilitates calcium-dependent interactions with PC exposed on the membranes of apoptotic and inflamed cells and bacterial cell walls [Thompson D, Pepys M B, Wood S P. Structure, 1999, 7(2):169-177.]. Co-crystallization and site-directed mutagenesis studies of CRP with PC have shown that Phe-66 and Glu-81, located in a hydrophobic pocket, are two key residues mediating the binding of PC to CRP [Agrawal A, Simpson M J, Black S, et al. The Journal of Immunology, 2002, 169(6):3217-3222. Black S, Agrawal A, Samols D. Molecular Immunology, 2003, 39(16):1045-1054.]. The exposed surface of the Phe-66 residue within the hydrophobic pocket provides hydrophobic interactions with the methyl group of PC. The Glu-81 residue is located at the other end of the hydrophobic pocket, where it interacts with the positively charged N. These residues are highly conserved among a large number of species, emphasizing the importance of these residues in mediating the interaction between PC and CRP [Shrive A K, Gheetham G M T, Holden D, et al. Nature structural biology, 1996, 3(4):346-354.].

[0005] To more deeply study the mechanism of the interaction between CRP and diseases, more precisely analyze the signaling pathway, it is extremely important to obtain highly pure CRP with complete structure and function. Methods reported for purifying CRP include starch block preparative electrophoresis, affinity chromatography, thin-film molecularly imprinted polymers (MIP), lecithin precipitation, negative affinity filtration chromatography, cell membrane biomimetic polymers. Among them, the commonly used method for purifying human CRP is affinity chromatography. Currently, the main method for purifying CRP is Ca 2+Affinity chromatography using phospholipid materials as ligands [Soler L, Garcia N, Unzueta A, et al. Veterinary immunology and immunopathology, 2016, 179: 26-31.], but these methods usually face the problem of non-specific adsorption. For example, introducing additional elution steps (ion exchange chromatography, size exclusion chromatography) will lead to a decrease in recovery rate or the introduction of new impurities. Therefore, it is imperative to develop new purification materials to address the challenges of CRP purification [Gisladottir B, Gudmundsdottir S, Brown L, et al. Fish & Shellfish Immunology, 2009, 26(2): 210-219.]. Summary of the Invention

[0006] In order to overcome the deficiencies and drawbacks of the prior art, the primary object of the present invention is to provide a method for preparing a C-reactive protein (CRP) thin film solid-phase extraction column.

[0007] The second object of the present invention is to provide a C-reactive protein thin film solid-phase extraction column prepared by the above preparation method.

[0008] The third object of the present invention is to provide the application of the above C-reactive protein thin film solid-phase extraction column in the purification of C-reactive protein.

[0009] The primary object of the present invention is achieved by the following technical solutions:

[0010] A method for preparing a C-reactive protein thin film solid-phase extraction column, comprising the following steps:

[0011] S1. Prepare the first hydrophilic monomer, crosslinking agent, initiator, ligand complex, reducing agent and solvent according to the mass-volume ratio of 53.65 - 300.44 mg: 0.03 - 0.3 ml: 50 μL: 250 μL: 18.53 - 103.75 mg: 10 mL. Immerse the polypropylene film in the mixed solution of the first hydrophilic monomer, crosslinking agent, initiator, ligand complex, reducing agent and solvent, and carry out a polymerization reaction at room temperature under closed and nitrogen protection. Modify the polypropylene film through atom transfer radical reaction to obtain film product I;

[0012] S2. Prepare the second hydrophilic monomer, ligand complex, reducing agent and solvent according to the mass-volume ratio of 148.5 - 445.5 mg: 250 μL: 25.02 - 75.05 mg: 2 mL. Immerse film product I in the mixed solution of the second hydrophilic monomer, ligand complex, reducing agent and solvent, and carry out a polymerization reaction at room temperature under closed and nitrogen protection. Modify the functional monomer through atom transfer radical polymerization to obtain film product II;

[0013] S3. Configure a second hydrophilic monomer, ligand complex, reducing agent and solvent with a mass volume ratio of 240 - 720 mg: 50 μL: 6.6 - 19.8 mg: 2.8 mL. Immerse the film product II into the mixed solution of the second hydrophilic monomer, ligand complex, reducing agent and solvent, seal it and protect it with nitrogen, and carry out a polymerization reaction at a temperature of 30 - 70 °C. Modify the functional monomer again through atom transfer radical polymerization to obtain the film product III;

[0014] S4. Tightly curl the film product III and insert it into a syringe, and use a push rod to insert it to the bottom to obtain a film column, that is, the film solid-phase extraction column for C-reactive protein.

[0015] Wherein, after the polymerization reaction is completed, rinse with ethanol to remove the ligand complex, unreacted monomers, crosslinking agent, initiator and reducing agent.

[0016] Preferably, the first hydrophilic monomer is 2-hydroxyethyl methacrylate monomer (HEMA), and the structure of the HEMA is as follows:

[0017]

[0018] The crosslinking agent is N,N'-methylenebisacrylamide (MBA);

[0019] The initiator is ethyl 2-bromoisobutyrate;

[0020] The ligand complex is prepared from copper bromide, N,N,N',N,'N”-pentamethyldiethylenetriamine and N,N-dimethylformamide according to a mass ratio of 0.068:2.5:99.35;

[0021] The reducing agent is L-ascorbic acid;

[0022] The solvent is N,N-dimethylformamide (DMF) or methanol-water with a mass fraction of 20%;

[0023] The second hydrophilic monomer is a phosphorylcholine functional monomer.

[0024] Preferably, the polymerization reaction time in step S1 is 1 - 8 h; the polymerization reaction time in step S2 is 4 - 8 h; the polymerization reaction time in step S3 is 4 - 8 h.

[0025] Preferably, the polymerization reaction time in step S1 is 2 - 4 h.

[0026] Preferably, the optimal polymerization reaction time in steps S2 and S3 is 4 h.

[0027] Preferably, the molar concentration of the first hydrophilic monomer is 0.04 - 0.22 mmol / L.

[0028] Preferably, the molar concentration of the first hydrophilic monomer is 0.14 mmol / mL.

[0029] Preferably, in step S1, the mass of the reducing agent L-ascorbic acid is 34.53% of the mass of the first hydrophilic monomer.

[0030] Preferably, the molar concentration of the second hydrophilic monomer is 0.04 - 0.08 mmol / L.

[0031] Preferably, the phosphorylcholine functional monomer is 2-methacryloyloxyethyl phosphorylcholine (MPC); the structure of the MPC is as follows:

[0032]

[0033] Preferably, the molar ratio of the first hydrophilic monomer to the crosslinking agent is 3:1, 2:1, 1:1, 1:2 or 1:3.

[0034] Preferably, the optimal molar ratio of the first hydrophilic monomer to the crosslinking agent is 1:1.

[0035] Preferably, in step S2, the mass of the reducing agent L-ascorbic acid is 16.85% of the mass of the second hydrophilic monomer.

[0036] Preferably, in step S3, the mass of the reducing agent L-ascorbic acid is 2.75% of the mass of the second hydrophilic monomer.

[0037] Preferably, in steps S2 and S3, the concentration of the second hydrophilic monomer MPC is 0.04 - 0.12 mmol / mL.

[0038] Preferably, in steps S2 and S3, the optimal concentration of the second hydrophilic monomer MPC is 0.08 mmol / mL.

[0039] The second object of the present invention is achieved by the following technical solutions:

[0040] A thin film solid phase extraction column for C-reactive protein prepared by the above preparation method.

[0041] Preferably, the thin film solid phase extraction column for C-reactive protein sequentially includes a PP base film, a HEMA polymerization layer, a MPC polymerization layer 1, and a MPC polymerization layer 2 from inside to outside.

[0042] The third object of the present invention is achieved by the following technical solutions:

[0043] An application of a thin film solid phase extraction column for C-reactive protein in the purification of C-reactive protein.

[0044] The advantages and beneficial effects of the present invention compared with the prior art are as follows:

[0045] (1) The present invention reports for the first time a new method based on purified CRP, the thin-film solid-phase extraction coil column of C-reactive protein.

[0046] (2) The thin-film solid-phase extraction coil column of C-reactive protein prepared by the present invention intersects with the traditional affinity chromatography, and it exhibits many advantages: having higher non-specific adsorption, not introducing impurities, simple steps, and high recovery rate.

[0047] (3) The thin-film solid-phase extraction coil column of C-reactive protein prepared by the present invention can be applied to the purification of CRP in actual samples such as human serum and mouse plasma, and high-purity CRP with complete structure and function can be obtained. Description of the Drawings

[0048] Figure 1 is the preparation flow chart of the thin-film solid-phase extraction coil column of C-reactive protein;

[0049] Figure 2 is the scanning electron microscope image of the surface morphology of the PP-(HEMA) membrane material obtained in Example 1;

[0050] Figure 3 is the scanning electron microscope image of the surface morphology of the PP-(HEMA-co-MPC) membrane material obtained in Example 1;

[0051] Figure 4 is the scanning electron microscope image of the surface morphology of the PP-(HEMA-co-MPC-MPC) membrane material obtained in Example 1;

[0052] Figure 5 is the gel electrophoresis detection of the PP-(HEMA-co-MPC-MPC) membrane. Detailed Embodiments

[0053] The present invention will be further described in detail below in conjunction with the embodiments and the drawings, but the embodiments of the present invention are not limited thereto.

[0054] Example 1

[0055] As Figure 1 shown is the preparation flow chart of the thin-film solid-phase extraction coil column of C-reactive protein, and the specific preparation steps are as follows:

[0056] a. Place the glass plate A in the culture medium. With the polypropylene film using the glass plate as the substrate, drop and immerse a mixed solution of 0.18 mL (193.14 mg) of functional monomer No. 1 hydrophilic monomer (2-hydroxyethyl methacrylate (HEMA)), 0.10 mL of crosslinking agent (N,N'-methylenebisacrylamide (MBA)), 50 μL of ethyl 2-bromoisobutyrate, 250 μL of ligand complex (composed of 66.7 mg of copper bromide, 618 μL of N,N,N',N',N”-pentamethyldiethylenetriamine (PMDETA) and 10 mL of N,N-dimethylformamide) and 66.7 mg of reducing agent (L-ascorbic acid) on the polypropylene film. Press the film with the glass plate B, purge the culture medium with nitrogen for 10 min, and react it hermetically at room temperature for 4 h to obtain the film product I, namely the PP-(HEMA) film;

[0057] b. On the surface of the film product I, add 297 mg of hydrophilic monomer No. 2 (2-methacryloyloxyethyl phosphorylcholine (MPC)), 2 mL of solvent (N,N-dimethylformamide), 250 μL of ligand complex (composed of 66.7 mg of copper bromide, 618 μL of N,N,N',N',N”-pentamethyldiethylenetriamine (PMDETA) and 10 mL of N,N-dimethylformamide), mix and stir for 5 min, purge with nitrogen for 10 min, immediately add 50.03 mg of reducing agent (ascorbic acid), shake for 30 seconds, take 3 mL and drop it into the polypropylene film, purge with nitrogen for 10 min, and react it hermetically at room temperature for 8 h to obtain the film product II, namely the PP-(HEMA-co-MPC) film;

[0058] c. Put the film product II into a beaker, then add 50 μL of ligand complex, 2.8 mL of 20% methanol-water, and 480 mg of MPC into the beaker, stir for 5 min, purge with nitrogen for 10 min, immediately add 13.2 mg of reducing agent (ascorbic acid) and shake for 30 s, purge with nitrogen again for 10 min, react hermetically at 30 °C for 4 h, wash the film with methanol and water multiple times, and dry it for standby to obtain the film product III, namely the PP-(HEMA-co-MPC-MPC) film;

[0059] d. Insert the cylindrical sieve plate of the film product III with a diameter of 0.95 cm and a height of 0.2 cm horizontally at the bottom of a 1 mL syringe. Start curling from one side of the long strip-shaped film, roll it into a cylinder, and ensure tight contact between layers. Insert it into the lumen of the 1 mL syringe, and use an injection push rod to push it to the bottom of the syringe to complete the preparation of the C-reactive protein film solid-phase extraction column.

[0060] Figures 2 to 4Scanning electron micrographs of the surface morphologies of the PP-(HEMA) membrane, PP-(HEMA-co-MPC) membrane, and PP-(HEMA-co-MPC-MPC) membrane materials obtained in this example, respectively.

[0061] Example 2

[0062] As Figure 1 shown is the flow chart for the preparation of a thin film solid phase extraction column for C-reactive protein. The specific preparation steps are as follows:

[0063] a. Place glass plate A in the culture medium. Using the polypropylene film with the glass plate as the substrate, add a mixed solution of 0.18 mL (193.14 mg) of functional monomer No. 1 hydrophilic monomer (2-hydroxyethyl methacrylate (HEMA)), 0.10 mL of crosslinking agent (N,N'-methylenebisacrylamide (MBA)), 50 μL of ethyl 2-bromoisobutyrate, 250 μL of ligand complex (composed of 66.7 mg of copper bromide, 618 μL of N,N,N',N',N''-pentamethyldiethylenetriamine (PMDETA), and 10 mL of N,N-dimethylformamide), and 66.7 mg of reducing agent (L-ascorbic acid) dropwise onto the polypropylene film. Press the film with glass plate B, purge the culture medium with nitrogen for 10 min, and react it hermetically at room temperature for 2 h to obtain film product I, namely the PP-(HEMA) membrane;

[0064] b. On the surface of film product I, add 297 mg of No. 2 hydrophilic monomer (2-methacryloyloxyethyl phosphorylcholine (MPC)), 2 mL of solvent (N,N-dimethylformamide), and 250 μL of ligand complex (composed of 66.7 mg of copper bromide, 618 μL of N,N,N',N',N''-pentamethyldiethylenetriamine (PMDETA), and 10 mL of N,N-dimethylformamide), stir for 5 min, purge with nitrogen for 10 min, immediately add 50.03 mg of reducing agent (ascorbic acid), shake for 30 s, take 3 mL and drop it onto the polypropylene film, purge with nitrogen for 10 min, and react it hermetically at room temperature for 8 h to obtain film product II, namely the PP-(HEMA-co-MPC) membrane;

[0065] c. Put film product II into a beaker, then add 50 μL of ligand complex, 2.8 mL of 20% methanol-water, and 480 mg of MPC into the beaker, stir for 5 min, purge with nitrogen for 10 min, immediately add 13.2 mg of reducing agent (ascorbic acid) and shake for 30 s, purge with nitrogen again for 10 min, react it hermetically at 30 °C for 4 h, wash the film with methanol and water multiple times, and dry it for standby to obtain film product III, namely the PP-(HEMA-co-MPC-MPC) membrane;

[0066] d. Place the cylindrical sieve plate of the thin film product Ⅲ with a diameter of 0.95 cm and a height of 0.2 cm horizontally at the bottom of a 1 mL syringe. Start curling from one side of the strip-shaped thin film, roll it into a cylinder, ensure tight contact between layers, insert it into the lumen of the 1 mL syringe, and use an injection plunger to push it to the bottom of the syringe to complete the preparation of the C-reactive protein thin film solid-phase extraction column.

[0067] Example 3

[0068] As Figure 1 shown is the flow chart for the preparation of the C-reactive protein thin film solid-phase extraction column. The specific preparation steps are as follows:

[0069] a. Place glass plate A in the culture medium. Using the polypropylene film as the substrate, add a mixed solution of 0.18 mL (193.14 mg) of functional monomer No. 1 hydrophilic monomer (2-hydroxyethyl methacrylate (HEMA)), 0.10 mL of cross-linking agent (N,N'-methylenebisacrylamide (MBA)), 50 μL of ethyl 2-bromoisobutyrate, 250 μL of ligand complex (composed of 66.7 mg of copper bromide, 618 μL of N,N,N',N',N''-pentamethyldiethylenetriamine (PMDETA) and 10 mL of N,N-dimethylformamide) and 66.7 mg of reducing agent (L-ascorbic acid) dropwise onto the polypropylene film. Press the film with glass plate B, purge the culture medium with nitrogen for 10 min, and react at room temperature for 2 h in a sealed state to obtain the thin film product Ⅰ, i.e., PP-(HEMA) film.

[0070] b. On the surface of the thin film product Ⅰ, add 148.5 mg (0.04 mmol / mL) of No. 2 hydrophilic monomer (2-methacryloyloxyethyl phosphorylcholine (MPC)), 2 mL of solvent (N,N-dimethylformamide), 250 μL of ligand complex (composed of 66.7 mg of copper bromide, 618 μL of N,N,N',N',N''-pentamethyldiethylenetriamine (PMDETA) and 10 mL of N,N-dimethylformamide), mix and stir for 5 min, purge with nitrogen for 10 min, immediately add 25.02 mg of reducing agent (ascorbic acid), shake for 30 seconds, take 3 mL and drop it onto the polypropylene film, purge with nitrogen for 10 min, and react at room temperature for 8 h in a sealed state to obtain the thin film product Ⅱ, i.e., PP-(HEMA-co-MPC) film.

[0071] c. Place the thin film product II into a beaker. Then add 50 μL of ligand complex, 2.8 mL of 20% methanol-water, and 240 mg (0.04 mmol / mL) of MPC into the beaker and stir for 5 min. Purge with nitrogen for 10 min. Immediately add 6.6 mg of reducing agent (ascorbic acid) and shake for 30 s. Purge with nitrogen again for 10 min. React under closed conditions at 30 °C for 4 h. Wash the thin film with methanol and water multiple times, dry it for later use, and obtain the thin film product III, namely the PP-(HEMA-co-MPC-MPC) membrane.

[0072] d. Insert the cylindrical sieve plate of the thin film product III with a diameter of 0.95 cm and a height of 0.2 cm horizontally at the bottom of a 1 mL syringe. Start curling from one side of the strip-shaped thin film, roll it into a cylinder, and ensure close contact between layers. Insert it into the lumen of the 1 mL syringe, and use a syringe plunger to push it to the bottom of the syringe to complete the preparation of the C-reactive protein thin film solid-phase extraction column.

[0073] Example 4

[0074] As Figure 1 shown in the flowchart for the preparation of the C-reactive protein thin film solid-phase extraction column, the specific preparation steps are as follows:

[0075] a. Place glass plate A in the culture medium. Using the polypropylene thin film with the glass plate as the substrate, add a mixed solution of 0.18 mL (193.14 mg) of functional monomer No. 1 hydrophilic monomer (2-hydroxyethyl methacrylate (HEMA)), 0.10 mL of cross-linking agent (N,N'-methylenebisacrylamide (MBA)), 50 μL of ethyl 2-bromoisobutyrate, 250 μL of ligand complex (composed of 66.7 mg of copper bromide, 618 μL of N,N,N',N',N''-pentamethyldiethylenetriamine (PMDETA), and 10 mL of N,N-dimethylformamide), and 66.7 mg of reducing agent (L-ascorbic acid) dropwise onto the polypropylene thin film. Press the thin film with glass plate B. Purge the culture medium with nitrogen for 10 min. React under closed conditions at room temperature for 4 h to obtain the thin film product I, namely the PP-(HEMA) membrane.

[0076] b. On the surface of the thin film product I, add 148.5 mg (0.04 mmol / mL) of the second hydrophilic monomer (2-methacryloyloxyethyl phosphorylcholine (MPC)), 2 mL of solvent (N,N-dimethylformamide), and 250 μL of ligand complex (composed of 66.7 mg of copper bromide, 618 μL of N,N,N',N',N''-pentamethyldiethylenetriamine (PMDETA), and 10 mL of N,N-dimethylformamide), mix and stir for 5 min, purge with nitrogen for 10 min, immediately add 25.02 mg of reducing agent (ascorbic acid), shake for 30 s, take 3 mL and drop it onto the polypropylene film, purge with nitrogen for 10 min, seal and react at room temperature for 8 h to obtain the thin film product II, namely the PP-(HEMA-co-MPC) film;

[0077] c. Put the thin film product II into a beaker, then add 50 μL of ligand complex, 2.8 mL of 20% methanol-water, and 240 mg (0.04 mmol / mL) of MPC into the beaker, stir for 5 min, purge with nitrogen for 10 min, immediately add 6.6 mg of reducing agent (ascorbic acid) and shake for 30 s, purge with nitrogen again for 10 min, seal and react at 30 °C for 4 h, wash the thin film with methanol and water multiple times, dry and reserve to obtain the thin film product III, namely the PP-(HEMA-co-MPC-MPC) film;

[0078] d. Insert the cylindrical sieve plate of the thin film product III with a diameter of 0.95 cm and a height of 0.2 cm into the bottom of a 1 mL syringe horizontally, start curling from one side of the strip-shaped thin film, roll it into a cylinder, ensure tight contact between layers, insert it into the lumen of the 1 mL syringe, and use an injection push rod to push it to the bottom of the syringe to complete the preparation of the C-reactive protein thin film solid-phase extraction column.

[0079] Test conditions

[0080] Test the C-reactive protein thin film solid-phase extraction column obtained in Example 1.

[0081] I. Using the PP-(HEMA-co-MPC-MPC) film column obtained in Example 1, investigate its recovery rate for CRP samples:

[0082] Adsorbents: PP-(HEMA) film, PP-(HEMA-co-MPC) film, PP-(HEMA-co-MPC-MPC) film;

[0083] Samples: 0.1 mg / mL cytochrome C;

[0084] Eluent: Buffer solution A (: Buffer solution A (composed of 10 mM tris(hydroxymethyl)aminomethane (Tris), 140 mM sodium chloride (NaCl), 50 mM calcium chloride (CaCl2 ), pH = 8.0);

[0085] Eluent: Buffer solution B (composed of 10 mM tris(hydroxymethyl)aminomethane (Tris), 140 mM sodium chloride (NaCl), 50 mM disodium ethylenediaminetetraacetate (EDTA-2Na), pH = 8.0);

[0086] Number of suction cycles: 10 times

[0087] The recovery rates of the obtained PP-(HEMA) membrane, PP-(HEMA-co-MPC) membrane, and PP-(HEMA-co-MPC-MPC) membrane are shown in Table 1. From the results in Table 1, it can be seen that the recovery rates of the thin films modified with phosphorylcholine are all > 85%, and the phosphatidylcholine functional layer has the ability to specifically capture CRP protein. Moreover, the extraction efficiency is further improved after secondary grafting, indicating that the secondary grafted phosphatidylcholine functional layer can effectively improve the capture rate of the base membrane for CRP protein.

[0088] Table 1 Influence of biomimetic phosphatidylcholine

[0089]

[0090] II. Using the PP-(HEMA-co-MPC-MPC) membrane column obtained in Example 1, investigate its stability and reusability during continuous purification of samples:

[0091] Adsorbent: PP-(HEMA-co-MPC-MPC) membrane column

[0092] Sample: 0.1 mg / mL cytochrome C, HSA, β-L actog L obuL in, MYO mixed protein solution;

[0093] Washing solution: Buffer solution A (: Buffer solution A (composed of 10 mM tris(hydroxymethyl)aminomethane (Tris), 140 mM sodium chloride (NaCl), 50 mM calcium chloride (CaCl 2 ), pH = 8.0);

[0094] Eluent: Buffer solution B (composed of 10 mM tris(hydroxymethyl)aminomethane (Tris), 140 mM sodium chloride (NaCl), 50 mM disodium ethylenediaminetetraacetate (EDTA-2Na), pH = 8.0);

[0095] Number of suction cycles: 10 times

[0096] The recovery rate of CRP by the obtained PP-(HEMA-co-MPC-MPC) membrane column is shown in Table 2. The absolute recovery rates of 10 membranes are mostly between 85% and 95%, indicating that the single membrane column has good reusability and can maintain good purification effect after multiple uses. Considering the 10 repeated experiments, it shows that the single biomimetic phospholipid choline-based membrane prepared by this method has good repeatability, with small differences in results between different batches, presenting good repeatability.

[0097] Table 2 Repeatability investigation

[0098]

[0099]

[0100] III. Using the PP-(HEMA-co-MPC-MPC) membrane column obtained in Example 1 for the purification of CRP in actual biological samples:

[0101] The purification conditions are as follows:

[0102] Adsorbent: PP-(HEMA-co-MPC-MPC) membrane column;

[0103] Samples: a: Serum of inflammatory patients; b: Mouse plasma;

[0104] Washing solution: Buffer solution A;

[0105] Elution solution: Buffer solution B;

[0106] The purification result diagram of the PP-(HEMA-co-MPC-MPC) membrane column for the serum of inflammatory patients (a) and mouse plasma (b) is as Figure 5 shown. From the Figure 5 results, it can be seen that there is only one band of CRP protein in the gel block, which is consistent with the expectation. This more intuitively shows that the method using the biomimetic phosphatidylcholine-based membrane can effectively avoid non-specific adsorption of miscellaneous proteins on its surface, and at the same time has the ability to specifically capture CRP protein. CRP in the sample can be enriched by this adsorbent, while other proteins cannot be adsorbed on this membrane column, proving that the membrane column prepared by the present invention can be successfully applied to the purification of CRP in actual samples.

[0107] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A preparation method of a thin-film solid-phase extraction coil column for C-reactive protein, characterized in that, it comprises the following steps: S1. Prepare a first hydrophilic monomer, a crosslinking agent, an initiator, a ligand complex, a reducing agent and a solvent according to a mass-volume ratio of 53.65 - 300.44 mg: 0.03 - 0.3 mL: 50 μL: 250 μL: 18.53 - 103.75 mg: 10 mL. Immerse a polypropylene film in the mixed solution of the first hydrophilic monomer, the crosslinking agent, the initiator, the ligand complex, the reducing agent and the solvent, and carry out a polymerization reaction at room temperature under sealing and nitrogen protection. Modify the polypropylene film through an atom transfer radical reaction to obtain film product I; S2. Prepare a second hydrophilic monomer, a ligand complex, a reducing agent and a solvent according to a mass-volume ratio of 148.5 - 445.5 mg: 250 μL: 25.02 - 75.05 mg: 2 mL. Immerse film product I in the mixed solution of the second hydrophilic monomer, the ligand complex, the reducing agent and the solvent, and carry out a polymerization reaction at room temperature under sealing and nitrogen protection. Modify the functional monomer through an atom transfer radical polymerization to obtain film product II; S3. Prepare a second hydrophilic monomer, a ligand complex, a reducing agent and a solvent according to a mass-volume ratio of 240 - 720 mg: 50 μL: 6.6 - 19.8 mg: 2.8 mL. Immerse film product II into the mixed solution of the second hydrophilic monomer, the ligand complex, the reducing agent and the solvent, seal and protect with nitrogen, and carry out a polymerization reaction at a temperature of 30 - 70 °C. Modify the functional monomer again through an atom transfer radical polymerization to obtain film product III; S4. Tightly curl film product III and insert it into a syringe, and use a push rod to insert it to the bottom to obtain a film coil column, that is, a thin-film solid-phase extraction coil column for C-reactive protein; the first hydrophilic monomer is 2-hydroxyethyl methacrylate monomer, and the structure of the 2-hydroxyethyl methacrylate monomer is as follows: The ligand complex is prepared from copper bromide, N,N,N',N,'N”-pentamethyldiethylenetriamine and N,N-dimethylformamide according to a mass ratio of 0.068:2.5:99.35; the second hydrophilic monomer is a phosphorylcholine functional monomer.

2. The preparation method of the thin-film solid-phase extraction coil column for C-reactive protein according to claim 1, characterized in that, the crosslinking agent is N,N'-methylenebisformamide; the initiator is ethyl 2-bromoisobutyrate; the reducing agent is L-ascorbic acid; the solvent is N,N-dimethylformamide or methanol-water with a mass fraction of 20%.

3. The preparation method of the thin-film solid-phase extraction coil column for C-reactive protein according to claim 1, characterized in that, the polymerization reaction time in step S1 is 1 - 8 h; the polymerization reaction time in step S2 is 4 - 8 h; the polymerization reaction time in step S3 is 4 - 8 h.

4. The preparation method of the thin-film solid-phase extraction coil column for C-reactive protein according to claim 1, characterized in that, the phosphorylcholine functional monomer is 2-methacryloyloxyethyl phosphorylcholine; the structure of the 2-methacryloyloxyethyl phosphorylcholine is as follows:

5. A C-reactive protein thin film solid phase extraction column prepared by the preparation method according to any one of claims 1 to 4.

6. The C-reactive protein thin film solid phase extraction column according to claim 5, characterized in that the C-reactive protein thin film solid phase extraction column sequentially comprises a PP base film, a HEMA polymerization layer, a 2-methacryloyloxyethyl phosphorylcholine polymerization layer I and a 2-methacryloyloxyethyl phosphorylcholine polymerization layer II from inside to outside.

7. Application of the C-reactive protein thin film solid phase extraction column according to claim 5 in purification of C-reactive protein.

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