Preparation method and application of cit-ra33 peptide@pei-fe3o4 magnetic nanoparticles

Abstract

The application discloses a preparation method of Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles and application thereof, and belongs to the technical field of protein separation and enrichment and biological sample pretreatment. After PEI-Fe3O4 magnetic nanoparticles are activated by 6-(maleimide) hexanoic acid succinimidyl ester, the Cit-RA33 peptide containing a cysteine residue at a terminal end is coupled, so that the Cit-RA33 peptide is fixed on the surface of the magnetic nanoparticles. The obtained magnetic nanoparticles can be used for in-vitro separation and enrichment of anti-RA33 antibodies, and can be rapidly separated with the aid of an external magnetic field, and can be used for sample treatment before subsequent protein analysis.

Description

Technical Field

[0001] This invention relates to the field of protein separation and enrichment and biological sample pretreatment technology, specifically to a method for preparing Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles and their application. Background Technology

[0002] Rheumatoid arthritis is a systemic autoimmune disease characterized by chronic synovitis, joint destruction, and the production of autoantibodies. Commonly used serological markers in clinical practice include rheumatoid factor and anti-cyclic citrullinated peptide antibodies. However, the autoantibody profile varies among patients, and single markers still have limitations in early disease diagnosis, subtyping evaluation, and monitoring of treatment efficacy.

[0003] Anti-RA33 antibody is one of the autoantibodies associated with rheumatoid arthritis, and its detection has certain research value for the analysis of rheumatoid arthritis-related autoantibody profiles. However, in actual serum or plasma samples, due to the complex sample matrix, strong background of high-abundance proteins, and differences in the abundance of target antibodies, direct analysis of anti-RA33 antibody is easily affected by the complex sample matrix, which may lead to problems such as insufficient detection sensitivity, strong background interference, or complex sample pretreatment.

[0004] Therefore, it is of practical significance to separate and enrich anti-RA33 antibodies in complex serum or plasma samples to reduce the impact of complex sample matrices on subsequent analysis.

[0005] Magnetic nanoparticles possess advantages such as large specific surface area, easy surface modification, and good magnetic responsiveness. By immobilizing functional ligands that can interact with target substances on the surface of magnetic nanoparticles, the target substances can be trapped on the surface of the magnetic nanoparticles. Furthermore, an external magnetic field can be used to collect the magnetic nanoparticles, thereby achieving rapid separation between the magnetic solid-phase material and the sample liquid phase.

[0006] Citrullinated RA33 antigenic peptides can serve as antigenic peptides recognized by anti-RA33 antibodies. By introducing cysteine ​​residues at the ends of citrullinated RA33 antigenic peptides, the reaction between the thiol group of the cysteine ​​residue and the maleimide group can be utilized to immobilize the citrullinated RA33 antigenic peptides on the surface of magnetic nanoparticles.

[0007] Based on this, the present invention constructs a Cit-RA33 peptide-functionalized PEI-Fe3O4 magnetic nanoparticle, enabling the Cit-RA33 peptide immobilized on the surface of the magnetic nanoparticle to interact with anti-RA33 antibody, and utilizes the magnetic responsiveness of PEI-Fe3O4 magnetic nanoparticle to achieve magnetic separation between the adsorbed component and the sample liquid phase, for the separation and enrichment of anti-RA33 antibody and sample processing before subsequent analysis. Summary of the Invention

[0008] The technical problem to be solved by this invention is to address the issue that high abundance proteins in complex serum or plasma samples and complex sample matrices affect the direct analysis of anti-RA33 antibodies. This invention provides a method for preparing Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles, which enable the obtained functionalized magnetic nanoparticles to adsorb anti-RA33 antibodies and to be separated by an external magnetic field, for use in the in vitro separation and enrichment of anti-RA33 antibodies and sample pretreatment.

[0009] To address the aforementioned technical problems, this invention provides a method for preparing Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles, comprising the following steps: Maleimide activation of S1, PEI-Fe3O4 magnetic nanoparticles.

[0010] PEI-Fe3O4 magnetic nanoparticles were dispersed in PBS buffer and reacted with 6-(maleimide)hexanoic acid succinimide ester to obtain maleimide-activated PEI-Fe3O4 magnetic nanoparticles.

[0011] Preferably, 55 mg of PEI-Fe3O4 magnetic nanoparticles were taken, dispersed thoroughly in PBS buffer, and then DMSO solution containing 11 mg of 6-(maleimide)hexanoate succinimide ester was added. The mixture was reacted at room temperature in the dark for 60 min. After the reaction was completed, magnetic separation was performed, the supernatant was discarded, and the mixture was washed three times with PBS buffer.

[0012] The 6-(maleimide)hexanoic acid succinimide ester is a heterobifunctional crosslinking agent containing maleimide active groups.

[0013] Preferably, the PEI-Fe3O4 magnetic nanoparticles are prepared by the following method: Add 0.135 g FeCl3·6H2O, 1.8 g sodium acetate and 0.5 g polyethyleneimine to 20.0 mL ethylene glycol and stir vigorously at room temperature for 45 min. Take 18.0 mL of the obtained mixture and transfer it to a 20 mL polytetrafluoroethylene-lined high-pressure reactor, and react at 200 °C for 12 h; After the reaction was completed and the product was allowed to cool naturally, it was washed twice with anhydrous ethanol and then twice with deionized water to obtain PEI-Fe3O4 magnetic nanoparticles.

[0014] Coupling of S2 and Cit-RA33 peptides.

[0015] The Cit-RA33 peptide was coupled with maleimide-activated PEI-Fe3O4 magnetic nanoparticles to immobilize the Cit-RA33 peptide on the surface of the PEI-Fe3O4 magnetic nanoparticles.

[0016] The Cit-RA33 peptide is a citrullinated RA33 antigen peptide with a cysteine ​​residue at the end. During the coupling process, the thiol group in the cysteine ​​residue reacts with the maleimide group on the surface of the PEI-Fe3O4 magnetic nanoparticles, thereby immobilizing the Cit-RA33 peptide on the surface of the PEI-Fe3O4 magnetic nanoparticles.

[0017] Preferably, the Cit-RA33 peptide contains a flexible spacer arm composed of three glycine residues, which helps to reduce the spatial influence of the magnetic carrier surface on the antigen peptide recognition site.

[0018] Preferably, 11 mg of Cit-RA33 peptide is weighed, added to 10.978 mL of PBS buffer and 22 μL of 0.5 M EDTA solution to prepare a peptide solution with a total volume of 11 mL, wherein the concentration of Cit-RA33 peptide is 1 mg / mL and the final concentration of EDTA is 1 mM.

[0019] The obtained peptide solution was added to maleimide-activated PEI-Fe3O4 magnetic nanoparticles and reacted with shaking at room temperature in the dark for 2 h, followed by an overnight reaction at 4 °C.

[0020] Preferably, after the coupling reaction is completed, 5.5 mL of 10 mM L-cysteine ​​solution is added, and the reaction is carried out at room temperature in the dark for 30 min to block the remaining maleimide groups.

[0021] Magnetic separation was then performed, followed by washing three times with PBS buffer and five times with deionized water. The resulting magnetic nanoparticles were resuspended in a small amount of deionized water, pre-frozen, and freeze-dried to obtain Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticle powder.

[0022] The present invention also provides a Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticle, comprising PEI-Fe3O4 magnetic nanoparticles and a Cit-RA33 peptide fixed on its surface.

[0023] The Cit-RA33 peptide is a citrullinated RA33 antigen peptide with a cysteine ​​residue at the end, and is fixed on the surface of PEI-Fe3O4 magnetic nanoparticles by a coupling structure formed by the reaction of the thiol group of the cysteine ​​residue with the maleimide group introduced into the surface of PEI-Fe3O4 magnetic nanoparticles via 6-(maleimide)hexanoic acid succinimide ester.

[0024] This invention also provides the application of the above-mentioned Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles in the in vitro isolation and enrichment of anti-RA33 antibodies.

[0025] During the in vitro separation and enrichment of anti-RA33 antibodies, the sample containing anti-RA33 antibodies was contacted with Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles, so that the Cit-RA33 peptide immobilized on the surface of the magnetic nanoparticles interacts with the anti-RA33 antibodies in the sample. Subsequently, the Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles were collected using an external magnetic field, and the supernatant was removed.

[0026] Preferably, the sample is diluted with PBS buffer at pH 5.0 and then contacted with Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles for 30 min.

[0027] Preferably, the sample is a serum sample from a rheumatoid arthritis patient or a serum sample from a rheumatoid arthritis patient spiked with anti-RA33 antibody.

[0028] Preferably, the Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles obtained by magnetic separation are washed, followed by elution and magnetic separation, and the eluent is collected.

[0029] The obtained samples can be used for electrophoretic analysis of anti-RA33 antibodies, protein quantification analysis, or sample pretreatment before mass spectrometry detection.

[0030] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: The present invention uses PEI-Fe3O4 magnetic nanoparticles as magnetic carriers, performs surface activation through 6-(maleimide)hexanoic acid succinimide ester, and utilizes the reaction between the thiol group in the terminal cysteine ​​residue of the Cit-RA33 peptide and the maleimide group to immobilize the Cit-RA33 peptide, which serves as the recognition ligand for anti-RA33 antibodies, on the surface of PEI-Fe3O4 magnetic nanoparticles, thereby constructing a functionalized magnetic nanomaterial for the separation and enrichment of anti-RA33 antibodies.

[0031] Experimental results showed that, under pH 5 conditions, the adsorption efficiency of the obtained Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles against RA33 antibody was approximately 93.2%, while the adsorption efficiencies for HSA and ordinary IgG were approximately 8.2% and 6.1%, respectively. Under the investigated experimental conditions, the obtained Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles exhibited high adsorption capacity against RA33 antibody and demonstrated a certain degree of selective adsorption.

[0032] Under the same adsorption conditions, the adsorption efficiency of PEI-Fe3O4 magnetic nanoparticles without Cit-RA33 peptide modification against RA33 antibody was approximately 49.4%, while the adsorption efficiency of Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles against RA33 antibody was approximately 93.2%, indicating that Cit-RA33 peptide modification helps to enhance the adsorption and enrichment capacity of PEI-Fe3O4 magnetic nanoparticles against RA33 antibody.

[0033] In addition, the obtained Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles have magnetic responsiveness and can perform rapid solid-liquid separation under the action of an external magnetic field. They can be used for the separation and enrichment of anti-RA33 antibodies in complex biological samples and for sample processing before subsequent protein analysis. Attached Figure Description

[0034] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 The images show SEM and TEM images of PEI-Fe3O4 magnetic nanoparticles and Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles, where A and B are morphology images of PEI-Fe3O4 magnetic nanoparticles, and C and D are morphology images of Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles.

[0035] Figure 2 Fourier transform infrared (FTIR) spectra and thermogravimetric analysis (TGA) diagrams of PEI-Fe3O4 magnetic nanoparticles and Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles are shown, where A is the FTIR spectrum and B is the TGA diagram.

[0036] Figure 3 The images show the X-ray photoelectron spectra of PEI-Fe3O4 magnetic nanoparticles, where A is the XPS Survey full spectrum, B is the C 1s high-resolution spectrum, C is the Fe 2p high-resolution spectrum, D is the N 1s high-resolution spectrum, and E is the O 1s high-resolution spectrum.

[0037] Figure 4 The images show the X-ray photoelectron spectra of Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles, where A is the XPS Survey full spectrum, B is the C 1s high-resolution spectrum, C is the Fe 2p high-resolution spectrum, D is the N 1s high-resolution spectrum, E is the O 1s high-resolution spectrum, and F is the S 2p high-resolution spectrum.

[0038] Figure 5The graphs show the adsorption performance of Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles. A represents a comparison of the adsorption efficiencies of Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles for HSA, anti-RA33 antibody, and IgG at pH 5; B represents a comparison of the adsorption efficiencies of Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles for HSA and anti-RA33 antibody at pH 7, pH 5, and pH 9; and C represents a comparison of the adsorption efficiencies of PEI-Fe3O4 magnetic nanoparticles and Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles for anti-RA33 antibody.

[0039] Figure 6 The results are SDS-PAGE assays. A shows the SDS-PAGE analysis results of HSA, anti-RA33 antibody, and mixed samples of HSA and anti-RA33 antibody before and after treatment with Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles. B shows the SDS-PAGE analysis results of serum samples from rheumatoid arthritis patients and serum samples spiked with anti-RA33 antibody after treatment with Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] The present invention will be further illustrated below with reference to examples, experimental examples, and comparative examples. Examples are used to illustrate the preparation of Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles; experimental examples are used to illustrate the structure, adsorption, and separation properties of the obtained magnetic nanoparticles; and comparative examples are used to illustrate the effect of Cit-RA33 peptide modification on the adsorption performance of anti-RA33 antibodies.

[0042] Unless otherwise stated, all reagents used in the experiment were of analytical grade and were not subjected to any other pretreatment before use; the water used in the experiment was deionized water.

[0043] Example 1: Preparation of Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles 1. Preparation of PEI-Fe3O4 magnetic nanoparticles Weigh 0.135 g FeCl3·6H2O, 1.8 g sodium acetate and 0.5 g polyethyleneimine, add them to 20.0 mL ethylene glycol, and stir vigorously at room temperature for 45 min.

[0044] Take 18.0 mL of the obtained mixture and transfer it to a 20 mL polytetrafluoroethylene-lined high-pressure reactor, and react at 200 °C for 12 h.

[0045] After the reaction was completed and the product was allowed to cool naturally, it was washed twice with anhydrous ethanol and then twice with deionized water to obtain PEI-Fe3O4 magnetic nanoparticles.

[0046] 2. Maleimide activation of PEI-Fe3O4 magnetic nanoparticles Take 55 mg of the obtained PEI-Fe3O4 magnetic nanoparticles and disperse them thoroughly in PBS buffer.

[0047] Add a DMSO solution containing 11 mg of 6-(maleimide)hexanoic acid succinimide ester and react at room temperature in the dark for 60 min.

[0048] After the reaction was completed, magnetic separation was performed, the supernatant was discarded, and the mixture was washed three times with PBS buffer to obtain maleimide-activated PEI-Fe3O4 magnetic nanoparticles.

[0049] 3. Conjugation of Cit-RA33 peptide Weigh 11 mg of Cit-RA33 peptide, add 10.978 mL of PBS buffer and 22 μL of 0.5 M EDTA solution to prepare a peptide solution with a total volume of 11 mL, wherein the concentration of Cit-RA33 peptide is 1 mg / mL and the final concentration of EDTA is 1 mM.

[0050] The obtained peptide solution was added to maleimide-activated PEI-Fe3O4 magnetic nanoparticles and reacted with shaking at room temperature in the dark for 2 h, followed by an overnight reaction at 4 °C.

[0051] The Cit-RA33 peptide contains a cysteine ​​residue at its terminal. During the above coupling reaction, the thiol group in the cysteine ​​residue reacts with the maleimide group on the surface of the PEI-Fe3O4 magnetic nanoparticles, thereby immobilizing the Cit-RA33 peptide on the surface of the PEI-Fe3O4 magnetic nanoparticles.

[0052] 4. Sealing and post-treatment After the coupling reaction was completed, 5.5 mL of 10 mM L-cysteine ​​solution was added, and the reaction was carried out at room temperature in the dark for 30 min to block the remaining maleimide groups.

[0053] Magnetic separation was then performed, followed by washing three times with PBS buffer and then five times with deionized water.

[0054] The obtained magnetic nanoparticles were resuspended in a small amount of deionized water, pre-frozen, and then freeze-dried to obtain Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticle powder.

[0055] Experimental Example 1: Structural Characterization of Cit-RA33 Peptide@PEI-Fe3O4 Magnetic Nanoparticles 1. Morphological characteristics The morphology of PEI-Fe3O4 magnetic nanoparticles and Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles was observed using scanning electron microscopy and transmission electron microscopy. The results are as follows: Figure 1 As shown.

[0056] Depend on Figure 1 A and Figure 1 As shown in Figure B, the PEI-Fe3O4 magnetic nanoparticles exhibit a near-spherical or aggregated granular structure with a relatively rough surface, revealing a surface structure formed by the aggregation of smaller nanoparticles. Due to the magnetic properties of Fe3O4 material and the fact that the sample was dried before electron microscopy observation, some aggregation between particles is evident.

[0057] Depend on Figure 1 C and Figure 1 As can be seen from D, after modification with Cit-RA33 peptide, the resulting Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles still maintain a particulate morphology, and the surface rough structure is not significantly damaged, indicating that the Cit-RA33 peptide modification process did not significantly affect the basic morphology and structure of PEI-Fe3O4 magnetic nanoparticles.

[0058] 2. Fourier transform infrared spectroscopy and thermogravimetric analysis Fourier transform infrared spectroscopy and thermogravimetric analysis results of PEI-Fe3O4 magnetic nanoparticles and Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles are as follows: Figure 2 As shown.

[0059] Depend on Figure 2 As can be seen in A, PEI-Fe3O4 magnetic nanoparticles are located at approximately 584 cm⁻¹. -1 The presence of a characteristic Fe-O absorption peak at approximately 3428 cm⁻¹ indicates the presence of Fe₃O₄ magnetic components in the material; the peak at approximately 3428 cm⁻¹ further supports this characteristic absorption. -1 and 1629 cm -1 The nearby absorption peaks can be attributed to surface hydroxyl groups, adsorbed water, and PEI-related functional groups.

[0060] After modification with Cit-RA33 peptide, the sample still retains the characteristic Fe-O absorption peak, and at approximately 1033 cm⁻¹... -1Absorption peaks associated with or enhanced by organic functional groups such as CN and CO appear nearby, and are located at approximately 3427 cm⁻¹. -1 and 1631 cm -1 The presence of overlapping OH, NH and amide-related signals in the vicinity suggests that the organic functional groups on the material surface changed after Cit-RA33 peptide modification.

[0061] Depend on Figure 2 As shown in Figure B, the total weight loss of PEI-Fe3O4 magnetic nanoparticles within the test temperature range was approximately 6.86%, while the total weight loss of Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles was approximately 7.55%. The increased total weight loss of the Cit-RA33 peptide-modified sample indicates an increase in the content of organic components on the material surface.

[0062] 3. X-ray photoelectron spectroscopy analysis X-ray photoelectron spectroscopy results of PEI-Fe3O4 magnetic nanoparticles are as follows: Figure 3 As shown.

[0063] XPS Survey full spectrum analysis revealed that Fe, O, C, and N elements were mainly detected on the surface of PEI-Fe3O4 magnetic nanoparticles. Among them, the Fe 2p and O 1s signals originated from the Fe3O4 magnetic components, while the C 1s and N 1s signals were related to the organic layer on the PEI surface.

[0064] The C 1s high-resolution spectrum can be decomposed into related peaks such as CC / CH, CN / CO, and C=O / OC=O; the Fe 2p high-resolution spectrum shows characteristic peaks of Fe 2p3 / 2 and Fe 2p1 / 2; the N 1s spectrum shows CN / -NH2 related peaks; and the O 1s spectrum shows Fe-O and OH / CO related peaks.

[0065] X-ray photoelectron spectroscopy results of Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles are as follows: Figure 4 As shown.

[0066] Compared with PEI-Fe3O4 magnetic nanoparticles, the Cit-RA33 peptide-modified sample also showed a weak S 2p correlation signal in the full Survey spectrum, in addition to Fe, O, C and N elements.

[0067] In the C 1s spectrum, peaks such as CC / CH, CN / CO, and OC=O / COO⁻ can be observed; in the N 1s spectrum, CN / -NH2 / amide N and protonated nitrogen-related peaks can be observed; in the O 1s spectrum, Fe-O, OH / O=CN, and CO / OC=O-related peaks can be observed.

[0068] The S 2p high-resolution spectrum shows a set of weak double peaks around 163.6 eV and 164.8 eV, which can be attributed to S 2p3 / 2 and S 2p1 / 2, suggesting that the Cit-RA33 peptide containing cysteine ​​residues may have been introduced onto the material surface.

[0069] The results of Fourier transform infrared spectroscopy, thermogravimetric analysis, and X-ray photoelectron spectroscopy all support the modification of Cit-RA33 peptide on the surface of PEI-Fe3O4 magnetic nanoparticles.

[0070] Experimental Example 2: Adsorption performance of Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles 1. Experimental Methods Anti-RA33 antibody standard was used as the target antibody, and human serum albumin HSA and human IgG were used as control proteins.

[0071] A certain amount of Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles were added to a 1.5 mL centrifuge tube, mixed with the corresponding protein solution and shaken for 30 min. Magnetic separation was performed by applying an external magnetic field and the supernatant was collected.

[0072] The changes in protein content before and after adsorption were analyzed using Coomassie brilliant blue staining, ultraviolet absorption, or SDS-PAGE methods, and the protein concentration before and after adsorption was calculated using a standard working curve.

[0073] Protein adsorption efficiency Calculate according to the following formula: In the formula, This represents the initial concentration of the protein prepared for the experiment. This represents the concentration of protein in the solution after adsorption.

[0074] After the adsorbed Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles were washed with buffer, eluent or non-reducing SDS-PAGE loading buffer was added, and magnetic separation was performed after heating. The eluent was then collected.

[0075] Protein elution efficiency Calculate according to the following formula: In the formula, This represents the initial concentration of the protein prepared for the experiment. This represents the concentration of protein in the solution after adsorption. This represents the concentration of protein in the solution after elution.

[0076] 2. Adsorption properties of different proteins At pH 5, the adsorption properties of Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles against RA33 antibody, HSA, and IgG were measured, and the results are as follows: Figure 5 As shown in A in the diagram.

[0077] The adsorption efficiency of the anti-RA33 antibody was approximately 93.2%, which was significantly higher than that of HSA and ordinary IgG; the adsorption efficiencies of HSA and ordinary IgG were approximately 8.2% and 6.1%, respectively.

[0078] The results showed that, under the same conditions, Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles had a high adsorption capacity for anti-RA33 antibodies, but a low adsorption capacity for HSA and ordinary IgG, exhibiting a certain degree of selective adsorption.

[0079] 3. Effect of pH on adsorption performance The adsorption performance of Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles against RA33 antibody and HSA was investigated at pH 7, pH 5, and pH 9, respectively. The results are as follows: Figure 5 As shown in B in the diagram.

[0080] The adsorption efficiency of Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles against anti-RA33 antibodies varied under different pH conditions. The highest adsorption efficiency against anti-RA33 antibodies was observed at pH 5, while the adsorption efficiency of HSA was relatively low under this condition.

[0081] The above results indicate that, among the pH 7, pH 5 and pH 9 conditions investigated, pH 5 is more favorable for the adsorption and enrichment of anti-RA33 antibodies by Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles.

[0082] Comparative Example 1: PEI-Fe3O4 magnetic nanoparticles without Cit-RA33 peptide modification Using unmodified PEI-Fe3O4 magnetic nanoparticles as a control, they were incubated with anti-RA33 antibody solution under the same conditions as in Experimental Example 2. After magnetic separation, the supernatant was collected and the protein content was determined. The results are as follows: Figure 5 As shown in C.

[0083] The adsorption efficiency of PEI-Fe3O4 magnetic nanoparticles without Cit-RA33 peptide modification against RA33 antibody was approximately 49.4%; the adsorption efficiency of Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles against RA33 antibody was approximately 93.2%.

[0084] The above results indicate that PEI-Fe3O4 magnetic nanoparticles without Cit-RA33 peptide modification exhibit certain non-specific adsorption, while the adsorption efficiency against RA33 antibody is further improved after Cit-RA33 peptide modification, suggesting that Cit-RA33 peptide modification helps enhance the adsorption and enrichment capacity of PEI-Fe3O4 magnetic nanoparticles against RA33 antibody.

[0085] Experimental Example 3: Adsorption of anti-RA33 antibody in a mixed protein system Mixed protein samples were prepared using HSA and anti-RA33 antibody.

[0086] The mixed protein sample was incubated with Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles at pH 5 for 30 min, and then magnetically separated by an external magnetic field and the supernatant was collected.

[0087] The samples were analyzed using SDS-PAGE, and the results are as follows: Figure 6 As shown in A in the diagram.

[0088] Figure 6 In lane A, M is a protein molecular weight standard; lane 1 is HSA with a concentration of 0.1 mg / mL; lane 2 is an anti-RA33 antibody standard; lane 3 is a mixed sample of HSA and anti-RA33 antibody; lane 4 is the supernatant of the mixed sample after adsorption by Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles at pH 5 for 30 min.

[0089] The results showed that after the HSA and anti-RA33 antibody mixed sample was treated with Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles, the anti-RA33 antibody-related bands in the supernatant were weakened compared with those before treatment, suggesting that some anti-RA33 antibody was adsorbed by Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles.

[0090] Experimental Example 4: Isolation and Enrichment of Serum Samples from Patients with Rheumatoid Arthritis Serum samples from patients with rheumatoid arthritis or serum samples from patients with rheumatoid arthritis spiked with anti-RA33 antibody were diluted with PBS buffer at pH 5.0.

[0091] A certain volume of diluted serum sample was taken, mixed with Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles and shaken for 30 min. The Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles were collected by applying an external magnetic field and the supernatant was removed.

[0092] The collected Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles were washed with PBS buffer at pH 5.0, then non-reducing SDS-PAGE loading buffer was added. After heating, magnetic separation was performed, and the eluent was collected.

[0093] SDS-PAGE analysis was performed on the sample before adsorption, the supernatant after adsorption, and the eluent of magnetic nanoparticles, respectively. The results are as follows: Figure 6 As shown in B in the diagram.

[0094] Figure 6 In the diagram, B represents protein molecular weight standards; lane 1 contains 500-fold diluted serum from rheumatoid arthritis patients; lane 2 contains serum from rheumatoid arthritis patients spiked with anti-RA33 antibody; lane 3 contains the eluent from serum from rheumatoid arthritis patients treated with Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles; and lane 4 contains the eluent from serum from rheumatoid arthritis patients spiked with anti-RA33 antibody treated with Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles.

[0095] SDS-PAGE results showed that protein bands were observed in the eluent after treatment with Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles, indicating that Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles can adsorb and recover some protein components from serum samples.

[0096] The results of the anti-RA33 antibody adsorption performance experiment and the mixed protein system experiment indicate that Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles have the potential for application in the adsorption and enrichment of anti-RA33 antibodies and the pretreatment of complex serum samples.

[0097] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles, characterized in that, Includes the following steps: S1. PEI-Fe3O4 magnetic nanoparticles were dispersed in PBS buffer and reacted with 6-(maleimide)hexanoic acid succinimide ester to obtain maleimide-activated PEI-Fe3O4 magnetic nanoparticles. S2. The maleimide-activated PEI-Fe3O4 magnetic nanoparticles are coupled with Cit-RA33 peptide to fix the Cit-RA33 peptide on the surface of the PEI-Fe3O4 magnetic nanoparticles. After the reaction, Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles are obtained by magnetic separation and washing. The Cit-RA33 peptide is a citrullinated RA33 antigen peptide with a cysteine ​​residue at the end, and the thiol group of the cysteine ​​residue is coupled to the maleimide group on the surface of the maleimide-activated PEI-Fe3O4 magnetic nanoparticles.

2. The preparation method according to claim 1, characterized in that, The preparation method of the PEI-Fe3O4 magnetic nanoparticles includes: Add 0.135g of ferric chloride hexahydrate, 1.8g of sodium acetate and 0.5g of polyethyleneimine to 20.0mL of ethylene glycol and stir at room temperature for 45min. Take 18.0 mL of the obtained mixture and place it in a 20 mL polytetrafluoroethylene-lined high-pressure reactor, and react at 200 °C for 12 h; After the reaction was completed, the mixture was allowed to cool naturally, washed twice with anhydrous ethanol, and then washed twice with deionized water to obtain the PEI-Fe3O4 magnetic nanoparticles.

3. The preparation method according to claim 1 or 2, characterized in that, In step S1, 55 mg of the PEI-Fe3O4 magnetic nanoparticles were dispersed in PBS buffer, and DMSO solution containing 11 mg of the 6-(maleimide)hexanoic acid succinimide ester was added. The mixture was reacted at room temperature in the dark for 60 min. After the reaction was completed, magnetic separation was performed, and the mixture was washed three times with PBS buffer.

4. The preparation method according to claim 1, characterized in that, In step S2, 11 mg of the Cit-RA33 peptide was added to 10.978 mL of PBS buffer and 22 μL of 0.5 M EDTA solution to prepare a peptide solution with a total volume of 11 mL, a Cit-RA33 peptide concentration of 1 mg / mL and a final EDTA concentration of 1 mM. The peptide solution was added to the maleimide-activated PEI-Fe3O4 magnetic nanoparticles and reacted with shaking at room temperature in the dark for 2 hours, followed by an overnight reaction at 4°C.

5. The preparation method according to claim 1, characterized in that, After the coupling reaction was completed, 5.5 mL of 10 mM L-cysteine ​​solution was added, and the mixture was reacted at room temperature in the dark for 30 min to block the remaining maleimide groups. Subsequently, magnetic separation was performed, and the particles were washed three times with PBS buffer and then five times with deionized water. The resulting magnetic nanoparticles were resuspended in deionized water, pre-frozen, and freeze-dried to obtain Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticle powder.

6. A Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticle, characterized in that, It includes PEI-Fe3O4 magnetic nanoparticles and Cit-RA33 peptide fixed on the surface of the PEI-Fe3O4 magnetic nanoparticles; The Cit-RA33 peptide is a citrullinated RA33 antigen peptide with a cysteine ​​residue at the end. The Cit-RA33 peptide is fixed on the surface of the PEI-Fe3O4 magnetic nanoparticles by a coupling structure formed by the reaction of the thiol group of the cysteine ​​residue with the maleimide group introduced into the surface of the PEI-Fe3O4 magnetic nanoparticles via 6-(maleimide)hexanoic acid succinimide ester.

7. The Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles according to claim 6, characterized in that, The Cit-RA33 peptide contains a flexible spacer arm composed of three glycine residues.

8. The use of the Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles according to claim 6 or 7 in the in vitro isolation and enrichment of anti-RA33 antibodies.

9. The application according to claim 8, characterized in that, The sample containing the anti-RA33 antibody was contacted with the Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles in PBS buffer at pH 5.0 for 30 min, and the Cit-RA33 peptide@PEI-Fe3O4 magnetic nanoparticles were collected by applying an external magnetic field.

10. The application according to claim 9, characterized in that, The sample is a serum sample from a rheumatoid arthritis patient or a serum sample from a rheumatoid arthritis patient spiked with anti-RA33 antibody; the application is sample pretreatment before electrophoretic analysis, protein quantification analysis, or mass spectrometry detection of anti-RA33 antibody.

Images