Preparation method of hydrophilic and hydrophobic balance magnetic beads and application of hydrophilic and hydrophobic balance magnetic beads in low-abundance protein enrichment

Abstract

The invention discloses a preparation method of hydrophilic and hydrophobic balance magnetic beads and application of the hydrophilic and hydrophobic balance magnetic beads in low-abundance protein enrichment. The hydrophilic and hydrophobic balance magnetic beads are selected from HLB, HLB-WCX, HLB-MCX, HLB-WAX and HLB-MAX. The surfaces of the prepared hydrophilic and hydrophobic balanced magnetic beads contain benzene rings (hydrophobic) and acylamino (hydrophilic) at the same time, balanced adsorption of proteins with different hydrophilic and hydrophobic characteristics is achieved, the enrichment efficiency and selectivity of low-abundance proteins are remarkably improved, and non-specific interference of high-abundance proteins is reduced.

Description

Technical Field

[0001] This invention belongs to the field of bio-separation materials technology, specifically relating to a method for preparing hydrophilic-hydrophobic balanced magnetic beads and their application in the enrichment of low-abundance proteins. Background Technology

[0002] This invention belongs to the field of bioseparation materials technology, specifically involving the surface functionalization modification of magnetic nanoparticles and their application in proteomics pretreatment, particularly achieving efficient enrichment of low-abundance proteins in complex biological samples by regulating hydrophilicity / hydrophobicity and ion exchange properties.

[0003] Background Technology: Biological samples (such as serum, plasma, urine, cell lysates, etc.) have complex protein compositions, with high-abundance proteins (such as albumin and immunoglobulins in serum) accounting for an extremely high proportion. This severely interferes with the detection and analysis of low-abundance proteins (such as tumor markers and signaling proteins), making it difficult for conventional detection methods (such as mass spectrometry and electrophoresis) to directly identify low-abundance proteins. Low-abundance proteins (such as tumor markers, signaling proteins, cytokines, etc.) often participate in important physiological regulatory processes in the body and are core biomarkers for early disease diagnosis, pathological mechanism research, and drug target screening. They have important biological functions and clinical diagnostic value. Therefore, achieving efficient enrichment of low-abundance proteins is a key technical challenge in the field of bioanalysis. In the early stages of many diseases (such as cancer and autoimmune diseases), changes in high-abundance proteins in the blood are not obvious, while low-abundance proteins show characteristic expression differences. Enriching low-abundance proteins can improve the early detection rate of diseases and provide support for precision medicine.

[0004] Low-abundance protein enrichment techniques primarily achieve this by removing high-abundance proteins or directly enriching low-abundance proteins. Commonly used methods include ultrafiltration, precipitation, electrophoresis, affinity chromatography, chromatography, and novel material technologies. Ultrafiltration is low-cost but prone to losing low-abundance proteins, while affinity chromatography offers high specificity but is expensive and has limited sample loading capacity. Currently, before conducting proteomics studies, samples need to be de-enriched. Immunoaffinity chromatography is commonly used to remove high-abundance proteins and reduce their impact on the identification of low-abundance proteins, but it still cannot achieve high depth and high throughput levels.

[0005] Kenneth A. Dawson et al. proposed the concept of "protein corona (PC)" in their research on protein-particle interactions. Nanoparticles (NPs) encounter many components in the physiological environment and engage in various interactions with them, thereby altering the behavior and properties of the NPs. These substances then coat the surface of the nanoparticles, forming a protein corona. Studies have shown that the formation of the protein corona is related to the properties of the NPs; therefore, by rationally designing NPs, the target protein corona can be obtained, leading to more precise proteomics research. SEER Corporation in the United States offers products such as Proteonano™, a magnetic nanoprobe that improves sensitivity by 1 million times, suitable for large-scale biomarker discovery. However, the magnetic beads prepared by SEER only consider the regulation of surface groups to achieve protein enrichment, without considering the regulation of surface hydrophilicity / hydrophobicity properties to enrich low-abundance proteins. Since the amino acids that make up proteins have different side chain hydrophilicity / hydrophobicity properties, designing magnetic beads with different surface hydrophilicity / hydrophobicity properties has a significant impact on the enrichment of low-abundance proteins.

[0006] Magnetic beads, due to their advantages such as large specific surface area, rapid and simple separation, and ease of functionalization, have been widely used in the field of protein enrichment. Currently, magnetic beads used for enriching low-abundance proteins mainly achieve specific or non-specific enrichment of target proteins through surface modification with hydrophilic groups, ion exchange groups, antibodies, aptamers, etc. Existing magnetic bead materials suffer from an imbalance of hydrophilicity and hydrophobicity: purely hydrophilic modified magnetic beads have weak binding capacity for hydrophobic low-abundance proteins, resulting in low enrichment efficiency; purely hydrophobic modified magnetic beads are prone to non-specific adsorption, leading to co-enrichment of high-abundance proteins and poor selectivity. Simultaneously, some magnetic beads exhibit uneven density of surface functional groups, poor stability, insufficient magnetic responsiveness, and weak resistance to contamination in complex biological samples, limiting their application in low-abundance protein enrichment. Achieving a hydrophilicity-hydrophobicity balance by precisely controlling the ratio of hydrophilicity to hydrophobicity on the magnetic bead surface can balance high binding capacity and high selectivity for low-abundance proteins, reduce non-specific adsorption of high-abundance proteins, and improve the resistance to contamination in complex biological samples. This is the core approach to overcoming the shortcomings of existing technologies. Summary of the Invention

[0007] The purpose of this invention is to overcome the defects of the prior art and provide a method for preparing hydrophilic-hydrophobic balanced magnetic beads.

[0008] Another object of the present invention is to provide the application of the hydrophilicity-hydrophobicity balanced magnetic beads prepared by the above preparation method in the enrichment of low-abundance proteins.

[0009] The technical solution of the present invention is as follows:

[0010] A method for preparing hydrophilic-hydrophobic balanced magnetic beads, characterized in that: the hydrophilic-hydrophobic balanced magnetic beads are selected from HLB, HLB-WCX, HLB-MCX, HLB-WAX and HLB-MAX obtained as follows;

[0011] Specifically, the steps include the following:

[0012] (1) FeCl3·6H2O and anhydrous sodium acetate were added to ethylene glycol to react and Fe3O4 was prepared.

[0013] (2) Fe3O4 was dispersed in deionized water, and ammonia and TEOS were added to react and prepare Fe3O4@SiO2;

[0014] (3) Fe3O4@SiO2 was dispersed in 80% ethanol, and ammonia and propyl 3-(trimethoxysilyl)methacrylate (MPS) were added to react and Fe3O4@SiO2@MPS was prepared.

[0015] (4) Fe3O4@SiO2@MPS was ultrasonically treated with citric acid solution and then dispersed in acetonitrile. DVB, NVP and AIBN were added to react and prepare hydrophilic-hydrophobic balanced magnetic beads HLB.

[0016] (5) HLB was dispersed in glacial acetic acid and H2O2 and heated to react, thus preparing HLB-WCX;

[0017] (6) HLB was dispersed in glacial acetic acid and concentrated sulfuric acid and reacted at 0°C to prepare HLB-MCX;

[0018] (7) Fe3O4@SiO2@MPS was dispersed in acetonitrile, and NVP, DVB, VBC and AIBN were added to react and VBC magnetic beads were prepared.

[0019] (8) Disperse VBC magnetic beads in 1,2-dichloroethane, add N,N-dimethylbutylamine to react, and prepare HLB-MAX;

[0020] (9) Disperse VBC magnetic beads in toluene, add piperazine to react, and prepare HLB-WAX.

[0021] In a preferred embodiment of the present invention

[0022] The reaction conditions for step (1) are: heating to 150 ℃ for 1 h, then heating to 190 ℃ for 8 h. In this step, the ratio of FeCl3·6H2O, anhydrous sodium acetate and ethylene glycol is 16-17 g: 29-30 g: 1200 mL.

[0023] The reaction conditions for step (2) are: stirring at room temperature followed by treatment at 180 °C for 24 h;

[0024] The reaction conditions for step (3) include: stirring the reaction at 50 °C. In this step, the ratio of Fe3O4@SiO2, 80% ethanol, ammonia and propyl 3-(trimethoxysilyl)methacrylate is 1g: 50 mL: 3 mL: 4 mL.

[0025] More preferably, the reaction conditions in step (4) include: heating to 80 °C and stirring the reaction, wherein the ratio of Fe3O4@SiO2@MPS, acetonitrile, DVB, NVP and AIBN is 1 g: 200 mL: 2.4 mL: 6 mL: 0.1 g.

[0026] More preferably, the reaction conditions in step (5) include: heating to 80 °C and reacting for 72 h, wherein the ratio of HLB, glacial acetic acid and hydrogen peroxide is 1 g: 3 mL: 1 mL.

[0027] More preferably, the reaction conditions for step (6) include: reaction at 0 °C for 20 min, wherein the ratio of HLB, glacial acetic acid and concentrated sulfuric acid is 1 g: 1 mL: 6 mL.

[0028] More preferably, the reaction conditions in step (7) include: heating to 120 °C and stirring the reaction, wherein the ratio of Fe3O4@SiO2@MPS, acetonitrile, NVP, DVB, VBC and AIBN is 1 g: 100 mL: 2 mL: 1 mL: 1 mL: 0.1 g.

[0029] More preferably, the reaction conditions for step (8) include: heating to 80 °C and reacting for 24 h, wherein the ratio of VBC magnetic beads, 1,2-dichloroethane and N,N-dimethylbutylamine is 1 g: 40 mL: 50 mg.

[0030] More preferably, the reaction conditions for step (9) include: heating to 80 °C and reacting for 12 h, wherein the ratio of VBC magnetic beads, toluene and piperazine is 1 g: 40 mL: 50 mg.

[0031] A method for enriching low-abundance proteins in a sample to be tested, using hydrophilic-hydrophobic balanced magnetic beads prepared by the above-mentioned method.

[0032] The above preparation method is used for the enrichment of low-abundance proteins.

[0033] The beneficial effects of this invention are:

[0034] 1. The hydrophilic-hydrophobic balanced magnetic beads prepared by this invention contain both benzene rings (hydrophobic) and amide groups (hydrophilic) on their surface, which achieves balanced adsorption of proteins with different hydrophilic and hydrophobic properties, significantly improving the enrichment efficiency and selectivity of low-abundance proteins and reducing non-specific interference from high-abundance proteins.

[0035] 2. This invention further modifies ion exchange groups such as carboxyl groups, sulfonic acid groups, quaternary ammonium groups, and piperazine groups to form various magnetic beads such as HLB-WCX, HLB-MCX, HLB-MAX, and HLB-WAX. When used in combination, these beads can enhance protein crown formation through multi-mode interactions (such as hydrophobicity, electrostatics, and hydrogen bonding), thereby achieving high-throughput enrichment of low-abundance proteins in complex biological samples (such as serum and plasma) and improving protein coverage depth.

[0036] 3. The hydrophilic-hydrophobic balanced magnetic beads prepared by this invention have good magnetic responsiveness and stability, uniform particle size (100-200nm), suitable zeta potential, and are easy to rapidly separate magnetically and reuse. They are suitable for high-throughput proteomics analysis, which improves the detection rate of early disease diagnosis (such as cancer) and the support capability for precision medicine.

[0037] 4. The preparation method of the present invention is simple and controllable, with a reasonable dosage range, optimized reaction conditions, and easy to scale up production. At the same time, it has strong anti-pollution ability and maintains high efficiency performance in physiological environment, solving the problems of imbalance between hydrophilicity and hydrophobicity and uneven surface function of existing magnetic beads.

[0038] 5. The enrichment method of the present invention, combined with specific pretreatment and enzymatic digestion procedures (such as incubation at 37 °C for 1 h and trypsin digestion for 16 h), can obtain a supernatant containing low-abundance protein peptides, which is suitable for LC-MS / MS analysis. Experimental results show that the number of proteins identified is significantly increased and better matches low-concentration proteins in human protein maps, demonstrating excellent sensitivity and practical value. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the preparation process in Example 1 of the present invention.

[0040] Figure 2 Scanning electron microscope (SEM) images of Fe3O4 magnetic beads (a), Fe3O4@SiO2 (b), and Fe3O4@SiO2@MPS (c) prepared in Example 1 of this invention show that these magnetic beads are spherical particles with a diameter of 100-200 nm.

[0041] Figure 3Scanning electron microscope images of HLB (a), HLB-WCX (b), HLB-MCX (c), VBC magnetic beads (d), HLB-WAX (e) and HLB-MAX (f) prepared for Example 1 of the present invention show that these magnetic beads are all spherical particles of 100-200 nm.

[0042] Figure 4 This shows the Zeta potential of the hydrophilic-hydrophobic balanced magnetic beads prepared in Example 1 of the present invention.

[0043] Figure 5 This shows the number of proteins identified by serum protein enrichment using hydrophilic-hydrophobic balanced magnetic beads in Examples 2 to 17 of the present invention.

[0044] Figure 6 This demonstrates the matching of serum protein enrichment by the hydrophilic-hydrophobic balanced magnetic beads prepared in Example 1 of the present invention with the corresponding absolute blood protein concentrations in the human protein map. Detailed Implementation

[0045] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0046] Example 1: Preparation of hydrophilic-hydrophobic balanced magnetic beads

[0047] 1. Experimental materials

[0048] Ferric chloride hexahydrate, anhydrous sodium acetate, ethylene glycol, ammonia, tetraethyl orthosilicate, ethanol, glacial acetic acid, and acetonitrile were all purchased from Xilong Scientific; propyl 3-(trimethoxysilyl)methacrylate was purchased from Merck Life Sciences; N-vinylpyrrolidone, divinylbenzene, and 4-vinylbenzyl chloride were all purchased from Maclean's; hydrogen peroxide, sulfuric acid, toluene, and piperazine were all purchased from Sinopharm; and 1,2-dichloroethane and N,N-dimethylbutylamine were all purchased from Aladdin.

[0049] 2. Experimental methods (e.g.) Figure 1 (As shown)

[0050] (1) Preparation of Fe3O4: Weigh 16.2 g of ferric chloride hexahydrate in a three-layer self-sealing bag, place the bag under a book, and crush the solid into powder with a hammer. Weigh 29.6 g of anhydrous sodium acetate in a new self-sealing bag. Pour the solid into a 3 L beaker, add 1200 mL of ethylene glycol, and stir until transparent. Pour all the above materials into a reaction vessel, being careful not to let them stick to the walls. Lower the reaction vessel lid and seal the reaction vessel (vacuum check). Set the reaction vessel temperature to 150 ℃ and react for 1 h. Raise the temperature to 190 ℃ and react for 12 h. After the reaction vessel cools down, release the material and sequentially perform magnetic separation and hot water washing five times. Then disperse it in 1000 mL of water for storage and later use. Figure 2 Fe3O4 magnetic beads as shown in figure a;

[0051] (2) Preparation of Fe3O4@SiO2: 50 g of Fe3O4 magnetic beads obtained in step (1) were dispersed in 3 L of deionized water, stirred, and 120 mL of concentrated ammonia was added. The mixture was stirred for 10 min to make it uniform. 60 mL of TEOS was added, and the mixture was stirred for 24 h. Then, 20 mL of TEOS was added, and the mixture was stirred for another 24 h. After magnetic separation, the resulting material was dispersed in 400 mL of ethanol, 4 mL of ammonia was added, and the mixture was treated at 180 °C for 24 h. The mixture was then washed until neutral and stored in 20% ethanol-water solution to obtain the desired product. Figure 2 b shows Fe3O4@SiO2;

[0052] (3) Preparation of Fe3O4@SiO2@MPS: Weigh 1 g of Fe3O4@SiO2 obtained in step (2), rinse twice with 50 mL of 80% ethanol, ultrasonically disperse in 50 mL of 80% ethanol, add 3 mL of ammonia water, stir at 50 °C for 10 min, slowly add 4 mL of MPS, stir at 250 r / min and 50 °C for 24 h, after the reaction is complete, wash three times with ethanol, and store in anhydrous ethanol to obtain the product as shown in the figure. Figure 2 c shows Fe3O4@SiO2@MPS;

[0053] (4) Preparation of HLB: Weigh 1 g of Fe3O4@SiO2@MPS obtained in step (3), add it to 20 mL of 2% citric acid solution and sonicate for 10 min; add it to 200 mL of acetonitrile, heat to 80 ℃, stir at 300 r / min for 30 min, then add 2.4 mL of vinylpyrrolidone (10 mmol), 6 mL of divinylbenzene (10 mmol) and 0.1 g of AIBN, and react for 12 h; after the reaction, magnetic separation is used to obtain the product and it is repeatedly washed with ethanol. Store it in 50% isopropanol aqueous solution to obtain the product as shown in the figure. Figure 3 The HLB shown in figure a has a Zeta potential as follows: Figure 4 As shown;

[0054] (5) Preparation of HLB-WCX: Weigh 1 g of the HLB magnetic beads obtained in step (4), add 3 mL of glacial acetic acid and 1 mL of hydrogen peroxide, heat to 80 °C and react for 72 h, cool and then magnetically separate to obtain the product, wash with deionized water until neutral, and then obtain the product as shown in the figure. Figure 3 The HLB-WCX shown in c has a zeta potential as follows: Figure 4 As shown;

[0055] (6) Preparation of HLB-MCX: Weigh 1 g of the HLB magnetic beads obtained in step (4), add 1 mL of glacial acetic acid and 6 mL of concentrated sulfuric acid, react at 0 °C for 20 min, and after magnetic separation, wash with deionized water until neutral to obtain the product. Figure 3 The HLB-MCX shown in d has a Zeta potential as follows: Figure 4 As shown;

[0056] (7) Preparation of VBC magnetic beads: Weigh 1 g of Fe3O4@SiO2@MPS obtained in step (3) and add it to 100 mL of acetonitrile. Heat to 120 °C, stir at 300 r / min for 30 min, add 2 mL of vinylpyrrolidone, 1 mL of 4-vinylbenzyl chloride, 1 mL of divinylbenzene and 0.1 g of AIBN, and react for 12 h. After the reaction is complete, magnetic separation is performed, and the product is washed several times with ethanol to obtain the following: Figure 3 The VBC bead shown in b has a Zeta potential as follows: Figure 4 As shown;

[0057] (8) Preparation of HLB-MAX: 1 g of VBC magnetic beads obtained in step (7) was added to 40 mL of 1,2-dichloroethane, followed by 50 mg of N,N-dimethylbutylamine. The mixture was reacted at 80 °C for 24 h. After the reaction was completed, the product was obtained by magnetic separation and washed with deionized water until neutral. Figure 3 The HLB-MAX shown in f has a Zeta potential as follows: Figure 4 As shown;

[0058] (9) Preparation of HLB-WAX: Weigh 1 g of the VBC magnetic beads obtained in step (7), add them to 40 mL of toluene, then add 50 mg of piperazine, and react at 80 °C for 12 h. After the reaction is complete, magnetically separate and wash several times with methanol / water to obtain the following product. Figure 3 The HLB-WAX shown in e has a zeta potential as follows: Figure 4 As shown.

[0059] Example 2

[0060] Hydrophilic-hydrophobic balanced magnetic beads for enrichment of low-abundance proteins in blood and proteomics analysis

[0061] (1) The prepared hydrophilic-hydrophobic balanced magnetic beads were used for the enrichment of low-abundance proteins in blood and proteomics analysis. The specific experimental procedure is as follows: 1 mg of HLB, HLB-WCX, HLB-MCX, HLB-WAX and HLB-MAX magnetic beads were mixed in equal proportions and washed 3 times with 200 μL of diluent (10 mM Tris-HCl (pH 7.4), 150 mM KCl, 0.05% CHAPS), magnetically separated, the supernatant was removed, and the beads were resuspended with 200 μL of diluent. The solution was added to diluted serum (40 μL serum + 160 μL diluent) and incubated at 37 °C for 1 h. After incubation, the protein was magnetically separated, the supernatant was discarded, and the protein was washed three times with 200 μL of diluent. After magnetic separation and discarding the supernatant, 100 μL of lysis buffer (8 M Urea, 1% SDS), 10 μL of enzyme digestion buffer 1 (1 M TEAB), and 2 μL of enzyme digestion buffer 2 (0.5 M TCEP) were added. The mixture was incubated at 37 °C with shaking for 1 h. Then, 4 μL of enzyme digestion buffer 3 (1 M IAM) was added, and the mixture was incubated at room temperature in the dark for 40 min. 800 μL of precipitation buffer (acetone solution) was added, and the protein was precipitated at -20 °C for 2 h. After magnetic separation and discarding the supernatant, the protein was washed with 800 μL of washing buffer A (90% acetone solution, 20 mM NaCl) and dried in a fume hood for 5 min. Finally, 100 μL of enzyme digestion buffer (50 mM ammonium bicarbonate) and 1 μg of trypsin were added, and the mixture was incubated at 37 °C with shaking for 16 h. Add 2 μL of TFA, bring the volume to 200 μL with enzyme digestion solution, centrifuge at 10000 g for 10 min at 4 ℃, collect the supernatant, desalt it using a three-layer tip column, and heat dry it.

[0062] (2) LC-MS / MS analysis

[0063] DIA mass spectrometry analysis was performed using an ASTRAL mass spectrometer. The liquid chromatography conditions were as follows: C18 analytical column (C18, 15 cm × 100 μm, 1.7 μm). Mobile phase A consisted of 98% distilled water, 2% acetonitrile, and 0.1% formic acid; mobile phase B consisted of 80% acetonitrile, 20% distilled water, and 0.1% formic acid. The gradient length was dynamically adjusted as follows: 0–1 min (8.0–17.0% B); 1–5.5 min (17.0–55.0% B); 5.5–7 min (55.0–99.0% B); 7–8 min (99.0% B). DIA data acquisition mode was used, with a primary scan range of 380–980 m / z and a secondary scan range of 150–2000 m / z. Positive ion detection was used, and the ion source voltage was set to 2.2 kV.

[0064] (3) Data Analysis

[0065] The .RAW files generated by the ASTRAL mass spectrometer were searched using the DIA-NN database. The target database was Uniprot.human. Search parameters were set to trypsin ring digestion, two protease cleavage sites were specified, and Carbamidomethylati (C) was selected as the protein fixation modification, with variable modifications of Ox (M) and Ac (N-term). The mass spectrometry first-stage mass error was set to 15 ppm, the second-stage mass error to 15 ppm, and the false positive rate to 1%.

[0066] Examples 3 to 17

[0067] The hydrophilic-hydrophobic balanced magnetic beads prepared in Example 1 were used for the enrichment of low-abundance proteins in blood and proteomics analysis. The specific differences between Examples 3 to 17 and Example 2 are shown in Table 1 below.

[0068] Example Differences from Example 2 Magnetic bead type and dosage Serum samples and diluent Diluent composition Washing conditions Other notes 2 Standard Procedure Five types of magnetic beads—HLB, HLB-WCX, HLB-MCX, HLB-WAX, and HLB-MAX—were mixed in equal proportions. One mg of the mixture was then washed with 200 μL of diluent. 40 μL serum + 160 μL diluent 10mM Tris-HCl (pH 7.4), 150mM KCl, 0.05% CHAPS Wash three times with 200 μL of diluent (after incubation). The subsequent LC-MS / MS analysis and data analysis are the same. 3 Reduced serum volume and amount of magnetic beads After mixing the five types of magnetic beads in equal proportions, take 0.5 mg of the mixture and wash with 100 μL of diluent. 20 μL serum + 80 μL diluent Same as Example 2 Same as Example 2 - 4 Adjust the serum to diluent ratio and reduce the amount of magnetic beads used. After mixing the five types of magnetic beads in equal proportions, take 0.5 mg of the mixture and wash with 100 μL of diluent. 40 μL serum + 60 μL diluent Same as Example 2 Same as Example 2 - 5 Increased serum volume Same as Example 2 80 μL serum + 120 μL diluent Same as Example 2 Same as Example 2 - 6 Changes in diluent composition Same as Example 2 Same as Example 2 0.05% CHAPS in PBS (10mM PBS, 150mM NaCl, 0.05% CHAPS) Same as Example 2 - 7 Changes in diluent composition Same as Example 2 Same as Example 2 50mM Bis-Trispropane (containing 150mM NaCl) Same as Example 2 - 8 Changes in washing solution after incubation Same as Example 2 Same as Example 2 Same as Example 2 Wash with 0.07% ammonia solution after incubation. - 9 Changes in diluent and washing solution after incubation Same as Example 6 Same as Example 6 Same as Example 6 Wash with 0.07% ammonia solution after incubation. Based on the variant of Example 6 10 Changes in diluent and washing solution after incubation Same as Example 7 Same as Example 7 Same as Example 7 Wash with 0.07% ammonia solution after incubation. Based on the variant of Example 7 11 Single magnetic bead type Use 200 μL of HLB (1 mg) Same as Example 2 Same as Example 2 Same as Example 2 - 12 Single magnetic bead type Use 200 μL of HLB-WCX (1 mg) Same as Example 2 Same as Example 2 Same as Example 2 - 13 Single magnetic bead type Use 200 μL of HLB-MCX (1 mg) Same as Example 2 Same as Example 2 Same as Example 2 - 14 Single magnetic bead type Use 200 μL of HLB-WAX (1 mg) Same as Example 2 Same as Example 2 Same as Example 2 - 15 Single magnetic bead type Use 200 μL of HLB-MAX (1 mg) Same as Example 2 Same as Example 2 Same as Example 2 - 16 Two types of magnetic beads mixed Take 200 μL of a mixture of HLB-WCX and HLB-MCX in equal proportions (total 1 mg). Same as Example 2 Same as Example 2 Same as Example 2 - 17 Two types of magnetic beads mixed Mix HLB-WAX and HLB-MAX in equal proportions (total 1 mg) and take 200 μL. Same as Example 2 Same as Example 2 Same as Example 2 -

[0069] from Figure 5 and Figure 6 It can be seen that the hydrophilicity-hydrophobicity balanced magnetic beads prepared in Example 1 of this invention exhibit efficient enrichment of low-abundance proteins. It should be noted that... Figure 6 The diagram illustrates the distribution curves of serum and plasma samples enriched by the mixed magnetic beads used in Example 2 according to the standard procedure of Example 2, arranged from high to low Log10 values ​​of the enriched proteins. A long-tailed plateau appears in the latter part of the curves, indicating that the signal intensity of a large number of low-abundance proteins is stable and continuous, and their Log10 values ​​are still higher than the instrument's background detection value, indicating that the signal of low-abundance proteins has been enhanced to a detectable range. Furthermore, the curve trends of serum and plasma are basically consistent, demonstrating the reproducibility of this method for different samples.

[0070] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for preparing hydrophilic-hydrophobic balanced magnetic beads, characterized in that: The hydrophilic-hydrophobic balanced magnetic beads are selected from the following HLB, HLB-WCX, HLB-MCX, HLB-WAX and HLB-MAX; Specifically, the steps include the following: (1) FeCl3·6H2O and anhydrous sodium acetate were added to ethylene glycol to react and Fe3O4 was prepared. (2) Fe3O4 was dispersed in deionized water, and ammonia and TEOS were added to react and prepare Fe3O4@SiO2; (3) Fe3O4@SiO2 was dispersed in 80% ethanol, and ammonia and propyl 3-(trimethoxysilyl)methacrylate were added to react and Fe3O4@SiO2@MPS was prepared. (4) Fe3O4@SiO2@MPS was ultrasonically treated with citric acid solution and then dispersed in acetonitrile. DVB, NVP and AIBN were added to react and prepare hydrophilic-hydrophobic balanced magnetic beads HLB. (5) HLB was dispersed in glacial acetic acid and H2O2 and heated to react, thus preparing HLB-WCX; (6) HLB was dispersed in glacial acetic acid and concentrated sulfuric acid and reacted at 0°C to prepare HLB-MCX; (7) Fe3O4@SiO2@MPS was dispersed in acetonitrile, and NVP, DVB, VBC and AIBN were added to react and VBC magnetic beads were prepared. (8) Disperse VBC magnetic beads in 1,2-dichloroethane, add N,N-dimethylbutylamine to react, and prepare HLB-MAX; (9) Disperse VBC magnetic beads in toluene, add piperazine to react, and prepare HLB-WAX.

2. The preparation method according to claim 1, characterized in that: The reaction conditions for step (1) are: heating to 150 ℃ for 1 h, then heating to 190 ℃ for 8 h. In this step, the ratio of FeCl3·6H2O, anhydrous sodium acetate and ethylene glycol is 16-17 g: 29-30 g: 1200 mL. The reaction conditions for step (2) are: stirring at room temperature followed by treatment at 180 °C for 24 h; The reaction conditions for step (3) include: stirring the reaction at 50 °C. In this step, the ratio of Fe3O4@SiO2, 80% ethanol, ammonia and propyl 3-(trimethoxysilyl)methacrylate is 1g: 50 mL: 3 mL: 4 mL.

3. The preparation method according to claim 2, characterized in that: The reaction conditions in step (4) include: heating to 80°C and stirring the reaction. In this step, the ratio of Fe3O4@SiO2@MPS, acetonitrile, DVB, NVP and AIBN is 1 g: 200 mL: 2.4 mL: 6 mL: 0.1 g.

4. The preparation method according to claim 3, characterized in that: The reaction conditions for step (5) include: heating to 80°C and reacting for 72 h. In this step, the ratio of HLB, glacial acetic acid and hydrogen peroxide is 1 g: 3 mL: 1 mL.

5. The preparation method according to claim 3, characterized in that: The reaction conditions for step (6) include: reaction at 0 °C for 20 min, and the ratio of HLB, glacial acetic acid and concentrated sulfuric acid in this step is 1 g: 1 mL: 6 mL.

6. The preparation method according to claim 2, characterized in that: The reaction conditions in step (7) include: heating to 120 °C and stirring the reaction. In this step, the ratio of Fe3O4@SiO2@MPS, acetonitrile, NVP, DVB, VBC and AIBN is 1 g: 100 mL: 2 mL: 1 mL: 1 mL: 0.1 g.

7. The preparation method according to claim 6, characterized in that: The reaction conditions for step (8) include: heating to 80°C and reacting for 24 h. In this step, the ratio of VBC magnetic beads, 1,2-dichloroethane and N,N-dimethylbutylamine is 1 g: 40 mL: 50 mg.

8. The preparation method according to claim 6, characterized in that: The reaction conditions for step (9) include: heating to 80°C and reacting for 12 h. In this step, the ratio of VBC magnetic beads, toluene and piperazine is 1 g: 40 mL: 50 mg.

9. A method for enriching low-abundance proteins in a sample to be tested, characterized in that: The preparation was carried out using hydrophilic-hydrophobic balanced magnetic beads prepared by the preparation method described in any one of claims 1 to 8.

10. The application of hydrophilic-hydrophobic balanced magnetic beads prepared by the preparation method according to any one of claims 1 to 8 in the enrichment of low-abundance proteins.

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