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CN-121972149-A - Preparation method of affinity-hydrophobicity balance magnetic beads and application of affinity-hydrophobicity balance magnetic beads in enrichment of low-abundance proteins

CN121972149ACN 121972149 ACN121972149 ACN 121972149ACN-121972149-A

Abstract

The invention discloses a preparation method of affinity-hydrophobicity balance magnetic beads and application thereof in low-abundance protein enrichment, wherein the affinity-hydrophobicity balance magnetic beads are selected from HLB, HLB-WCX, HLB-MCX, HLB-WAX and HLB-MAX. The surface of the affinity-hydrophobicity balance magnetic bead prepared by the invention contains benzene ring (hydrophobic) and amido (hydrophilic), so that the equilibrium adsorption of proteins with different affinity-hydrophobicity characteristics is realized, the enrichment efficiency and selectivity of low-abundance proteins are obviously improved, and the non-specific interference of high-abundance proteins is reduced.

Inventors

  • CHEN GUO
  • SI HONGJUN
  • Chi Changbiao

Assignees

  • 华侨大学

Dates

Publication Date
20260505
Application Date
20260129

Claims (10)

  1. 1. A method for preparing affinity-hydrophobicity water balance magnetic beads is characterized in that the affinity-hydrophobicity water balance magnetic beads are selected from HLB, HLB-WCX, HLB-MCX, HLB-WAX and HLB-MAX prepared by the following steps; The method specifically comprises the following steps: (1) Adding FeCl 3 ·6H 2 O and anhydrous sodium acetate into ethylene glycol to react, and preparing Fe 3 O 4 ; (2) Dispersing Fe 3 O 4 in deionized water, adding ammonia water and TEOS for reaction, and preparing Fe 3 O 4 @SiO 2 ; (3) Dispersing Fe 3 O 4 @SiO 2 in 80% ethanol, adding ammonia water and 3- (trimethoxysilyl) propyl methacrylate for reaction, and preparing Fe 3 O 4 @SiO 2 @MPS; (4) Dispersing Fe 3 O 4 @SiO 2 @MPS in acetonitrile after ultrasonic treatment of citric acid solution, and adding DVB, NVP and AIBN for reaction to prepare hydrophilic-hydrophobic balance magnetic bead HLB; (5) Dispersing HLB in glacial acetic acid and H 2 O 2 , and heating to react to obtain HLB-WCX; (6) Dispersing HLB in glacial acetic acid and concentrated sulfuric acid, and reacting at 0 ℃ to obtain HLB-MCX; (7) Dispersing Fe 3 O 4 @SiO 2 @MPS in acetonitrile, adding NVP, DVB, VBC and AIBN for reaction, and preparing VBC magnetic beads; (8) Dispersing VBC magnetic beads in 1, 2-dichloroethane, adding N, N-dimethylbutylamine to react, and preparing HLB-MAX; (9) Dispersing VBC magnetic beads in toluene, adding piperazine for reaction, and preparing HLB-WAX.
  2. 2. The method of manufacturing according to claim 1, wherein: the reaction condition of the step (1) is that the temperature is raised to 150 ℃ for reaction for 1h, and then the temperature is raised to 190 ℃ for reaction for 8h, wherein the ratio of FeCl 3 ·6H 2 O, anhydrous sodium acetate and ethylene glycol in the step is 16-17: 17 g:29-30: 30 g:1200 mL; The reaction condition of the step (2) is that the reaction is stirred at room temperature and then is treated at 180 ℃ for 24 h; The reaction conditions of the step (3) include stirring reaction at 50 ℃, wherein the ratio of Fe 3 O 4 @SiO 2 , 80% ethanol, ammonia water and 3- (trimethoxysilyl) propyl methacrylate in the step is 1g of 50: 50 mL:3 mL:4 mL.
  3. 3. The method according to claim 2, wherein the reaction condition in the step (4) comprises heating to 80 ℃ and stirring to react, wherein the ratio of Fe 3 O 4 @SiO 2 @MPS, acetonitrile, DVB, NVP and AIBN in the step is 1 g:200 mL:2.4 mL:6 mL:0.1 g.
  4. 4. The method according to claim 3, wherein the reaction condition in the step (5) comprises a reaction at a temperature of 80℃of 72h, wherein the ratio of HLB, glacial acetic acid and hydrogen peroxide is 1 g/3 mL/1 mL.
  5. 5. The method according to claim 3, wherein the reaction condition in the step (6) comprises a reaction at 0℃of 20 min, wherein the ratio of HLB, glacial acetic acid and concentrated sulfuric acid is 1 g/1 mL/6 mL.
  6. 6. The method according to claim 2, wherein the reaction condition in the step (7) comprises heating to 120 ℃ and stirring to react, wherein the ratio of Fe 3 O 4 @SiO 2 @MPS, acetonitrile, NVP, DVB, VBC and AIBN in the step is 1 g:100: 100 mL:2 mL:1 mL:1 mL:0.1 g.
  7. 7. The process according to claim 6, wherein the reaction conditions in the step (8) comprise a reaction at a temperature of 80℃of 24h, in which the ratio of VBC beads, 1, 2-dichloroethane and N, N-dimethylbutylamine is 1g, 40: 40 mL:50: 50 mg.
  8. 8. The method according to claim 6, wherein the reaction condition in the step (9) comprises a reaction of 12 h at 80℃with a ratio of VBC beads, toluene and piperazine of 1g to 40: 40 mL:50: 50 mg.
  9. 9. A method for enriching low-abundance proteins in a sample to be detected is characterized by adopting the affinity-hydrophobicity water balance magnetic beads prepared by the preparation method according to any one of claims 1 to 8.
  10. 10. Use of affinity/hydrophobicity balance magnetic beads prepared by the preparation method of any one of claims 1 to 8 in low abundance protein enrichment.

Description

Preparation method of affinity-hydrophobicity balance magnetic beads and application of affinity-hydrophobicity balance magnetic beads in enrichment of low-abundance proteins Technical Field The invention belongs to the technical field of biological separation materials, and particularly relates to a preparation method of affinity-hydrophobicity balance magnetic beads and application of the affinity-hydrophobicity balance magnetic beads in enrichment of low-abundance proteins. Background The invention belongs to the technical field of biological separation materials, and particularly relates to surface functionalization modification of magnetic nanoparticles and application thereof in pretreatment of proteomics, in particular to efficient enrichment of low-abundance proteins in complex biological samples by regulating and controlling hydrophilicity and hydrophobicity and ion exchange characteristics. The background technology is that the protein composition in biological samples (such as serum, plasma, urine, cell lysate and the like) is complex, the ratio of high-abundance proteins (such as albumin and immunoglobulin in serum) is extremely high, and the detection and analysis of low-abundance proteins (such as tumor markers and signal proteins) are seriously interfered, so that the low-abundance proteins are difficult to directly identify by conventional detection means (such as mass spectrum and electrophoresis). The low-abundance proteins (such as tumor markers, signal proteins, cytokines and the like) are often involved in important physiological regulation and control processes of organisms, are core biomarkers for early diagnosis of diseases, pathological mechanism research and drug target screening, have important biological functions and clinical diagnostic values, and therefore, the realization of high-efficiency enrichment of the low-abundance proteins is a key technical problem in the field of biological analysis. In the early stage of many diseases (such as cancer and autoimmune diseases), the change of high-abundance proteins in blood is not obvious, and characteristic expression difference of low-abundance proteins is generated, so that the early detection rate of the diseases can be improved by enriching the low-abundance proteins, and the support is provided for accurate medical treatment. The low-abundance protein enrichment technology is mainly realized by removing high-abundance proteins or directly enriching low-abundance proteins, and common methods include ultrafiltration, precipitation, electrophoresis, affinity, chromatography and novel material technologies. The ultrafiltration method has low cost, is easy to lose low-abundance proteins, has high specificity and high cost, and has limited sample loading capacity. At present, the sample needs to be subjected to high abundance removal before proteomics research, and high abundance proteins are removed by using an immunoaffinity method to reduce the influence of the high abundance proteins on low abundance protein identification, but still the high depth and high flux level cannot be achieved. Kenneth a. Dawson et al, when studying Protein interactions with particles, set forth the concept of "Protein Corona (PC)", nanoparticles (NPs) encounter and interact with a number of components in the physiological environment, thereby altering the behavior and properties of NPs, which coat the nanoparticle surface to form Protein crowns. Research shows that the formation of protein crowns is related to the characteristics of NPs, so that the target protein crowns can be obtained through reasonable design of NPs, and proteomics research is more accurate. The sensitivity is improved by 100 ten thousand times based on the magnetic nano probe as ProteonanoTM which is proposed by the American SEER company, and the method is suitable for large-scale biomarker discovery. But the prepared magnetic beads only consider the regulation and control of surface groups to realize the enrichment of proteins, and the regulation and control of the hydrophilic-hydrophobic characteristics of the surfaces are not considered to enrich low-abundance proteins. The amino acids forming the protein have different side chain hydrophile-hydrophobic characteristics, so that the magnetic beads with different surface hydrophile-hydrophobic characteristics are designed, and the magnetic beads have important influence on the enrichment of low-abundance proteins. The magnetic beads have the advantages of large specific surface area, rapid and simple separation, easy functional modification and the like, and are widely applied to the field of protein enrichment. The magnetic beads for enriching low-abundance proteins at present realize specific or non-specific enrichment of target proteins mainly through surface modification of hydrophilic groups, ion exchange groups, antibodies, aptamers and the like. The existing magnetic bead material has the problems of poor affinity and hydro