A macroporous agarose boron affinity material, a preparation method and application thereof
By preparing macroporous agarose boric acid affinity material, the low-capacity problem of neomycin separation and purification in complex biological samples was solved by utilizing the high density of boron affinity ligands and the overall macroporous structure of agarose, thus achieving a highly efficient neomycin separation effect.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- QUANZHOU MARINE BIOTECHNOLOGY IND RES INST
- Filing Date
- 2023-03-13
- Publication Date
- 2026-07-21
AI Technical Summary
Existing technologies cannot effectively, rapidly, and sensitively isolate and purify neomycin from complex biological samples, especially due to the low capacity of macroporous gel materials and interference from complex substances, which makes detection difficult.
A macroporous agarose boric acid affinity material was prepared by combining hydrophilic polyethyleneimine-modified silicon nanoparticles with high-performance boron affinity ligands. The high density of the boron affinity ligands and the macroporous structure of the agarose were utilized to achieve selective binding to neomycin.
It significantly improves the binding ability to neomycin, solves the problem of separating and purifying neomycin in complex aquatic products, and provides an efficient separation method.
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Figure CN117205895B_ABST
Abstract
Description
Technical fields:
[0001] This invention belongs to the field of bioengineering technology, specifically relating to a macroporous agarose-boronic acid affinity material, its preparation method, and its application. Background technology:
[0002] Neomycin belongs to the aminoglycoside antibiotic family. It is a glycoside compound formed by linking an aminocyclic alcohol and an amino sugar via an oxygen bridge. It can inhibit the release of synthetic proteins by bacteria or induce the synthesis of incorrect proteins, ultimately leading to bacterial death. Due to its broad-spectrum growth inhibitory effect on both Gram-positive and Gram-negative bacteria, neomycin is widely used to treat bacterial infections in animals. However, neomycin has potential ototoxicity and nephrotoxicity in both humans and animals. To protect consumer health, the European Union has set maximum residue limits for neomycin in animal tissues: 500 μg / kg for pork, liver, and eggs; 1500 μg / kg for milk; and 5000 μg / kg for kidneys. Therefore, it is crucial to develop an effective, rapid, and sensitive method for detecting neomycin residues in food, which has promoted the development of new detection strategies, such as immunoassays, biochemical sensors, and chromatographic methods. Most reported methods can achieve detection limits down to the nanogram level in certain cases. However, this is still insufficient. The trace amounts of neomycin in food and the large presence of interfering substances severely affect the identification of neomycin, making direct detection very difficult. Therefore, effective, reliable, and simple pretreatment is essential before analyzing neomycin.
[0003] Extensive research has been conducted on the isolation and purification of neomycin from complex biological samples. Acidic solvent extraction coupled with solid-phase extraction is the most common method for pretreatment of neomycin, and these pretreatment methods have shown good results through optimization of the purification process. However, substances present in the actual biological sample matrix can easily interfere with ion exchange modes. Furthermore, the eluent of ion exchange columns often contains a large amount of hydrophobic substances, which is detrimental to subsequent analysis. To address this issue, researchers have developed an immunoaffinity column for purifying neomycin from aquatic products. Studies have found that after treatment with the immunoaffinity column, the recovery rate of neomycin added to actual aquatic products is 75.58%–117.80%. However, the application of antibody-integrated affinity columns typically suffers from high cost and poor stability to complex interfering substances. Therefore, exploring an ideal method for the separation and purification of residual neomycin in food has become an interesting problem.
[0004] Macroporous gel materials, such as monolithic agarose gel columns and cryogenic gel columns, have been proven to effectively overcome the drawbacks of interference from complex substances during the separation and purification of target analytes. The pore structure of macroporous gel materials enables high flow rates and rapid mass transfer, eliminating the need for complex pretreatment processes and even allowing direct separation of target analytes from complex biological samples. However, a major challenge in using macroporous gel materials for bioseparation is the low capacity of the composite material, caused by its macroporous structure and limited effective surface area. Therefore, the technical effect to be achieved in this invention is to prepare a macroporous agarose material with high capacity capable of separating neomycin from complex aquatic products. Summary of the Invention:
[0005] The technical problem to be solved by the present invention is that current methods for separating and purifying neomycin from complex biological samples cannot meet the requirements for ideal separation and purification of residual neomycin in food.
[0006] To address the aforementioned problems, this invention utilizes hydrophilic polyethyleneimine-modified silicon nanoparticles and high-performance boron affinity ligands to develop a novel composite agarose affinity monolithic column AG@silica-PEI. 70000 @DFFPBA was successfully used for the separation of neomycin from aquatic products. The boron affinity material synthesized in this invention utilizes the high density of boron affinity ligands and the macroporous structure of the agarose monolith to exhibit significant selective binding ability to neomycin. This also demonstrates the feasibility of using a boron affinity monolithic column for the separation of neomycin from complex aquatic products.
[0007] To achieve the above objectives, the present invention provides a method for preparing a macroporous agarose-boronic acid affinity material, comprising the following steps:
[0008] (1) Preparation of polyethyleneimine-functionalized silicon nanoparticles;
[0009] (2) Preparation of monolithic agarose column;
[0010] (3) Preparation of epoxy-functionalized agarose monolithic column;
[0011] (4) Preparation of agarose monolithic column modified with polyethyleneimine-functionalized silicon nanoparticles;
[0012] (5) Preparation of a monolithic agarose column functionalized with phenylboronic acid.
[0013] Furthermore, step (1) of functionalizing silicon nanoparticles with polyethyleneimine includes the following steps:
[0014] (1-1) Preparation of silicon nanoparticles: Ammonia and methanol were added to deionized water and stirred to obtain solution A; tetraethyl silicate was dissolved in methanol to obtain solution B; solution A and solution B were mixed together and stirred for 8-12 hours; the obtained particles were washed with deionized water and methanol respectively, and then vacuum dried to obtain silicon nanoparticles. After the silicon nanoparticles entered the interior of the agarose monolithic column through the pores, they were firmly bound to the inner wall of the monolithic column by covalent bonds, which increased the internal specific surface area of the monolithic column and provided more reaction sites for the binding of affinity ligands, thereby improving the purification efficiency of affinity materials.
[0015] (1-2) Preparation of amino-functionalized silicon nanoparticles: Silicon nanoparticles were dissolved in an ethanol solution of 3-aminopropyltriethoxysilane and stirred; then the obtained particles were washed with ethanol and dried under vacuum to obtain amino-functionalized silicon nanoparticles. The amino-functionalized silicon nanoparticles can be combined with glutaraldehyde through a Schiff base reaction, preparing for further modification of PEI.
[0016] (1-3) Preparation of aldehyde-functionalized silicon nanoparticles: Amino-functionalized silicon nanoparticles were dissolved in glutaraldehyde solution and stirred continuously; then the obtained particles were washed with deionized water and methanol, and dried under vacuum to obtain aldehyde-functionalized silicon nanoparticles. Glutaraldehyde, through a Schiff base reaction, binds to the amino group on one end of the silicon nanoparticle and to PEI at the other end, acting as a "bridge".
[0017] (1-4) Preparation of polyethyleneimine-functionalized silicon nanoparticles: Aldehyde-functionalized silicon nanoparticles were dissolved in a polyethyleneimine salt solution and stirred continuously; then the obtained particles were washed with ethanol and dried under vacuum to obtain polyethyleneimine-functionalized silicon nanoparticles. The large number of amino groups in polyethyleneimine provides a large number of reaction sites for the binding of boron-based ligands, which increases the affinity group density, reduces the steric hindrance effect, and improves the reaction efficiency.
[0018] Furthermore, the preparation method of the monolithic agarose column in step (2) is as follows: Dissolve dry agarose powder in deionized water, stir and heat until boiling to form a transparent gel, which is the aqueous phase; add Tween 80 to liquid paraffin and stir at 70-90°C, which is the oil phase; add the oil phase to the stirring aqueous phase, and continue stirring at 75°C until an emulsion is formed, stop heating and pour the emulsion into a constant-temperature hollow plastic solid-phase extraction column; after cooling the extraction column at 1-4°C, pour the monolithic agarose column out of the extraction column and wash it with deionized water to obtain the monolithic agarose column.
[0019] Furthermore, the preparation method of the epoxy-functionalized agarose monolithic column in step (3) is as follows: the agarose monolithic column is mixed with sodium hydroxide solution, dimethyl sulfoxide, epichlorohydrin and sodium borohydride and placed on a shaker for continuous shaking activation at 30-50°C; then the agarose monolithic column is rinsed with deionized water until neutral, and the standard is that no color is developed after adding sodium thiosulfate and phenolphthalein to the rinsed water, thus obtaining the epoxy-functionalized agarose monolithic column.
[0020] Furthermore, the preparation method of the polyethyleneimine-functionalized silicon nanoparticle-modified agarose monolithic column in step (4) is as follows: the epoxy-functionalized agarose monolithic column and the polyethyleneimine-functionalized silicon nanoparticles are immersed in sodium carbonate buffer and placed on a shaker and continuously shaken at 130-180 rpm; then the agarose monolithic column is rinsed with deionized water to obtain the polyethyleneimine-functionalized silicon nanoparticle-modified agarose monolithic column.
[0021] Furthermore, the preparation method of the phenylboronic acid-functionalized agarose monolithic column in step (5) is as follows: 3,5-difluoro-4-formylphenylboronic acid and sodium cyanoboronide are dissolved in a mixture of acetonitrile and methanol, and continuously shaken at 130-180 rpm for 24-48 h in a light-protected environment; then the agarose monolithic column is rinsed with deionized water to obtain the phenylboronic acid-functionalized agarose monolithic column, which is the macroporous agarose boric acid affinity material of the present invention.
[0022] A macroporous agarose boric acid affinity material prepared by the above method is a macroporous gel material with high-performance boron affinity ligands. Polyethyleneimine-functionalized silicon nanoparticles are added to the material, making it a macroporous agarose-based multiple boric acid affinity material based on hyperbranched polyethyleneimine.
[0023] The above-mentioned macroporous agarose-boronic acid affinity material is used in the separation of neomycin.
[0024] Boron affinity materials typically exist in the form of monolithic, particulate, and molecularly imprinted polymers, showing great potential for selective recognition, bioseparation, and immobilization of cis-dihydroxyl-containing substances. The reversible covalent binding of pH-mediated boron affinity ligands to cis-dihydroxyl-containing substances in aqueous solutions makes boron affinity materials ideal for the specific recognition of such substances. The boron affinity ligands in the macroporous agarose boric acid affinity material prepared in this invention form the basis for recognizing the cis-dihydroxyl group on neomycin aminoglycosides.
[0025] Furthermore, boron affinity ligands possess high density, which can address the low capacity issue in composite materials. To further address the low affinity of individual boric acid ligands and enhance their binding ability to the target analyte, dendritic macromolecules or polymers are used as scaffolds, multiplying the number and density of boron affinity ligands immobilized on the matrix, forming synergistic, multiple boron ester bonds. In particular, highly branched polyethyleneimine-assisted multiple boron affinity materials are more suitable for application in this invention due to their water solubility, resistance to organic solvents, high stability, low structural rigidity, and lower cost.
[0026] Besides increasing the density of borate groups to enhance affinity for target substances, ligands with relatively low pKa also play a crucial role in improving affinity. Commonly used boron affinity ligands, such as amination and vinylphenylboronic acid, have high pKa and therefore lower affinity in neutral environments. Literature indicates that aminophenylboronic acid and 3-acrylamide polyphenylboronic acid-based composites can bind to cis-dihydroxy compounds at neutral pH due to their low pKa. However, the binding process of these ligands to cis-dihydroxy compounds is time-consuming, which is not conducive to rapid purification of target substances. Among various alternative excellent ligands, fluorophenylboronic acid and 3,5-difluoro-4-carboxyphenylboronic acid show good binding ability to target cis-dihydroxy compounds. Therefore, the combination of fluorophenylboronic acid and polyethyleneimine can significantly enhance the binding ability of affinity materials to neomycin in biological samples.
[0027] The beneficial effects of this invention are as follows:
[0028] (1) Introducing high-performance boron affinity ligands into agarose macroporous gel materials solves the problem of low capacity of composite materials by utilizing the high density of boron affinity ligands. On the other hand, the boron affinity ligands recognize the cis dihydroxy group on the aminoglycoside of neomycin, providing feasibility for separating neomycin from complex aquatic products using the macroporous gel materials of this invention.
[0029] (2) Hydrophilic polyethyleneimine-modified silicon nanoparticles were added to the agarose macroporous gel material. On the one hand, the addition of nanoparticles enhanced the macroporous structure of the gel and the limited effective surface area; on the other hand, the combination of phenylboronic acid and polyethyleneimine significantly enhanced the affinity of the material to bind neomycin in biological samples.
[0030] (3) A new boron affinity monolithic column is provided that can separate neomycin from complex aquatic products. Attached Figure Description
[0031] Figure 1Figure 1 shows the FT-IR characterization results of the boron affinity materials. (A) Silica-PEI70000; (B) Monolith@Silica-PEI70000@DFFPBA with different functionalized agarose materials; (C) SEM image of the Silica-PEI70000 affinity material; (D) SEM image of the Monolith@Silica-PEI70000@DFFPBA affinity material.
[0032] Figure 2 The effect of polyethyleneimine of different molecular weights on the adsorption of neomycin by Monolith@Silica-PEI70000@DFFPBA;
[0033] Figure 3 The effect of different levels of functionalization on the adsorption of neomycin on agarose monolithic column;
[0034] Figure 4 The effect of PBS buffers with different pH and ionic strength on the adsorption of neomycin by Monolith@Silica-PEI70000@DFFPBA;
[0035] Figure 5 The adsorption kinetics and adsorption isotherms of neomycin on the affinity monolithic column are shown. (A) Adsorption kinetics of neomycin on the affinity monolithic column; (B) Adsorption isotherms of neomycin on the affinity monolithic column.
[0036] Figure 6 The number of cycles for neomycin adsorption by Monolith@Silica-PEI70000@DFFPBA;
[0037] Figure 7 This invention relates to the product forms and practical applications of the boron affinity material. Detailed implementation method:
[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, 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.
[0039] Example 1:
[0040] A method for preparing a macroporous agarose-boronic acid affinity material includes the following steps:
[0041] (1) Preparation of silicon nanoparticles: 11.2 mL of 25% ammonia water and 50 mL of methanol were added to 16.5 mL of deionized water and stirred continuously at 700-900 rpm for 15-30 min at room temperature, and named as solution A; 6.9 mL of tetraethyl silicate was dissolved in 65 mL of methanol and named as solution B; solution A and solution B were mixed together and then stirred continuously at 400-600 rpm for 8-12 h at 20-30℃; the obtained particles were washed with deionized water and methanol respectively, and then vacuum dried at 20-30℃ to obtain silicon nanoparticles.
[0042] (2) Preparation of amino-functionalized silicon nanoparticles: 0.4 g of silicon nanoparticles were dissolved in 83 mL of 4% (v / v) 3-aminopropyltriethoxysilane ethanol solution and stirred continuously at 200-400 rpm for 1.5-2.5 h at 35-45 °C; then the obtained particles were washed with ethanol and dried under vacuum at 20-30 °C to obtain amino-functionalized silicon nanoparticles.
[0043] (3) Preparation of aldehyde-functionalized silicon nanoparticles: 0.2 g of amino-functionalized silicon nanoparticles were dissolved in 30 mL of 10% glutaraldehyde solution (v / v, dissolved in 0.1 M pH 5.0 phosphate buffer) and stirred continuously at 200-400 rpm for 12 h at 20-30 °C; then the obtained particles were washed with deionized water and methanol and then vacuum dried at 20-30 °C to obtain aldehyde-functionalized silicon nanoparticles.
[0044] (4) Preparation of polyethyleneimine-functionalized silicon nanoparticles: 0.4 g of aldehyde-functionalized silicon nanoparticles were dissolved in 30 mL of 0.1 M, pH 5.0 buffer solution containing 200 mg of polyethyleneimine (molecular weight 70000) carbonate and stirred continuously at 200-400 rpm for 24 h at 20-30 °C; then the obtained particles were washed with ethanol and dried under vacuum at 20-30 °C to obtain polyethyleneimine-functionalized silicon nanoparticles, named Silica-PEI.
[0045] (5) Preparation of monolithic agarose column: Based on 5g of agarose dry powder, dissolve 5g of agarose dry powder in 80-150mL of deionized water, stir and heat at 350-550rpm until boiling to form a transparent gel, as the aqueous phase; add 3-6mL of Tween 80 to 30-50mL of liquid paraffin, stir at 300-500rpm for 10-20min at 70-90℃, as the oil phase; add the oil phase to the stirring aqueous phase, stir continuously at 350-550rpm for 30-50min at 75℃ until an emulsion is formed, stop heating and pour the emulsion into a constant temperature hollow plastic solid phase extraction column; after cooling the extraction column at 1-4℃ for 2-4h, pour the monolithic agarose column out of the extraction column, and wash with deionized water, naming it Monolith.
[0046] (6) Preparation of epoxy-functionalized agarose monolith: Based on 1g of agarose monolith, 1g Monolith was mixed with 150mL of 0.7-1.1M sodium hydroxide solution, 60-80mL of dimethyl sulfoxide, 60-80mL of epichlorohydrin and 100-120mg of sodium borohydride and placed on a shaker. The mixture was continuously shaken at 130-180rpm at 30-50℃ for 10-20h. Monolith was then rinsed with deionized water until neutral. The standard was that no color development was observed after adding sodium thiosulfate and phenolphthalein to the rinsed water. The epoxy-functionalized agarose monolith was obtained and named Monolith@epoxy.
[0047] (7) Preparation of agarose monolith column modified with polyethyleneimine-functionalized silicon nanoparticles: 1 g Monolith@epoxy and 50 mg Silica-PEI were immersed in sodium carbonate buffer (0.1 M, pH 5.0) and placed on a shaker. The column was shaken continuously at 130-180 rpm for 12 h at 20-30 °C. The column was then rinsed with deionized water and named Monolith@Silica-PEI.
[0048] (8) Preparation of phenylboronic acid-functionalized agarose monolith column: 1g Monolith@Silica-PEI, 1g 3,5-difluoro-4-carboxyphenylboronic acid and 1.5g sodium cyanoboronide were dissolved in a mixture of 150mL acetonitrile and 300mL methanol. The mixture was shaken continuously at 130-180rpm for 24-48h at 20-30℃ in the dark. The agarose monolith column was then rinsed with deionized water and named Monolith@Silica-PEI@DFFPBA.
[0049] Example 2:
[0050] The specific steps for preparing silicon nanoparticles are as follows: Accurately weigh 11.2 mL of 25% ammonia, 50 mL of methanol, and 16.5 mL of deionized water and mix them thoroughly. Stir continuously at room temperature for 30 min and name this solution A. Accurately weigh 6.9 mL of tetraethyl silicate and dissolve it in 65 mL of methanol and name this solution B. Mix solution A and solution B together and then stir continuously at 20 °C for 10 h. After the reaction is complete, wash the obtained particles three times with deionized water and methanol, and then dry them under vacuum at 30 °C to obtain silicon nanoparticles.
[0051] Everything else is the same as in Example 1.
[0052] Example 3:
[0053] The specific steps for preparing aminated silicon nanoparticles are as follows: accurately weigh 0.4 g of silicon nanoparticles and dissolve them in 83 mL of 4% (v / v) 3-aminopropyltriethoxysilane ethanol solution, and stir continuously at 45 °C for 1.5 h; then wash the obtained particles three times with ethanol and dry them under vacuum at 30 °C to obtain aminated silicon nanoparticles.
[0054] The rest are the same as any of Examples 1-2.
[0055] Example 4:
[0056] The specific steps for preparing aldehyde-based silicon nanoparticles are as follows: 0.2 g of aminated silicon nanoparticles are accurately weighed and dissolved in 30 mL of 10% glutaraldehyde solution (v / v, dissolved in 0.1 M pH 5.0 phosphate buffer), and stirred continuously at 30 °C for 12 h; then the obtained particles are washed three times with deionized water and methanol, and then dried under vacuum at 30 °C to obtain aldehyde-based silicon nanoparticles.
[0057] The rest are the same as any of Examples 1-3.
[0058] Example 5:
[0059] The specific steps for preparing polyethyleneimine-functionalized silicon nanoparticles are as follows: 0.4 g of aldehyde-modified silicon nanoparticles were accurately weighed and dissolved in 30 mL of sodium carbonate buffer (0.1 M, pH 5.0) containing 200 mg of polyethyleneimine (molecular weight 70000). The solution was stirred continuously at 30 °C for 24 h. The resulting particles were then washed three times with ethanol and dried under vacuum at 30 °C to obtain polyethyleneimine-functionalized silicon nanoparticles.
[0060] The rest are the same as any of Examples 1-4.
[0061] Example 6:
[0062] The specific steps for preparing the monolithic agarose column are as follows: Accurately weigh 5g of agarose powder and dissolve it in 150mL of deionized water. Boil the solution while stirring until it becomes a transparent gel, which is the aqueous phase. Weigh 4mL of Tween 80 and add it to 50mL of liquid paraffin. Stir at 85℃ for 20min until the solution is fully mixed, which is the oil phase. Add the oil phase to the stirring aqueous phase and stir continuously at 75℃ and 550rpm for 40min until an emulsion is formed. Stop heating and pour the emulsion into a thermostatic plastic solid phase extraction column. After cooling the extraction column at 4℃ for 2h, pour the monolithic agarose column out of the extraction column and wash it with deionized water to obtain the monolithic agarose column.
[0063] The rest are the same as any of Examples 1-5.
[0064] Example 7:
[0065] The specific steps for preparing an epoxidized agarose monolithic column are as follows: Accurately weigh 1g of the agarose monolithic column and place it in an Erlenmeyer flask. Add 150mL of 1M sodium hydroxide solution, 75mL of dimethyl sulfoxide, 75mL of epichlorohydrin, and 110mg of sodium borohydride. Place the column on a shaker and activate it by continuous shaking at 45℃ for 12h. Then, rinse the agarose monolithic column with deionized water until it is neutral. The standard is that no color development occurs after adding sodium thiosulfate and phenolphthalein to the rinse water. This is the epoxidized agarose monolithic column.
[0066] The rest are the same as any of Examples 1-6.
[0067] Example 8:
[0068] The specific steps for preparing the monolithic agarose column modified with polyethyleneimine-functionalized silicon nanoparticles are as follows: Accurately weigh 1g of epoxidized agarose monolithic column and 50mg of polyethyleneimine-functionalized silicon nanoparticles and place them in an Erlenmeyer flask. Add 150mL of 0.1M sodium carbonate buffer (pH 5.0) and place the flask on a shaker. Shake continuously at 30℃ for 12h. Then rinse the monolithic agarose column with deionized water to obtain the monolithic agarose column modified with polyethyleneimine-functionalized silicon nanoparticles.
[0069] The rest are the same as any of Examples 1-7.
[0070] Example 9:
[0071] The specific steps for preparing the phenylboronic acid-functionalized agarose monolithic column are as follows: Accurately weigh 1 g of the agarose monolithic column modified with polyethyleneimine-functionalized silicon nanoparticles, 1 g of 3,5-difluoro-4-carboxyphenylboronic acid, and 1.5 g of sodium cyanoboronide, and dissolve them in a mixture of 150 mL acetonitrile and 300 mL methanol. Shake continuously at 30 °C in the dark for 48 h. Then rinse the agarose monolithic column with deionized water to obtain the phenylboronic acid-functionalized agarose monolithic column, soak it in 20% ethanol and place it at 4 °C for later use.
[0072] The rest are the same as any of Examples 1-8.
[0073] Result verification:
[0074] (1) The boron affinity materials prepared according to the steps of Examples 2-8 were characterized by FT-IR and SEM, respectively. Figure 1 As shown, the prepared Silica-PEI 70000 and Monolith@Silica-PEI 70000 FT-IR characterization of @DFFPBA material. The FT-IR characterization image shows that at 1114 cm⁻¹... -1 The characteristic infrared peak of the asymmetric vibration of Si-O, 950 cm⁻¹ -1 A distinct silica adsorption band can be observed at the infrared peak of the asymmetric vibration characteristic of Si-OH. Figure 1 A) After further introducing PEI, at 3381 and 3291 cm -1 A relatively wide adsorption band was observed at this location. Figure 1 A) indicates that PEI was successfully incorporated; after the introduction of 3,5-difluoro-4-formylphenylboronic acid, the concentration at 1413 cm⁻¹ was [missing information]. -1 ~1572cm -1 A benzene ring vibration signal was observed at 1313 cm⁻¹. -1 The characteristic infrared band of -B(OH)2 appeared at that location. Figure 1 B), the benzene ring vibration signal and the boron hydroxyl signal indicate that the boric acid group was successfully modified on the agarose monolithic column material. After aldehyde modification, the silicon nanoparticles are cross-linked with each other. After the introduction of PEI, the sphericity of the particles becomes smaller, and the polymer layer on the inorganic silicon nanoparticles can be observed. Figure 1 C) The observation of cross-linked silicon nanoparticles on the agarose surface indicates that Silica-PEI... 70000 Successfully modified into agarose monolithic column ( Figure 1 D).
[0075] (2) To demonstrate the influence of borate affinity ligands and polyethyleneimine-modified silica nanoparticles on the binding affinity of neomycin, the binding affinity of monolithic agarose columns with different degrees of functionalization to neomycin was investigated. For example... Figure 2 As shown, the functionalized agarose monolithic columns AG@PEI@DFFPBA and AG@silica-PEI@DFFPBA exhibit significantly higher neomycin binding capacity than AG@epoxy, AG@PEI, and AG@silica-PEI, indicating that the specific affinity interaction between borate affinity ligands and neomycin is dominant. Compared with AG@PEI@DFFPBA, which uses polyethyleneimine as a scaffold to immobilize borate ligands, AG@silica-PEI@DFFPBA shows a higher neomycin binding capacity. This can be attributed to the immobilization of PEI-functionalized silica nanoparticles, which increases the effective surface area for reaction with borate groups, allowing more borate affinity ligands to be immobilized in AG@silica-PEI@DFFPBA.
[0076] (3) The effectiveness of PEIs with different molecular weights was verified using the adsorption capacity for neomycin as the standard. PEIs were used to... 600 PEI 1800 PEI 10000 PEI 70000 The prepared boron affinity material was equilibrated with 0.01M phosphate buffer for 15 min. The prepared neomycin solution was then circulated in the column at 37℃ for 15 min. The eluent and the original solution were collected and analyzed by liquid chromatography. The adsorption capacity of the agarose monolithic column for neomycin was calculated. Figure 3 As shown, the binding ability of AG@silica-PEI@DFFPBA to neomycin gradually increases with the increase of the molecular weight of polyethyleneimine. This can be explained by the fact that the molecular volume of neomycin is small and the open three-dimensional structure of the hydrophilic polyethyleneimine molecule is conducive to the penetration of neomycin into the binding site.
[0077] (4) Prepare phosphate buffer solutions with pH values of 4.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0, and concentrations of 0 M, 0.05 M, 0.1 M, 0.3 M, 0.6 M, 0.8 M, 1.0 M, 2.0 M, and 4.0 M. The boron affinity material was equilibrated sequentially with the above phosphate buffer solutions for 15 min. Then, a neomycin solution prepared with the corresponding buffer solution was circulated at 37 °C for 15 min. The eluent and the original solution were collected and analyzed by liquid chromatography. The adsorption capacity of the agarose monolithic column for neomycin was calculated, and the effects of pH and buffer ionic strength on the adsorption effect of the boron affinity material were evaluated. The results are as follows: Figure 4As shown, when the pH of the solution is below 8.0, the binding capacity of boron affinity materials to neomycin gradually increases with increasing pH in nine phosphate buffer solutions of 0M, 0.05M, 0.1M, 0.3M, 0.6M, 0.8M, 1.0M, 2.0M, and 4.0M. This is consistent with previous studies on the binding mechanism of boronic acid groups to cis-dihydroxy compounds under alkaline conditions. However, when the pH is above 8.0, the binding capacity of boron affinity materials to neomycin does not significantly increase, which is related to the saturation of the binding sites. When the ionic strength increases to 0.6M, the binding capacity of boron affinity materials to neomycin gradually increases, which is consistent with previous reports on the effect of ionic strength on the capture of glycoproteins by boron affinity materials. When the ionic strength exceeds 0.6M, the binding capacity of boron affinity materials to neomycin decreases significantly. This may be because when the ambient pH is greater than the pKa of the boronic acid ligand, the boronic acid ligand carries a negative charge and therefore interacts electrostatically with the charged analyte. In summary, AG@silica-PEI 70000 The @DFFPBA monolithic column exhibits the best adsorption performance in 0.6M phosphate buffer (pH 8.0). Excessive ionic strength and pH can lead to strong hydrophobic and electrostatic interactions, which in turn inhibit the binding of boron-affinity materials to neomycin. Therefore, 0.6M phosphate buffer at pH 8.0 is selected as the optimal loading condition.
[0078] (5) Accurately weigh 1g of boron affinity material and equilibrate it with 0.6M, pH 8 phosphate buffer for 15min. Then add neomycin solution sequentially and adsorb at 37℃ for 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, and 45.0min respectively. Calculate the amount of neomycin adsorbed by the boron affinity material at the corresponding time.
[0079] Accurately weigh 1g of boron affinity material and equilibrate it with 0.6M, pH 8 phosphate buffer for 15 min. Then add neomycin solution at concentrations of 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 1.0, 1.25, and 1.5 mg / mL, respectively. Adsorb at 37℃ for 15 min and calculate the adsorption capacity of the boron affinity material for neomycin at the corresponding concentrations.
[0080] The binding properties of boron affinity materials in capturing cis-dihydroxy groups were investigated using AG@silica-PEI. 70000 Adsorption kinetics of @DFFPBA. For example... Figure 5As shown in Figure A, neomycin exhibited a significantly high binding rate on AG@silica-PEI@DFFPBA in the initial stage (~2.5 min), reaching over 70% of the maximum binding capacity. At 15 min, the binding rate of the agarose monolithic column slowed down, approaching equilibrium. After 30 min, the binding capacity of the boron affinity material decreased slightly, but the difference in binding capacity for neomycin between 30 min and 45 min was not significant. Pseudo-first-order and pseudo-second-order kinetic models were used to analyze the dynamic process. AG@silica-PEI 70000 @DFFPBA R in a pseudo-first-order model of neomycin binding 2 The value is 0.9355, which is higher than the R-value of the pseudo-second-order model. 2 The value of 0.8677 indicates that the pseudo-first-order model better describes the adsorption behavior of neomycin. The results show that the pseudo-first-order model can fit the adsorption of neomycin well, further confirming the effectiveness of AG@silica-PEI. 70000 The boric acid group on @DFFPBA exhibits a strong binding affinity to the cis-dihydroxy group of neomycin. AG@silica-PEI 70000 @DFFPBA equilibrium binding isotherm for neomycin, as shown Figure 5 As shown in Figure B, the binding capacity of the boron-affinity monolithic column for neomycin was calculated to be 2.02 mg / g. The adsorption equilibrium isotherms were further fitted using the Langmuir and Freundlich models. The results showed that the Langmuir model provided a better fit for the neomycin adsorption capacity than the Freundlich model.
[0081] (6) Accurately weigh 1 g of boron affinity material and equilibrate it in 0.6 M, pH 8 phosphate buffer for 15 min. Then add neomycin solution and adsorb at 37 °C for 15 min. Elute with 5 mL of acetic acid (10 mM), then equilibrate with 0.6 M, pH 8 phosphate buffer for 15 min before the next adsorption cycle. After 20 cycles, calculate the amount of neomycin adsorbed by the boron affinity material each time.
[0082] Structure as Figure 6 As shown, in the first 15 iterations, AG@silica-PEI 70000 The binding capacity of @DFFPBA remained highly reproducible, with a recovery rate exceeding 97% of the neomycin stock solution, indicating good stability of the boron affinity material in neomycin binding. Subsequently, the binding capacity gradually decreased, reaching approximately 82% of the neomycin stock solution by the 20th cycle. This may be due to the loss of some boric acid affinity ligands or incomplete elution of the affinity-adsorbed neomycin in the earlier cycles.
[0083] Application Example 1:
[0084] Muscle tissue samples of silver pomfret, spotted gudgeon, whiteleg shrimp, squid, and Manila clams were collected and placed in 50 mL centrifuge tubes. Neomycin solutions with concentrations of 300 ng / kg, 500 ng / kg, and 700 ng / kg were added, respectively. After vortexing for 5 min, the samples were allowed to stand at room temperature for 2 h. 2 g of the spiked sample was mixed with 4 mL of sulfosalicylic acid solution (5%), dispersed and homogenized for 2 min, and centrifuged at room temperature for 15 min. The supernatant was then collected, and the precipitate was retreated with another 3 mL of sulfosalicylic acid solution (2%) following the same procedure. The supernatants from the two extractions were combined, and the pH was adjusted to 8.0. 1 g of boron affinity material was accurately weighed and equilibrated in 0.6 M, pH 8 phosphate buffer for 15 min, then added to the mixed supernatant. Adsorption was performed at 37 °C for 15 min, and the adsorption capacity of the boron affinity material for neomycin was calculated.
[0085] The results showed that the recovery rate of neomycin was 94.34%–98.88% within the spiked concentration range of 400–700 ng / kg. Given the presence of complex interfering substances, especially ions, during the extraction process of aquatic products, the synthesized composite borate affinity material has shown excellent effectiveness in monitoring neomycin in aquatic products.
[0086] Application Example 2:
[0087] Product format: such as Figure 7 As shown, the product is available in two forms: a storage form and a finished product application form. The storage form is a solid, sealed packing material that can be directly assembled into a column according to the required volume when needed. The finished product is a 1g sterilized packing solution that is sealed onto the top of the column, temporarily stored at 4°C after vacuuming, and can be directly activated and used when needed.
[0088] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.
[0089] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.
Claims
1. The application of a macroporous agarose-boronic acid affinity material in the separation of neomycin, characterized in that... The preparation method includes the following steps: (1) Preparation of polyethyleneimine-functionalized silicon nanoparticles: (1-1) Preparation of silicon nanoparticles: Ammonia and methanol were added to deionized water and stirred to obtain solution A; tetraethyl silicate was dissolved in methanol to obtain solution B; solution A and solution B were mixed together and stirred for 8-12 hours; the obtained particles were washed with deionized water and methanol respectively and dried under vacuum to obtain silicon nanoparticles. (1-2) Preparation of amino-functionalized silicon nanoparticles: Dissolve silicon nanoparticles in an ethanol solution of 3-aminopropyltriethoxysilane and stir; then wash the obtained particles with ethanol and dry them under vacuum to obtain amino-functionalized silicon nanoparticles. (1-3) Preparation of aldehyde-functionalized silicon nanoparticles: Dissolve amino-functionalized silicon nanoparticles in glutaraldehyde solution and stir continuously; then wash the obtained particles with deionized water and methanol, and dry them under vacuum to obtain aldehyde-functionalized silicon nanoparticles. (1-4) Preparation of polyethyleneimine-functionalized silicon nanoparticles: Aldehyde-functionalized silicon nanoparticles were dissolved in a polyethyleneimine salt solution and stirred continuously; then the obtained particles were washed with ethanol and dried under vacuum to obtain polyethyleneimine-functionalized silicon nanoparticles. (2) Preparation of monolithic agarose column; (3) Preparation of epoxy-functionalized agarose monolithic column; (4) Preparation of agarose monolithic column modified with polyethyleneimine-functionalized silicon nanoparticles; (5) Preparation of a monolithic agarose column functionalized with phenylboronic acid; The preparation method of the polyethyleneimine-functionalized silicon nanoparticle-modified agarose monolithic column in step (4) is as follows: the epoxy-functionalized agarose monolithic column and the polyethyleneimine-functionalized silicon nanoparticles are immersed in sodium carbonate buffer and placed on a shaker and continuously shaken at 130-180 rpm; then the agarose monolithic column is rinsed with deionized water to obtain the polyethyleneimine-functionalized silicon nanoparticle-modified agarose monolithic column. The preparation method of the phenylboronic acid-functionalized agarose monolithic column in step (5) is as follows: 3,5-difluoro-4-formylphenylboronic acid and sodium cyanoborohydrin are dissolved in a mixture of acetonitrile and methanol and continuously shaken at 130-180 rpm for 24-48 h in a light-protected environment; then the agarose monolithic column is washed with deionized water to obtain the phenylboronic acid-functionalized agarose monolithic column, i.e., macroporous agarose boric acid affinity material.
2. The application as described in claim 1, characterized in that... The preparation method of the monolithic agarose column in step (2) is as follows: Dissolve agarose powder in deionized water, stir and heat until boiling to form a transparent gel, which is the aqueous phase; add Tween 80 to liquid paraffin and stir at 70-90℃, which is the oil phase; add the oil phase to the stirring aqueous phase, and continue stirring at 75℃ until an emulsion is formed, stop heating and pour the emulsion into a constant temperature hollow plastic solid phase extraction column; after cooling the extraction column at 1-4℃, pour the monolithic agarose column out of the extraction column and wash it with deionized water to obtain the monolithic agarose column.
3. The application as described in claim 1, characterized in that... The preparation method of the epoxy-functionalized agarose monolithic column in step (3) is as follows: the agarose monolithic column is mixed with sodium hydroxide solution, dimethyl sulfoxide, epichlorohydrin and sodium borohydride and placed on a shaker for continuous shaking activation at 30-50℃; then the agarose monolithic column is rinsed with deionized water until neutral, and the standard is that no color is developed after adding sodium thiosulfate and phenolphthalein to the rinsed water, thus obtaining the epoxy-functionalized agarose monolithic column.