技术领域technical field
本发明属于磁性复合纳米材料领域，特别涉及pH响应型磁性金属有机框架复合纳米材料、其制备方法以及在糖蛋白捕获和释放方面的应用。The invention belongs to the field of magnetic composite nanomaterials, and in particular relates to a pH-responsive magnetic metal-organic frame composite nanomaterial, a preparation method thereof and an application in glycoprotein capture and release.
背景技术Background technique
蛋白糖基化，即在糖基转移酶作用下将糖转移至蛋白质，与蛋白质上的氨基酸形成糖苷键，是蛋白最普遍的翻译后修饰的一种形式，在许多生物学活动中扮演了重要角色，比如细胞粘附、分子识别、信号传导、蛋白折叠和代谢途径等。不正常的糖基化也与许多疾病有关，例如糖尿病、癌症、神经退行性疾病、心血管疾病等。为了更好地理解这些生物过程，或者发现新的疾病标志物，首先需要对糖蛋白进行捕获和释放。Protein glycosylation, that is, the transfer of sugar to proteins under the action of glycosyltransferases, and the formation of glycosidic bonds with amino acids on proteins, is the most common form of post-translational modification of proteins and plays an important role in many biological activities roles, such as cell adhesion, molecular recognition, signal transduction, protein folding, and metabolic pathways. Abnormal glycosylation is also associated with many diseases, such as diabetes, cancer, neurodegenerative diseases, cardiovascular diseases, etc. To better understand these biological processes, or to discover new disease markers, glycoproteins first need to be captured and released.
在过去十年内，根据不同的机理，已有一些方法提出来特异性的识别或分离糖蛋白/糖肽，比如凝集素亲和色谱法(参见文献Wu,J.；Zhu,J.；Yin,H.；Buckanovich,R.J.；Lubman,D.M.Analysis of glycan variation on glycoproteins from serum by thereverse lectin-based ELISA assay.J.Proteome Res.2014,13,2197-2204)，酰肼化学法(参见文献Liu,L.；Yu,M.；Zhang,Y.；Wang,C.；Lu,H.Hydrazide functionalized core-shell magnetic nanocomposites for highly specific enrichment of N-glycopeptides.ACS Appl.Mater.Interfaces 2014,6,823-7832)，亲水相互作用色谱法(参见文献Zhu,J.；Sun,Z.；Cheng,K.；Chen,R.；Ye,M.；Xu,B.；Sun,D.；Wang,L.；Liu,J.；Wang,F.；Zou,H.Comprehensive mapping of protein N-glycosylation in human liverby combining hydrophilic interaction chromatography and hydrazidechemistry.J.Proteome Res.2014,13,1713-1721)和硼酸亲和色谱法(参加文献Li,D.；Chen,Y.；Liu,Z.Boronate affinity materials for separation and molecularrecognition:structure,properties and applications.Chem.Soc.Rev.2015,44,8097-8123)。目前，引起广泛关注的实现糖蛋白捕获和分离的方法是将具有pH开关性能的硼酸亲和色谱与具有独特磁性能的磁性纳米粒子结合的策略，一般是在磁性纳米材料表面修饰上小分子的硼酸配体，首先，磁性纳米材料通过多步操作被预功能化活性基团(如羧基、氨基、叠氮基等)，然后这些活性基团再与小分子硼酸配体的活性基团(如氨基、炔基等)反应，从而嫁接上小分子硼酸配体(参见文献hang,L.；Xu,Y.；Yao,H.；Xie,L.；Yao,J.；Lu,H.；Yang,P.Boronic acid functionalized core-satellite composite nanoparticles foradvanced enrichment of glycopeptides and glycoproteins.Chem.Eur.J.2009,15,10158-10166)；然而，其捕获或释放糖蛋白的过程中，所需的缓冲液pH值是强酸性或强碱性，不利于保持糖蛋白的活性，而且，硼酸功能化的磁性复合纳米材料的制备过程较为繁琐，极大增加了制备成本，加之小分子硼酸配体嫁接量因为其较大的空间位阻使得硼酸嫁接效率较低，从而限制其在生物医学方面的应用。因此，研发一种能简单有效的在温和的pH环境下高选择性地捕获和释放糖蛋白的策略，是一项非常有意义且迫切的工作。In the past ten years, according to different mechanisms, some methods have been proposed to specifically identify or separate glycoproteins/glycopeptides, such as lectin affinity chromatography (see literature Wu, J.; Zhu, J.; Yin, H.; Buckanovich, R.J.; Lubman, D.M.Analysis of glycan variation on glycoproteins from serum by thereby lectin-based ELISA assay.J.Proteome Res.2014,13,2197-2204), hydrazide chemical method (see literature Liu, L .; Yu, M.; Zhang, Y.; Wang, C.; Lu, H. Hydrazide functionalized core-shell magnetic nanocomposites for highly specific enrichment of N-glycopeptides. ACS Appl. Mater. Interfaces 2014, 6, 823-7832), Hydrophilic interaction chromatography (see literature Zhu, J.; Sun, Z.; Cheng, K.; Chen, R.; Ye, M.; Xu, B.; Sun, D.; Wang, L.; Liu , J.; Wang, F.; Zou, H.Comprehensive mapping of protein N-glycosylation in human liverby combining hydraulic interaction chromatography and hydrazidechemistry.J.Proteome Res.2014,13,1713-1721) and boronic acid affinity chromatography ( Participate in the literature Li, D.; Chen, Y.; Liu, Z. Boronate affinity materials for separation and molecular recognition: structure, properties and applications. Chem. Soc. Rev. 2015, 44, 8097-8123). At present, the method that has attracted widespread attention to achieve glycoprotein capture and separation is the strategy of combining boric acid affinity chromatography with pH switch properties with magnetic nanoparticles with unique magnetic properties, generally small molecules on the surface of magnetic nanomaterials. For boronic acid ligands, firstly, the magnetic nanomaterials are pre-functionalized with active groups (such as carboxyl, amino, azido, etc.) Amino groups, alkynyl groups, etc.) to graft small molecule boronic acid ligands (see literature: hang, L.; Xu, Y.; Yao, H.; Xie, L.; Yao, J.; Lu, H.; Yang , P.Boronic acid functionalized core-satellite composite nanoparticles for advanced enrichment of glycopeptides and glycoproteins.Chem.Eur.J.2009,15,10158-10166); however, in the process of capturing or releasing glycoproteins, the required buffer The pH value is strongly acidic or alkaline, which is not conducive to maintaining the activity of glycoproteins. Moreover, the preparation process of boric acid functionalized magnetic composite nanomaterials is relatively cumbersome, which greatly increases the preparation cost. In addition, the amount of small molecule boric acid ligand grafting is due to Its large steric hindrance makes the grafting efficiency of boronic acid low, thus limiting its application in biomedicine. Therefore, it is a very meaningful and urgent work to develop a simple and effective strategy to capture and release glycoproteins with high selectivity in a mild pH environment.
金属有机框架(Metal-Organic Frameworks，MOFs)是由金属离子和有机配体通过配位键或分子间作用力形成的。MOFs以金属离子为中心，具有较高的孔隙率，优良的机械稳定性和易于调整的表面性能，且其具有丰富的芳香配体，由于以上独特的性质，金属有机框架被广泛应用于不同的领域，如储能、催化、小分子受体、苯同系物的分离等。最近，MOFs也被广泛应用于生物医学领域，例如药物释放、光动力学治疗、显影、酶催化和蛋白富集等。在有关于蛋白的应用中，科学家们主要集中于提高蛋白质的捕获效率，而忽略了蛋白的释放过程，被MOFs捕获的蛋白质很难从材料上洗脱下来，即使能够被洗脱下来，由于洗脱过程需要加入对蛋白活性有影响的物质，如2-甲基咪唑或三氟乙酸(参见文献W.L.Liu,S.H.Lo,B.Singco,C.C.Yang,H.Y.Huang and C.H.Lin,J.Mater.Chem.B,2013,1,928；M.Zhao,C.Deng and X.Zhang,Chem.Commun.,2014,50,6228；N.Sun,X.Zhang and C.Deng,Nanoscale,2015,7,6487；M.Zhao,X.Zhang and C.Deng,Chem.Commun.,2015,51,8116；Y.Chen,Z.Xiong,L.Peng,Y.Gan,Y.Zhao,J.Shen,J.Qian,L.Zhang and W.Zhang,ACSAppl.Mater.Interfaces,2015,7,16338；J.Zheng,Z.Lin,G.Lin,H.Yang and L.Zhang,J.Mater.Chem.B,2015,5,2185.)，会严重影响对释放后的蛋白质的进一步分析或应用。Metal-Organic Frameworks (MOFs) are formed by metal ions and organic ligands through coordination bonds or intermolecular forces. MOFs are centered on metal ions, have high porosity, excellent mechanical stability and easy-to-adjust surface properties, and they are rich in aromatic ligands. Due to the above unique properties, metal-organic frameworks are widely used in different Fields, such as energy storage, catalysis, small molecule acceptors, separation of benzene homologues, etc. Recently, MOFs have also been widely used in biomedical fields, such as drug release, photodynamic therapy, imaging, enzyme catalysis, and protein enrichment, etc. In protein-related applications, scientists mainly focus on improving the capture efficiency of proteins, while ignoring the release process of proteins. It is difficult for proteins captured by MOFs to be eluted from the material, even if they can be eluted, due to the The removal process needs to add substances that have an impact on protein activity, such as 2-methylimidazole or trifluoroacetic acid (see literature W.L.Liu, S.H.Lo, B.Singco, C.C.Yang, H.Y.Huang and C.H.Lin, J.Mater.Chem. B, 2013, 1,928; M. Zhao, C. Deng and X. Zhang, Chem. Commun., 2014, 50, 6228; N. Sun, X. Zhang and C. Deng, Nanoscale, 2015, 7, 6487; M .Zhao, X.Zhang and C.Deng, Chem.Commun., 2015, 51, 8116; Y.Chen, Z.Xiong, L.Peng, Y.Gan, Y.Zhao, J.Shen, J.Qian, L. Zhang and W. Zhang, ACS Appl. Mater. Interfaces, 2015, 7, 16338; J. Zheng, Z. Lin, G. Lin, H. Yang and L. Zhang, J. Mater. Chem. B, 2015, 5,2185.), will seriously affect the further analysis or application of the released protein.
发明内容Contents of the invention
本发明的目的旨在针对上述现有技术中存在的问题，提供一种pH响应型磁性金属有机框架复合纳米材料，可以在温和的pH环境下可逆的、高选择性的捕获或释放糖蛋白。The purpose of the present invention is to solve the problems in the above-mentioned prior art, and provide a pH-responsive magnetic metal-organic framework composite nanomaterial, which can reversibly and highly selectively capture or release glycoproteins in a mild pH environment.
本发明另一目的旨在提供一种反应条件温和、制备成本低的制备方法，用于制备上述pH响应型磁性金属有机框架复合纳米材料。Another object of the present invention is to provide a preparation method with mild reaction conditions and low preparation cost for preparing the pH-responsive magnetic metal-organic framework composite nanomaterial.
本发明再一目的旨在提供上述pH响应型磁性复合纳米球在糖蛋白捕获或释放中的应用。Another object of the present invention is to provide the application of the above pH-responsive magnetic composite nanospheres in the capture or release of glycoproteins.
为了达到上述目的，本发明采取以下技术方案来实现。In order to achieve the above object, the present invention adopts the following technical solutions to achieve.
本发明提供了一种pH响应型磁性金属有机框架复合纳米材料，该复合纳米材料由Fe3O4纳米粒子，包覆在Fe3O4纳米粒子表面的高分子层以及生长于高分子层上的金属有机框架构成；所述高分子层为螯合金属离子的第一聚合物或者由螯合金属离子的第一聚合物和亲水性的第二聚合物组成，当高分子层为由螯合金属离子的第一聚合物和亲水性的第二聚合物组成时，所述第一聚合物与第二聚合物的质量比为1：(0.3～1)；所述金属有机框架是由Fe3+和含有至少一个羧基或者至少两个硼酸基的苯硼酸衍生物通过配位键形成。The invention provides a pH-responsive magnetic metal-organic framework composite nanomaterial, which consists ofFe3O4 nanoparticles, a polymer layer coatedon the surface of theFe3O4 nanoparticles, and a polymer layer grownon the polymer layer Metal-organic framework; the polymer layer is a first polymer that chelates metal ions or consists of a first polymer that chelates metal ions and a second hydrophilic polymer. When the polymer layer is composed of chelating When composed of the first polymer containing metal ions and the second hydrophilic polymer, the mass ratio of the first polymer to the second polymer is 1: (0.3～1); the metal organic framework is composed of Fe3+ and phenylboronic acid derivatives containing at least one carboxyl group or at least two boronic acid groups are formed through coordinate bonds.
上述pH响应型磁性金属有机框架复合纳米材料，第一聚合物为聚乙烯吡咯烷酮或聚多巴胺；所述第二聚合物为聚醚酰亚胺或聚丙烯酸；含有至少一个羧基的苯硼酸衍生物为5-硼酸基间苯二甲酸(5-Boronoisophthalic acid)、3-羧基苯硼酸或4-羧基苯硼酸，所述含有至少两个硼酸基的苯硼酸衍生物为对苯二硼酸或间苯二硼酸。For the above pH-responsive magnetic metal-organic framework composite nanomaterial, the first polymer is polyvinylpyrrolidone or polydopamine; the second polymer is polyetherimide or polyacrylic acid; the phenylboronic acid derivative containing at least one carboxyl group is 5-boronoisophthalic acid (5-Boronoisophthalic acid), 3-carboxyphenylboronic acid or 4-carboxyphenylboronic acid, the phenylboronic acid derivative containing at least two boronic acid groups is terephthalic diboronic acid or isophthalic diboronic acid .
上述pH响应型磁性金属有机框架复合纳米材料的平均粒径分布窄，约为300nm～500nm。该磁性金属有机框架复合纳米材料由Fe3O4纳米粒子作为核，具有高的磁饱和强度，从而提供较好的磁响应性能；包裹在Fe3O4纳米粒子表面的高分子层作为过渡层，不仅改进了材料的亲水性，而且能够螯合金属离子，使金属有机框架(MOFs)生长于该高分子层上；最外层的金属有机框架由Fe3+和含有至少一个羧基或者至少两个硼酸基的苯硼酸衍生物通过配位键形成，其中含有至少一个羧基或者至少两个硼酸基的苯硼酸衍生物既作为金属有机框架的有机配体，又作为具有pH响应性和捕获蛋白质的功能分子，从而赋予材料pH响应性能以及选择性捕获蛋白质的能力。The average particle size distribution of the pH-responsive magnetic metal-organic framework composite nanomaterial is narrow, about 300nm-500nm. The magnetic metal-organic framework composite nanomaterial is composed of Fe3 O4 nanoparticles as the core, which has high magnetic saturation intensity, thus providing better magnetic response performance; the polymer layer wrapped on the surface of Fe3 O4 nanoparticles is used as a transition layer , not only improves the hydrophilicity of the material, but also can chelate metal ions, so that metal-organic frameworks (MOFs) grow on the polymer layer; the outermost metal-organic framework is composed of Fe3+ and contains at least one carboxyl group or at least Phenylboronic acid derivatives with two boronic acid groups are formed through coordination bonds, and phenylboronic acid derivatives containing at least one carboxyl group or at least two boronic acid groups are used not only as organic ligands of metal-organic frameworks, but also as pH-responsive and capture proteins. Functional molecules, which endow the material with pH-responsive properties and the ability to selectively capture proteins.
本发明进一步提供了上述pH响应型磁性金属有机框架复合纳米材料的制备方法，该制备方法的基本原理是首先利用溶剂热法制备Fe3O4纳米粒子；再在制得的Fe3O4纳米粒子表面包覆由第一聚合物和亲水性的第二聚合物混合而成高分子层，制得高分子层包覆的Fe3O4/Polymer纳米粒子；然后将可溶性三价铁盐作为金属源、含有至少一个羧基或者至少两个硼酸基的苯硼酸衍生物作为有机配体，通过一锅法将MOFs生长在Fe3O4/Polymer纳米粒子表面得到pH响应型磁性金属有机框架复合纳米材料。The present invention further provides a preparation method of the above-mentioned pH-responsive magnetic metal-organic framework composite nanomaterial. The basic principle of the preparation method is to first prepare Fe3 O4 nanoparticles by solvothermal method; The surface of the particles is coated with a polymer layer mixed with the first polymer and the second hydrophilic polymer to obtain Fe3 O4 /Polymer nanoparticles coated with the polymer layer; then the soluble ferric salt is used as Metal sources, phenylboronic acid derivatives containing at least one carboxyl group or at least two boronic acid groups were used as organic ligands, and MOFs were grown on the surface of Fe3 O4 /Polymer nanoparticles by a one-pot method to obtain pH-responsive magnetic metal-organic framework composite nanoparticles. Material.
基于上述原理，本发明提供的pH响应型磁性金属有机框架复合纳米材料的制备方法，步骤如下：Based on the above principles, the preparation method of the pH-responsive magnetic metal-organic framework composite nanomaterial provided by the present invention has the following steps:
(1)配制Fe3O4纳米粒子悬浮液和高分子水溶液(1) Preparation of Fe3 O4 nanoparticle suspension and polymer aqueous solution
将Fe3O4纳米粒子分散到去离子水中，配制Fe3O4纳米粒子浓度为40～80mg/mL的Fe3O4纳米粒子悬浮液；将第一聚合物或第一聚合物与第二聚合物溶于去离子水中，配制第一聚合物浓度为55～85mg/ml或第一聚合物与第二聚合物总浓度为55～85mg/ml的高分子水溶液，当将第一聚合物与第二聚合物溶于去离子水时，第一聚合物与第二聚合物的质量比为1：(0.3～1)；Dispersing Fe3 O4 nanoparticles into deionized water to prepare Fe3 O4 nano particle suspension with a Fe3 O4 nano particle concentration of 40-80 mg/mL; mix the first polymer or the first polymer with the second The polymer is dissolved in deionized water, and the concentration of the first polymer is prepared to be 55-85 mg/ml or the total concentration of the first polymer and the second polymer is 55-85 mg/ml. When the first polymer and the When the second polymer is dissolved in deionized water, the mass ratio of the first polymer to the second polymer is 1: (0.3-1);
(2)制备Fe3O4/Polymer纳米粒子(2) Preparation of Fe3 O4 /Polymer nanoparticles
将步骤(1)配制的Fe3O4纳米粒子悬浮液和高分子水溶液按照体积比1：(2～6)计量并加入到反应容器中，在搅拌下于室温反应至少8小时得第一反应液，然后用磁铁收集第一反应液中的固体产物，再依次用去离子水、乙醇和N,N-二甲基甲酰胺对固体产物洗涤得到Fe3O4/Polymer纳米粒子；The Fe3 O4 nanoparticle suspension and polymer aqueous solution prepared in step (1) are measured according to the volume ratio 1: (2-6) and added to the reaction vessel, and reacted at room temperature for at least 8 hours under stirring to obtain the first reaction liquid, and then use a magnet to collect the solid product in the first reaction solution, and then wash the solid product with deionized water, ethanol and N,N-dimethylformamide to obtain Fe3 O4 /Polymer nanoparticles;
(3)制备pH响应型磁性金属有机框架复合纳米材料(3) Preparation of pH-responsive magnetic metal-organic framework composite nanomaterials
将步骤(2)所得洗涤后的Fe3O4/Polymer纳米粒子分散到N,N-二甲基甲酰胺中，得到浓度为65～95mg/ml的Fe3O4/Polymer纳米粒子悬浮液；将乙腈和N,N-二甲基甲酰胺等体积混合均匀得到第一混合液；按Fe3O4/Polymer纳米粒子悬浮液与第一混合液中N,N-二甲基甲酰胺的体积比为1︰(1～16)计量Fe3O4/Polymer纳米粒子悬浮液和第一混合液，在搅拌下将Fe3O4/Polymer纳米粒子悬浮液加入到第一混合液中，并搅拌至Fe3O4/Polymer纳米粒子分散均匀得到第二混合液；在搅拌下，向第二混合液中加入可溶性三价铁盐和含有至少一个羧基或者至少两个硼酸基的苯硼酸衍生物并搅拌均匀得到第三混合液，所述苯硼酸衍生物的加入量以第二混合液中N,N-二甲基甲酰胺的含量为基准，每6～10ml N,N-二甲基甲酰胺加入1毫摩尔苯硼酸衍生物，所述可溶性三价铁盐与苯硼酸衍生物的摩尔比为(0.5～2):1；在搅拌下将第三混合液升温至100～130℃反应至少1小时得到第二反应液，之后用磁铁收集第二反应液中的固体产物，再依次用N,N-二甲基甲酰胺、乙醇和去离子水对固体产物洗涤即得到pH响应型磁性金属有机框架复合纳米材料。Dispersing the washed Fe3 O4 /Polymer nanoparticles obtained in step (2) into N,N-dimethylformamide to obtain a suspension of Fe3 O4 /Polymer nanoparticles with a concentration of 65-95 mg/ml; Mix equal volumes of acetonitrile and N,N- dimethylformamide to obtain the first mixed solution; The ratio is 1: (1~16) measure the Fe3 O4 /Polymer nano particle suspension and the first mixed liquid, add the Fe3 O4 /Polymer nano particle suspension to the first mixed liquid under stirring, and stir until the Fe3 O4 /Polymer nanoparticles are uniformly dispersed to obtain a second mixed solution; under stirring, add soluble ferric salt and a phenylboronic acid derivative containing at least one carboxyl group or at least two boronic acid groups to the second mixed solution and Stir evenly to obtain the third mixed solution, the amount of the phenylboronic acid derivative added is based on the content of N,N-dimethylformamide in the second mixed solution, every 6-10ml N,N-dimethylformamide Add 1 millimole of phenylboronic acid derivatives, the molar ratio of the soluble ferric salt to phenylboronic acid derivatives is (0.5-2):1; heat the third mixed solution to 100-130°C under stirring for at least 1 hour to obtain the second reaction solution, then use a magnet to collect the solid product in the second reaction solution, and then wash the solid product with N,N-dimethylformamide, ethanol and deionized water in order to obtain a pH-responsive magnetic metal organic Frame composite nanomaterials.
上述pH响应型磁性金属有机框架复合纳米材料的制备方法，Fe3O4纳米粒子可以参考现有技术中已经披露的常规制备方法得到，参见Zhao,M.；Zhang,X.；Deng,C.Rationalsynthesis of novel recyclable Fe3O4@MOF nanocomposites for enzymaticdigestion.Chem.Commun.2015,51,8116-8119和Zhang,Y.；Yang,Y.；Ma,W.；Guo,J.；Lin,Y.；Wang,C.Uniform Magnetic Core/Shell Microspheres Functionalized with Ni2+-Iminodiacetic Acid for One Step Purification and Immobilization of His-TaggedEnzymes.ACS Appl.Mater.Interfaces 2013,5,2626-2633。本发明中采用的制备方法为：将原料氯化铁、柠檬酸钠和醋酸铵，按摩尔比1：(0.33～0.79)：10加入到盛有氯化铁质量(33～36)倍的溶剂乙二醇的反应容器中，在搅拌下使原料溶解并混合均匀；然后将反应温度升至180～220℃，反应不少于15小时；反应结束后再将反应温度冷却至室温，用磁铁收集反应液中的Fe3O4纳米粒子；然后依次用乙醇和去离子水对Fe3O4纳米粒子进行洗涤除去未反应的原料。The preparation method of the above pH-responsive magnetic metal-organic framework composite nanomaterials, Fe3 O4 nanoparticles can be obtained by referring to the conventional preparation methods disclosed in the prior art, see Zhao, M.; Zhang, X.; Deng, C. Rationalsynthesis of novel recyclable Fe3O4@MOF nanocomposites for enzymatic digestion. Chem.Commun.2015, 51, 8116-8119 and Zhang, Y.; Yang, Y.; Ma, W.; Guo, J.; Lin, Y.; Wang, C.Uniform Magnetic Core/Shell Microspheres Functionalized with Ni2+ -Iminodiacetic Acid for One Step Purification and Immobilization of His-Tagged Enzymes.ACS Appl.Mater.Interfaces 2013,5,2626-2633. The preparation method adopted in the present invention is: the raw materials ferric chloride, sodium citrate and ammonium acetate are added to the solvent containing ferric chloride mass (33-36) times in molar ratio 1: (0.33-0.79): 10 In the reaction vessel of ethylene glycol, dissolve and mix the raw materials evenly under stirring; then raise the reaction temperature to 180-220°C, and react for not less than 15 hours; after the reaction, cool the reaction temperature to room temperature and collect it with a magnet Fe3 O4 nanoparticles in the reaction solution; then, the Fe3 O4 nanoparticles were washed with ethanol and deionized water in sequence to remove unreacted raw materials.
上述pH响应型磁性金属有机框架复合纳米材料的制备方法，步骤(2)的目的是利用螯合金属离子的第一聚合物或螯合金属离子的第一聚合物与亲水性的第二聚合物一起将Fe3O4纳米粒子包覆完全，得到高分子层包裹的Fe3O4纳米粒子(即Fe3O4/Polymer纳米粒子)，以便在Fe3O4纳米粒子表面能进一步生长MOFs，其中，第一聚合物作为过渡层，螯合MOFs层的金属离子，使MOFs层在Fe3O4/Polymer纳米粒子表面生长，第二聚合物用于增加材料的亲水性，第二聚合物在本材料中并不是必须的，但是为了提高最终材料在水中的分散性，进而提高对蛋白质捕获和释放效率，第一聚合物与第二聚合物一起组成高分子层时为较佳的选择，此时第一聚合物与第二聚合物的质量比优选为1：(0.3～1)，优选为1：(0.3～0.5)。当Fe3O4纳米粒子悬浮液和高分子水溶液体积比为1：(2～6)时，基本可以保证Fe3O4纳米粒子被高分子层包覆完全。步骤(2)用磁铁收集第一反应液中的固体产物之后，需要进一步用去离子水、乙醇和N,N-二甲基甲酰胺对固体产物洗涤除去吸附在固体产物表面、未反应的第一聚合物或第二聚合物，一般每种洗液洗涤三至五次即可。The preparation method of the above-mentioned pH-responsive magnetic metal-organic framework composite nanomaterial, the purpose of step (2) is to utilize the first polymer of chelated metal ions or the first polymer of chelated metal ions and the second polymer of hydrophilicity materials together to completely cover Fe3 O4 nanoparticles to obtain Fe3 O4 nanoparticles wrapped by polymer layers (ie Fe3 O4 /Polymer nanoparticles), so that MOFs can be further grown on the surface of Fe3 O4 nanoparticles , in which, the first polymer is used as a transition layer to chelate the metal ions of the MOFs layer, so that the MOFs layer grows on the surface of Fe3 O4 /Polymer nanoparticles, the second polymer is used to increase the hydrophilicity of the material, and the second polymer The material is not necessary in this material, but in order to improve the dispersibility of the final material in water, thereby improving the efficiency of protein capture and release, it is a better choice when the first polymer and the second polymer form a polymer layer together In this case, the mass ratio of the first polymer to the second polymer is preferably 1:(0.3-1), preferably 1:(0.3-0.5). When the volume ratio of the Fe3 O4 nanoparticle suspension to the polymer aqueous solution is 1: (2-6), it can basically ensure that the Fe3 O4 nanoparticles are completely covered by the polymer layer. Step (2) After collecting the solid product in the first reaction liquid with a magnet, it is necessary to further wash the solid product with deionized water, ethanol and N,N-dimethylformamide to remove the unreacted second product adsorbed on the surface of the solid product. For the first polymer or the second polymer, it is generally sufficient to wash three to five times with each lotion.
上述pH响应型磁性金属有机框架复合纳米材料的制备方法，步骤(3)的目的是在步骤(2)得到的Fe3O4/Polymer纳米粒子表面生长MOFs层，得到pH响应型磁性金属有机框架复合纳米材料(即Fe3O4/Polymer/MOFs复合纳米材料)。可溶性三价铁盐用于提供Fe3+，其可以为Fe(NO)3或FeCl3，当可溶性三价铁盐与含有至少一个羧基或者至少两个硼酸基的苯硼酸衍生物摩尔比为(0.5～2):1，特别是(0.8～1):1时，形成的MOFs层表现出良好的pH响应性能和蛋白质选择吸附性能。为了能够提高Fe3O4/Polymer纳米粒子表面螯合金属离子效率，Fe3O4/Polymer纳米粒子悬浮液与混合液的添加比例应按照中Fe3O4/Polymer纳米粒子悬浮液与N,N-二甲基甲酰胺体积比1：(1～16)添加，其优选比例为1：(6～10)，当Fe3O4/Polymer纳米粒子添加量过少，不利于Fe3O4/Polymer纳米粒子表面形成MOFs层，而当Fe3O4/Polymer纳米粒子添加量过大时，容易造成Fe3O4/Polymer纳米粒子的聚集，同样不利于Fe3O4/Polymer纳米粒子表面形成MOFs层。步骤(3)用磁铁收集第二反应液中的固体产物之后，需要进一步用N,N-二甲基甲酰胺、乙醇和去离子水对固体产物洗涤除去吸附在固体产物表面的混合液以及未反应的苯硼酸衍生物等，一般每种洗液洗涤三至五次即可。In the preparation method of the pH-responsive magnetic metal-organic framework composite nanomaterial, the purpose of step (3) is to grow a MOFs layer on the surface of the Fe3 O4 /Polymer nanoparticles obtained in step (2) to obtain a pH-responsive magnetic metal-organic framework Composite nanomaterials (ie Fe3 O4 /Polymer/MOFs composite nanomaterials). The soluble ferric salt is used to provide Fe3+ , which can be Fe(NO)3 or FeCl3 , when the molar ratio of the soluble ferric salt to the phenylboronic acid derivative containing at least one carboxyl group or at least two boronic acid groups is ( 0.5～2):1, especially (0.8～1):1, the formed MOFs layer showed good pH response performance and protein selective adsorption performance. In order to improve the efficiency of chelating metal ions on the surface of Fe3 O4 /Polymer nanoparticles, the addition ratio of the Fe3 O4 /Polymer nanoparticle suspension and the mixed solution should be in accordance with the Fe3 O4 /Polymer nanoparticle suspension and N, N-dimethylformamide is added at a volume ratio of 1: (1~16), and the preferred ratio is 1: (6~10). When the amount of Fe3 O4 /Polymer nanoparticles added is too small, it is not conducive to Fe3 O4 /Polymer nanoparticles surface to form a MOFs layer, and when Fe3 O4 /Polymer nanoparticles are added too much, it is easy to cause the aggregation of Fe3 O4 /Polymer nanoparticles, which is also not conducive to the surface of Fe3 O4 /Polymer nanoparticles Form the MOFs layer. Step (3) After collecting the solid product in the second reaction solution with a magnet, it is necessary to further wash the solid product with N,N-dimethylformamide, ethanol and deionized water to remove the mixed solution adsorbed on the surface of the solid product and unused For the reacted phenylboronic acid derivatives, etc., it is generally sufficient to wash three to five times with each lotion.
上述pH响应型磁性金属有机框架复合纳米材料的制备方法，所述第一聚合物为聚乙烯吡咯烷酮或聚多巴胺；所述第二聚合物为聚醚酰亚胺或聚丙烯酸；所述可溶性三价铁盐为Fe(NO)3或FeCl3；所述含有至少一个羧基的苯硼酸衍生物为5-硼酸基间苯二甲酸、3-羧基苯硼酸或4-羧基苯硼酸，所述含有至少两个硼酸基的苯硼酸衍生物为对苯二硼酸或间苯二硼酸。The preparation method of the above-mentioned pH-responsive magnetic metal-organic framework composite nanomaterial, the first polymer is polyvinylpyrrolidone or polydopamine; the second polymer is polyetherimide or polyacrylic acid; the soluble trivalent The iron salt is Fe(NO)3 or FeCl3 ; the phenylboronic acid derivative containing at least one carboxyl group is 5-boronic isophthalic acid, 3-carboxyphenylboronic acid or 4-carboxyphenylboronic acid, and the phenylboronic acid derivative containing at least two The derivatives of phenylboronic acid with a boronic acid group are terephthalic diboronic acid or isophthalic diboronic acid.
本发明进一步提供了一种上述pH响应型磁性金属有机框架复合纳米材料在糖蛋白捕获或糖蛋白释放中的应用；在pH为中性环境下，所述磁性金属有机框架复合纳米材料表面的苯硼酸能和糖蛋白的顺式二醇共价形成环醚，而实现对糖蛋白的捕获；而当环境pH转变到8～9的碱性环境时，就能够可逆的释放糖蛋白，即所述磁性金属有机框架复合纳米材料将捕获的糖蛋白释放。The present invention further provides an application of the pH-responsive magnetic metal-organic framework composite nanomaterial in glycoprotein capture or glycoprotein release; in a neutral pH environment, the benzene on the surface of the magnetic metal-organic framework composite nanomaterial Boric acid can covalently form a cyclic ether with the cis-diol of glycoproteins to capture glycoproteins; and when the environmental pH changes to an alkaline environment of 8-9, it can reversibly release glycoproteins, that is, the Magnetic metal-organic framework composite nanomaterials release trapped glycoproteins.
上述糖蛋白的捕获和释放方法，可以用于捕获和释放任何一种类型的糖蛋白，例如转铁蛋白、辣根过氧化物酶、甲胎蛋白等。The above glycoprotein capture and release method can be used to capture and release any type of glycoprotein, such as transferrin, horseradish peroxidase, alpha-fetoprotein and the like.
与现有技术相比，本发明具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
1、本发明提供的pH响应型磁性金属有机框架复合纳米材料，以Fe3O4纳米粒子作为核，具有高的磁饱和强度，从而提供较好的磁响应性能，可以在较短的时间内完成磁分离；此外，该复合纳米材料表面的MOFs层包含苯硼酸衍生物，在作为有机配体的同时，可以在不同pH环境下可逆的、高选择性的捕获和释放糖蛋白。1. The pH-responsive magnetic metal-organic framework composite nanomaterial provided by the present invention uses Fe3 O4 nanoparticles as the core, and has high magnetic saturation intensity, thereby providing better magnetic response performance, and can Complete magnetic separation; in addition, the MOFs layer on the surface of the composite nanomaterial contains phenylboronic acid derivatives, which can reversibly and highly selectively capture and release glycoproteins under different pH environments while serving as organic ligands.
2、本发明提供的pH响应型磁性金属有机框架复合纳米材料，能够实现在pH为中性环境下有效地捕获糖蛋白，并在pH为8～9的碱性环境下可逆的释放糖蛋白，捕获和释放糖蛋白的pH环境相对温和，避免对糖蛋白活性产生影响。2. The pH-responsive magnetic metal-organic framework composite nanomaterial provided by the present invention can effectively capture glycoproteins in a neutral pH environment and reversibly release glycoproteins in an alkaline environment with a pH of 8-9, The pH environment for capturing and releasing glycoproteins is relatively mild to avoid affecting the activity of glycoproteins.
3、本发明提供的pH响应型磁性金属有机框架复合纳米材料的制备方法，操作简单、反应条件温和，中间产物Fe3O4/Polymer纳米粒子在室温条件下便可得到，而Fe3O4/Polymer纳米粒子、可溶性三价铁盐和含有至少一个羧基或者至少两个硼酸基的苯硼酸衍生物采用一锅法在短时间内就可以制备得到该复合纳米材料，因此易于在生物医药行业领域内推广。3. The preparation method of the pH-responsive magnetic metal-organic framework composite nanomaterial provided by the present invention has simple operation and mild reaction conditions, and the intermediate product Fe3 O4 /Polymer nanoparticles can be obtained at room temperature, while Fe3 O4 /Polymer nanoparticles, soluble ferric salt and phenylboronic acid derivatives containing at least one carboxyl group or at least two boronic acid groups can be prepared in a short period of time to obtain the composite nanomaterial, so it is easy to be used in the field of biomedical industry Promotion within.
附图说明Description of drawings
图1为本发明实施例Fe3O4/Polymer/MOFs复合纳米材料的制备流程图。Fig. 1 is a flow chart of the preparation of Fe3 O4 /Polymer/MOFs composite nanomaterials according to the embodiment of the present invention.
图2为本发明实施例使用Fe3O4/Polymer/MOFs复合纳米材料对蛋白混合体系中糖蛋白的捕获和分离流程图。Fig. 2 is a flowchart of the capture and separation of glycoproteins in protein mixed systems using Fe3 O4 /Polymer/MOFs composite nanomaterials according to an embodiment of the present invention.
图3为本发明实施例Fe3O4纳米粒子、Fe3O4/Polymer纳米粒子、Fe3O4/Polymer/MOFs复合纳米材料的结构表征示意图，其中(A)、(D)和(G)为Fe3O4纳米粒子的扫描电镜(SEM)图、透射电镜(TEM)图和尺寸分布图(DLS)，(B)、(E)和(H)为Fe3O4/Polymer纳米粒子的扫描电镜(SEM)图、透射电镜(TEM)图和尺寸分布图(DLS)，(C)、(F)和(I)为Fe3O4/Polymer/MOFs复合纳米材料的扫描电镜(SEM)图、透射电镜(TEM)图和尺寸分布图(DLS)。Figure 3 is a schematic diagram of the structural characterization of Fe3 O4 nanoparticles, Fe3 O4 /Polymer nanoparticles, and Fe3 O4 /Polymer/MOFs composite nanomaterials of the present invention, where (A), (D) and (G ) is the scanning electron microscope (SEM) image, transmission electron microscope (TEM) image and size distribution (DLS) image of Fe3 O4 nanoparticles, (B), (E) and (H) are Fe3 O4 /Polymer nanoparticles The scanning electron microscope (SEM) image, transmission electron microscope (TEM) image and size distribution image (DLS), (C), (F) and (I) are the scanning electron microscope (SEM) of Fe3 O4 /Polymer/MOFs composite nanomaterials ) map, transmission electron microscope (TEM) map and size distribution map (DLS).
图4为本发明实施例Fe3O4纳米粒子、Fe3O4/Polymer纳米粒子、Fe3O4/Polymer/MOFs复合纳米材料的Zeta电位图。Fig. 4 is a Zeta potential diagram of Fe3 O4 nanoparticles, Fe3 O4 /Polymer nanoparticles, and Fe3 O4 /Polymer/MOFs composite nanomaterials of the embodiments of the present invention.
图5为Fe3O4纳米粒子(A)、Fe3O4/Polymer纳米粒子(B)、Fe3O4/Polymer/MOFs复合纳米材料(C)的能量弥散X射线(EDX)能谱图。Figure 5 is the energy dispersive X-ray (EDX) spectrum of Fe3 O4 nanoparticles (A), Fe3 O4 /Polymer nanoparticles (B), Fe3 O4 /Polymer/MOFs composite nanomaterials (C) .
图6为本发明实施例PVP(a)、PEI(b)、PBA(c)、Fe3O4纳米粒子(d)、Fe3O4/Polymer纳米粒子(e)、Fe3O4/Polymer/MOFs复合纳米材料(f)在波数4000～500(cm-1)之间的红外图谱。Figure 6 is the embodiment of the present invention PVP (a), PEI (b), PBA (c), Fe3 O4 nanoparticles (d), Fe3 O4 /Polymer nanoparticles (e), Fe3 O4 /Polymer nanoparticles Infrared spectra of /MOFs composite nanomaterials (f) at wavenumbers between 4000 and 500 (cm-1 ).
图7为本发明实施例Fe3O4/Polymer/MOFs复合纳米材料的氮气的吸附-解吸附等温曲线(A)以及根据氮气吸附-解吸附等温曲线利用Barrett–Joyner–Halenda(BJH)估算的Fe3O4/Polymer/MOFs复合纳米材料的孔径尺寸分布曲线。Fig. 7 is the nitrogen adsorption-desorption isotherm curve (A) of the Fe3 O4 /Polymer/MOFs composite nanomaterial of the embodiment of the present invention and estimated by Barrett-Joyner-Halenda (BJH) according to the nitrogen adsorption-desorption isotherm curve Pore size distribution curve of Fe3 O4 /Polymer/MOFs composite nanomaterials.
图8为本发明实施例PVP(a)、PBA(b)、Fe3O4纳米粒子(c)、Fe3O4/Polymer纳米粒子(d)、Fe3O4/Polymer/MOFs复合纳米材料(e)、MOFs(f)的X射线光衍射(XRD)图谱；其中最下方标识的是Fe3O4的标准XRD衍射峰(JCPDS号19-06290)。Figure 8 shows the examples of PVP (a), PBA (b), Fe3 O4 nanoparticles (c), Fe3 O4 /Polymer nanoparticles (d), Fe3 O4 /Polymer/MOFs composite nanomaterials of the present invention (e), X-ray diffraction (XRD) pattern of MOFs (f); the bottom mark is the standard XRD diffraction peak of Fe3 O4 (JCPDS No. 19-06290).
图9为本发明实施例PVP(a)、PBA(b)、Fe3O4纳米粒子(c)、Fe3O4/Polymer纳米粒子(d)、Fe3O4/Polymer/MOFs复合纳米材料(e)的热重分析(TGA)图。Figure 9 shows the examples of PVP (a), PBA (b), Fe3 O4 nanoparticles (c), Fe3 O4 /Polymer nanoparticles (d), Fe3 O4 /Polymer/MOFs composite nanomaterials of the present invention (e) Thermogravimetric analysis (TGA) profile.
图10为本发明实施例Fe3O4纳米粒子(a)、Fe3O4/Polymer纳米粒子(b)、Fe3O4/Polymer/MOFs复合纳米材料(d)的磁滞回线图(A)以及所得磁滞回线在低场区域的展开图(B)。Fig. 10 is the hysteresis loop diagram of Fe3 O4 nanoparticles (a), Fe3 O4 /Polymer nanoparticles (b), Fe3 O4 /Polymer/MOFs composite nanomaterials (d) of the present invention ( A) and the resulting hysteresis loop expansion in the low-field region (B).
图11为本发明实施例Fe3O4/Polymer/MOFs复合纳米材料对无磁铁状态以及有磁铁响应的状态图。Fig. 11 is a state diagram of Fe3 O4 /Polymer/MOFs composite nanomaterials responding to the non-magnet state and the magnetism state according to the embodiment of the present invention.
图12为本发明实施例分子量标样(marker)，蛋白混合液(Mix)与Fe3O4/Polymer/MOFs复合纳米材料在pH＝7环境下孵育前后的蛋白混合液的标样(Mix)、上层清液(S)、键合蛋白的材料(C)，以及在pH＝7或pH＝9环境下洗脱后的洗脱液(E)、洗脱后的材料(C)的SDS-PAGE分析图；其中，孵育过程用I表示，洗脱过程用E表示。Figure 12 is the molecular weight standard sample (marker) of the present invention, the standard sample (Mix) of the protein mixture solution (Mix) before and after incubation with Fe3 O4 /Polymer/MOFs composite nanomaterials in a pH=7 environment , the supernatant (S), the protein-binding material (C), and the SDS- PAGE analysis diagram; wherein, the incubation process is represented by I, and the elution process is represented by E.
图13为分子量标样(marker)、蛋白混合样品(Mix)与商用磁珠在pH＝7环境下孵育前后的蛋白混合液的标样(Mix)、上层清液(S)、键合蛋白的材料(C)，以及在pH＝7或pH＝9环境下洗脱后的洗脱液(E)、洗脱后的材料(C)的SDS-PAGE分析图；其中，孵育过程用I表示，洗脱过程用E表示。Figure 13 shows the molecular weight standard sample (marker), protein mixed sample (Mix) and commercial magnetic beads before and after incubation of the protein mixture standard (Mix), supernatant (S), binding protein Material (C), and the SDS-PAGE analysis figure of the eluate (E) after elution under the environment of pH=7 or pH=9, and the material (C) after elution; wherein, the incubation process is represented by I, The elution process is represented by E.
具体实施方式detailed description
以下将结合附图对本发明各实施例的技术方案进行清楚、完整的描述，显然，所描述实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施例，都属于本发明所保护的范围。The technical solutions of the various embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Apparently, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.
实施例1制备Fe3O4/Polymer/MOFs复合纳米材料Example 1 Preparation of Fe3 O4 /Polymer/MOFs Composite Nanomaterials
本实施例中，高分子层为聚乙烯吡咯烷酮(PVP)，金属有机框架以硝酸铁为金属源，以1,4-对苯二硼酸(PBA)作为有机配体。In this embodiment, the polymer layer is polyvinylpyrrolidone (PVP), the metal-organic framework uses iron nitrate as a metal source, and 1,4-terephthalenediboronic acid (PBA) as an organic ligand.
图1给出了Fe3O4/Polymer/MOFs复合纳米材料的制备流程图，首先利用溶剂热法制备表面显负电性的Fe3O4纳米粒子；再依据静电作用、范德华力等相互作用，在制得的Fe3O4纳米粒子表面包覆一层高分子层，得到高分子层包覆的Fe3O4纳米球(Fe3O4/Polymer纳米粒子)；然后将硝酸铁为金属源、1,4-对苯二硼酸(PBA)作为有机配体，与Fe3O4/Polymer纳米粒子一起通过一锅法制备得到Fe3O4/Polymer/MOFs复合纳米材料。Figure 1 shows the flow chart of the preparation of Fe3 O4 /Polymer/MOFs composite nanomaterials. Firstly, Fe3 O4 nanoparticles with negatively charged surface are prepared by solvothermal method; Coating a polymer layer on the surface of the obtained Fe3 O4 nanoparticles to obtain Fe3 O4 nanospheres (Fe3 O4 /Polymer nanoparticles) covered by the polymer layer; then use iron nitrate as a metal source , 1,4-terephthalic diboronic acid (PBA) as an organic ligand, and Fe3 O4 /Polymer nanoparticles were prepared by a one-pot method to obtain Fe3 O4 /Polymer/MOFs composite nanomaterials.
结合上述流程，本实施例Fe3O4/Polymer/MOFs复合纳米材料的制备步骤如下：Combining the above process, the preparation steps of Fe3 O4 /Polymer/MOFs composite nanomaterials in this example are as follows:
(1)制备Fe3O4纳米粒子(1) Preparation of Fe3 O4 nanoparticles
将原料1.157g六水合氯化铁、1.0g柠檬酸钠和3.303g醋酸铵加入到盛有57mL溶剂乙二醇的聚四氟乙烯不锈钢反应釜中，磁力搅拌1小时使上述原料溶解；然后移除搅拌子，将反应釜温度升至180℃，反应15小时；再将反应釜冷却至室温，用磁铁收集反应得到的反应液中产物；然后依次用乙醇对产物重复洗涤五次(10mL×5)、用去离子水对产物重复洗涤五次(10mL×5)得到Fe3O4纳米粒子；Add the raw materials 1.157g ferric chloride hexahydrate, 1.0g sodium citrate and 3.303g ammonium acetate into a polytetrafluoroethylene stainless steel reaction kettle filled with 57mL solvent ethylene glycol, and magnetically stir for 1 hour to dissolve the above raw materials; then transfer In addition to the stirring bar, the temperature of the reactor was raised to 180°C and reacted for 15 hours; then the reactor was cooled to room temperature, and the product in the reaction solution obtained by the reaction was collected with a magnet; then the product was repeatedly washed five times with ethanol (10mL×5 ), the product was repeatedly washed five times with deionized water (10mL × 5) to obtain Fe3 O4 nanoparticles;
(2)配制Fe3O4纳米粒子悬浮液和高分子水溶液(2) Preparation of Fe3 O4 nanoparticle suspension and polymer aqueous solution
将步骤(1)所得洗涤后的Fe3O4纳米粒子分散到18.75ml去离子水中，配制Fe3O4纳米粒子浓度为40mg/mL的Fe3O4纳米粒子悬浮液；将聚乙烯吡咯烷酮(PVP)溶于去离子水中，配制PVP浓度为55mg/ml的高分子水溶液；TheFe3O4 nanoparticle after step (1) gained washing is dispersed in18.75ml deionized water, preparationFe3O4 nanoparticle concentration is theFe3O4 nanoparticle suspension of 40mg/mL; Polyvinylpyrrolidone ( PVP) is dissolved in deionized water, and the preparation PVP concentration is the polymer aqueous solution of 55mg/ml;
(3)制备Fe3O4/Polymer纳米粒子(3) Preparation of Fe3 O4 /Polymer nanoparticles
取步骤(2)配制的6.25mlFe3O4纳米粒子悬浮液和12.5ml高分子水溶液加入到反应容器中，在搅拌下于室温反应8小时得第一反应液，然后用磁铁收集第一反应液中的固体产物，再依次用去离子水对产物重复洗涤五次(10mL×5)、用乙醇对产物重复洗涤五次(10mL×5)和用N,N-二甲基甲酰胺对产物洗涤五次(10mL×5)得到Fe3O4/Polymer纳米粒子；Get 6.25ml of Fe3 O4 nano particle suspension and 12.5ml polymer aqueous solution prepared in step (2) and join in the reaction vessel, react at room temperature under stirring for 8 hours to obtain the first reaction solution, then collect the first reaction solution with a magnet The solid product in the solution was washed with deionized water five times (10mL×5), with ethanol five times (10mL×5) and with N,N-dimethylformamide. Five times (10mL×5) to obtain Fe3 O4 /Polymer nanoparticles;
(4)制备Fe3O4/Polymer/MOFs(4) Preparation of Fe3 O4 /Polymer/MOFs
将步骤(3)所得洗涤后的Fe3O4/Polymer纳米粒子分散到2.45ml N,N-二甲基甲酰胺中，得到浓度为65.3mg/ml的Fe3O4/Polymer纳米粒子悬浮液；将6ml乙腈和6ml N,N-二甲基甲酰胺混合得到第一混合液；在搅拌下将1ml Fe3O4/Polymer纳米粒子悬浮液加入到第一混合液中，并搅拌至Fe3O4/Polymer纳米粒子分散均匀得到第二混合液；在搅拌下，向第二混合液中加入202mgFe(NO3)3·9H2O和160mg PBA并搅拌均匀得到第三混合液；在搅拌下将第三混合液升温至100℃，反应1小时得到第二反应液，之后用磁铁收集第二反应液中的固体产物，再依次用N,N-二甲基甲酰胺对产物重复洗涤五次(10mL×5)、用乙醇对产物重复洗涤五次(10mL×5)和用去离子水对产物重复洗涤五次(10mL×5)得到Fe3O4/Polymer/MOFs复合纳米材料。Disperse the washed Fe3 O4 /Polymer nanoparticles obtained in step (3) into 2.45ml N,N-dimethylformamide to obtain a Fe3 O4 /Polymer nanoparticle suspension with a concentration of 65.3mg/ml ; Mix 6ml acetonitrile and 6ml N,N-dimethylformamide to obtain the first mixed solution; add 1ml Fe3 O4 /Polymer nanoparticle suspension to the first mixed solution under stirring, and stir until Fe3 O4 /Polymer nanoparticles are uniformly dispersed to obtain the second mixed solution; under stirring, add 202mgFe(NO3 )3 ·9H2 O and 160mg PBA to the second mixed solution and stir evenly to obtain the third mixed solution; Raise the temperature of the third mixed solution to 100°C, react for 1 hour to obtain the second reaction solution, then use a magnet to collect the solid product in the second reaction solution, and then wash the product repeatedly with N,N-dimethylformamide for five times (10mL×5), washed the product five times with ethanol (10mL×5) and washed the product five times with deionized water (10mL×5) to obtain Fe3 O4 /Polymer/MOFs composite nanomaterials.
实施例2制备Fe3O4/Polymer/MOFs复合纳米材料Example 2 Preparation of Fe3 O4 /Polymer/MOFs Composite Nanomaterials
本实施例中，高分子层为聚乙烯吡咯烷酮(PVP)和聚醚酰亚胺(PEI)，金属有机框架以硝酸铁为金属源，以1,4-对苯二硼酸(PBA)作为有机配体。In this embodiment, the polymer layer is polyvinylpyrrolidone (PVP) and polyetherimide (PEI), the metal-organic framework uses ferric nitrate as the metal source, and 1,4-terephenylboronic acid (PBA) as the organic ligand. body.
本实施例Fe3O4/Polymer/MOFs复合纳米材料的制备步骤如下：The preparation steps of Fe3 O4 /Polymer/MOFs composite nanomaterials in this example are as follows:
(1)制备Fe3O4纳米粒子(1) Preparation of Fe3 O4 nanoparticles
将原料1.157g六水合氯化铁、0.4g柠檬酸钠和3.303g醋酸铵加入到盛有60mL溶剂乙二醇的聚四氟乙烯不锈钢反应釜中，磁力搅拌1小时使上述原料溶解；然后移除搅拌子，将反应釜温度升至200℃，反应16小时；再将反应釜冷却至室温，用磁铁收集反应得到的反应液中产物；然后依次用乙醇对产物重复洗涤三次(15mL×3)、用去离子水对产物重复洗涤三次(15mL×3)得到Fe3O4纳米粒子；Add raw materials 1.157g ferric chloride hexahydrate, 0.4g sodium citrate and 3.303g ammonium acetate to a polytetrafluoroethylene stainless steel reaction kettle filled with 60mL solvent ethylene glycol, and magnetically stir for 1 hour to dissolve the above raw materials; then transfer Remove the stirrer, raise the temperature of the reactor to 200°C, and react for 16 hours; then cool the reactor to room temperature, and collect the product in the reaction solution obtained by the reaction with a magnet; then repeatedly wash the product three times with ethanol (15mL×3) 1. The product was repeatedly washed three times with deionized water (15mL×3) to obtain Fe3 O4 nanoparticles;
(2)配制Fe3O4纳米粒子悬浮液和高分子水溶液(2) Preparation of Fe3 O4 nanoparticle suspension and polymer aqueous solution
将步骤(1)所得洗涤后的Fe3O4纳米粒子分散到15ml去离子水中，配制Fe3O4纳米粒子浓度为50mg/mL的Fe3O4纳米粒子悬浮液；将聚乙烯吡咯烷酮(PVP)和聚醚酰亚胺(PEI)按照质量比1：0.4溶于去离子水中，配制PVP和PEI总浓度为70mg/ml的高分子水溶液；TheFe3O4 nanoparticle after step (1 ) gained washing is dispersed in 15ml deionized water, preparationFe3O4 nanoparticle concentration is theFe3O4 nanoparticle suspension of 50mg/mL; Polyvinylpyrrolidone (PVP ) and polyetherimide (PEI) were dissolved in deionized water at a mass ratio of 1:0.4 to prepare a polymer aqueous solution with a total concentration of PVP and PEI of 70 mg/ml;
(3)制备Fe3O4/Polymer纳米粒子(3) Preparation of Fe3 O4 /Polymer nanoparticles
取步骤(2)配制的5mlFe3O4纳米粒子悬浮液和20ml高分子水溶液加入到反应容器中，在搅拌下于室温反应12小时得第一反应液，然后用磁铁收集第一反应液中的固体产物，再依次用去离子水对产物重复洗涤三次(15mL×3)、用乙醇对产物重复洗涤三次(15mL×3)和用N,N-二甲基甲酰胺对产物洗涤三次(15mL×3)得到Fe3O4/Polymer纳米粒子；Get step (2) 5mlFe3 O4 nanoparticle suspension and 20ml polymer aqueous solution are added in the reaction vessel, under stirring, react at room temperature for 12 hours to obtain the first reaction solution, then collect the first reaction solution with a magnet. The solid product was washed three times with deionized water (15mL×3), three times with ethanol (15mL×3) and three times with N,N-dimethylformamide (15mL×3). 3) obtaining Fe3 O4 /Polymer nanoparticles;
(4)制备Fe3O4/Polymer/MOFs复合纳米材料(4) Preparation of Fe3 O4 /Polymer/MOFs composite nanomaterials
将步骤(3)所得洗涤后的Fe3O4/Polymer纳米粒子分散到2ml N,N-二甲基甲酰胺中，得到浓度为80mg/ml的Fe3O4/Polymer纳米粒子悬浮液；将8ml乙腈和8ml N,N-二甲基甲酰胺混合得到第一混合液；在搅拌下将1ml Fe3O4/Polymer纳米粒子悬浮液加入到第一混合液中，并搅拌至Fe3O4/Polymer纳米粒子分散均匀得到第二混合液；在搅拌下，向第二混合液中加入320mgFe(NO3)3·9H2O和160mg PBA并搅拌均匀得到第三混合液；在搅拌下将第三混合液升温至120℃，反应2小时得到第二反应液，之后用磁铁收集第二反应液中的固体产物，再依次用N,N-二甲基甲酰胺对产物重复洗涤三次(15mL×3)、用乙醇对产物重复洗涤三次(15mL×3)和用去离子水对产物重复洗涤三次(15mL×3)得到Fe3O4/Polymer/MOFs复合纳米材料。Disperse the washed Fe3 O4 /Polymer nanoparticles obtained in step (3) into 2ml N,N-dimethylformamide to obtain a Fe3 O4 /Polymer nanoparticle suspension with a concentration of 80mg/ml; Mix 8ml of acetonitrile and 8ml of N,N-dimethylformamide to obtain the first mixed solution; add 1ml of Fe3 O4 /Polymer nanoparticle suspension to the first mixed solution with stirring, and stir until Fe3 O4 /Polymer nanoparticles are uniformly dispersed to obtain the second mixed solution; under stirring, add 320mgFe(NO3 )3 9H2 O and 160mg PBA to the second mixed solution and stir evenly to obtain the third mixed solution; The temperature of the three mixtures was raised to 120°C and reacted for 2 hours to obtain the second reaction solution. After that, the solid product in the second reaction solution was collected with a magnet, and the product was repeatedly washed three times with N,N-dimethylformamide (15 mL× 3) The product was repeatedly washed three times with ethanol (15 mL×3) and three times with deionized water (15 mL×3) to obtain Fe3 O4 /Polymer/MOFs composite nanomaterials.
实施例3制备Fe3O4/Polymer/MOFs复合纳米材料Example 3 Preparation of Fe3 O4 /Polymer/MOFs Composite Nanomaterials
本实施例中，高分子层为聚多巴胺(PDA)和聚丙烯酸(PAA)，金属有机框架以氯化铁为金属源，以3-羧基苯硼酸作为有机配体。In this embodiment, the polymer layer is polydopamine (PDA) and polyacrylic acid (PAA), the metal-organic framework uses ferric chloride as the metal source, and 3-carboxyphenylboronic acid as the organic ligand.
本实施例Fe3O4/Polymer/MOFs复合纳米材料的制备步骤如下：The preparation steps of the Fe3O4/Polymer/MOFs composite nanomaterial in this example are as follows:
(1)制备Fe3O4纳米粒子(1) Preparation of Fe3 O4 nanoparticles
将原料1.157g六水合氯化铁、0.7g柠檬酸钠和3.303g醋酸铵加入到盛有60mL溶剂乙二醇的聚四氟乙烯不锈钢反应釜中，磁力搅拌1小时使上述原料溶解；然后移除搅拌子，将反应釜温度升至220℃，反应17小时；再将反应釜冷却至室温，用磁铁收集反应得到的反应液中产物；然后依次用乙醇对产物重复洗涤三次(20mL×3)、用去离子水对产物重复洗涤三次(20mL×3)得到Fe3O4纳米粒子；Add raw materials 1.157g ferric chloride hexahydrate, 0.7g sodium citrate and 3.303g ammonium acetate to a polytetrafluoroethylene stainless steel reaction kettle filled with 60mL solvent ethylene glycol, and stir magnetically for 1 hour to dissolve the above raw materials; then transfer Remove the stirring bar, raise the temperature of the reactor to 220°C, and react for 17 hours; then cool the reactor to room temperature, and collect the product in the reaction liquid obtained by the reaction with a magnet; then repeatedly wash the product three times with ethanol (20mL×3) 1. The product was repeatedly washed three times (20mL×3) with deionized water to obtain Fe3 O4 nanoparticles;
(2)配制Fe3O4纳米粒子悬浮液和高分子水溶液(2) Preparation of Fe3 O4 nanoparticle suspension and polymer aqueous solution
将步骤(1)所得洗涤后的Fe3O4纳米粒子分散到9.4ml去离子水中，配制Fe3O4纳米粒子浓度为80mg/mL的Fe3O4纳米粒子悬浮液；将聚多巴胺(PDA)和聚丙烯酸按照质量比1：1溶于去离子水中，配制PVP和PEI总浓度为85mg/ml的高分子水溶液；TheFe3O4 nanoparticle after step (1) gained washing is dispersed in9.4ml deionized water, preparationFe3O4 nanoparticle concentration is theFe3O4 nanoparticle suspension of 80mg/mL; polydopamine (PDA ) and polyacrylic acid were dissolved in deionized water at a mass ratio of 1:1 to prepare a polymer aqueous solution with a total concentration of PVP and PEI of 85 mg/ml;
(3)制备Fe3O4/Polymer纳米粒子(3) Preparation of Fe3 O4 /Polymer nanoparticles
取步骤(2)配制的3.5mlFe3O4纳米粒子悬浮液和21ml高分子水溶液加入到反应容器中，在搅拌下于室温反应24小时得第一反应液，然后用磁铁收集第一反应液中的固体产物，再依次用去离子水对产物重复洗涤三次(15mL×3)、用乙醇对产物重复洗涤三次(15mL×3)和用N,N-二甲基甲酰胺对产物洗涤三次(15mL×3)得到Fe3O4/Polymer纳米粒子；Get the3.5mlFe3O4nanoparticle suspension and21ml polymer aqueous solution prepared in step (2) and join in the reaction vessel, react at room temperature under stirring for 24 hours to obtain the first reaction solution, and then use a magnet to collect the first reaction solution The solid product was washed three times with deionized water (15mL×3), three times with ethanol (15mL×3) and three times with N,N-dimethylformamide (15mL ×3) Fe3 O4 /Polymer nanoparticles were obtained;
(4)制备Fe3O4/Polymer/MOFs复合纳米材料(4) Preparation of Fe3 O4 /Polymer/MOFs composite nanomaterials
将步骤(3)所得洗涤后的Fe3O4/Polymer纳米粒子分散到2ml N,N-二甲基甲酰胺中，得到浓度为95mg/ml的Fe3O4/Polymer纳米粒子悬浮液；将16ml乙腈和16ml N,N-二甲基甲酰胺混合得到第一混合液；在搅拌下将1ml Fe3O4/Polymer纳米粒子悬浮液加入到第一混合液中，并搅拌至Fe3O4/Polymer纳米粒子分散均匀得到第二混合液；在搅拌下，向第二混合液中加入650mgFeCl3和332mg 3-羧基苯硼酸并搅拌均匀得到第三混合液；在搅拌下将第三混合液升温至130℃，反应8小时得到第二反应液，之后用磁铁收集第二反应液中的固体产物，再依次用N,N-二甲基甲酰胺对产物重复洗涤三次(15mL×3)、用乙醇对产物重复洗涤三次(15mL×3)和用去离子水对产物重复洗涤三次(15mL×3)得到Fe3O4/Polymer/MOFs复合纳米材料。Disperse the washed Fe3 O4 /Polymer nanoparticles obtained in step (3) into 2ml N,N-dimethylformamide to obtain a Fe3 O4 /Polymer nanoparticle suspension with a concentration of 95mg/ml; Mix 16ml of acetonitrile and 16ml of N,N-dimethylformamide to obtain the first mixed solution; add 1ml of Fe3 O4 /Polymer nanoparticle suspension to the first mixed solution with stirring, and stir until Fe3 O4 /Polymer nanoparticles are uniformly dispersed to obtain the second mixed solution; under stirring, add 650mgFeCl3 and 332mg3 -carboxyphenylboronic acid to the second mixed solution and stir to obtain the third mixed solution; under stirring, the third mixed solution is heated to 130°C, reacted for 8 hours to obtain the second reaction solution, and then collected the solid product in the second reaction solution with a magnet, and then washed the product repeatedly with N,N-dimethylformamide three times (15mL×3), with The product was repeatedly washed with ethanol three times (15mL×3) and with deionized water (15mL×3) to obtain Fe3 O4 /Polymer/MOFs composite nanomaterials.
实施例4制备Fe3O4/Polymer/MOFs复合纳米材料Example 4 Preparation of Fe3 O4 /Polymer/MOFs Composite Nanomaterials
本实施例中，高分子层为聚乙烯吡咯烷酮(PVP)和聚醚酰亚胺(PEI)，金属有机框架以硝酸铁为金属源，以4-羧基苯硼酸作为有机配体。In this embodiment, the polymer layer is polyvinylpyrrolidone (PVP) and polyetherimide (PEI), the metal-organic framework uses iron nitrate as a metal source, and 4-carboxyphenylboronic acid as an organic ligand.
本实施例Fe3O4/Polymer/MOFs复合纳米材料的制备步骤如下：The preparation steps of Fe3 O4 /Polymer/MOFs composite nanomaterials in this example are as follows:
(1)制备Fe3O4纳米粒子(1) Preparation of Fe3 O4 nanoparticles
将原料1.157g六水合氯化铁、0.4g柠檬酸钠和3.303g醋酸铵加入到盛有60mL溶剂乙二醇的聚四氟乙烯不锈钢反应釜中，磁力搅拌1小时使上述原料溶解；然后移除搅拌子，将反应釜温度升至200℃，反应16小时；再将反应釜冷却至室温，用磁铁收集反应得到的反应液中产物；然后依次用乙醇对产物重复洗涤三次(15mL×3)、用去离子水对产物重复洗涤三次(15mL×3)得到Fe3O4纳米粒子；Add raw materials 1.157g ferric chloride hexahydrate, 0.4g sodium citrate and 3.303g ammonium acetate to a polytetrafluoroethylene stainless steel reaction kettle filled with 60mL solvent ethylene glycol, and magnetically stir for 1 hour to dissolve the above raw materials; then transfer Remove the stirrer, raise the temperature of the reactor to 200°C, and react for 16 hours; then cool the reactor to room temperature, and collect the product in the reaction solution obtained by the reaction with a magnet; then repeatedly wash the product three times with ethanol (15mL×3) 1. The product was repeatedly washed three times with deionized water (15mL×3) to obtain Fe3 O4 nanoparticles;
(2)配制Fe3O4纳米粒子悬浮液和高分子水溶液(2) Preparation of Fe3 O4 nanoparticle suspension and polymer aqueous solution
将步骤(1)所得洗涤后的Fe3O4纳米粒子分散到15ml去离子水中，配制Fe3O4纳米粒子浓度为50mg/mL的Fe3O4纳米粒子悬浮液；将聚乙烯吡咯烷酮(PVP)和聚醚酰亚胺(PEI)按照质量比1：0.3溶于去离子水中，配制PVP和PEI总浓度为70mg/ml的高分子水溶液；TheFe3O4 nanoparticle after step (1 ) gained washing is dispersed in 15ml deionized water, preparationFe3O4 nanoparticle concentration is theFe3O4 nanoparticle suspension of 50mg/mL; Polyvinylpyrrolidone (PVP ) and polyetherimide (PEI) were dissolved in deionized water at a mass ratio of 1:0.3 to prepare a polymer aqueous solution with a total concentration of PVP and PEI of 70 mg/ml;
(3)制备Fe3O4/Polymer纳米粒子(3) Preparation of Fe3 O4 /Polymer nanoparticles
取步骤(2)配制的5mlFe3O4纳米粒子悬浮液和15ml高分子水溶液加入到反应容器中，在搅拌下于室温反应10小时得第一反应液，然后用磁铁收集第一反应液中的固体产物，再依次用去离子水对产物重复洗涤三次(15mL×3)、用乙醇对产物重复洗涤三次(15mL×3)和用N,N-二甲基甲酰胺对产物洗涤三次(15mL×3)得到Fe3O4/Polymer纳米粒子；Get step (2) 5mlFe3 O4 nanoparticle suspension and 15ml polymer aqueous solution are added in the reaction vessel, under stirring, react at room temperature for 10 hours to obtain the first reaction solution, then collect the first reaction solution with a magnet. The solid product was washed three times with deionized water (15mL×3), three times with ethanol (15mL×3) and three times with N,N-dimethylformamide (15mL×3). 3) obtaining Fe3 O4 /Polymer nanoparticles;
(4)制备Fe3O4/Polymer/MOFs复合纳米材料(4) Preparation of Fe3 O4 /Polymer/MOFs composite nanomaterials
将步骤(3)所得洗涤后的Fe3O4/Polymer纳米粒子分散到2ml N,N-二甲基甲酰胺中，得到浓度为80mg/ml的Fe3O4/Polymer纳米粒子悬浮液；将10ml乙腈和10ml N,N-二甲基甲酰胺混合得到第一混合液；在搅拌下将1ml Fe3O4/Polymer纳米粒子悬浮液加入到第一混合液中，并搅拌至Fe3O4/Polymer纳米粒子分散均匀得到第二混合液；在搅拌下，向第二混合液中加入404mg Fe(NO3)3·9H2O和166mg 4-羧基苯硼酸并搅拌均匀得到第三混合液；在搅拌下将第三混合液升温至120℃，反应4小时得到第二反应液，之后用磁铁收集第二反应液中的固体产物，再依次用N,N-二甲基甲酰胺对产物重复洗涤三次(15mL×3)、用乙醇对产物重复洗涤三次(15mL×3)和用去离子水对产物重复洗涤三次(15mL×3)得到Fe3O4/Polymer/MOFs复合纳米材料。Disperse the washed Fe3 O4 /Polymer nanoparticles obtained in step (3) into 2ml N,N-dimethylformamide to obtain a Fe3 O4 /Polymer nanoparticle suspension with a concentration of 80mg/ml; Mix 10ml acetonitrile and 10ml N,N-dimethylformamide to obtain the first mixed solution; add 1ml Fe3 O4 /Polymer nanoparticle suspension to the first mixed solution under stirring, and stir until Fe3 O4 /Polymer nanoparticles are uniformly dispersed to obtain a second mixed solution; under stirring, add 404mg Fe(NO3 )3 9H2 O and 166mg 4-carboxyphenylboronic acid to the second mixed solution and stir evenly to obtain a third mixed solution; Under stirring, the temperature of the third mixed solution was raised to 120°C and reacted for 4 hours to obtain the second reaction solution. After that, the solid product in the second reaction solution was collected with a magnet, and the product was repeated with N,N-dimethylformamide in turn. Washing three times (15mL×3), washing the product repeatedly with ethanol three times (15mL×3) and washing the product three times with deionized water (15mL×3) obtained Fe3 O4 /Polymer/MOFs composite nanomaterials.
实施例5制备Fe3O4/Polymer/MOFs复合纳米材料Example 5 Preparation of Fe3 O4 /Polymer/MOFs Composite Nanomaterials
本实施例中，高分子层为聚乙烯吡咯烷酮(PVP)和聚丙烯酸(PAA)，金属有机框架以氯化铁为金属源，以间苯二硼酸作为有机配体。In this embodiment, the polymer layer is polyvinylpyrrolidone (PVP) and polyacrylic acid (PAA), the metal-organic framework uses ferric chloride as a metal source, and isophthalic acid as an organic ligand.
本实施例Fe3O4/Polymer/MOFs复合纳米材料的制备步骤如下：The preparation steps of Fe3 O4 /Polymer/MOFs composite nanomaterials in this example are as follows:
(1)制备Fe3O4纳米粒子(1) Preparation of Fe3 O4 nanoparticles
将原料1.157g六水合氯化铁、0.4g柠檬酸钠和3.303g醋酸铵加入到盛有60mL溶剂乙二醇的聚四氟乙烯不锈钢反应釜中，磁力搅拌1小时使上述原料溶解；然后移除搅拌子，将反应釜温度升至200℃，反应16小时；再将反应釜冷却至室温，用磁铁收集反应得到的反应液中产物；然后依次用乙醇对产物重复洗涤三次(15mL×3)、用去离子水对产物重复洗涤三次(15mL×3)得到Fe3O4纳米粒子；Add raw materials 1.157g ferric chloride hexahydrate, 0.4g sodium citrate and 3.303g ammonium acetate to a polytetrafluoroethylene stainless steel reaction kettle filled with 60mL solvent ethylene glycol, and magnetically stir for 1 hour to dissolve the above raw materials; then transfer Remove the stirrer, raise the temperature of the reactor to 200°C, and react for 16 hours; then cool the reactor to room temperature, and collect the product in the reaction solution obtained by the reaction with a magnet; then repeatedly wash the product three times with ethanol (15mL×3) 1. The product was repeatedly washed three times with deionized water (15mL×3) to obtain Fe3 O4 nanoparticles;
(2)配制Fe3O4纳米粒子悬浮液和高分子水溶液(2) Preparation of Fe3 O4 nanoparticle suspension and polymer aqueous solution
将步骤(1)所得洗涤后的Fe3O4纳米粒子分散到15ml去离子水中，配制Fe3O4纳米粒子浓度为50mg/mL的Fe3O4纳米粒子悬浮液；将聚乙烯吡咯烷酮(PVP)和聚丙烯酸(PAA)按照质量比1：0.5溶于去离子水中，配制PVP和PAA总浓度为70mg/ml的高分子水溶液；TheFe3O4 nanoparticle after step (1 ) gained washing is dispersed in 15ml deionized water, preparationFe3O4 nanoparticle concentration is theFe3O4 nanoparticle suspension of 50mg/mL; Polyvinylpyrrolidone (PVP ) and polyacrylic acid (PAA) are dissolved in deionized water according to the mass ratio of 1:0.5, and the polymer aqueous solution whose total concentration of PVP and PAA is 70mg/ml is prepared;
(3)制备Fe3O4/Polymer纳米粒子(3) Preparation of Fe3 O4 /Polymer nanoparticles
取步骤(2)配制的5mlFe3O4纳米粒子悬浮液和25ml高分子水溶液加入到反应容器中，在搅拌下于室温反应14小时得第一反应液，然后用磁铁收集第一反应液中的固体产物，再依次用去离子水对产物重复洗涤三次(15mL×3)、用乙醇对产物重复洗涤三次(15mL×3)和用N,N-二甲基甲酰胺对产物洗涤三次(15mL×3)得到Fe3O4/Polymer纳米粒子；Get step (2) 5mlFe3 O4 nanoparticle suspension and 25ml polymer aqueous solution are added in the reaction container, under stirring, react at room temperature for 14 hours to obtain the first reaction solution, then collect the first reaction solution with a magnet. The solid product was washed three times with deionized water (15mL×3), three times with ethanol (15mL×3) and three times with N,N-dimethylformamide (15mL×3). 3) obtaining Fe3 O4 /Polymer nanoparticles;
(4)制备Fe3O4/Polymer/MOFs复合纳米材料(4) Preparation of Fe3 O4 /Polymer/MOFs composite nanomaterials
将步骤(3)所得洗涤后的Fe3O4/Polymer纳米粒子分散到2ml N,N-二甲基甲酰胺中，得到浓度为80mg/ml的Fe3O4/Polymer纳米粒子悬浮液；将8ml乙腈和8ml N,N-二甲基甲酰胺混合得到第一混合液；在搅拌下将1ml Fe3O4/Polymer纳米粒子悬浮液加入到第一混合液中，并搅拌至Fe3O4/Polymer纳米粒子分散均匀得到第二混合液；在搅拌下，向第二混合液中加入160mg FeCl3和160mg间苯二硼酸并搅拌均匀得到第三混合液；在搅拌下将第三混合液升温至120℃，反应6小时得到第二反应液，之后用磁铁收集第二反应液中的固体产物，再依次用N,N-二甲基甲酰胺对产物重复洗涤三次(15mL×3)、用乙醇对产物重复洗涤三次(15mL×3)和用去离子水对产物重复洗涤三次(15mL×3)得到Fe3O4/Polymer/MOFs复合纳米材料。Disperse the washed Fe3 O4 /Polymer nanoparticles obtained in step (3) into 2ml N,N-dimethylformamide to obtain a Fe3 O4 /Polymer nanoparticle suspension with a concentration of 80mg/ml; Mix 8ml of acetonitrile and 8ml of N,N-dimethylformamide to obtain the first mixed solution; add 1ml of Fe3 O4 /Polymer nanoparticle suspension to the first mixed solution with stirring, and stir until Fe3 O4 /Polymer nanoparticles are uniformly dispersed to obtain the second mixed solution; under stirring, add 160mg FeCl3 and 160mg isophthalic diboronicacid to the second mixed solution and stir to obtain the third mixed solution; the third mixed solution is heated up under stirring to 120°C, reacted for 6 hours to obtain the second reaction solution, and then collected the solid product in the second reaction solution with a magnet, and then washed the product repeatedly with N,N-dimethylformamide three times (15mL×3), with The product was repeatedly washed with ethanol three times (15mL×3) and with deionized water (15mL×3) to obtain Fe3 O4 /Polymer/MOFs composite nanomaterials.
对比例comparative example
本对比例是制备无Fe3O4的金属有机框架材料，首先将320mgFe(NO3)3·9H2O和160mg PBA加入到由8ml乙腈和8ml N,N-二甲基甲酰胺中，搅拌至硝酸铁和PBA溶解完全，其次将所得溶液转移至不锈钢聚四氟乙烯乙烯反应釜中，于120℃反应3天，然后将所得反应产物于10000rpm离心15min收集红棕色产物，再将收集到的红棕色产物依次用N,N-二甲基甲酰胺对产物重复洗涤三次(15mL×3)、用乙醇对产物重复洗涤三次(15mL×3)和用去离子水对产物重复洗涤三次(15mL×3)，最好将洗涤所得产物干燥得到金属有机框架材料。This comparative example is to prepare metal-organic framework materials without Fe3 O4 . First, 320mgFe(NO3 )3 9H2 O and 160mg PBA are added to 8ml acetonitrile and 8ml N,N-dimethylformamide, and stirred After ferric nitrate and PBA are completely dissolved, the resulting solution is transferred to a stainless steel polytetrafluoroethylene reactor, reacted at 120°C for 3 days, and then the resulting reaction product is centrifuged at 10,000 rpm for 15 minutes to collect the reddish-brown product, and then the collected The reddish-brown product was repeatedly washed three times with N,N-dimethylformamide (15mL×3), three times with ethanol (15mL×3) and three times with deionized water (15mL×3). 3), preferably drying the washed product to obtain a metal organic framework material.
实验例1结构表征Experimental Example 1 Structural Characterization
为了探究高分子层以及金属框架MOFs已经成功复合到Fe3O4纳米粒子上，本实验例对实施例2给出的Fe3O4/Polymer/MOFs复合纳米材料制备过程中得到的Fe3O4纳米粒子、Fe3O4/Polymer纳米粒子和Fe3O4/Polymer/MOFs复合纳米材料的形貌尺寸和微观结构进行了表征如图3至图8所示。In order to explore the successful compounding of polymer layer and metal framework MOFs on Fe3 O4 nanoparticles, this experimental example is based on the Fe3 O4 /Polymer/MOFs composite nanomaterials obtained in Example 2. Fe3 O4 The morphology, size and microstructure of nanoparticles, Fe3 O4 /Polymer nanoparticles and Fe3 O4 /Polymer/MOFs composite nanomaterials were characterized, as shown in Fig. 3 to Fig. 8 .
(1)形貌与尺寸分布(1) Morphology and size distribution
从图3(A)-(F)可以看出，Fe3O4纳米粒子、Fe3O4/Polymer纳米粒子、Fe3O4/Polymer/MOFs复合纳米材料均为球形，大小均一，数据表面所包裹的高分子层及MOF层并没有对Fe3O4纳米粒子的形貌和尺寸产生过多影响；Fe3O4/Polymer/MOFs复合纳米材料平均粒径为300nm～500nm；通过动态光散射检测得到Fe3O4纳米粒子、Fe3O4/Polymer纳米粒子、Fe3O4/Polymer/MOFs复合纳米材料的平均粒径峰值分别为299±4nm,317±22nm，340±61nm(图3G-I)，此结果与扫描电镜和透射电镜结果一致。From Figure 3(A)-(F), it can be seen that Fe3 O4 nanoparticles, Fe3 O4 /Polymer nanoparticles, and Fe3 O4 /Polymer/MOFs composite nanomaterials are all spherical, uniform in size, and the data surface The wrapped polymer layer and MOF layer did not have too much influence on the morphology and size of Fe3 O4 nanoparticles; the average particle size of Fe3 O4 /Polymer/MOFs composite nanomaterials was 300nm-500nm; The average particle diameter peaks of Fe3 O4 nanoparticles, Fe3 O4 /Polymer nanoparticles, and Fe3 O4 /Polymer/MOFs composite nanomaterials obtained by scattering detection are 299±4nm, 317±22nm, 340±61nm respectively (Fig. 3G-I), this result is consistent with the results of SEM and TEM.
(2)微结构研究(2) Microstructure research
如图4所示，Fe3O4纳米粒子、Fe3O4/Polymer纳米粒子、Fe3O4/Polymer/MOFs复合纳米材料在去离子水中的zeta电位分别为-15mV(由于Fe3O4纳米粒子表面附着的柠檬酸钠存在羧基，从而使Fe3O4纳米粒子显负电)、+34mV(由于Fe3O4纳米粒子包裹PVP和PEI后，PEI存在大量氨基，从而使Fe3O4/Polymer纳米粒子显正电)和-12mV(由于MOFs层中有机配体PBA存在硼酸基团，从而使Fe3O4/Polymer/MOFs复合纳米材料显负电)；这种电荷翻转进一步证明了高分子层及MOFs层已成功被合成到Fe3O4纳米粒子和Fe3O4/Polymer纳米粒子上。As shown in Figure 4, the zeta potentials of Fe3 O4 nanoparticles, Fe3 O4 /Polymer nanoparticles, and Fe3 O4 /Polymer/MOFs composite nanomaterials in deionized water were -15mV (due to the Fe3 O4 The sodium citrate attached to the surface of the nanoparticles has a carboxyl group, which makes the Fe3 O4 nanoparticles negatively charged), +34mV (because after the Fe3 O4 nanoparticles wrap PVP and PEI, there are a large number of amino groups in the PEI, so that the Fe3 O4 /Polymer nanoparticles are positively charged) and -12mV (the Fe3 O4 /Polymer/MOFs composite nanomaterials are negatively charged due to the presence of boronic acid groups in the organic ligand PBA in the MOFs layer); this charge reversal further proves the high Molecular layers and MOFs layers have been successfully synthesized onto Fe3 O4 nanoparticles and Fe3 O4 /Polymer nanoparticles.
如图5所示，通过能量弥散X射线(EDX)检测得出，Fe3O4纳米粒子中，C元素重量百分比为10.04％、原子百分比为21.69％，O元素重量百分比为31.59％、原子百分比为51.21％，Fe元素重量百分比为58.36％、原子百分比27.10％；Fe3O4/Polymer纳米粒子中，C元素重量百分比为8.20％、原子百分比为14.92％，N元素重量百分比为12.04％、原子百分比为18.80％，O元素重量百分比为35.94％、原子百分比为49.12％，Fe元素重量百分比为43.82％、原子百分比17.16％；Fe3O4/Polymer/MOFs复合纳米材料中，B元素重量百分比为9.55％、原子百分比为16.02％，C元素重量百分比为12.20％、原子百分比为18.42％，N元素重量百分比为13.98％、原子百分比为18.10％，O元素重量百分比为32.88％、原子百分比为37.27％，Fe元素重量百分比为31.39％、原子百分比10.19％。从上述检测结果可以看出，Fe3O4/Polymer/MOFs复合纳米材料中存在N元素和B元素，进一步证明高分子层(高分子层中PEI中含有氨基，氨基中含有N元素)和MOFs层(MOFs层中的有机配体PBA含有B元素)已经成功在Fe3O4纳米粒子表面形成。As shown in Figure5 , through energy dispersive X-ray (EDX) detection, inFe3O4 nanoparticles, the weight percentage of C element is 10.04%, the atomic percentage is 21.69%, the O element weight percentage is 31.59%, atomic percentage is 51.21%, Fe element weight percentage is 58.36%, atomic percentage is 27.10%; in Fe3 O4 /Polymer nanoparticles, C element weight percentage is 8.20%, atomic percentage is 14.92%, N element weight percentage is 12.04%, atomic The percentage is 18.80%, the O element weight percentage is 35.94%, the atomic percentage is 49.12%, the Fe element weight percentage is 43.82%, the atomic percentage is 17.16%; in the Fe3 O4 /Polymer/MOFs composite nanomaterial, the B element weight percentage is 9.55%, atomic percentage is 16.02%, C element weight percentage is 12.20%, atomic percentage is 18.42%, N element weight percentage is 13.98%, atomic percentage is 18.10%, O element weight percentage is 32.88%, atomic percentage is 37.27% , Fe element weight percentage is 31.39%, atomic percentage is 10.19%. From the above test results, it can be seen that there are N elements and B elements in the Fe3 O4 /Polymer/MOFs composite nanomaterials, which further proves that the polymer layer (the PEI in the polymer layer contains amino groups, and the amino groups contain N elements) and MOFs layer (the organic ligand PBA in the MOFs layer contains B element) has been successfully formedon the surface ofFe3O4 nanoparticles.
本实验例采用PE spectrometer型傅里叶转换红外光谱仪(FTIR)分别检测PVP、PEI、PBA、Fe3O4纳米粒子、Fe3O4/Polymer纳米粒子、Fe3O4/Polymer/MOFs复合纳米材料在500-4000cm-1波数范围内的红外吸收光谱，检测步长为4cm-1，检测结果如图6所示，Fe3O4/Polymer/MOFs复合纳米材料的吸收峰含有Fe-O(600cm-1)、C-N(1436cm-1)和PBA(633cm-1)的特征吸收峰，说明高分子层和MOFs层已经成功在Fe3O4纳米粒子表面形成。然而，C-N和PBA的特征峰较弱，可能是因为PVP、PEI和PBA的含量较少的原因。In this experimental example, a PE spectrometer type Fourier transform infrared spectrometer (FTIR) was used to detect PVP, PEI, PBA, Fe3 O4 nanoparticles, Fe3 O4 /Polymer nanoparticles, Fe3 O4 /Polymer/MOFs composite nanoparticles The infrared absorption spectrum of the material in the range of 500-4000cm-1 wave number, the detection step length is 4cm-1 , the detection results are shown in Figure 6, the absorption peak of the Fe3 O4 /Polymer/MOFs composite nanomaterial contains Fe-O( 600cm-1 ), CN (1436cm-1 ) and PBA (633cm-1 ) characteristic absorption peaks, indicating that the polymer layer and MOFs layer have been successfully formed on the surface of Fe3 O4 nanoparticles. However, the characteristic peaks of CN and PBA were weaker, which may be due to the less content of PVP, PEI and PBA.
如图7所示，通过获得的N2吸附/解吸附等温曲线表明Fe3O4/Polymer/MOFs复合纳米材料的Langumir表面积及孔容分别为159m2/g、0.6cm3/g，根据Barrett–Joyner–Halenda(BJH)估算的Fe3O4/Polymer/MOFs复合纳米材料孔径约为11.8埃～27.3埃，这种多孔的性质说明在Fe3O4/Polymer纳米粒子表面形成了网络结构；而且产物的颜色从原始的黑色变为红棕色，证明这种网络结构确实是由Fe3+和PBA共同形成的金属有机框架结构(MOFs)，而非无色的仅有PBA自身脱水形成的共价有机框架(Covalent Organic Frameworks，简称COFs)。As shown in Figure 7, the obtained N2 adsorption/desorption isotherm curve shows that the Langumir surface area and pore volume of Fe3 O4 /Polymer/MOFs composite nanomaterials are 159m2 /g and 0.6cm3 /g, respectively. According to Barrett –Joyner–Halenda (BJH) estimates that the pore size of Fe3 O4 /Polymer/MOFs composite nanomaterials is about 11.8 angstroms to 27.3 angstroms. This porous property indicates that a network structure is formed on the surface of Fe3 O4 /Polymer nanoparticles; Moreover, the color of the product changed from the original black to reddish brown, proving that the network structure is indeed a metal-organic framework (MOFs) formed by Fe3+ and PBA, rather than a colorless co-formed by dehydration of PBA itself. Covalent Organic Frameworks (COFs for short).
本实验例采用X’Pert Pro MPD型X射线衍射(XRD)仪分别检测了Fe3O4纳米粒子、Fe3O4/Polymer纳米粒子、Fe3O4/Polymer/MOFs复合纳米材料的X射线衍射图谱，如图8所示，Fe3O4纳米粒子、Fe3O4/Polymer纳米粒子、Fe3O4/Polymer/MOFs复合纳米材料均与标准Fe3O4的衍射峰位置一致，说明Fe3O4/Polymer/MOFs复合纳米材料保留了Fe3O4纳米粒子的晶体结构；同时，Fe3O4/Polymer/MOFs复合纳米材料在25°出现了一个新的宽的衍射峰，而对比例3所得到无Fe3O4的金属有机框架产物的XRD在25°同样存在一个衍射峰，这说明该衍射峰是由于受所制备MOFs壳层的影响而出现的，实施例2所得产物表面的MOFs层是由Fe3+和PBA通过不规则的配位而形成的无定型的金属有机框架结构。In this experimental example, X'Pert Pro MPD X-ray diffraction (XRD) instrument was used to detect the X-rays of Fe3 O4 nanoparticles, Fe3 O4 /Polymer nanoparticles, and Fe3 O4 /Polymer/MOFs composite nanomaterials. Diffraction patterns, as shown in Figure 8, Fe3 O4 nanoparticles, Fe3 O4 /Polymer nanoparticles, Fe3 O4 /Polymer/MOFs composite nanomaterials are all consistent with the diffraction peak positions of standard Fe3 O4 , indicating that Fe3 O4 /Polymer/MOFs composite nanomaterials retain the crystal structure of Fe3 O4 nanoparticles; at the same time, Fe3 O4 /Polymer/MOFs composite nanomaterials have a new broad diffraction peak at 25°, while The XRD of the metal-organic framework product obtained in Comparative Example 3 without Fe3 O4 also has a diffraction peak at 25 °, which shows that the diffraction peak is due to the effect of the prepared MOFs shell. The product obtained in Example 2 The surface MOFs layer is an amorphous metal-organic framework structure formed by Fe3+ and PBA through irregular coordination.
综上所述，Fe3O4/Polymer/MOFs复合纳米材料的壳层具有金属有机框架结构，且该结构并没有明显影响复合纳米材料的磁晶体结构，这种独特的MOFs壳层将有利于在糖蛋白捕获和释放中的应用。In summary, the shell of Fe3 O4 /Polymer/MOFs composite nanomaterials has a metal-organic framework structure, and this structure does not significantly affect the magnetic crystal structure of composite nanomaterials. This unique MOFs shell will benefit Applications in glycoprotein capture and release.
实验例2磁性能研究Experimental Example 2 Study on Magnetic Properties
为了探究Fe3O4/Polymer/MOFs复合纳米材料的磁性能，本实验例采用STA 449CJupiter型热重分析(TGA)仪对实施例2给出的Fe3O4纳米粒子、Fe3O4/Polymer纳米粒子、Fe3O4/Polymer/MOFs复合纳米材料在氮气保护下从35℃升温到850℃的重量损失，结果如图9所示；通过热重分析数据计算得出Fe3O4纳米粒子、Fe3O4/Polymer纳米粒子、Fe3O4/Polymer/MOFs复合纳米材料的质量百分含量(磁含量)分别约为83％，82％，81％，说明Fe3O4纳米粒子、Fe3O4/Polymer纳米粒子、Fe3O4/Polymer/MOFs复合纳米材料均具有较好的磁性能，有助于提高后续蛋白分离速率。In order to explore the magnetic properties of Fe3 O4 /Polymer/MOFs composite nanomaterials, this experimental example uses STA 449CJupiter type thermogravimetric analysis (TGA) instrument to analyze the Fe3 O4 nanoparticles, Fe3 O4 / The weight loss of Polymer nanoparticles and Fe3 O4 /Polymer/MOFs composite nanomaterials from 35°C to 850°C under nitrogen protection, the results are shown in Figure 9; the Fe3 O4nm The mass percentages (magnetic content) of particles, Fe3 O4 /Polymer nanoparticles, and Fe3 O4 /Polymer/MOFs composite nanomaterials are about 83%, 82%, and 81%, respectively, indicating that Fe3 O4 nanoparticles , Fe3 O4 /Polymer nanoparticles, and Fe3 O4 /Polymer/MOFs composite nanomaterials all have good magnetic properties, which help to improve the subsequent protein separation rate.
进一步的，本实验例采用Model BHV-525型振动样品磁强计(VSM)分别检测了Fe3O4纳米粒子、Fe3O4/Polymer纳米粒子、Fe3O4/Polymer/MOFs复合纳米材料在-18000到18000Oe范围内的磁滞回线(见图10(A))和所得磁滞回线在磁场-100～100Oe之间的扩展图(见图10(B))，所有样品的磁滞回线均经过原点，无剩磁和矫顽力，说明Fe3O4纳米粒子、Fe3O4/Polymer纳米粒子、Fe3O4/Polymer/MOFs复合纳米材料都具有超顺磁性，且所制备的Fe3O4纳米粒子、Fe3O4/Polymer纳米粒子、Fe3O4/Polymer/MOFs复合纳米材料磁饱和强度分别为64emu/g，63emu/g，38emu/g，因此Fe3O4/Polymer/MOFs复合纳米材料具有较好的磁响应性，10s内即可完成磁分离(如图11所示)。Further, in this experimental example, the Model BHV-525 vibrating sample magnetometer (VSM) was used to detect Fe3 O4 nanoparticles, Fe3 O4 /Polymer nanoparticles, Fe3 O4 /Polymer/MOFs composite nanomaterials The hysteresis loop in the range of -18000 to 18000Oe (see Figure 10(A)) and the expansion diagram of the resulting hysteresis loop in the magnetic field between -100 and 100Oe (see Figure 10(B)), the magnetic properties of all samples The hysteresis loops all pass through the origin, without remanence and coercive force, indicating that Fe3 O4 nanoparticles, Fe3 O4 /Polymer nanoparticles, and Fe3 O4 /Polymer/MOFs composite nanomaterials have superparamagnetism, and The prepared Fe3 O4 nanoparticles, Fe3 O4 /Polymer nanoparticles, and Fe3 O4 /Polymer/MOFs composite nanomaterials have magnetic saturation strengths of 64emu/g, 63emu/g, and 38emu/g, respectively, so Fe3 The O4 /Polymer/MOFs composite nanomaterial has good magnetic response, and the magnetic separation can be completed within 10s (as shown in Figure 11).
应用例糖蛋白分离研究Application example Glycoprotein separation research
本应用例所使用的Fe3O4/Polymer/MOFs复合纳米材料按照以下方法处理备用：将实施例2得到的Fe3O4/Polymer/MOFs复合纳米材料分散到1.0mL去离子水中得到Fe3O4/Polymer/MOFs复合纳米材料浓度为30mg/mL的悬浮液，备用。The Fe3 O4 /Polymer/MOFs composite nanomaterials used in this application example are treated as follows: Disperse the Fe3 O4 /Polymer/MOFs composite nanomaterials obtained in Example 2 into 1.0 mL deionized water to obtain Fe3 O4 /Polymer/MOFs composite nanomaterial suspension with a concentration of 30 mg/mL, for later use.
转铁蛋白(transferrin，TRF)为单链糖蛋白，其含糖量约6％，是血浆中主要的含铁蛋白质，负责运载由消化管吸收的铁和由红细胞降解释放的铁；在血液中不正常的转铁蛋白含量与许多疾病有关，例如心脏衰竭、缺铁性贫血、营养不良、转铁蛋白缺乏症等。因此，本应用例中，以转铁蛋白为模型靶向糖蛋白，牛血清白蛋白(BSA)为模型非糖蛋白来研究材料对糖蛋白的选择性分离研究。Transferrin (TRF) is a single-chain glycoprotein with a sugar content of about 6%. It is the main iron-containing protein in plasma and is responsible for carrying iron absorbed by the digestive tract and released by red blood cell degradation; in the blood Abnormal transferrin levels are associated with many diseases such as heart failure, iron deficiency anemia, malnutrition, transferrin deficiency, etc. Therefore, in this application example, transferrin is used as a model to target glycoproteins, and bovine serum albumin (BSA) is used as a model non-glycoprotein to study the selective separation of glycoproteins by materials.
利用Fe3O4/Polymer/MOFs复合纳米材料在pH＝7的含乙腈(体积浓度为20％)的BR(Britton-Robinson)缓冲溶液(作为孵育液)中捕获混合蛋白体系中的TRF和在pH＝9的BR缓冲溶液中释放TRF的过程参见图2，操作过程为：将转铁蛋白(TRF)、牛血清白蛋白(BSA)分别溶于pH＝7的含乙腈(体积浓度为20％)的BR缓冲溶液中，制得两种浓度为1000μg/mL的相应蛋白溶液，并把每种蛋白溶液等体积混匀得到蛋白混合液(TRF+BSA)；取所得30μL Fe3O4/Polymer/MOFs复合纳米材料的悬浮液和30μL蛋白混合液，将两者于反应容器中混合并在震荡条件下于室温孵育60min，然后用磁铁进行分离，分别收集上层清液及沉淀，再将沉淀用孵育液洗涤三次(每次100μL)，得到沉淀材料。Using Fe3 O4 /Polymer/MOFs composite nanomaterials in BR (Britton-Robinson) buffer solution containing acetonitrile (volume concentration 20%) at pH = 7 (as an incubation solution) to capture TRF in a mixed protein system and to capture TRF in a mixed protein system The process of releasing TRF in the BR buffer solution of pH=9 is referring to Fig. 2, and operating process is: transferrin (TRF), bovine serum albumin (BSA) are respectively dissolved in the acetonitrile containing acetonitrile of pH=7 (volume concentration is 20% ) in the BR buffer solution, prepare two corresponding protein solutions with a concentration of 1000μg/mL, and mix each protein solution in equal volume to obtain a protein mixture (TRF+BSA); take the obtained 30μL Fe3 O4 /Polymer /MOFs composite nanomaterial suspension and 30 μL protein mixture were mixed in a reaction vessel and incubated at room temperature under shaking conditions for 60 min, then separated by a magnet, and the supernatant and precipitate were collected, and the precipitate was used The incubation solution was washed three times (100 μL each time) to obtain the precipitated material.
将沉淀材料分散到20μL孵育液中得到沉淀悬浮液，分别取12μL孵育前的蛋白混合液、上层清液和沉淀悬浮液进行SDS-PAGE(十二烷基硫酸钠聚丙烯酰胺凝胶电泳)分析，结果如图12所示。Disperse the precipitation material into 20 μL of incubation solution to obtain a precipitation suspension, and take 12 μL of the protein mixture, supernatant and precipitation suspension before incubation for SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis , the result is shown in Figure 12.
将上述键合了蛋白质的沉淀材料分别分散到40μL pH＝7和pH＝9的BR缓冲溶液中在震荡条件下于室温洗脱60min，然后用磁铁进行分离，分别收集洗脱液和洗脱后得到的沉淀材料，并对洗脱液和洗脱后得到的沉淀材料进行SDS-PAGE分析，结果如图12所示。Disperse the above-mentioned protein-bound precipitation materials into 40 μL of BR buffer solutions with pH=7 and pH=9, and elute at room temperature under shaking conditions for 60 minutes, then separate with a magnet, collect the eluate and the eluted solution respectively. The obtained precipitation material was subjected to SDS-PAGE analysis on the eluate and the precipitation material obtained after elution, and the results are shown in FIG. 12 .
如图12所示，上层清液中只有BSA残余，且键合了蛋白的材料只展示了TRF条带，说明溶液中的TRF已被选择性吸附到Fe3O4/Polymer/MOFs复合纳米材料表面。当用pH＝4的孵育液洗脱蛋白质，被吸附到材料表面的糖蛋白TRF并不能被洗脱下来，说明TRF和Fe3O4/Polymer/MOFs复合纳米材料的结合作用是比较强的特异性结合作用。然而，当pH调至9时，蛋白质很容易地从材料表面洗脱下来，说明Fe3O4/Polymer/MOFs复合纳米材料能够pH响应性地可逆得捕获和释放TRF。As shown in Figure 12, only BSA remains in the supernatant, and the protein-bound material only shows TRF bands, indicating that TRF in the solution has been selectively adsorbed to Fe3 O4 /Polymer/MOFs composite nanomaterials surface. When the protein was eluted with pH=4 incubation solution, the glycoprotein TRF adsorbed on the surface of the material could not be eluted, indicating that the binding effect of TRF and Fe3 O4 /Polymer/MOFs composite nanomaterials is relatively strong and specific. sexual bonding. However, when the pH was adjusted to 9, the protein was easily eluted from the surface of the material, indicating that the Fe3 O4 /Polymer/MOFs composite nanomaterials can reversibly capture and release TRF in a pH-responsive manner.
作为对比，将900μg羧化的商用磁珠加入到30μL蛋白混合液，将两者于反应容器中混合并在震荡条件下于室温孵育60min，然后用磁铁进行分离，分别收集上层清液及沉淀，再将沉淀用孵育液洗涤三次(每次100μL)，得到沉淀材料。然后将沉淀材料分散到20μL孵育液中得到沉淀悬浮液，分别取12μL孵育前的蛋白混合液、孵育后磁分离后的上层清液和沉淀悬浮液进行SDS-PAGE(十二烷基硫酸钠聚丙烯酰胺凝胶电泳)分析，结果如图13所示。再将上述沉淀材料分别分散到40μL pH＝7和pH＝9的BR缓冲溶液中在震荡条件下于室温洗脱60min，然后用磁铁进行分离，分别收集洗脱液和洗脱后得到的沉淀材料，并对洗脱液和洗脱后得到的沉淀材料进行SDS-PAGE分析，结果如图13所示。As a comparison, add 900 μg of carboxylated commercial magnetic beads to 30 μL of protein mixture, mix the two in a reaction vessel and incubate at room temperature for 60 minutes under shaking conditions, then separate with a magnet, collect the supernatant and precipitate, respectively, The precipitate was then washed three times with the incubation solution (100 μL each time) to obtain the precipitated material. Then, the precipitation material was dispersed into 20 μL of incubation solution to obtain a precipitation suspension, and 12 μL of the protein mixture before incubation, the supernatant after magnetic separation after incubation, and the precipitation suspension were respectively subjected to SDS-PAGE (sodium dodecyl sulfate poly Acrylamide gel electrophoresis) analysis, the results are shown in Figure 13. Disperse the above precipitated material into 40 μL of BR buffer solutions with pH=7 and pH=9, elute at room temperature for 60 minutes under shaking conditions, then separate with a magnet, and collect the eluent and the precipitated material obtained after elution respectively , and SDS-PAGE analysis was performed on the eluate and the precipitated material obtained after elution, and the results are shown in Figure 13.
如图13所示，当采用羧化的商用磁珠时，上清液和键合了蛋白的材料上均包括TRF和BSA条带，且键合了蛋白的材料对应的条带更明显，说明商用磁珠已经捕获了蛋白质，但没有表现出任何的选择性；而当用pH＝7和pH＝9的BR缓冲溶液对键合了蛋白的材料进行洗脱时，两种洗脱液均不能将TRF或BSA从商用磁珠上洗脱下来，说明商用磁珠没有表现出pH响应性能。As shown in Figure 13, when carboxylated commercial magnetic beads are used, both the supernatant and the protein-bound material include TRF and BSA bands, and the bands corresponding to the protein-bound material are more obvious, indicating that Commercial magnetic beads have captured proteins but did not show any selectivity; while protein-bound material was eluted with pH=7 and pH=9 BR buffer solutions, neither eluent could The elution of TRF or BSA from the commercial magnetic beads shows that the commercial magnetic beads do not exhibit pH responsiveness.
本应用例中的转铁蛋白可以替换为辣根过氧化物酶(HRP)或甲胎蛋白(AFP)等糖蛋白；牛血清白蛋白(BSA)可以替换为溶菌酶(LYZ)、糜蛋白酶(CTP)，细胞色素C(Cyt C)等非糖蛋白，且上述糖蛋白和非糖蛋白可以以任意比例搭配。Transferrin in this application example can be replaced by glycoproteins such as horseradish peroxidase (HRP) or alpha-fetoprotein (AFP); bovine serum albumin (BSA) can be replaced by lysozyme (LYZ), chymotrypsin ( CTP), cytochrome C (Cyt C) and other non-glycoproteins, and the above-mentioned glycoproteins and non-glycoproteins can be matched in any ratio.
本应用例中采用的pH＝7的含乙腈(体积浓度为20％)的BR缓冲溶液可以替换为pH＝7的含乙腈(体积浓度为20％)的磷酸氢二钠–磷酸二氢钠缓冲液，或pH＝7的含乙腈(体积浓度为20％)的磷酸氢二钾–磷酸二氢钾缓冲液；采用的pH＝9的BR缓冲溶液可以替换为pH为8～9的甘氨酸–氢氧化钠缓冲液，或Tris–盐酸缓冲液。The BR buffer solution containing acetonitrile (volume concentration 20%) at pH=7 used in this application example can be replaced by disodium hydrogen phosphate-sodium dihydrogen phosphate buffer containing acetonitrile (volume concentration 20%) at pH=7 solution, or dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution containing acetonitrile (volume concentration 20%) at pH=7; the BR buffer solution with pH=9 can be replaced by glycine-hydrogen at pH 8-9 Sodium oxide buffer, or Tris–HCl buffer.
总之，所制备的Fe3O4/CMCS/PAAPBA纳米球在生物样本中可以pH响应地对糖蛋白的选择性捕获，富集和释放，展示了其在诊断和蛋白组学中的应用潜力。In summary, the as-prepared Fe3 O4 /CMCS/PAAPBA nanospheres can selectively capture, enrich and release glycoproteins in pH-responsively in biological samples, demonstrating their potential applications in diagnostics and proteomics.
本领域的普通技术人员将会意识到，这里的实施例是为了帮助读者理解本发明的原理，应被理解为本发明的保护范围并不局限于这样的特别陈述和实施例。本领域的普通技术人员可以根据本发明公开的这些技术启示做出各种不脱离本发明实质的其它各种具体变形和组合，这些变形和组合仍然在本发明的保护范围内。Those skilled in the art will appreciate that the embodiments herein are to help readers understand the principles of the present invention, and it should be understood that the protection scope of the present invention is not limited to such specific statements and embodiments. Those skilled in the art can make various other specific modifications and combinations based on the technical revelations disclosed in the present invention without departing from the essence of the present invention, and these modifications and combinations are still within the protection scope of the present invention.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610994774.XACN106552603B (en) | 2016-11-11 | 2016-11-11 | pH-responsive magnetic metal-organic framework composite nanomaterials and their preparation methods and applications |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610994774.XACN106552603B (en) | 2016-11-11 | 2016-11-11 | pH-responsive magnetic metal-organic framework composite nanomaterials and their preparation methods and applications |
| Publication Number | Publication Date |
|---|---|
| CN106552603Atrue CN106552603A (en) | 2017-04-05 |
| CN106552603B CN106552603B (en) | 2019-02-05 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610994774.XAActiveCN106552603B (en) | 2016-11-11 | 2016-11-11 | pH-responsive magnetic metal-organic framework composite nanomaterials and their preparation methods and applications |
| Country | Link |
|---|---|
| CN (1) | CN106552603B (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107607640A (en)* | 2017-08-30 | 2018-01-19 | 复旦大学 | A kind of glycopeptide enrichment of nano composite material of boric acid modified and Mass Spectrometry detection method |
| CN108548480A (en)* | 2018-05-09 | 2018-09-18 | 电子科技大学 | Three layers of selfreparing flexibility strain transducer of one kind and preparation method thereof |
| CN108636359A (en)* | 2018-05-16 | 2018-10-12 | 湘潭大学 | A kind of magnetic metal organic framework microballoon of phenyl boric acid functionalization and its preparation method and application |
| CN109012630A (en)* | 2018-08-30 | 2018-12-18 | 珠海健帆生物科技股份有限公司 | Magnetic composite adsorbent for blood perfusion and preparation method thereof and perfusion device |
| CN109265709A (en)* | 2018-08-17 | 2019-01-25 | 西南交通大学 | It is a kind of can slow releasing pharmaceutical and the factor conductive hydrogel preparation method and application |
| CN109433158A (en)* | 2018-09-29 | 2019-03-08 | 四川大学 | Magnetic nanometer composite material and the preparation method and application thereof for the enrichment of multi-mode peptide fragment |
| WO2019141152A1 (en)* | 2018-01-17 | 2019-07-25 | 南开大学 | Novel composite biological agent based on porous frame material |
| CN111514308A (en)* | 2020-03-10 | 2020-08-11 | 西南民族大学 | PH-induced charge-inversion antibacterial gold nanorod and preparation method and application thereof |
| CN112675821A (en)* | 2020-11-26 | 2021-04-20 | 四川大学 | Magnetic covalent organic framework material for glycopeptide enrichment based on amphiphilic site and preparation method and application thereof |
| CN113121835A (en)* | 2019-12-31 | 2021-07-16 | 华东师范大学 | Iron-based metal organic framework material and preparation and application thereof |
| CN113980926A (en)* | 2021-10-22 | 2022-01-28 | 南京师范大学 | Magnetic nanoparticle-glycosyltransferase-amorphous metal-organic framework composite catalytic material and its preparation method and application |
| CN114223041A (en)* | 2019-06-05 | 2022-03-22 | 巴特尔纪念研究院 | Polymer-functionalized magnetic particle embodiments for solute separation and devices and systems using the same |
| CN115746230A (en)* | 2022-08-31 | 2023-03-07 | 江苏大学 | Preparation method and application of boric acid functionalized magnetic porous covalent organic polymer |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103894161A (en)* | 2014-04-09 | 2014-07-02 | 复旦大学 | Synthesis method of magnetic metal organic framework composite material and application of material |
| CN104722274A (en)* | 2015-01-26 | 2015-06-24 | 北京化工大学 | Preparation and application of magnetic MOF-5 nano compound adsorbing agent |
| CN105312038A (en)* | 2014-07-29 | 2016-02-10 | 中国科学院大连化学物理研究所 | Formylphenylboronic acid modified magnetic nanoparticles and preparation and application thereof |
| CN105879043A (en)* | 2016-04-01 | 2016-08-24 | 江苏大学 | Preparation method of dual-targeting drug carrier based on magnetic metal organic framework material |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103894161A (en)* | 2014-04-09 | 2014-07-02 | 复旦大学 | Synthesis method of magnetic metal organic framework composite material and application of material |
| CN105312038A (en)* | 2014-07-29 | 2016-02-10 | 中国科学院大连化学物理研究所 | Formylphenylboronic acid modified magnetic nanoparticles and preparation and application thereof |
| CN104722274A (en)* | 2015-01-26 | 2015-06-24 | 北京化工大学 | Preparation and application of magnetic MOF-5 nano compound adsorbing agent |
| CN105879043A (en)* | 2016-04-01 | 2016-08-24 | 江苏大学 | Preparation method of dual-targeting drug carrier based on magnetic metal organic framework material |
| Title |
|---|
| TIAN JIN等: "Promoting desulfurization capacity and separation efficiency simultaneously by the novel magnetic Fe3O4@PAA@MOF-199", 《RSC ADVANCES》* |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107607640A (en)* | 2017-08-30 | 2018-01-19 | 复旦大学 | A kind of glycopeptide enrichment of nano composite material of boric acid modified and Mass Spectrometry detection method |
| WO2019141152A1 (en)* | 2018-01-17 | 2019-07-25 | 南开大学 | Novel composite biological agent based on porous frame material |
| CN108548480A (en)* | 2018-05-09 | 2018-09-18 | 电子科技大学 | Three layers of selfreparing flexibility strain transducer of one kind and preparation method thereof |
| CN108636359A (en)* | 2018-05-16 | 2018-10-12 | 湘潭大学 | A kind of magnetic metal organic framework microballoon of phenyl boric acid functionalization and its preparation method and application |
| CN109265709B (en)* | 2018-08-17 | 2021-01-29 | 西南交通大学 | Preparation method and application of conductive hydrogel capable of sustained release of drugs and factors |
| CN109265709A (en)* | 2018-08-17 | 2019-01-25 | 西南交通大学 | It is a kind of can slow releasing pharmaceutical and the factor conductive hydrogel preparation method and application |
| CN109012630A (en)* | 2018-08-30 | 2018-12-18 | 珠海健帆生物科技股份有限公司 | Magnetic composite adsorbent for blood perfusion and preparation method thereof and perfusion device |
| CN109012630B (en)* | 2018-08-30 | 2021-06-08 | 健帆生物科技集团股份有限公司 | Magnetic composite adsorbent for blood perfusion, preparation method thereof and perfusion apparatus |
| CN109433158A (en)* | 2018-09-29 | 2019-03-08 | 四川大学 | Magnetic nanometer composite material and the preparation method and application thereof for the enrichment of multi-mode peptide fragment |
| CN114223041A (en)* | 2019-06-05 | 2022-03-22 | 巴特尔纪念研究院 | Polymer-functionalized magnetic particle embodiments for solute separation and devices and systems using the same |
| CN113121835A (en)* | 2019-12-31 | 2021-07-16 | 华东师范大学 | Iron-based metal organic framework material and preparation and application thereof |
| CN111514308A (en)* | 2020-03-10 | 2020-08-11 | 西南民族大学 | PH-induced charge-inversion antibacterial gold nanorod and preparation method and application thereof |
| CN111514308B (en)* | 2020-03-10 | 2023-02-17 | 西南民族大学 | PH-induced charge-inversion antibacterial gold nanorod and preparation method and application thereof |
| CN112675821A (en)* | 2020-11-26 | 2021-04-20 | 四川大学 | Magnetic covalent organic framework material for glycopeptide enrichment based on amphiphilic site and preparation method and application thereof |
| CN113980926A (en)* | 2021-10-22 | 2022-01-28 | 南京师范大学 | Magnetic nanoparticle-glycosyltransferase-amorphous metal-organic framework composite catalytic material and its preparation method and application |
| CN113980926B (en)* | 2021-10-22 | 2024-03-26 | 南京师范大学 | Magnetic nanoparticle-glycosyltransferase-amorphous metal organic framework composite catalytic material and its preparation method and application |
| CN115746230A (en)* | 2022-08-31 | 2023-03-07 | 江苏大学 | Preparation method and application of boric acid functionalized magnetic porous covalent organic polymer |
| Publication number | Publication date |
|---|---|
| CN106552603B (en) | 2019-02-05 |
| Publication | Publication Date | Title |
|---|---|---|
| CN106552603A (en) | PH response type magnetic metal organic frame composite nano materials and preparation method and application | |
| Ma et al. | Superparamagnetic silica nanoparticles with immobilized metal affinity ligands for protein adsorption | |
| Zhang et al. | A self-assembled polydopamine film on the surface of magnetic nanoparticles for specific capture of protein | |
| Yang et al. | pH-Responsive magnetic metal–organic framework nanocomposites for selective capture and release of glycoproteins | |
| Gao et al. | Preparation and characterization of uniformly sized molecularly imprinted polymers functionalized with core–shell magnetic nanoparticles for the recognition and enrichment of protein | |
| Gao et al. | A facile method for protein imprinting on directly carboxyl-functionalized magnetic nanoparticles using non-covalent template immobilization strategy | |
| Liu et al. | Synthesis of amino-silane modified superparamagnetic silica supports and their use for protein immobilization | |
| Ma et al. | Recent advances in preparation and applications of magnetic framework composites | |
| CN104475030B (en) | A kind of preparation method and applications of magnetic metal organic framework material | |
| Jian et al. | Click chemistry: a new facile and efficient strategy for the preparation of Fe 3 O 4 nanoparticles covalently functionalized with IDA-Cu and their application in the depletion of abundant protein in blood samples | |
| Cao et al. | Facile synthesis of a Ni (ii)-immobilized core–shell magnetic nanocomposite as an efficient affinity adsorbent for the depletion of abundant proteins from bovine blood | |
| Gao et al. | Specific recognition of bovine serum albumin using superparamagnetic molecularly imprinted nanomaterials prepared by two-stage core–shell sol–gel polymerization | |
| Hao et al. | A facile and general approach for preparation of glycoprotein-imprinted magnetic nanoparticles with synergistic selectivity | |
| CN110665465B (en) | Magnetic covalent organic framework material for glycopeptide enrichment and preparation method and application thereof | |
| CN103143331A (en) | Synthesis and application of [Cu3(btc)2] metal-organic framework composites coated on the surface of magnetic microspheres ferric oxide | |
| CN107486177B (en) | Magnetic metal with how affine site-organic backbone nanosphere and the preparation method and application thereof | |
| CN101613694A (en) | A kind of magnetic/functionalized silica composite microsphere immobilized enzyme and preparation method thereof | |
| Zhang et al. | Magnetic silica‐coated sub‐microspheres with immobilized metal ions for the selective removal of bovine hemoglobin from bovine blood | |
| Wei et al. | Ionic liquid modified magnetic microspheres for isolation of heme protein with high binding capacity | |
| TW201323491A (en) | A magnetic ion-exchange microspheres and method for preparing the same | |
| CN104307481A (en) | Magnetic MOFs solid-phase extractant, and preparation method and application thereof | |
| Mohapatra et al. | Boronic acid functionalized superparamagnetic iron oxide nanoparticle as a novel tool for adsorption of sugar | |
| Yang et al. | pH-Responsive magnetic nanospheres for the reversibly selective capture and release of glycoproteins | |
| CN112675821B (en) | Magnetic covalent organic framework material for glycopeptide enrichment based on amphiphilic site and preparation method and application thereof | |
| Wang et al. | Synthesis of hierarchical nickel anchored on Fe 3 O 4@ SiO 2 and its successful utilization to remove the abundant proteins (BHb) in bovine blood |
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |