新型磁性分子印迹纳米复合体系用于生物大分子识别的研究
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摘要
蛋白质印迹是制备对模板蛋白具有特异选择性功能的分子印迹技术。因受蛋白质性能的限制,进展相对缓慢,技术尚不成熟,仍需要改进常规的制备和分析方法来适应蛋白质的物理性质(如大小,相容性、构象灵活性、水溶性),以提高其稳定性,选择性,位点可接近性,和降低成本效率等。
本工作结合磁性分离速度快和分子印迹技术的预定选择性的优点,以及纳米结构材料比表面积大传质速度快的优势,采用表面印迹技术首次合成了四种新型的平均粒径100～400nm核—壳型的磁性分子印迹纳米复合体系,并用于生物大分子的分离识别研究。四种新型的磁性分子印迹纳米复合体系包括:①识别牛血红蛋白的核—壳型磁性分子印迹聚合物纳米粒子,即采用溶胶—凝胶法制备二氧化硅包覆壳的磁性四氧化三铁纳米粒子,以间氨基苯硼酸作为功能单体和交联剂,通过过硫酸铵氧化,在二氧化硅修饰的四氧化三铁表面形成聚间氨基苯硼酸分子印迹聚合物薄层;②新型牺牲共价键合牛血红蛋白超顺磁性纳米印迹聚合物,即以共沉淀法合成的Fe_3O_4磁性纳米粒子为载体,将其表面硅烷化修饰氨基基团,利用戊二醛双醛基的特性将其修饰到磁性纳米粒子表面,而另一端醛基与蛋白质上的氨基基团以亚胺共价键键合,再利用间氨基苯硼酸进行聚合,合成表面印迹有蛋白质分子的核—壳型超顺磁性纳米聚合物纳米球;③新型磁性牛血红蛋白表面印迹聚苯乙烯纳米粒子,即在包覆二氧化硅壳的磁性四氧化三铁纳米粒子的表面通过硅烷化试剂接枝聚合聚苯乙烯,通过表面印迹技术合成出较大粒径的特殊表面形态的超顺磁性表面印迹聚苯乙烯纳米粒子;④超顺磁性的高分散性聚苯乙烯,即用油酸和十二烷基苯磺酸钠为双层表面活性剂表面修饰Fe_3O_4磁性纳米粒子,制成稳定的水分散性纳米Fe_3O_4可聚合磁流体,在Fe_3O_4磁流体存在下,将苯乙烯与甲基丙烯酸通过乳液聚合方法制备了磁性高分子纳米球,再使用间氨基苯硼酸对其进行表面印迹。
通过透射电子显微镜、X射线衍射、热重分析、振动样品磁强计研究了分子印迹磁性纳米材料的形态、吸附、识别性能,探讨了分子印迹聚合物对模板蛋白的识别机理。吸附结果表明聚间氨基苯硼酸包覆分子印迹聚合物的磁性纳米粒子对模板蛋白质具有高吸附能力和相对低的非特异吸附。这些印迹磁性纳米粒子克服了整体印迹材料阻碍蛋白质分子扩散的缺点,壳层上具有特异识别位点的分子印迹纳米粒子对模板蛋白质有特殊的选择性和高稳定性,并且能够使蛋白质分子在较短时间内达到扩散吸附平衡,并可在外加磁场下实现快速磁性分离。这些结果证明表面为印迹聚合物修饰的超顺磁性分子印迹纳米复合物在化学和生化分离、细胞筛选、生物传感器、药物传输等领域中具有很好的应用前景,并能够实现蛋白质组学中高丰度蛋白质的去除和低丰度蛋白质的富集。
基于上述,本工作结合磁性纳米技术、分子印迹技术、电磁学、分析化学等诸多领域的优势,为研究新型多功能性分子印迹聚合物提供了新的研究思路,并对磁性分子印迹纳米粒子在蛋白质分离和富集的应用作了初步探讨。
Protein molecular imprinting is an effective technique for the creation of specific recognition sites for template protein in a polymer network. It has only limited success and its progress has been slow due to some inherent limitations of protein template. Therefore, more investigations are required to further improve selectivity, stability, accessibility to recognition site and cost-effectiveness of the polymer, and the conventional preparation and analysis of molecular imprinted polymers (MIP) has to be modified to accommodate the physical properties of protein (e.g. size, complexity, flexibility and solubility).
In this work, combining speediness of magnetic separation, selectivity of molecular imprinting technique and high surface-to-volume ratio and good mass transport of nano-structured materials, four kinds of novel core-shell magnetic molecularly imprinted nano-composite system with an average diameter of approximately 100～400 nm were synthesized for separation and recognition of biomacromolecules for the first time using surface imprinting technique. Four kinds of novel magnetic molecularly imprinted nano-composite system include:①core-shell magnetic molecularly imprinted polymer nanoparticles for recognition of bovine hemoglobin. The magnetic nanoparticles were synthesized by the chemical coprecipitation of Fe~(2+) and Fe~(3+) in an ammonia solution. Subsequently, silica was coated on the Fe_3O_4 nanoparticles using a sol-gel method to obtain silica shell magnetic nanoparticles. Subsequently, 3-aminophenylboronic acid (APBA), which is the functional and cross-linking monomer, and poly(APBA) thin films were coated onto the silica-modified Fe_3O_4 surface through oxidation with ammonium persulfate in an aqueous solution in the presence or absence of protein. (2) novel nano-imprinted polymer using covalently bonded template method. Fe_3O_4 magnetic nanoparticles synthesized by chemical coprecipitation were surface modified. Aminopropylsilanzed magnetic nanoparticles was first aldehyde functionalized with glutaradehyde and then reacted with BHb by a covalently bond through imine groups. Core-shell superparamagnetic nano-polymer nanospheres were synthesized by poly(APBA)-coated thin film;③novel bovine hemoglobin surface-imprinted polystyrene nanoparticles with magnetic susceptibility. polystyrene was grafted on the the surface of silica-coated magnetic iron oxide nanoparticles through silylation reagent. The larger particle size of superparamagnetic surface-imprinted polystyrene nanoparticles with the special surface morphology were prepared through surface imprinting technique;④superparamagnetic molecularly imprinted polystyrene nanoparticles with high dispersion. Surface-modified Fe_3O_4 magnetic nanoparticles were synthesized using oleic acid and sodium dodecylbenzenesulfonate (SDBS) as double-surface surfactant, which can prepare nano-sized Fe_3O_4 magnetic fluid with a stable aqueous dispersion. The magnetic polymer nanospheres were prepared by emulsion polymerization by using styrene and methylacrylic acid as the monomer with the existence of magnetic fluid and then imprinted thin layer of poly(APBA) was coated on their surface.
The morphology, adsorption, and recognition properties of the magnetic molecularly imprinted nanomaterial were investigated by transmission electron microscopy (TEM), X-ray diffraction (XRD), thermogravimetric analysis (TGA), and vibrating sample magnetometer (VSM), and the recognition mechanism of template protein identification was discussed. The protein adsorption results showed that poly(APBA) MlP-coated magnetic nanoparticles have high adsorption capacity for template protein and comparatively low non-specific adsorption. Due to specific recognition sites for template protein on the imprinted shell make this nanoparticles with specific selectivity and high stability, the imprinted magnetic nanoparticles could easily reach the adsorption equilibrium and magnetic separation under an external magnetic field, thus overcoming the drawback of monolith materials of hindering protein molecules from diffusion. All these results demonstrate that the the imprinted polymer coated magnetic nanoparticles can be one of the most promising candidates for various applications, which include chemical and biochemical separation, cell sorting, recognition elements in biosensors, and drug delivery and has potential applications in the separation and detection of biomacromolecules so as to enable high abundance of protein to be removed and low abundance of protein to be enriched in proteomics.
Based on the above, combining the advantages of magnetic nanotechnology, molecular imprinting technology, electromagnetics, analytical chemistry and so forth, this work provides new research ideas for the study of novel versatile molecularly imprinted polymer. In this work, the application of magnetic molecularly imprinted nanoparticles in the field of protein separation and enrichment were initially attempted.
本工作结合磁性分离速度快和分子印迹技术的预定选择性的优点,以及纳米结构材料比表面积大传质速度快的优势,采用表面印迹技术首次合成了四种新型的平均粒径100～400nm核—壳型的磁性分子印迹纳米复合体系,并用于生物大分子的分离识别研究。四种新型的磁性分子印迹纳米复合体系包括:①识别牛血红蛋白的核—壳型磁性分子印迹聚合物纳米粒子,即采用溶胶—凝胶法制备二氧化硅包覆壳的磁性四氧化三铁纳米粒子,以间氨基苯硼酸作为功能单体和交联剂,通过过硫酸铵氧化,在二氧化硅修饰的四氧化三铁表面形成聚间氨基苯硼酸分子印迹聚合物薄层;②新型牺牲共价键合牛血红蛋白超顺磁性纳米印迹聚合物,即以共沉淀法合成的Fe_3O_4磁性纳米粒子为载体,将其表面硅烷化修饰氨基基团,利用戊二醛双醛基的特性将其修饰到磁性纳米粒子表面,而另一端醛基与蛋白质上的氨基基团以亚胺共价键键合,再利用间氨基苯硼酸进行聚合,合成表面印迹有蛋白质分子的核—壳型超顺磁性纳米聚合物纳米球;③新型磁性牛血红蛋白表面印迹聚苯乙烯纳米粒子,即在包覆二氧化硅壳的磁性四氧化三铁纳米粒子的表面通过硅烷化试剂接枝聚合聚苯乙烯,通过表面印迹技术合成出较大粒径的特殊表面形态的超顺磁性表面印迹聚苯乙烯纳米粒子;④超顺磁性的高分散性聚苯乙烯,即用油酸和十二烷基苯磺酸钠为双层表面活性剂表面修饰Fe_3O_4磁性纳米粒子,制成稳定的水分散性纳米Fe_3O_4可聚合磁流体,在Fe_3O_4磁流体存在下,将苯乙烯与甲基丙烯酸通过乳液聚合方法制备了磁性高分子纳米球,再使用间氨基苯硼酸对其进行表面印迹。
通过透射电子显微镜、X射线衍射、热重分析、振动样品磁强计研究了分子印迹磁性纳米材料的形态、吸附、识别性能,探讨了分子印迹聚合物对模板蛋白的识别机理。吸附结果表明聚间氨基苯硼酸包覆分子印迹聚合物的磁性纳米粒子对模板蛋白质具有高吸附能力和相对低的非特异吸附。这些印迹磁性纳米粒子克服了整体印迹材料阻碍蛋白质分子扩散的缺点,壳层上具有特异识别位点的分子印迹纳米粒子对模板蛋白质有特殊的选择性和高稳定性,并且能够使蛋白质分子在较短时间内达到扩散吸附平衡,并可在外加磁场下实现快速磁性分离。这些结果证明表面为印迹聚合物修饰的超顺磁性分子印迹纳米复合物在化学和生化分离、细胞筛选、生物传感器、药物传输等领域中具有很好的应用前景,并能够实现蛋白质组学中高丰度蛋白质的去除和低丰度蛋白质的富集。
基于上述,本工作结合磁性纳米技术、分子印迹技术、电磁学、分析化学等诸多领域的优势,为研究新型多功能性分子印迹聚合物提供了新的研究思路,并对磁性分子印迹纳米粒子在蛋白质分离和富集的应用作了初步探讨。
Protein molecular imprinting is an effective technique for the creation of specific recognition sites for template protein in a polymer network. It has only limited success and its progress has been slow due to some inherent limitations of protein template. Therefore, more investigations are required to further improve selectivity, stability, accessibility to recognition site and cost-effectiveness of the polymer, and the conventional preparation and analysis of molecular imprinted polymers (MIP) has to be modified to accommodate the physical properties of protein (e.g. size, complexity, flexibility and solubility).
In this work, combining speediness of magnetic separation, selectivity of molecular imprinting technique and high surface-to-volume ratio and good mass transport of nano-structured materials, four kinds of novel core-shell magnetic molecularly imprinted nano-composite system with an average diameter of approximately 100～400 nm were synthesized for separation and recognition of biomacromolecules for the first time using surface imprinting technique. Four kinds of novel magnetic molecularly imprinted nano-composite system include:①core-shell magnetic molecularly imprinted polymer nanoparticles for recognition of bovine hemoglobin. The magnetic nanoparticles were synthesized by the chemical coprecipitation of Fe~(2+) and Fe~(3+) in an ammonia solution. Subsequently, silica was coated on the Fe_3O_4 nanoparticles using a sol-gel method to obtain silica shell magnetic nanoparticles. Subsequently, 3-aminophenylboronic acid (APBA), which is the functional and cross-linking monomer, and poly(APBA) thin films were coated onto the silica-modified Fe_3O_4 surface through oxidation with ammonium persulfate in an aqueous solution in the presence or absence of protein. (2) novel nano-imprinted polymer using covalently bonded template method. Fe_3O_4 magnetic nanoparticles synthesized by chemical coprecipitation were surface modified. Aminopropylsilanzed magnetic nanoparticles was first aldehyde functionalized with glutaradehyde and then reacted with BHb by a covalently bond through imine groups. Core-shell superparamagnetic nano-polymer nanospheres were synthesized by poly(APBA)-coated thin film;③novel bovine hemoglobin surface-imprinted polystyrene nanoparticles with magnetic susceptibility. polystyrene was grafted on the the surface of silica-coated magnetic iron oxide nanoparticles through silylation reagent. The larger particle size of superparamagnetic surface-imprinted polystyrene nanoparticles with the special surface morphology were prepared through surface imprinting technique;④superparamagnetic molecularly imprinted polystyrene nanoparticles with high dispersion. Surface-modified Fe_3O_4 magnetic nanoparticles were synthesized using oleic acid and sodium dodecylbenzenesulfonate (SDBS) as double-surface surfactant, which can prepare nano-sized Fe_3O_4 magnetic fluid with a stable aqueous dispersion. The magnetic polymer nanospheres were prepared by emulsion polymerization by using styrene and methylacrylic acid as the monomer with the existence of magnetic fluid and then imprinted thin layer of poly(APBA) was coated on their surface.
The morphology, adsorption, and recognition properties of the magnetic molecularly imprinted nanomaterial were investigated by transmission electron microscopy (TEM), X-ray diffraction (XRD), thermogravimetric analysis (TGA), and vibrating sample magnetometer (VSM), and the recognition mechanism of template protein identification was discussed. The protein adsorption results showed that poly(APBA) MlP-coated magnetic nanoparticles have high adsorption capacity for template protein and comparatively low non-specific adsorption. Due to specific recognition sites for template protein on the imprinted shell make this nanoparticles with specific selectivity and high stability, the imprinted magnetic nanoparticles could easily reach the adsorption equilibrium and magnetic separation under an external magnetic field, thus overcoming the drawback of monolith materials of hindering protein molecules from diffusion. All these results demonstrate that the the imprinted polymer coated magnetic nanoparticles can be one of the most promising candidates for various applications, which include chemical and biochemical separation, cell sorting, recognition elements in biosensors, and drug delivery and has potential applications in the separation and detection of biomacromolecules so as to enable high abundance of protein to be removed and low abundance of protein to be enriched in proteomics.
Based on the above, combining the advantages of magnetic nanotechnology, molecular imprinting technology, electromagnetics, analytical chemistry and so forth, this work provides new research ideas for the study of novel versatile molecularly imprinted polymer. In this work, the application of magnetic molecularly imprinted nanoparticles in the field of protein separation and enrichment were initially attempted.
引文
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