CN107867723A - 一种聚阳离子电解质修饰的磁性纳米粒子及其制备方法与应用 - Google Patents
一种聚阳离子电解质修饰的磁性纳米粒子及其制备方法与应用 Download PDFInfo
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Abstract
本发明的目的在于提供一种聚阳离子电解质修饰的磁性纳米粒子及其制备方法。首先采用沉淀法制备四氧化三铁纳米粒子,然后利用聚烯丙基胺盐酸盐对四氧化三铁纳米颗粒进行包裹修饰。经聚阳离子电解质修饰的四氧化三铁纳米颗粒仍然具有较强的磁性,同时,其表面荷有一定量的正电荷,可以基于静电作用原理应用于基因、微生物菌体、细胞等的分离、筛选、回收等生物学领域。本发明提供的磁性材料制备方法简单,电荷容易调变,稳定性好。
Description
发明领域
本发明属于纳米材料与生物工程技术领域,具体涉及一种聚阳离子电解质修饰的磁性纳米粒子及其制备方法与应用。
背景技术
在纳米新材料的研究应用中,磁性纳米材料因具有特殊的超顺磁性、磁导向性、表面易修饰等特性而被广为应用,其中四氧化三铁(Fe3O4)纳米颗粒是近几年发展起来的一种新型磁性材料,在核酸分析、靶向药物、酶和细胞固定化以及微生物分离筛选等生物领域受到广泛的关注。以共沉淀法制备Fe3O4纳米颗粒最为简单实用,但因其容易发生团聚,限制了它的应用。表面修饰成为改善Fe3O4纳米颗粒应用性能的一种有效的方法。
经典的修饰以在Fe3O4纳米颗粒表面共价接枝各类官能团为基础,需要调变反应参数以控制修饰程度。由于蛋白等生物样本具有两亲性,可利用其荷电特性,针对性的进行功能化修饰。采用阳离子聚电解质-聚烯丙基胺盐酸盐(PAAH)对Fe3O4纳米颗粒进行功能化修饰,在维持纳米颗粒磁性的同时,其表面较高的电荷使得纳米颗粒的分散性与稳定性得以提高。不仅如此,PAAH具有无毒以及生物相容性良好等特点,在微生物及细胞表面修饰与功能化方面可进行拓展应用,实现蛋白、基因、微生物、细胞的快速筛选、分离以及循环使用。
发明内容
本发明通过简单的方法,制备出一种聚阳离子电解质修饰的磁性纳米粒子,本发明提供了其制备方法,基于电荷作用,使聚阳离子电解质修饰的磁性纳米粒子吸附在荷负电的蛋白、基因、微生物、细胞表面,在利用磁性作用,实现目标生物样本的快速分离、筛选和循环利用。
本发明所采用的技术方案如下:
一种聚阳离子电解质修饰的磁性纳米粒子及其制备方法与应用,步骤包括:
(1)采用沉淀法制备四氧化三铁纳米粒子
将三价铁盐溶液与二价铁盐溶液按照摩尔比例n(Fe3+):n(Fe2+)=(1.0-3.0):1.0充分振荡混匀,配制0.1-5mol/L的氨水溶液,在50-100℃水浴、惰性气体保护环境下,按照摩尔比例n(OH-):n(Fe3+)=(5.0-20.0):1.0逐滴滴加氨水溶液,100-1500rpm搅拌转速下反应0.5-4h。所得黑色四氧化三铁颗粒利用磁性分离后,用去离子水充分水洗至中性,于室温下真空干燥。
(2)利用PAAH对四氧化三铁纳米颗粒进行包裹修饰
配制1-100g/L的PAAH溶液,按照质量比m(PAAH):m(Fe3O4)=(0.1-500):1.0将步骤(1)制备得到的磁性纳米粒子置于PAAH溶液中超声分散0.5-4h,利用磁性分离分离后用0.22μm滤膜过滤,真空干燥得到PAAH修饰的磁性纳米颗粒。
所述步骤(1)中三价铁盐包括氯化铁、硫酸铁、硝酸铁及其混合物。
所述步骤(1)中二价铁盐包括氯化亚铁、硫酸亚铁及其混合物。
所述步骤(1)中惰性气体包括但不限于氮气、氩气、氦气。
所述步骤(2)中PAAH的分子量为1k-150kDa。
所述步骤(2)中超声条件为100-600W,10-90kHz。
本发明的有益效果是:
本发明制备的一种聚阳离子电解质修饰的磁性纳米粒子,具有分散均匀,饱和磁化强度高的特点,且制备条件温和,操作简便,可在酶或细胞参与的生物催化与转化过程,以及基因、微生物菌体、蛋白、细胞等的分离、筛选、回收等过程中应用。
附图说明
图1 PAAH修饰的Fe3O4纳米颗粒(Fe3O4-PAAH)粒径分布图
图2 C.lini ST-1菌体电镜照片
图3磁纳米修饰的亚麻刺盘孢C.lini ST-1菌体电镜照片
图4 Fe3O4-PAAH以及磁性复合物的室温磁滞回线
图5外加磁场对磁性复合物的回收
图6游离菌体磁性复合物批次转化去氢表雄酮的比较
具体实施方法
为了进一步介绍本发明,下面结合附图进行详细说明:
实施例1Fe3O4纳米颗粒的制备及PAAH修饰
Fe3O4纳米颗粒的制备:分别取1mol/L的FeCl3·6H2O溶液2mL,与2mol/L的FeCl2·4H2O溶液0.5mL充分振荡混匀。在N2环境下,于70℃水浴、900rpm转速条件下,滴加25mL、 1mol/L的氨水溶液,反应30min后用磁铁将得到的黑色Fe3O4颗粒分离,用去离子水充分水洗至中性。
PAAH修饰Fe3O4纳米颗粒的制备:配置10g/L的PAAH溶液,按照质量比m(PAAH): m(Fe3O4)=100:1将Fe3O4纳米颗粒加至PAAH溶液中并超声分散,超声条件120W、40kHz、60min。之后依次经10000r/min离心10min,0.22μm滤膜过滤、真空干燥后得到PAAH修饰的Fe3O4纳米颗粒,记为Fe3O4-PAAH。所得磁性纳米颗粒平均粒径为38.85nm,如图1所示。
实施例2磁纳米修饰的亚麻刺盘孢Colletotrichum lini ST-1磁性复合物的制备及其催化转化效果
游离菌液的制备与表征:从斜面固体培养基上取适量C.lini ST-1菌丝接入100mL/500mL 新鲜种子培养基,于30℃、220rpm摇床培养2~3d,获得种子。以10%的接种量将种子液转接至30mL/250mL的发酵培养基,30℃、220r/min摇床培养24h。离心收集菌丝体,用磷酸盐缓冲液洗涤2遍,8000r/min离心10min收集菌体。准确称取4.0g菌体,分散于30mL磷酸盐缓冲液中,获得游离菌液,4℃保藏备用。游离菌体扫描电镜图片如图2所示,菌体表面光滑。
磁性复合物的制备与表征:向游离菌液中加入1%的Fe3O4-PAAH,于室温振荡12h后,利用外加磁场去除未吸附磁性颗粒的游离菌体,以蒸馏水清洗磁性复合物表面多余的 Fe3O4-PAAH,得到的磁性复合物重悬于磷酸盐缓冲液中备用。磁性复合物扫描电镜图片如图 3所示。由图可以发现,菌体表面因吸附了磁性纳米颗粒而粗糙不平。游离菌体与磁性复合物的室温磁滞回线如图4所示。二者磁滞曲线均呈S形。Fe3O4-PAAH的饱和磁化强度为 69.01emu/g,磁性复合物的饱和磁化强度为46.20emu/g,且剩磁几乎为0,具有良好的超顺磁性。这种高超顺磁性和快速磁响应的特性使得菌丝可在外加磁场作用下方便回收与再利用。
磁性复合物在去氢表雄酮生物羟化的应用:将制备得到的磁性复合物投入到30mL磷酸钠缓冲液中,添加10g/L的去氢表雄酮作为底物进行转化,30℃、220rpm条件下转化48h后结束该批反应。用磁铁回收磁性复合物,经磷酸盐缓冲液洗涤后重置于磷酸盐缓冲液中,重新添加10g/L的DHEA进行后续批次的转化,转化条件相同。如图5所示,通过外加磁场,磁性复合物可方便、快捷的分离回收。批次转化结果如图6所示。游离菌体在转化后因与反应产物及残留底物形成混悬液,难以实现有效分离和回收,故仅转化1次,产物3α,7α,15α- 三羟基雄甾烯酮的浓度为4.31g/L。磁性复合物共进行了4个批次的转化,产物3α,7α,15α-三羟基雄甾烯酮的累积浓度为10.83g/L,是游离菌体的2.5倍。
本实施例中,采用简便的方法得到经聚阳离子电解质修饰的四氧化三铁纳米颗粒,具有较强顺磁性,表面荷有一定量的正电荷,可以基于电荷作用实现菌体的分离和回收。
Claims (9)
1.一种聚阳离子电解质修饰的磁性纳米粒子的制备方法,其特征在于,制备步骤包括:(1)采用沉淀法制备四氧化三铁纳米粒子;(2)利用聚烯丙基胺盐酸盐(PAAH)对四氧化三铁纳米颗粒进行包裹修饰。
2.权利要求1所述的方法,其特征在于,步骤(1)中,将三价铁盐溶液与二价铁盐溶液按照摩尔比例n(Fe3+):n(Fe2+)=(1.0-3.0):1.0充分振荡混匀,配制0.1-5mol/L的氨水溶液,在50-100℃水浴、惰性气体保护环境下,按照摩尔比例n(OH-):n(Fe3+)=(5.0-20.0):1.0逐滴滴加氨水溶液,100-1500rpm搅拌转速下反应0.5-4h;所得黑色四氧化三铁颗粒利用磁性分离后,用去离子水充分水洗至中性,于室温下真空干燥。
3.权利要求2所述的方法,其特征在于,所述步骤(1)中三价铁盐溶液包括氯化铁、硫酸铁、硝酸铁及其混合物的水溶液。
4.权利要求2所述的方法,其特征在于,所述步骤(1)中二价铁盐溶液包括氯化亚铁、硫酸亚铁及其混合物的水溶液。
5.权利要求2所述的方法,其特征在于,所述步骤(1)中惰性气体包括但不限于氮气、氩气、氦气。
6.权利要求1所述的方法,其特征在于,步骤(2)中,配制1-100g/L的PAAH溶液,按照质量比m(PAAH):m(Fe3O4)=(0.1-500):1.0将步骤(1)制备得到的磁性纳米粒子置于PAAH溶液中超声分散0.5-4h,利用磁性分离后用0.22μm滤膜过滤,真空干燥得到PAAH修饰的磁性纳米颗粒。
7.权利要求6所述的方法,其特征在于,所述步骤(2)中PAAH的分子量为1k-150kDa。
8.权利要求6所述的方法,其特征在于,所述步骤(2)中超声条件为100-600W,10-90kHz。
9.权利要求1所述的聚阳离子电解质修饰的磁性纳米粒子的应用方法,其特征为,可应用于酶或细胞参与的生物催化与转化过程,基因、微生物菌体、蛋白、细胞等的分离、筛选、回收等。
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