CN112237906A - Php修饰的磁性纳米微球、制备方法及其在dna分离中的应用 - Google Patents
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Abstract
本发明公开了一种PHP修饰的磁性纳米微球、制备方法及其在DNA分离中的应用。所述的磁性纳米微球以聚多巴胺为连接臂,将聚2‑羟基丙烯亚胺修饰在被聚多巴胺包覆的磁性Fe3O4纳米微球表面。本发明的PHP修饰的磁性纳米微球比表面积大,活性位点多,对DNA具有很好的结合能力,饱和吸附量为153.33μg/mg,并具有磁性,饱和磁化强度高达41.28emu/g,可在外界磁场条件下快速分离,实现循环使用,重复使用5次后,对DNA的结合率仍保持80%~90%。
Description
技术领域
本发明属于吸附材料技术领域,涉及一种聚2-羟基丙烯亚胺(PHP)修饰的磁性纳米微球、制备方法及其在DNA分离中的应用。
背景技术
高品质脱氧核糖核酸(DNA)的分离是分子生物学、法医学等多个应用领域的重要研究课题。生物样品由复杂的样品组成,其DNA浓度远低于蛋白质等其他成分。常用的传统提取或纯化DNA的溶剂方法需要消耗大量的有毒有机溶剂或动物源酶,并且需要反复离心。
为了减少或避免使用有机溶剂,固相萃取(SPE)已成为较传统方法更好的选择(C.A.Ghiorghita,F.Bucatariu,E.S.Dragan,Poly(N,N-dimethylamino)ethylmethacrylate/sodium alginate multilayers and their interaction with proteins/enzymes,Int.J.Biol.Macromol.107(2018)1584-1590.)。纳米复合材料被设计用于提取DNA,如氧化石墨烯、聚合离子液体、壳聚糖、纤维素和硅胶凝胶。然而,该工艺仍涉及固液相分离,需要重复离心,处理时间较长。磁性固相萃取(MSPE)技术作为SPE的升级版,在DNA分离过程中,由于磁性粒子(MPs)在外部磁场中容易被吸引,无需离心步骤,节省时间,因此在基因组DNA分离中得到了越来越广泛的应用(M.Ghaemi,G.Absalan,Study on theadsorption of DNA on Fe3O4nanoparticles and on ionic liquid-modifiedFe3O4nanoparticles,Microchim.Acta 181(2013)45-53.)。但是目前已知磁性粒子,比如FePO4@PEI(L.L.Hu,B.Hu,L.M.Shen,D.D.Zhang,X.W.Chen,J.H.Wang,Polyethyleneimine-iron phosphate nanocomposite as a promising adsorbent for the isolation ofDNA,Talanta 132(2015)857-863.)、氧化锆的磁性纳米复合材料(M.Saraji,S.Yousefi,M.Talebi,Plasmid DNA purification by zirconia magnetic nanocomposite,Anal.Biochem.539(2017)33-38.)等在DNA分离时均存在效率低下,磁响应性差、磁性结构不稳定、无法重复利用等情况。
发明内容
为解决现有的磁性粒子在分离DNA过程中效率低下、磁响应差、磁结构不稳定、无法重复利用的问题,本发明提供一种PHP修饰的磁性纳米微球、制备方法及其在DNA分离中的应用。
本发明的技术方案如下:
PHP修饰的磁性纳米微球的制备方法,具体步骤如下:
按聚多巴胺包覆的Fe3O4纳米粒子(Fe3O4@PDA)和聚2-羟基丙烯亚胺(PHP)的质量比为1:3~1:5,将聚多巴胺包覆的Fe3O4纳米粒子超声分散在pH=8.5的Tris缓冲溶液中,加入聚2-羟基丙烯亚胺,室温下搅拌反应,反应结束后,利用外加磁场分离得到沉淀产物,用水和乙醇重复洗涤,干燥后得到PHP修饰的磁性纳米微球(Fe3O4@PDA@PHP)。
优选地,所述的搅拌反应时间为6~10h。
优选地,所述的聚多巴胺包覆的Fe3O4纳米粒子和聚2-羟基丙烯亚胺的质量比为1:4。
本发明还提供上述制备方法得到的PHP修饰的磁性纳米微球。
进一步地,本发明提供上述PHP修饰的磁性纳米微球在DNA分离中的应用,具体方法为:在DNA溶液中加入PHP修饰的磁性纳米微球,调节pH=2~5,吸附5min以上。
优选地,调节pH=4。
具体地,在应用过程中,所述的PHP修饰的磁性纳米微球吸附饱和后,磁分离微球,置于pH=9的TE缓冲液中进行脱附,采用吸附-脱附进行循环利用。
与现有技术相比,本发明具有以下优点:
(1)PHP修饰的磁性纳米微球由于含有氨基和羟基,具有更好的DNA结合能力,对DNA提取率高达97%,饱和吸附量为153.33μg/mg;
(2)PHP修饰的磁性纳米微球具有磁性,饱和磁化强度高达41.28emu/g,可在外界磁场条件下快速分离,实现循环使用,重复使用5次后,对于DNA提取率仍保持80%~90%。
附图说明
图1为PHP修饰的磁性纳米微球的制备示意图及DNA的分离过程图。
图2为不同pH和Zeta电位分离效果图。
图3为不同接触时间的分离效果图。
图4为Fe3O4@PDA和Fe3O4@PDA@PHP对不同DNA初始浓度的分离效果图。
图5为PHP修饰的磁性纳米微球的循环吸附示意图。
具体实施方式
下面通过实施例和附图对本发明作进一步详述。
聚多巴胺包覆的Fe3O4纳米粒子的制备参考文献【S.Zhang,et al,Mussel-inspired polydopamine biopolymer decorated with magnetic nanoparticles formultiple pollutants removal,J Hazard Mater,270(2014)27-34.】。
实施例1
分别按Fe3O4@PDA与PHP的质量比为1:1,1:2,1:3,1:4,1:5,1:6将Fe3O4@PDA超声分散在100mLpH=8.5Tris缓冲溶液中,再加入PHP,然后室温搅拌反应10h。反应结束后,利用外加磁场分离得到沉淀产物,用水和乙醇重复洗涤,干燥后得到PHP修饰的磁性纳米微球(Fe3O4@PDA@PHP)。
将制得的Fe3O4@PDA@PHP加入到200ug/mL,pH=4的DNA溶液中,温和搅拌几分钟,然后在一定的时间间隔内过滤几滴上清液,测量分离效果。随着质量比的增大,Fe3O4@PDA@PHP复合纳米微球的分离能力也在逐渐增大,当质量比达到1:4时,分离能力达到最大值。质量比继续增大,分离能力没有变化。说明Fe3O4@PDA与PHP的最佳质量比为1:4。图5为PHP修饰的磁性纳米微球的循环吸附示意图,从图中可以看出,重复使用5次后,其对DNA的分离能力基本不变。
实施例2
取0.1g Fe3O4@PDA超声分散在100mLpH=8.5Tris缓冲溶液中,再加入0.4g PHP,然后室温搅拌反应10h。反应结束后,利用外加磁场分离得到沉淀产物,用水和乙醇重复洗涤,干燥后得到PHP修饰的磁性纳米微球(Fe3O4@PDA@PHP)。
取相同质量的Fe3O4@PDA@PHP分别加入到200ug/mL的pH=2、pH=3、pH=4、pH=5、pH=6、pH=7、pH=8、pH=9的DNA溶液中,温和搅拌几分钟,然后在一定的时间间隔内过滤几滴上清液,得到DNA分离效果如图2所示。从图2可以看出,随着溶液pH增加,Fe3O4@PDA@PHP的分离效果也逐渐降低。当pH≤5时,制备的纳米复合材料有较高的吸附能力。尽管如此,pH值超过5,Qe就会大幅下降。因此,通过测量Fe3O4@PDA@PHP和Fe3O4@PDA的zeta电位探索DNA吸附机理。结果表明,Fe3O4@PDA@PHP的zeta电位明显高于Fe3O4@PDA,这说明PHP的修饰引入了大量的氨基。当吸附过程在酸溶液中进行时,将氨基去质子化来增强磁性纳米复合材料与DNA之间的相互作用。此外,混合物中没有过量的OH—可以占据DNA前面的活性结合位点,从而增加了纳米粒子与DNA之间的相互作用。因此pH=4.0为最佳的DNA分离pH值。
实施例3
取0.1g Fe3O4@PDA超声分散在100mLpH=8.5Tris缓冲溶液中,再加入0.4gPHP,然后室温搅拌反应10h。反应结束后,利用外加磁场分离得到沉淀产物,用水和乙醇重复洗涤,干燥后得到PHP修饰的磁性纳米微球(Fe3O4@PDA@PHP)。
取一定质量的Fe3O4@PDA@PHP加入到200ug/mL的pH=4的DNA溶液中,温和搅拌,然后在5~240min时间间隔内过滤几滴上清液,得到DNA分离效果如图3所示。可以明显观察到,Qt在开始时急剧增强,然后缓慢变化,逐渐达到平衡。利用动力学模型模拟了提取时间对DNA分离的影响,拟二阶模型为结果如图3所示,阐明了吸附机理。
实施例4
取0.1g Fe3O4@PDA超声分散在100mLpH=8.5Tris缓冲溶液中,再加入0.4g PHP,然后室温搅拌反应10h。反应结束后,利用外加磁场分离得到沉淀产物,用水和乙醇重复洗涤,干燥后得到PHP修饰的磁性纳米微球(Fe3O4@PDA@PHP)。
取相同质量的Fe3O4@PDA@PHP与Fe3O4@PDA分别加入到pH=4的10ug/mL、25ug/mL、50ug/mL、100ug/mL、150ug/mL、200ug/mL、300ug/mL的DNA溶液中,温和搅拌几分钟,然后在一定的时间间隔内过滤几滴上清液,得到DNA分离效果如图4所示。从图4中可以看出,无论是在哪个DNA浓度下,Fe3O4@PDA@PHP的分离效果都远远优于Fe3O4@PDA的分离效果。
Claims (8)
1.PHP修饰的磁性纳米微球的制备方法,其特征在于,具体步骤如下:
按聚多巴胺包覆的Fe3O4纳米粒子和聚2-羟基丙烯亚胺的质量比为1:3~1:5,将聚多巴胺包覆的Fe3O4纳米粒子超声分散在pH=8.5的Tris缓冲溶液中,加入聚2-羟基丙烯亚胺,室温下搅拌反应,反应结束后,利用外加磁场分离得到沉淀产物,用水和乙醇重复洗涤,干燥后得到PHP修饰的磁性纳米微球。
2.根据权利要求1所述的制备方法,其特征在于,所述的搅拌反应时间为6~10h。
3.根据权利要求1所述的制备方法,其特征在于,所述的聚多巴胺包覆的Fe3O4纳米粒子和聚2-羟基丙烯亚胺的质量比为1:4。
4.根据权利要求1至3任一所述的制备方法制得的PHP修饰的磁性纳米微球。
5.根据权利要求4所述的PHP修饰的磁性纳米微球在DNA分离中的应用。
6.根据权利要求5所述的应用,其特征在于,具体方法为:在DNA溶液中加入PHP修饰的磁性纳米微球,调节pH=2~5,吸附5min以上。
7.根据权利要求6所述的应用,其特征在于,调节pH=4。
8.根据权利要求6所述的应用,其特征在于,在应用过程中,所述的PHP修饰的磁性纳米微球吸附饱和后,磁分离微球,置于pH=9的TE缓冲液中进行脱附,采用吸附-脱附进行循环利用。
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