CN112973621A - 一种新型磁珠及其制备方法 - Google Patents
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Abstract
本发明涉及一种新型磁珠及其制备方法，解决现有磁珠粒径大小不均匀，表面能小，核酸吸附效率低的问题。本发明提供一种Ag‑Fe3O4‑SiO2磁珠，其内核是银纳米球，核外是纳米Fe3O4磁性粒子吸附层，最外层是纳米二氧化硅胶体粒子。通过选择不同粒径的银纳米球和纳米Fe3O4磁性粒子，并控制反应时间，可控制获得的Ag‑Fe3O4‑SiO2磁珠的粒径大小，且制备Ag‑Fe3O4‑SiO2磁珠粒径均匀，磁响应强。
Description
技术领域
本发明涉及一种新型磁珠及磁珠的制备方法。
背景技术
随着分子生物学技术的不断广泛应用，分子生物学技术已从以往的小规模的实验室研究逐渐转变成诸如疾病诊断、基因组测序、疾病基因的流行病学调研等需要大规模的程序化的操作的实用手段。以人类基因组测序为例，需要年复一年的核酸提取、纯化、测序。许多优秀的高级科研人员的大量的时间被花费在简单的重复劳动之中，实在是一种巨大的浪费。同时手工操作的不可避免的不规范性容易造成误差，人类基因组测序一般要重复3次以上，又进一步加重工作强度。
目前游离DNA的提取方法主要有磁珠法，磁珠法提取核酸技术其原理为：采用超顺磁性纳米颗粒，利用在高盐条件低pH下吸附核酸，然后在低盐高pH下分离核酸的原理进行样本核酸的分离纯化，因其只需要在磁场作用下就高效简易地将核酸分离纯化出来，故其能实现高通量自动化标准化操作。
磁珠(ImpetiCbead，IMB)将固化试剂特有的优点与免疫学反应的高度特异性结合于一体，以免疫学为基础，渗透到病理、生理、药理、微生物、生化以及分子遗传学等各个领域，其在免疫检测、细胞分离、生物大分子纯化和分子生物学等方面得到了越来越广泛的应用。目前市面上用于核酸提取的磁珠大部分采用传统溶胀法制备而得。基本思路为采用高分子微球(例如：聚苯乙烯微球)在特定有机溶剂环境中，微球发生溶胀，通透性变好。此条件下，Fe3O4磁性纳米颗粒(粒径一般在几十纳米左右)进入微球中。随着有机溶剂不断挥发，微球通透性恢复正常，磁性纳米颗粒镶嵌其中。虽然此种方法制备过程相对简单，但通过此方法制备的磁珠用于生物样品中游离DNA的富集应用中存在一些缺点：1、粒径不均匀，且为微米级，一般在1-4μm范围内，导致磁珠在溶液中的悬浮性较差，容易沉降，影响和样品中游离DNA的相互作用。2、此种磁珠的比表面积较小，可供于DNA的结合位点较少，需增大磁珠用量，才能达到有效分离样品中DNA的目的，增加了成本。3、嵌入高分子球中的Fe3O4磁性纳米粒子粒径太小，磁响应不强，影响磁性分离过程。4、高分子基底材质容易造成较强的非特异性吸附，容易引入杂质，影响下游DNA的检测。
银纳米粒子，尤其是银纳米球具有优良的电学、光学性质和良好的生物相容性，易与生物大分子(如蛋白质、核酸、肽)稳定结合，在分子识别和生物标记方面有广泛应用。近年来，银纳米球的制备工艺趋近成熟，颗粒均已，尺寸可控的银纳米球已可以大批量制备，市场上也出现了不同尺寸的银纳米球销售。
将磁性粒子与银粒子结合起来的银-磁复合粒子，由于兼具了纳米磁与纳米银的优点而具有更为广阔的应用前景，目前已成为研究的热点。中国发明专利CN100460338C，“一种银/磁纳米颗粒的制备方法”公开的银/磁纳米颗粒的粒径为60±20nm，其内核为四氧化三铁、外壳为单质银；制备方法为在反相微乳液体系的水相中，硝酸银溶液以磁性四氧化三铁纳米颗粒为内核，通过还原反应，将单质银沉积在四氧化三铁纳米颗粒的表面，从而获得具有核壳型结构的最终产物。专利CN201210014091.5公开了一种银-四氧化三铁核壳纳米颗粒及其制备方法。颗粒为以银为核、四氧化三铁为壳的核壳结构纳米颗粒，使用硝酸铁、硝酸银和醋酸钠反应制得，其粒径为160～230nm，其中，银为粒径为60～90nm的银纳米颗粒，四氧化三铁壳的壳厚为50～70nm。
然而，现有技术中无论是银/磁纳米颗粒，还是其制备方法，都存在着欠缺之处，首先，单质银的生长不可控，大小不一，造成银/磁纳米颗粒形状、大小不均，影响其性能；其次，制备得到的银/磁纳米颗粒大小固定，不能适应不同核酸提取需求。
发明内容
本发明的目的在于提供一种新型核酸提取用磁珠，并提供磁珠的制备方法。解决现有磁珠粒径大小不均匀，表面能小，核酸吸附效率低的问题。
为了实现上述目的，本发明提供一种Ag-Fe3O4-SiO2磁珠，其特征是所述Ag-Fe3O4-SiO2磁珠为核壳型微球，其内核是银纳米球，核外是纳米Fe3O4磁性粒子吸附层，最外层是纳米二氧化硅胶体粒子。
本发明还提供了一种Ag-Fe3O4-SiO2磁珠制备方法，包括以下步骤：
步骤1，取适量银纳米球分散于去离子水中得到银纳米球溶液；
步骤2，取适量水性纳米Fe3O4磁性粒子分散于去离子水中得到纳米Fe3O4磁性粒子溶液；
步骤3，向所述银纳米球溶液中加入适量表面活性剂，混合均匀；
步骤4，再向银纳米球溶液中加入纳米Fe3O4磁性粒子溶液，震荡反应3-12h，离心分离得到Ag-Fe3O4核壳材料；
步骤5，使用硅烷水解法在Ag-Fe3O4核壳材料外包覆二氧化硅保护层，得到Ag-Fe3O4-SiO2磁珠。
其中，所述银纳米球的粒径为20-300nm；所述纳米Fe3O4磁性粒子的粒径为5-45nm；所述Ag-Fe3O4-SiO2磁珠的粒径为50-500nm。银纳米球和纳米Fe3O4磁性粒子均采用现有技术制备或直接购买现有产品。
使用硅烷水解法在Ag-Fe3O4核壳材料外包覆二氧化硅保护层的过程包括：取固相Ag-Fe3O4核壳材料分散于由异丙醇、去离子水和氨水组成的混合溶液中，然后再加入正硅酸乙酯(TEOS)，室温下搅拌反应后用去离子水和无水乙醇分别洗涤得固相，取固相用去离子水和无水乙醇洗涤、烘干，得到Ag-Fe3O4-SiO2磁珠。
其中，所述表面活性剂为十六烷基三甲基溴化铵或聚乙烯吡咯烷酮。
本发明提供一种新型Ag-Fe3O4-SiO2磁珠，并通过选择不同粒径的银纳米球和纳米Fe3O4磁性粒子，并控制反应时间，可控制获得的Ag-Fe3O4-SiO2磁珠的粒径大小，且制备Ag-Fe3O4-SiO2磁珠粒径均匀，磁响应强。
具体实施方式
下面通过实施例对本发明作进一步说明。
实施例1
步骤1，取适量粒径20nm的银纳米球分散于去离子水中得到10ml，浓度为5mM银纳米球溶液；
步骤2，取适量粒径5nm的水性纳米Fe3O4磁性粒子分散于去离子水中得到50ml，浓度为5mM的纳米Fe3O4磁性粒子溶液；
步骤3，向所述银纳米球溶液中加入100mM的CTAB(十六烷基三甲基溴化铵)20ml，混合均匀；
步骤4，再向银纳米球溶液中加入上述纳米Fe3O4磁性粒子溶液，震荡反应12h，离心分离得到Ag-Fe3O4核壳材料；
步骤5，将固相Ag-Fe3O4核壳材料分散于由9mL水、20mL异丙醇和1mL氨水组成的混合溶液中，搅拌30min后，然后再加入150μL正硅酸乙酯(TEOS)，室温下搅拌反应后用去离子水和无水乙醇分别洗涤得固相，取固相用去离子水和无水乙醇洗涤三遍、烘干，得到粒径为约100nm的Ag-Fe3O4-SiO2磁珠。
实施例2
步骤1，取适量粒径300nm的银纳米球分散于去离子水中得到10ml，浓度为1mM银纳米球溶液；
步骤2，取适量粒径45nm的水性纳米Fe3O4磁性粒子分散于去离子水中得到50ml，浓度为20mM的纳米Fe3O4磁性粒子溶液；
步骤3，向所述银纳米球溶液中加入100mM的CTAB(十六烷基三甲基溴化铵)10ml，混合均匀；
步骤4，再向银纳米球溶液中加入上述纳米Fe3O4磁性粒子溶液，震荡反应12h，离心分离得到Ag-Fe3O4核壳材料；
步骤5，将固相Ag-Fe3O4核壳材料分散于由9mL水、20mL异丙醇和0.5mL氨水组成的混合溶液中，搅拌30min后，然后再加入100μL正硅酸乙酯(TEOS)，室温下搅拌反应后用去离子水和无水乙醇分别洗涤得固相，取固相用去离子水和无水乙醇洗涤三遍、烘干，得到粒径为约0.5μm的Ag-Fe3O4-SiO2磁珠。
实施例3
步骤1，取适量粒径50nm的银纳米球分散于去离子水中得到10ml，浓度为10mM银纳米球溶液；
步骤2，称取0.9g油酸铁、0.156g油酸、5g 1-十八烯加入50mL三颈烧瓶中，加热至320℃(以18℃/min的升温速度)，反应1h后快速冷却至室温，取反应后固相以丙酮和正己烷的混合洗涤剂洗涤三次，得约为0.2g的表面油酸包覆的四氧化三铁颗粒。取0.2g表面油酸包覆的四氧化三铁颗粒分散于72mL四氢呋喃中，形成Fe3O4的四氢呋喃分散液。将0.05gDHCA分散至6mL四氢呋喃中，再向其中加入6mL的Fe3O4的四氢呋喃分散液，50℃下加热反应3h，冷却至室温后加入浓度为0.1mol/L的0.5mL NaOH水溶液，离心后将固体分散至50mL去离子水中，得到浓度为20mM的纳米Fe3O4磁性粒子溶液；
步骤3，向所述银纳米球溶液中加入100mM的CTAB(十六烷基三甲基溴化铵)20ml，混合均匀；
步骤4，再向银纳米球溶液中加入上述纳米Fe3O4磁性粒子溶液，震荡反应12h，离心分离得到Ag-Fe3O4核壳材料；
步骤5，将固相Ag-Fe3O4核壳材料分散于由9mL水、20mL异丙醇和0.5mL氨水组成的混合溶液中，搅拌30min后，然后再加入100μL正硅酸乙酯(TEOS)，室温下搅拌反应后用去离子水和无水乙醇分别洗涤得固相，取固相用去离子水和无水乙醇洗涤三遍、烘干，得到粒径为约200μm的Ag-Fe3O4-SiO2磁珠。
实施例4
步骤1，取适量粒径50nm的银纳米球分散于去离子水中得到10ml，浓度为5mM银纳米球溶液；
步骤2，取适量粒径45nm的水性纳米Fe3O4磁性粒子分散于去离子水中得到50ml，浓度为10mM的纳米Fe3O4磁性粒子溶液；
步骤3，向所述银纳米球溶液中加入5％wt的聚乙烯吡咯烷酮溶液(PVP)1ml，混合均匀；
步骤4，再向银纳米球溶液中加入上述纳米Fe3O4磁性粒子溶液，震荡反应6h，离心分离得到Ag-Fe3O4核壳材料；
步骤5，将固相Ag-Fe3O4核壳材料分散于由10mL水、25mL异丙醇和0.5mL氨水组成的混合溶液中，搅拌30min后，然后再加入100μL正硅酸乙酯(TEOS)，室温下搅拌反应后用去离子水和无水乙醇分别洗涤得固相，取固相用去离子水和无水乙醇洗涤三遍、烘干，得到粒径为约200nm的Ag-Fe3O4-SiO2磁珠。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (9)
1.一种Ag-Fe3O4-SiO2磁珠，其特征是所述Ag-Fe3O4-SiO2磁珠为核壳型微球，其内核是银纳米球，核外是纳米Fe3O4磁性粒子吸附层，最外层是纳米二氧化硅胶体粒子。
2.根据权利要求1所述的Ag-Fe3O4-SiO2磁珠，其特征在于，所述银纳米球的粒径为20-300nm；所述纳米Fe3O4磁性粒子的粒径为5-45nm；所述Ag-Fe3O4-SiO2磁珠的粒径为50-500nm。
3.一种Ag-Fe3O4-SiO2磁珠制备方法，其特征在于，包括以下步骤：
步骤1，取适量银纳米球分散于去离子水中得到银纳米球溶液；
步骤2，取适量水性纳米Fe3O4磁性粒子分散于去离子水中得到纳米Fe3O4磁性粒子溶液；
步骤3，向所述银纳米球溶液中加入适量表面活性剂，混合均匀；
步骤4，再向银纳米球溶液中加入纳米Fe3O4磁性粒子溶液，震荡反应3-12h，离心分离得到Ag-Fe3O4核壳材料；
步骤5，使用硅烷水解法在Ag-Fe3O4核壳材料外包覆二氧化硅保护层，得到Ag-Fe3O4-SiO2磁珠。
4.根据权利要求3所述的Ag-Fe3O4-SiO2磁珠制备方法，其特征在于，所述银纳米球与所述水性纳米Fe3O4磁性粒子的摩尔比为1:5-1:100。
5.根据权利要求3所述的Ag-Fe3O4-SiO2磁珠制备方法，其特征在于，所述银纳米球与所述表面活性剂的摩尔比为1:10-1:50。
6.根据权利要求3所述的Ag-Fe3O4-SiO2磁珠制备方法，其特征在于，使用硅烷水解法在Ag-Fe3O4核壳材料外包覆二氧化硅保护层的过程包括：取固相Ag-Fe3O4核壳材料分散于由异丙醇、去离子水和氨水组成的混合溶液中，然后再加入正硅酸乙酯(TEOS)，室温下搅拌反应后用去离子水和无水乙醇分别洗涤得固相，取固相用去离子水和无水乙醇洗涤、烘干，得到Ag-Fe3O4-SiO2磁珠。
7.根据权利要求3所述的Ag-Fe3O4-SiO2磁珠制备方法，其中所述表面活性剂为十六烷基三甲基溴化铵或聚乙烯吡咯烷酮。
8.根据权利要求3所述的Ag-Fe3O4-SiO2磁珠制备方法，其特征在于，所述银纳米球的粒径为20-300nm；所述纳米Fe3O4磁性粒子的粒径为5-45nm；所述Ag-Fe3O4-SiO2磁珠的粒径为50-500nm。
9.根据权利要求3所述的Ag-Fe3O4-SiO2磁珠制备方法，其特征在于，所述水性纳米Fe3O4磁性粒子制备方法为；将油相Fe3O4纳米颗粒分散至有机溶剂中，加入2，3-二巯基丁二酸的有机溶液对油相Fe3O4纳米颗粒进行表面配体交换反应，待反应完毕后对产物依次进行有机溶剂清洗、水清洗和调节pH，得到水性纳米Fe3O4磁性粒子。
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