CN110124633A - 组合低共熔溶剂单体和杂化单体的整体柱 - Google Patents
组合低共熔溶剂单体和杂化单体的整体柱 Download PDFInfo
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Abstract
本发明涉及一种组合低共熔溶剂单体和杂化单体的整体柱。以DES单体（氯化胆碱‑甲基丙烯酸）和杂化单体(3‑氨丙基三乙氧基硅烷‑甲基丙烯酸)为二元功能单体，二甲基丙烯酸乙二醇酯为交联剂，甲醇和PEG 20000作为二元致孔剂，利用原位热聚合技术制备新型整体柱。本发明成本低廉，合成过程简单，所制成的新型整体柱具有良好的通透性、较强的机械强度及生物相容性，可以成功分离牛血清白蛋白和细胞色素C，同时对这两种蛋白能实现较好的富集。所拟方法为蛋白质的分离纯化提供了一种新思路。
Description
技术领域
本发明属于分离分析技术领域,具体涉及一种新的组合低共熔溶剂单体和杂化单体整体柱。
背景技术
蛋白质是一种具有生物活性的生物大分子，与各种生命活动有着密切的关系。随着蛋白质组学和人类基因组计划的开展，人们希望能在分子水平上理解细胞的生物学。蛋白质生物化学试图揭示基因产物的作用和生物学功能。在保持蛋白质的生物活性和化学完整性的同时，将所需的蛋白质从复杂基体中分离出来，对于生命科学、分子生物学等领域的发展具有重大的意义。完整蛋白分离技术主要有聚丙烯酰胺凝胶电泳、毛细管电泳和液相色谱。然而，使用颗粒填充柱层析法进行蛋白质分离是有挑战性的，因为蛋白质与固定相的相互作用是复杂的，有时是不可逆的(大分子可以堵塞固定相的孔隙，使其失效)。这些分离常常受到分辨率低和/或重现性有限的影响。
在完整蛋白质分离领域，整体柱具有独特的高孔隙率和快速传质特性，能够选择性地调控中孔和大孔尺寸以及骨架直径，这为整体柱提供了几个关键优势，例如，比填充柱具有高的渗透性、更强的样品耐脏性和更高的分辨率，使其特别适合于完整蛋白质的分离。目前整体柱在蛋白质分离领域发展迅速，特别是在LCXLC模式下，可以通过完整的蛋白质图谱开发出独特的早期疾病诊断和/或快速治疗发展的应用。将多种目标化合物固定在整体柱上并实现亲和或伪亲和层析分离的能力，在高通量酶或抗体富集/纯化或蛋白质筛选方面具有潜在的市场应用前景。绝大多数整体柱的功能应用依赖功能单体来实现，然而，目前可供选择的功能单体的种类有限，限制了整体柱的发展。因此开发出对蛋白质具有良好的富集效率和选择性的新型功能单体是十分必要的。
低共熔溶剂(Deep Eutectic Solvents，DES)是指由一定化学计量比的氢键受体(如季铵盐)和氢键给体(如酰胺、羧酸和多元醇等化合物)组合而成的两组分或三组分低共熔混合物，其凝固点显著低于各个组分纯物质的熔点，能够在形成DES的同时进行自由基聚合的单体，即氢键给体或铵盐，我们称之为DES单体。DES单体具有成本低廉、可生物降解、合成过程简单、结构可设计等优势，同时具备优良的物化性质，在分离分析领域的应用前景日益受到研究者的关注。DES单体水溶性好，能与蛋白质发生氢键作用、静电作用、范德华力等相互作用，对蛋白质有较好的吸附效果；且作为一种绿色溶剂，对蛋白质无毒性，有很好的生物相容性，适合于完整蛋白质的分离。目前，尚未有文献报道将DES单体用于整体柱的制备，我们在实验过程中发现，用DES单体制备的整体柱较软，影响了整体柱的寿命及耐用性。
杂化单体(3-氨基丙基三乙氧基硅烷-甲基丙烯酸，APTES-MAA)能在分子水平上将有机无机成分通过化学键合到一起，形成有机无机杂化分子。分子内有硅氧骨架，这种骨架可以提供高渗透性、有机溶剂耐受性、良好的机械强度，因此将杂化单体引入制备整体柱，可以弥补DES单体的不足，利用杂化单体在分子水平的刚性来增强整体柱的性能。因此，将DES单体和杂化单体协同作用，制备了一种固相萃取整体柱，用于富集牛血清蛋白。
发明内容
本发明的目的在于提供一种组合低共熔溶剂单体和杂化单体的整体柱，它是以DES单体和杂化单体作为二元功能单体，利用原位热聚合技术制备新型整体柱的方法。本发明成本低廉,实验操作简单,反应条件容易控制,可以实现对牛血清蛋白较高的富集效果，同时可以将牛血清蛋白和细胞色素C成功分离。
本发明提供的组合低共熔溶剂单体和杂化单体的整体柱的制备方法包括的步骤：
1）DES单体的制备：将氯化胆碱（ChCl）与甲基丙烯酸（MAA）按照1：2的摩尔比均匀混合，置于90 ℃油浴加热1 h，可得均一透明的溶液即为DES单体ChCl-MAA，放置于干燥器中备用；
杂化单体的制备：取6.4 mmol的3-氨丙基三乙氧基硅烷（APTES）和8.1 mmol的甲基丙烯酸（MAA）放入反应瓶中混合，超声10 min溶解，然后将其放入60 ℃水浴锅中，反应24 h后取出，得到黄色透明的液体即为杂化单体APTES-MAA。
2）整体柱的制备：将甲醇和PEG 20000二元致孔剂中加入功能单体APTES-MAA和DES单体ChCl-MAA，交联剂二甲基丙烯酸乙二醇酯和引发剂偶氮二异丁腈，混合超声后形成预聚合液注入已处理好的毛细管中，用胶塞封住毛细管两端，置于60℃恒温水浴锅内，反2h后取出毛细管，用乙腈冲洗整体柱中未反应的物质和其他可溶性物质。截取25 cm的毛细管柱备用,其中有效长度为20 cm。
步骤2）中所述DES单体ChCl-MAA的体积为32-80 μL。
步骤2）中所述杂化单体APTES-MAA的体积为112-176 μL。
步骤2）中所述二元致孔剂甲醇的体积为670 μL, PEG 20000的质量为70 mg。
本发明提供了组合低共熔溶剂单体和杂化单体的整体柱的制备方法得到的整体柱，可用于富集牛血清蛋白，其突出的特点为：
1）本发明制备的整体柱将DES单体和杂化单体协同作用，DES单体水溶性好，能与蛋白质发生氢键作用、静电作用、范德华力等相互作用，对蛋白质有较好的吸附效果；且作为一种绿色溶剂，对蛋白质无毒性，有很好的生物相容性，适合于完整蛋白质的分离。
2）本发明制备的整体柱将DES单体和杂化单体协同作用，杂化单体分子内的硅氧骨架可以提供高渗透性、有机溶剂耐受性、良好的机械强度，利用杂化单体在分子水平的刚性来增强整体柱的性能。
3）本发明成本低廉,实验操作简单,反应条件容易控制,可以实现对牛血清蛋白较高的富集效果，同时可以将牛血清蛋白和细胞色素C成功分离。
附图说明
图１为实施例２所制得的组合DES单体和杂化单体的整体柱截面扫描电镜图。
图2为实施例２所制得的组合DES单体和杂化单体的整体柱扫描电镜图。
图3为不同单体制备的整体柱对牛血清蛋白的回收率。
图4为整体柱对牛血清蛋白和细胞色素C分离后的聚丙烯酰胺凝胶电泳图，图中，通道1：蛋白Marker；通道2：上样液；通道3：上样后流出液；通道4：清洗流出液；通道5：洗脱流出液；通道6：洗脱流出液。
具体实施方式
下面结合具体实施例，进一步详细阐述本发明。实施例中未注明具体条件的实验方法，通常按照常规条件以及手册中所述的条件，或按照制造厂商所建议的条件；所用的通用设备、材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
功能单体的制备
1.DES单体的制备：将氯化胆碱（60 mmol，8.4 g）与甲基丙烯酸（120 mmol，10.2 mL）按照1：2的摩尔比加入螺纹小瓶中，随后90 ℃油浴1 h，可得均一透明的溶液。放置于干燥器中备用；
2.杂化单体的制备：取1498 μL（6.4 mmol）的3-氨丙基三乙氧基硅烷（APTES）和688 μL（8.1 mmol）的甲基丙烯酸（MAA）放入反应瓶中混合，超声10 min溶解。然后将其放入60 ℃水浴锅中，反应24 h后取出，得到黄色透明的液体即为杂化单体APTES-MAA；
实施例２
整体柱的制备
在670 μL甲醇和70 mg PEG 2000二元致孔剂中加入功能单体APTES-MAA（64 μL）和DES单体ChCl-MAA（144 μL），交联剂二甲基丙烯酸乙二醇酯（62 μL）和引发剂偶氮二异丁腈（2.6 mg），混合超声后形成预聚合液注入已处理好的毛细管中，用胶塞封住毛细管两端，置于60℃恒温水浴锅内，反应2 h后取出毛细管，用乙腈冲洗整体柱中未反应的物质和其他可溶性物质。
实施例3
为了确定功能单体类型对蛋白质萃取效果的影响，合成单一单体（APTES-MAA整体柱、DES整体柱）及传统单体（MAA整体柱）的整体柱作为对照。具体操作步骤如下：
DES/APTES-MAA整体柱的制备即为实施例２所制备的整体柱；
对照组整体柱的制备
APTES-MAA整体柱的制备：除不加入DES单体外，用实施例2相同的方法和实验条件合成；DES整体柱的制备：除不加入APTES-MAA单体外，用实施例2相同的方法和实验条件合成；MAA整体柱的制备：除不加入APTES-MAA单体和DES单体外，加入MAA单体210μL，用实施例2相同的方法和实验条件合成。聚合完成后，用乙腈冲洗，以除去整体柱中残留的致孔剂和未反应的试剂。
将制得的整体柱与注射器连接后即得到本发明的整体柱萃取装置。该萃取装置制备简单、操作简便，实验过程中使用一台微量注射泵来推动注射器传输液体，从而对牛血清蛋白标准溶液进行分离富集。
整个萃取过程包括活化、萃取、清洗和解吸四个步骤。
（1）活化：用200 μL PBS（pH 5.0，20 mM）对整体柱进行活化；
（2）萃取：准确吸取牛血清蛋白100μg mL-1（200μL）的样品溶液以通过萃取柱进行萃取；
（3）清洗：用PBS（pH 5.0，20 mM）通过整体柱清洗残留的样品基质，以避免干扰；并用洁净的空注射器推出残留液；
（4）解吸：用100 μL PBS（pH 12.0，20 mM）通过萃取柱对目标物进行解吸，并在整体柱出口端收集洗脱液进分析。
整个萃取过程均以5 μL min-1的流速进行。以Bradford法测定蛋白质浓度。
结果如图3，表明利用APTES-MAA作为单体的制备的整体柱的回收率为52.5% 。用DES单体替代APTES-MAA单体后，萃取效果没有明显改善(62.1%)。而双单体(DES/APTES-MAA)制备的整体柱回收率(95.5%)约为单一单体制备的整体柱回收率的1.5倍。相比之下，传统单体MAA制备的整体柱所得回收率为15.3%，萃取效果远低于DES/APTES-MAA整体柱。这可能是因为APTES-MAA的高刚度提高了整体柱的传质效率和结构稳定性；DES单体含有丰富的氢键，可产生有效的结合位点，同时，DES可以提高整体柱的表面积，增强对目标蛋白吸附，二者协同作用提升整体柱的性能，从而提高了富集效果。因此，利用DES和APTES-MAA的二元单体作为功能单体。
实施例4
为了考察整体柱对混合蛋白的分离能力，以实施例2的DES/APTES-MAA整体柱对混合蛋白进行固相萃取并收集萃取过程中每一步的洗脱液进行聚丙烯酰胺凝胶电泳SDS-PAGE测定。
萃取过程同实施例3，固相萃取的上样溶液为牛血清蛋白（100 μg mL-1）与细胞色素C（100 μg mL-1）的混合液，在对牛血清蛋白洗脱完成后，再用1mol L-1 NaCl（50 μL）溶液对细胞色素C进行洗脱。
结果如图4。通道1为Marker，通道2、6中下方条带分子量大致为13 kDa的为细胞色素C，通道2、5中上方条带分子量大致为66 kDa的为牛血清白蛋白。通道3中无条带，说明上样液中的牛血清蛋白和细胞色素C已被完全吸附到固相萃取整体柱中；通道4中无蛋白条带，说明清洗流出液中无蛋白；通道5中有一条牛血清白蛋白的条带，且颜色比通道2对应的标品条带颜色深，说明洗脱液将牛血清白蛋白从整体柱中洗脱下来且达到富集效果。通道6中的条带为细胞色素C的条带，同样颜色比通道2对应的标品条带颜色深，说明洗脱液将细胞色素C从整体柱中洗脱下来且达到富集效果。以上结果说明该整体柱能根据蛋白质的特点成功分离牛血清蛋白与细胞色素C，由此证明该方法的可行性。
Claims (6)
1.一种组合低共熔溶剂单体和杂化单体整体柱的制备方法，其特征在于：
1）DES单体的制备：将氯化胆碱（ChCl）与甲基丙烯酸（MAA）按照1：2的摩尔比均匀混合，置于90 ℃油浴加热1 h，得到均一透明的溶液即为DES单体ChCl-MAA，放置于干燥器中备用；
杂化单体的制备：取6.4 mmol的3-氨丙基三乙氧基硅烷（APTES）和8.1 mmol的甲基丙烯酸（MAA）放入反应瓶中混合，超声10 min溶解，然后将其放入60 ℃水浴锅中，反应24 h后取出，得到黄色透明的液体即为杂化单体APTES-MAA；
2）整体柱的制备：将甲醇和PEG 20000二元致孔剂中加入功能单体APTES-MAA和DES单体ChCl-MAA，交联剂二甲基丙烯酸乙二醇酯和引发剂偶氮二异丁腈，混合超声后形成预聚合液注入已处理好的毛细管中，用胶塞封住毛细管两端，置于60℃恒温水浴锅内，反2 h后取出毛细管，用乙腈冲洗整体柱中未反应的物质和其他可溶性物质。
2.根据权利要求1所述的制备方法,其特征在于：步骤2）中所述DES单体ChCl-MAA的体积为32-80 μL。
3.根据权利要求1所述的制备方法,其特征在于：步骤2）中所述杂化单体APTES-MAA的体积为112-176 μL。
4.根据权利要求1所述的制备方法,其特征在于：步骤2）中所述二元致孔剂甲醇的体积为670 μL, PEG 20000的质量为70 mg。
5.权利要求1-4任一所述的制备方法得到的组合低共熔溶剂单体和杂化单体整体柱。
6.权利要求5所述的组合低共熔溶剂单体和杂化单体整体柱用于富集牛血清蛋白以及蛋白质的分离纯化。
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