CN108404855A - 一种固定化脂质体毛细管整体柱的制备方法和应用 - Google Patents
一种固定化脂质体毛细管整体柱的制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种固定化脂质体毛细管整体柱的制备方法及其应用。在毛细管硅胶整体柱的硅胶基质表面，通过物理涂覆的方法将脂质体固定。本发明固定化脂质体毛细管整体柱的制备方法重复性好、脂质体固定量大，所制整体柱的性能稳定、寿命较长。固定化脂质体毛细管整体柱对萘酚、苯系物、甾体药物、氨基甲酸酯等中性化合物表现出优越的分离性能；对溶菌酶、胰蛋白酶、牛血清白蛋白三种蛋白质能实现基线分离，显著改善了蛋白质在毛细管硅胶整体柱上的吸附现象；而且各物质在固定化脂质体毛细管整体柱上的保留因子都显著增大、保留行为的差异更明显。因而，固定化脂质体毛细管整体柱表征物质与生物膜之间相互作用强度的效果更好。
Description
技术领域
本发明属于分离分析技术领域，具体涉及一种固定化脂质体毛细管整体柱的制备方法和应用。
背景技术
毛细管电色谱(Capillary electrochromatography,CEC)是20世纪90年代发展起来的一种微分离分析技术。由于其结合了毛细管电泳的高效性和液相色谱的高选择性，在分离分析领域备受关注。毛细管电色谱柱是CEC系统的最重要组成部分。根据固定相在毛细管内的存在状态，毛细管电色谱柱可分为毛细管填充柱、毛细管开管柱和毛细管整体柱。毛细管填充柱是将固定相通过专业的烧塞和装填程序填充进入毛细管内而得；填充柱柱容量大且分离性能好，但是制柱程序复杂、分离过程中柱床和塞子附近易产生气泡而影响分离效果和稳定性。毛细管开管柱是通过物理涂覆、化学键合等方法将固定相固定在毛细管内壁制备而成的一类电色谱柱；相比于填充柱，开管柱制备方法简单，但是相比小、柱容量低。毛细管整体柱是将有机或者无机单体、引发剂、致孔剂等混合物通过原位聚合或者固化的方法在毛细管内形成均一、多孔结构的连续床固定相而得。毛细管整体柱具有多孔特性，比表面积大，因而能提供更大的相比和柱容量；与填充柱、开管柱相比，整体柱具有制备简单、通透性好、传质速度快、易于改性和柱容量高等优点，在分离科学中有良好的应用前景。
脂质体(liposome)，又称磷脂囊泡(vesicle)，是两性磷脂分子在水中自发形成的具有双分子层结构的囊泡。由于其与天然生物膜结构非常相似，可作为模拟生物膜用于研究物质与生物膜的相互作用。脂质体作为固定相与毛细管电色谱技术相结合，可以使中性化合物得以分离(化合物在脂质体和电泳缓冲液中的分配系数不同)、增加带电化合物的分离选择性、改善分离蛋白质和多肽时的严重吸附现象，并且在评价物质-生物膜相互作用时具有独特优势：分析速度快、脂质体量和样品量消耗少、可与质谱联用。目前，脂质体毛细管电色谱技术在中性化合物分离、蛋白质分离、药物-生物膜相互作用等方面得到广泛应用。
目前，脂质体毛细管电色谱主要使用的是固定化脂质体毛细管开管柱，是指将脂质体作为涂层剂，采用物理涂覆、共价键合等固定化方法，在毛细管内壁形成较稳定的脂质体涂层。但是，由于毛细管内壁的比表面积小而使固定于毛细管内壁的脂质体的量非常有限，从而导致相比小、柱容量小、物质在脂质体毛细管开管柱上的保留行为差异不显著，不利于化合物分离、物质-生物膜相互作用等应用。我们前期曾尝试用表面蚀刻法来增大毛细管柱内壁的比表面积，结果发现，固定的脂质体量虽有所提高，但增加幅度很有限。因此，如何制备相比大、柱容量大的脂质体毛细管柱仍是脂质体毛细管电色谱发展中的重点和难点。
发明内容
本发明提拱了一种固定化脂质体毛细管整体柱的制备方法和应用，目的在于提供一种脂质体固定量大、柱容量大，可应用于分离中性化合物和蛋白质、研究物质-生物膜相互作用的毛细管整体柱。
相比于固定化脂质体毛细管开管柱，固定化脂质体毛细管整体柱内脂质体的固定量更大，因而使物质在整体柱上的分离度和保留因子增大，最终使各物质之间更容易被分离，也能更真实地表征物质与生物膜之间的相互作用。脂质体冲洗时间和放置反应时间是比较重要的参数，关系到脂质体在整体柱内的涂覆量是否达到饱和。本发明对上述两个条件进行了优化使所制备的固定化脂质体毛细管整体柱达到最佳分离效果。
本发明提供的技术方案如下。
一种固定化脂质体毛细管整体柱的制备方法，包括以下步骤：
(1)制备毛细管硅胶整体柱；
(2)将制得的毛细管硅胶整体柱在加压下先用4-羟乙基哌嗪乙磺酸(HEPES)缓冲液第一次冲洗，然后用脂质体溶液冲洗，两端封口，室温放置至其反应完毕，最后再用4-羟乙基哌嗪乙磺酸缓冲液第二次冲洗毛细管柱，即完成制备。
上述步骤(1)中毛细管硅胶整体柱的制备方法，为常用的溶胶-凝胶法，包括以下步骤：
准确称取聚乙二醇和尿素，用醋酸溶液溶解后，再缓慢滴加四甲氧基硅烷，混合液于冰水浴中搅拌，待溶液成均匀的溶胶态后注入到预先处理过的毛细管中，毛细管两端封口后于40℃反应24h，再经扩中孔、除杂、干燥、老化处理后得到毛细管硅胶整体柱。
其中，所述醋酸溶液浓度为0.01mol/L；所述聚乙二醇分子量为10000；所述聚乙二醇、尿素、醋酸、四甲氧基硅烷的摩尔比为5:333:5:1344。
上述步骤(2)中HEPES缓冲液的浓度为25mmol/L，pH为7.40。加压冲洗的压力为50psi。
上述步骤(2)中HEPES缓冲液第一次冲洗时间为30min，脂质体溶液冲洗时间为30min,室温放置反应时间为30min，HEPES缓冲液第二次冲洗时间为10min。
上述步骤(2)中脂质体溶液是以磷脂酰胆碱为磷脂成分，以HEPES缓冲液为分散溶剂，采用薄膜分散-探针超声法制备而得。脂质体的平均粒径约为37nm，多分散系数约为0.34，磷脂含量为5mmol/L。
本发明的有益效果：
(1)固定化脂质体毛细管整体柱制备方法的重复性好，所制备的整体柱性能稳定、寿命较长、脂质体固定量大；
(2)固定化脂质体毛细管整体柱对中性化合物的分离能力良好；
(3)固定化脂质体毛细管整体柱可有效降低蛋白质在管壁的吸附作用，并对蛋白质实现良好分离；
(4)固定化脂质体毛细管整体柱能真实地表征物质与生物膜之间的相互作用。
附图说明
图1为萘酚的分离图谱，1-β-萘酚、2-α-萘酚；
图2为苯系物的分离图谱，1-苯、2-甲苯、3-乙苯、4-丙苯；
图3为甾体药物的分离图谱，1-氢化可的松、2-醋酸可的松、3-皮质酮、4-雄烯二酮、5-睾丸素；
图4为氨基甲酸酯的分离图谱，1-克多威、2-克百威、3-抗蚜威、4-异丙威、5-甲萘威；
图5为三种蛋白质的分离图谱，1-溶菌酶、2-胰蛋白酶、3-牛血清白蛋白。
附图标记：a-毛细管硅胶整体柱，b-固定化脂质体毛细管整体柱，c-固定化脂质体毛细管开管柱。
具体实施方式
下面结合具体实施方式对本发明进一步说明，本发明的内容完全不限于此。
实施例1
脂质体的制备。
1、配制4-羟乙基哌嗪乙磺酸(HEPES)缓冲液
精密称取1.4894g HEPES，加约220mL水完全溶解，用1mol/L NaOH调节pH至7.40，再加水定容至250mL，得25mmol/L HEPES缓冲液。
2、制备脂质体
(1)精密称取28.804mg磷脂酰胆碱，用17mL氯仿:甲醇溶液(2:1，v/v)溶解后，转移至圆底烧瓶中于35℃下减压旋转蒸发，直至烧瓶壁上形成一层均匀分散的薄膜，真空干燥12-16h，使残留的有机溶剂挥发完全。
(2)向烧瓶中加入约7.6mL上述配制的HEPES缓冲液，涡漩振荡使薄膜完全溶解，即可形成多室脂质体溶液。采用细胞超声破碎仪(200W；工作时间3s；间隙时间4s；超声次数99次)，在冰浴条件下将多室脂质体溶液反复超声破碎数次至澄清透明，即可制得小单室脂质体溶液。使用0.22μm微孔滤膜过滤小单室脂质体溶液，于4℃冰箱保存。
所得脂质体溶液的总磷脂含量为5mmol/L，平均粒径为36.7±1.27nm(n＝3)，多分散系数为0.338±0.036(n＝3)。
实施例2
固定化脂质体毛细管整体柱的制备(下述冲洗毛细管所加的压力均为50psi)。
1、取新毛细管，依次用甲醇冲洗30min、1mol/L的NaOH冲洗1h、1mol/L的HCl冲洗30min、二次蒸馏水冲洗直至流出液为中性，在N2氛围下120℃干燥2h，两端用硅橡皮封口备用。
2、称取50mg聚乙二醇和20mg尿素，用500μL 0.01mol/L的醋酸溶液溶解，再缓慢滴加200μL四甲氧基硅烷，于冰水浴中剧烈搅拌，待溶液成均匀的溶胶态后注入到毛细管中；40℃反应12-16h后，将毛细管柱于120℃加热3h，进行扩中孔；然后依次用甲醇、水冲洗干净毛细管，在N2氛围下50℃干燥6h后，以2℃/min的速率从50℃升至180℃并保持1h，最后330℃下热处理固化20h，得毛细管硅胶整体柱。
3、将制得的毛细管硅胶整体柱用实施例1所制备的HEPES缓冲液冲洗30min；然后用总磷脂含量为5mmol/L的脂质体HEPES溶液冲洗30min(此时整根毛细管硅胶整体柱内充满了脂质体溶液)，两端用硅橡皮封口，室温下放置反应30min；最后用HEPES缓冲液冲洗10min，以除去整体柱内未固定的脂质体；即得到固定化脂质体毛细管整体柱。
测定固定化脂质体毛细管整体柱内的脂质体固定量(以固定的磷脂量表示)，结果为395.98nmol/m，显著高于固定化脂质体毛细管开管柱的脂质体固定量(121.21nmol/m)，表明本发明制备的固定化脂质体毛细管整体柱的脂质体固定量大。
实施例3
固定化脂质体毛细管整体柱的性能测试。
电色谱分离条件：固定化脂质体毛细管整体柱总长为40.2cm，其中有效长度为30cm；电泳缓冲液为pH 7.4的20mM磷酸盐缓冲液；样品进样条件为5kV下进样3s；柱温为20℃，分离电压为20kV；分离时在毛细管两端分别加50psi的压力来防止气泡的产生。硫脲中性化合物作为样品用于测定电渗流(EOF)。
日内稳定性测试：在同一根固定化脂质体毛细管整体柱中，重复进样硫脲样品30次，测得EOF的相对标准偏差(RSD)为2.61％(n＝30)。结果表明，所制备的固定化脂质体毛细管整体柱具有良好的日内稳定性。
使用寿命测试：固定化脂质体毛细管整体在4℃下保存10天，测得EOF的RSD为14.8％(n＝10)，说明在4℃下柱稳定性较好，使用寿命至少在10天以上。
管间重复性测试：按相同制备方法制备4根固定化脂质体毛细管整体柱。分别测定4根整体柱的EOF，每根以重复测定10次所得的平均值作为其EOF。测得4根整体柱EOF的RSD为3.07％(n＝4)，表明管间重复性好。
以上结果表明：本发明制备的固定化脂质体毛细管整体柱的稳定性高，本发明提供的整体柱制备方法的重复性好。
实施例4
固定化脂质体毛细管整体柱对中性化合物的分离。
将实施例2制得的固定化脂质体毛细管整体柱用于分离四个系列的中性化合物。电色谱分离条件为：毛细管柱总长为40.2cm，其中有效长度为30cm；电泳缓冲液为pH 7.4的20mM磷酸盐缓冲液；分离电压20kV；柱温20±2℃；电动进样5kV×3s；检测波长214nm。
如图1～图4所示，固定化脂质体毛细管整体柱对四个系列的中性化合物均表现出了较强的分离能力，分离选择性显著优于毛细管硅胶整体柱和固定化脂质体毛细管开管柱。
毛细管硅胶整体柱柱容量高，但对中性物质的分离能力不强。固定化脂质体毛细管开管柱的脂质体固定量小，导致对物质的分离能力和保留能力都非常有限、各物质保留行为的差异也不显著。固定化脂质体毛细管整体柱不仅柱容量高，而且脂质体固定量大，因此对中性物质的分离性能和保留能力更优越，对各物质保留行为的差异更明显，表征物质与生物膜之间相互作用强弱的能力更强大。表1列出了中性物质分别在固定化脂质体毛细管开管柱(OT-ILCC)和整体柱(MS-ILCC)上的分离度和保留因子(k)。
表1中性物质在OT-ILCC和MS-ILCC上的分离度与保留因子
如图1～图4所示，两种萘酚异构体在毛细管硅胶整体柱上完全不能被分离，在固定化脂质体毛细管开管柱上仅能被部分分离，但在固定化脂质体毛细管整体柱上能达到基线分离。四种烷基苯在毛细管硅胶整体柱和固定化脂质体毛细管开管柱上均完全不能被分离，而在固定化脂质体毛细管整体柱上表现出较好的分离度。五种甾体激素类物质在毛细管硅胶整体柱和固定化脂质体毛细管开管柱上只能被部分分离，而在固定化脂质体毛细管整体柱上表现出较好的分离效果。五种氨基甲酸酯类在毛细管硅胶整体柱上不能完全被分离，在固定化脂质体毛细管开管柱上完全不能被分离，而在固定化脂质体毛细管整体柱上的分离效果较好。
实施例5
固定化脂质体毛细管整体柱对蛋白质的分离。
电色谱分离条件为：毛细管柱总长为40.2cm，其中有效长度为30cm；电泳缓冲液为pH值7.4的20mmol/L磷酸盐和68mmol/L NaCl的混合溶液；分离电压20kV；柱温20±2℃；电动进样5kV×3s；检测波长214nm。
如图5所示，本发明的固定化脂质体毛细管整体柱能对溶菌酶、胰蛋白酶、牛血清白蛋白三种蛋白实现基线分离。而毛细管硅胶整体柱对蛋白质有着强烈的吸附作用，三种蛋白质在毛细管硅胶整体柱上60min内都未能出峰，无法对蛋白质进行分离。结果表明固定化脂质体毛细管整体柱可以显著降低蛋白质在毛细管硅胶整体柱上的吸附作用，因而可实现对蛋白质的分离。
综上所述，本发明所制备的固定化脂质体毛细管整体柱的脂质体固定量大(可达395.98nmol/m)，性能稳定；本发明提供的固定化脂质体毛细管整体柱的制备方法重复性好，管间重复性为3.07％(RSD,n＝4)；分离中性化合物和蛋白质效果好。
以上所述，仅为本发明较佳的具体实施方式，但本发明保护的范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内所做的任何修改，等同替换和改进等，均应包含在本发明的保护范围之内。
Claims (7)
1.一种固定化脂质体毛细管整体柱的制备方法，其特征在于，包括以下步骤：
(1)制备毛细管硅胶整体柱；
(2)将制得的毛细管硅胶整体柱在加压下先用4-羟乙基哌嗪乙磺酸(HEPES)缓冲液第一次冲洗，然后用脂质体溶液冲洗，两端封口，室温放置至其反应完毕，最后再用4-羟乙基哌嗪乙磺酸缓冲液第二次冲洗毛细管柱，即完成制备。
2.根据权利要求1所述的固定化脂质体毛细管整体柱的制备方法，其特征在于：所述步骤(2)中HEPES缓冲液的浓度为25mmol/L，pH为7.40；所述加压冲洗的压力为50psi。
3.根据权利要求1所述的固定化脂质体毛细管整体柱的制备方法，其特征在于：所述步骤(2)中HEPES缓冲液第一次冲洗时间为30min。
4.根据权利要求1所述的固定化脂质体毛细管整体柱的制备方法，其特征在于：所述步骤(2)中脂质体溶液冲洗时间为30min,室温放置反应时间为30min。
5.根据权利要求1所述的固定化脂质体毛细管整体柱的制备方法，其特征在于：所述步骤(2)中HEPES缓冲液第二次冲洗时间为10min。
6.根据权利要求1所述的固定化脂质体毛细管整体柱的制备方法，其特征在于：所述步骤(2)中脂质体溶液是以磷脂酰胆碱为磷脂成分，以HEPES缓冲液为分散溶剂，采用薄膜分散-探针超声法制备而得；脂质体的平均粒径约为37nm，多分散系数约为0.34，磷脂含量为5mmol/L。
7.一种利用权利要求1所述的固定化脂质体毛细管整体柱的制备方法制备的脂质体毛细管整体柱，其特征在于：所述固定化脂质体毛细管整体柱用于中性化合物和蛋白质的分离。
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