CN1106403C - 纤维素醚和涂敷过的片状载体材料上固定生物分子的方法 - Google Patents

纤维素醚和涂敷过的片状载体材料上固定生物分子的方法 Download PDF

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CN1106403C
CN1106403C CN98808434A CN98808434A CN1106403C CN 1106403 C CN1106403 C CN 1106403C CN 98808434 A CN98808434 A CN 98808434A CN 98808434 A CN98808434 A CN 98808434A CN 1106403 C CN1106403 C CN 1106403C
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史蒂芬·西格尔
F·勒舍尔
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Abstract

本发明涉及多糖衍生物，它含有a)至少一个憎水的取代基和b)至少一个含氮的取代基，具体地，该衍生物是纤维素醚，它含有作为取代基a)的三烷基甲硅烷基和作为取代基b)的氨基烷基基团。此外，本发明还涉及在涂敷过的片状载体材料上固定生物分子的方法，其中生物分子固定于涂层上或处于涂层内，该涂敷过的片状载体材料在涂层内或之外含有至少一种这样的多糖衍生物。另外，在制备混合纤维素醚的过程中，三烷基甲硅烷基纤维素在有机溶剂内的溶液与不溶或仅微溶于该溶剂的反应性氨基烷衍生物混合，进行反应并分离最终产物。

Description

纤维素醚和在涂敷过的片状载体材料上固定生物分子的方法
本发明涉及新型多糖衍生物例如纤维素醚，即尤其氨基烷基三烷基甲硅烷基纤维素，涉及它们的制备和在限定分子层的表面涂敷方法中的应用。
固态载体上生物分子的固定化在许多现代分析和分离技术例如亲和色谱法、生物反应器技术和尤其生物与化学感应分析中起着决定性的作用。
例如，免疫学测试和杂交测试中的检测由吸附于固态基质表面上的受体分子与要确定的物种的特定反应来实行。尤其在单个DNA、RNA、抗原和/或蛋白质分子的高灵敏性检测领域，所研究的分子特定地和牢固地结合于基质表面上、从而阻止分子在这些基质表面上的非特定吸附，是很重要的。
在固态基质表面上结合生物分子的各种方法是已知的。
简单的选择是吸附固定，其中在基质表面上的结合方式，是单纯的经非共价键的相互作用而产生的吸附。此方法有多种缺点。该固定化限于其表面性能容许吸附性结合并可保证足够稳定性的基质。由于不完全涂敷或解吸附过程可能形成生物分子层的间隙。最终，对取向和受体分子数量两者的控制是不能令人满意的，这样，实现制备的重现性就很困难。
为了避免这些缺点，一直在寻求经共价作用的固定化方法，该方法可使生物分子通过官能团共价作用结合到固态基质上。这些共价方法以与基质表面和生物分子两者都反应的双官能桥试剂为基础。
这些通常分几个步骤进行的固定方法，很耗时间和材料。这些方法的另一缺点是形成不均匀的聚合物表面结构，例如操作中水分存在下所产生的硅烷膜，见例如Joachim Renken等人1996年发表于Anal.Chem.第68卷、第176-182页的文章“不同的免疫吸附剂在声波板模型传感器上的多频评价”。
生物分子固定在固态基质表面上的另一种技术是自组合技术(SA)(见例如Kevin L.Prime等人发表于J.Amer.Chem.Soc.杂志1993年115卷第10714-10721的文章“含有末端固定的环氧乙烷低聚物的表面上蛋白质的吸附：一种采用自组合单层的模型体系”)。这里，有机物质的稳态膜由吸附于固态基质时的分子自发产生的自组合而形成。相传已久的SA体系是金表面上的有机二硫化物和硫醇。该方法的缺点是它受限于仅有的几种基质类型，例如金属或特定的氧化物。
与SA技术有关的是Langmuir-Blodgett(LB，朗缪尔-布洛杰特)技术。如果将合适的物质铺展在水表面上，它们就铺展开形成单分子膜。借助于此种由Langmuir和Blodgett开发的技术，有可能将这些单分子膜转移到固态基质上(见Katharine B.Blodgett等人1937年6月发表于Physical Review第51卷、第964-982页的文章“硬脂酸钡形成的膜及其光学性能”)。此方法所需投入的时间和材料很有限。
采用所谓的非两性分子的Stab-Haar聚合物获得特别有序和稳态的膜(例如M.Schaub等人1992年发表于Thin Solid Films第210/211卷、第397-400页的文章“由X-射线反射研究毛发状的棒状聚合物的超分子结构”)。
在转移到固态基质上的期间里，聚合物棒自身沿平行于涂敷的方向定向。通过聚合物上存在的烯取代基在紫外光照射下发生[2+2]环加成反应而产生的交联，能够得到稳定性特别高的膜(Gerhard Wegner1992年发表于Thin SolidFilms第216卷、第105-116页的文章“聚合物的超薄膜：结构、表征和性能”)。
使用带有烯侧链的纤维素衍生物是极其成功的。在膜转移到基质上之后，它们能够以改进的Lemieux氧化方式转变成羰基，生物分子作为“西佛氏碱”经共价结合到该羰基上(WO-A 95/08770或者Frank Loscher等人1996年发表于Proc.SPIE第2928卷、第209-219的文章)。
另外，还有可能通过改变暴露的时间长短来改变涂层的密度。有可能通过采用双官能桥试剂例如氰尿酰氯，将生物分子经共价作用结合到含有游离氨基或羟基的其他纤维素衍生物上(见上述文献)。
此方法的缺点是它受限制于憎水的和憎水化的基质类型。因而，用憎水LB物质涂敷之前，例如亲水玻璃或石英基质必须在复杂的湿化学步骤中借助于例如硅烷衍生物进行憎水化。
因此，本发明的目的是提供一种方法和适用于该方法的化学化合物，该方法能将至少一层分子施加到种类繁多的表面上，与表面的亲水性无关。
该目的一方面是借助于多糖衍生物、尤其是聚合度≥5的纤维素衍生物来达到，该纤维素衍生物优选混合的包含a)至少一个憎水和b)至少一个含氮的取代基的纤维素醚。
在优选的实施方式中，该混合的纤维素醚带有作为取代基a)的三烷基甲具有1或2个碳原子、取代基b)中的具有2-8个碳原子。另外，多糖衍生物也可以包含c)至少一种另外的携带可由光化学交联的基团的取代基，该基团的交联是通过自由基反应或热进行。
根据本发明，优选的混合纤维素醚可理解为这样的化合物：其中纤维素骨架的各个OH基团的H由有机基团或有机甲硅烷基取代，即直接与O相邻的原子是C或Si。另外，此种表达也能够包括另外携带有其他取代基的衍生物(尤其是在OH基团的O上)，这样的其他取代基的例子是取代基c)。在实际分子中(作为基本资料，见Lothar Brandt发表于Ullmann′s Encyclopedia ofIndustrial Chemistry第A5卷、第二版、第461页以下，关键词“纤维素醚”)，在纤维素醚分子中并非每个单独分子单元(脱水葡萄糖单元)都需要在一个或更多的OH基团上被置换，而是化合物的标识是指整个分子或分子单元，即一个平均值；通常每个分子单元最多能有3个OH基团被取代。对于含有取代基a)或c)(但没有b)的纤维素衍生物的制备和/或性能，参考上述Frank Loscher等人的参考文献和Dieter Klemm等人1984年发表于Z.Chem.第24期第2卷第62页的文章“由有机可溶的三甲基纤维素进行有机可溶的纤维素酯合成的4-二甲基氨基-吡啶催化的纤维素酯的合成”。
本发明的目的进一步还可以这样达到：起始采用已知的在涂敷过的片状载体材料上固定生物分子的方法，在该方法中生物分子固定于涂层上或处于涂层中。在此情况下，本发明的方法的特征在于该涂敷过的片状载体材料在涂层内或涂层外含有至少一种上述的多糖衍生物，尤其一种混合的纤维素醚。
下面更详细地说明本方法的优选实施方式。
向固体表面施加至少一层多糖衍生物的单分子层，这里多糖衍生物优选包含作为取代基a)的憎水取代基，它带有烷基、烯基、芳基、烷基甲硅烷基、烯基甲硅烷基和/或芳基甲硅烷基基团，而且还含有其他的取代基，使得可能采用Langmuir-Blodgett(LB)和/或Langmuir-Blodgett-Schafer(LBS，朗缪尔-布洛杰特-谢弗)技术转移到表面上。
该层能够通过在溶液中温育、由自组合(SA)方法或优选采用Langmuir-Blodgett或Langmuir-Blodgett-Schafer技术施加而成。本发明的多糖衍生物能够结合亲水性和憎水性两种表面。因此，此类型的物质能够被施加和用作表面改性膜。
又可向分子内加入可光聚合或可热聚合的基团例如肉桂酰基，而且也可以加入所有化学行业已知的其他基团来稳定这些层，这是因为它们可在转移之前、期间和之后，通过聚合发生交联从而稳定这些层(开头所述的出版物连同它们所揭示的内容清楚地引入在这里)。
在此，可聚合基团可以连接在上述的多糖衍生物上、或者以与多糖衍生物混合的其它分子的形式存在、被施加于层上或层内。聚合能够在单层内发生，然而，如果许多单层互相叠加，聚合也能够发生于各层的分子之间。
本发明的多糖衍生物用于许多的应用。因此，它们能够用作位于载体材料表面与一层或多层的其他层之间的“粘合促进剂”。另外，能够采用所有已知的方法来施加更多的层，但优选采用Langmuir-Blodgett或Langmuir-Blodgett-Schafer技术施加。合适的更多的层具体地是非两性的Haar-Stab聚合物，特别是含多糖的分子，例如三烷基甲硅烷基纤维素自身如肉桂酸三甲基甲硅烷基纤维素酯，还有其他的衍生物。
这些多组分层的尤其重要的应用是用来在这些表面上固定分子。这里，至少施加到第一层上用作“粘合促进剂”的最上面层，具有使其他分子共价结合的官能团。这些包括例如氨基、醛基、羧酸衍生物和/或能够转化成活性基团的基团例如烯双键，肉桂酸衍生物等。
此外，该类型物质由于存在氨基烷基基团，也适用于分子的直接结合。氨基烷基基团用作亲核试剂，并与携带亲电基团的分子形成共价键。
如果上述类型物质的分子带有甲硅烷基例如三烷基-、三芳基-或三烯基甲硅烷基，那么表面性能能够改性，使甲硅烷基能够在涂敷后脱除，留下羟基。这能够例如通过酸的作用实现。
将本发明方法的实施方式总结一下，较佳的方面是：
-多糖衍生物在实施涂敷之前、期间和之后进行交联，
-通过加入交联剂，使涂层作为整体或单层进行交联，
-涂层含有一层或多层的单层，
-多糖衍生物存在于片状的载体材料以外所有的层中，
-含有多糖衍生物的层是仅有的涂层，
-含有多糖衍生物的层是片状材料与涂层之间的中间层，其中如果合适、涂层也可含有多糖衍生物，
—生物分子结合到多糖衍生物上，和
—多糖衍生物的取代基b)在完成涂敷之后脱除，OH基团重新建立。
本发明尤其好的是给出在载体材料上固定生物分子的方法，其中生物分子固定于涂层上或涂层内，涂敷由LB、LBS或SA技术实施，根据上述说明，给出可光化学交联的和非两性的分子，其中片状载体材料在涂层内或以外含有至少一种混合的纤维素醚。
本发明的混合的纤维素醚能够以这样的方式制备：有机溶剂内的三烷基甲硅烷基纤维素溶液与不溶或仅微溶于该溶剂的反应性氨基烷衍生物混合，进行反应并分离最终产物。
实施例
实施例1
氨基丙基三甲基甲硅烷基纤维素的合成
1克三甲基甲硅烷基纤维素(Tmsc)溶解于50毫升四氢呋喃(THF)和6毫升吡啶的混合物中。将2克固态的1-氨基-3-溴丙烷加入该溶液中。1-氨基-3-溴丙烷在THF中的溶解性差，这抑制了它的聚合反应，因为已溶解的部分立刻进行反应。反应在室温下经过20小时之后，抽真空将产物滤出，用甲醇和水洗涤，并重结晶。将该纤维素衍生物溶解于氯仿中(1微克/1微升)，并铺展于水表面。在25毫牛顿/米的表面压力下，它最后被转移到由玻璃制成的片状的载体上。
实施例2
氨基己基三甲基甲硅烷基纤维素的合成
3.28克6-氨基己酸溶于40毫升的10％(重量)浓度的Na2CO3水溶液和20毫升的二  烷中。在0℃下、15分钟内，搅拌加入6.47克氯甲酸9-芴基甲酯(为了保护氨基酸)的45毫升二烷的溶液。将反应产物加入600毫升水中，用乙醚萃取两次，每次用100毫升。用HCl处理水相，直至混合物轻微地显示酸性反应，接着每次用200毫升乙酸乙酯萃取，用水洗涤有机相，用硫酸镁干燥，并脱除溶剂。
1.04克4-二甲基氨基吡啶和0.5克三甲基甲硅烷基纤维素溶解于50毫升二氯甲烷中。接着快速加入已被保护的氨基酸(按上述方式制备)在40毫升二氯甲烷中的悬浮液，并滴加内含1.75克二环己基碳化二亚胺的10毫升二氯甲烷。室温下搅拌反应混合物2小时，然后浓缩，用200毫升甲醇沉淀出最终产物，用甲醇洗涤，并自二氯甲烷中重结晶。产率为89％。
形成的产物能够以与实施例1所述相似的方式通过形成斜面，即在表面上的流体动力流，或在保持表面压力恒定的同时通过拖出基质(例如玻璃)的方法使用。优选地，实际的涂敷施用到以如上所述的和参考文献例示的方式制成的单层(“粘合促进剂”)上。生物分子(即例如DNA、抗体、抗原、酶类、激素或其他肽类的分子)的固定化能够成功地实施。
下面的结构式旨在用例子说明本发明的产物，图1中，在(单)氨基烷基(二)三烷基甲硅烷基纤维素(在那里显示为脱水葡萄糖单元上完全被取代)中，m≥1(优选2-8)，R1、R2、R3相同或不同，优选为CH3或C2H5，1平均≥5。

Claims (14)

1.一种混合的纤维素醚，它含有a)至少一个憎水的取代基和b)至少含氮的取代基，其中所述的取代基a)是三烷基甲硅烷基，取代基b)是氨基烷基基团，所述纤维素醚的聚合度≥5。
2.如权利要求1所述的纤维素醚，其特征在于取代基a)中的烷基基团具有1或2个碳原子，取代基b)中的烷基基团具有2-8个碳原子。
3.如权利要求1或2所述的纤维素醚，其特征在于还含有c)至少一种另外的取代基，所述取代基携带有可由自由基反应进行光或热化学交联的基团。
4.在涂敷过的片状载体材料上固定生物分子的方法，其中生物分子固定于涂层上或处于涂层内，该方法的特征在于所述涂敷过的片状载体材料在涂层内或以外含有至少一种如权利要求1所述的纤维素醚。
5.如权利要求4所述的方法，其特征在于所述纤维素醚在涂敷实施之前、期间或之后交联。
6.如权利要求4所述的方法，其特征在于所述涂层通过加入交联剂进行交联。
7.如权利要求4或5所述的方法，其特征在于所述涂层含有一层或多层单个层。
8.如权利要求4或5所述的方法，其特征在于所述纤维素醚存在于片状载体材料之外的所有层中。
9.如权利要求4或5所述的方法，其特征在于所述的含有纤维素醚的层是仅有的涂层。
10.如权利要求4或5所述的方法，其特征在于所述的含有纤维素醚的层是片状载体材料与涂层之间的中间层。
11.如权利要求4或5所述的方法，其特征在于所述生物分子结合到纤维素醚上。
12.如权利要求4或5所述的方法，其特征在于所述的纤维素醚的取代基b)在涂敷完成后脱除，并重新建立OH基团。
13.如权利要求4所述的方法，其特征在于所述的涂敷由朗缪尔-布洛杰特、朗缪尔-布洛杰特-谢弗或自组合技术实施，并给出可光化学交联的和非两性的分子。
14.如权利要求10所述的方法，其特征在于所述涂层也可含有纤维素醚。
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