CN1232817C - 无载体的电泳装置 - Google Patents
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Abstract
本发明涉及在试样物质的分析物中分离试样物质的无载体的电泳方法。该方法将在多个阶段中进行,即在一个第一阶段中进行试样物质的粗分级及在至少一个第二阶段中进行被粗分级的试样物质的细分级。各阶段可用并列同时方式、串联方式或以并列同时方式与一个串联阶段相组合来实施。
Description
技术领域
本发明涉及在试样物质的分析物中分离试样物质的无载体的电泳方法及实施该方法的电泳装置。
背景技术
公知的无载体的电泳方法FFE通常用电泳装置工作,该电泳装置的分离室仅具有两个分离的电极空间及在这些电极空间之间具有一个分离空间。
然而当该FFE应用在蛋白质学(Proteomics)研究领域中时,必需能在短时间内以尽可能大的分离能力及尽可能多的试样通过量来分离许多不同的试样物质。
但在FFE中,如在通常的分离方法中那样,电泳装置在分离能力及试样通过量方面的同时优化仅在非常窄的界限内可以作到,因为试样物质量的增加将导致分离能力的下降。
分离能力的优化则需要具有尽可能窄及精确的分离室间隙及特殊的分离边界条件、如相对小的线性流速,尽可能长的分离时间及在分离空间的整个宽度上或在分离空间的区域上尽可能多的分级位置,在该分离空间中所感兴趣的试样物质将被分级。但因为线性流速不能任意地降低,需要延长分离时间即相应地增加电极长度。这又意味着同时增加分离室的外部尺寸,这就难以甚至不能作到以所需的精确度制造分离室间隙。
由DE 2215761A1公知了一种电泳装置,它按照电过滤方法工作。该公知的电泳装置具有一个分离室,在该分离室的两侧上设有电极、分级位置及试样配给位置,其中在分离室中设有多个膜片,它们将分离室分成许多彼此相连接的分离空间。这些膜片用作过滤器及可渗透各个待分离的试样种类。
此外由EP 0203713A2公知了在这种电泳装置上对于每个由膜片限定的分离空间设有一个自身的电极对。
发明内容
本发明的任务在于,提出一种无载体的电泳方法及实施该方法的电泳装置,它们允许获得高的分离能力、短的分离时间及大的通过量。
根据本发明,该任务的技术解决方案在于一种用于从分析物中无载体的电泳分离试样物质的电泳装置,所述电泳分离包括:
一个用于试样物质的粗分级的第一分离阶段,在该第一分离阶段中具有一介质入口和一分级出口,
至少一个第二分离阶段,用于试样物质的细分级;其中,至少一个分离阶段的分级出口与另一个分离阶段的介质入口相连,其特征在于:该电泳装置至少具有一个分离室,在该分离室中构成多个分开的分离空间,所述分离空间具有分开的电极、分开的分级出口及分开的试样入口,所述分离空间属于一个或多个分离阶段。
因此,根据本发明,使用两阶段的分离方法取代单阶段的分离方法作到同时优化分离能力及试样通过量,该方法在第一阶段中以达到尽可能多的试样通过量进行很快的预分离或粗分级及接着在至少一个第二阶段中进试样物质的粗分级部分流的合乎目标的细分级。
在粗分级的第一阶段中试样通过量的提高可这样来实现,即对于待分离的分析物借助FFE的分离过程在提高线性流速及同时缩短迁移距离的情况下进行。在此情况下,用于粗分级的分级位置的数目明显地减少,及在仅对一个部分感兴趣的情况下,设置单个分级位置。试样通过量的进一步提高在实施以并列同时的多重处理形式的两阶段方法时可以实现。
附图说明
以下将借助附图来详细描述本发明的特别优选的实施例。其中:
-图1,图2及图3是本发明的三个实施例中的分离室的构成的概要视图,
-图4及图5是在本发明的一个实施例中的分离室的概要剖面图,
-图6及图7是在介质输入及分级区域中的分离室前部分及分离室后部分的结构,
-图8,图9及图10是不同的分级位置及横向流的输入导管的相对空间布置,及
-图11,12及13是借助横向流的流断面的影响。
具体实施方式
在图1,2及3中概要地表示了根据本发明的电泳装置的不同实施例中分离室的三个例子。为了使分离室具有允许以必要的精确度加工分离室间隙的外部尺寸,在分离室中设置了多个分开的分离空间。根据图1,设有四个分开的分离空间,它们带有四个分开的介质输入口及四个分开的分级部分,每个分级部分具有n个分级位置,其中n小于15。图2表示一个具有两个分开的分离空间及两个分开的介质输入口的分离室,在两个分开的分级部分上各具有n个分级位置,其中n大于50。最后图3表示一个分离室,它具有三个分开的分离空间,三个分开的介质输入口,两个具有n1及n3个分级位置的分级部分,其中n1小于15及n3大于50以及另一个分开的具有n2个分级位置的分级部分,其中n2大于15。
视实施形式而定,图1至3中所示的分离空间具有独立的电极,或当在相应的电极空间中使用相同的介质时具有与相邻的分离空间共同的电极。
使用在图1至3中所示的分离室可进行无载体的电泳FFE,用于以至少两阶段方法的形式在其分析物中分离出试样物质,其中在第一阶段中进行试样物质的粗分级,及在至少一个第二阶段中对粗分级的试样物质进行细分级。
该方法能以并列的同时工作或串联工作来实现,其中在图1及2中所示的分离空间可用于并列的同时工作,它们作为用于粗分级的分离室(图1)及用于细分级的分离室(图2)。图3表示用于以三阶段方法形式串联工作的分离室,其中粗分级与两阶段的细分级相串联组合。
在并列工作方式中,可以同时对多个分离空间配给单一试样物质,或可以将不同的试样物质施加到分开的分离空间中。在并列的同时方法中试样物质的分离可实现试样物质通过量的提高试样物质数量的提高。
通过缩短分开的分离空间的宽度可缩短分析物的迁移距离及分离过程能以分离介质及试样物质的高流速来进行。随着分离空间数目的增加,分离室的宽度将明显变小,不过这将引起:仅是粗分级可具有明显高的试样物质通过量。
当具有完全分开的电极空间的分离空间串联地连接时,从一个分离空间到下个分离空间的分离级将在相同的分离条件下继续被分离,及可以由此实现高的分离功率。在串联的分离空间中也可在不同的条件下进行分离,这些条件涉及所使用的分离技术、分离介质和/或通常的电泳分离温度。
分离空间及各个分离空间的技术实施借助上述结构几乎可任意地组合,如下面将描述的。
如在图4及5中所示,一个分离室通常由两个结构单元、即分离室前部分及分离室后部分组成。但在根据本发明的构成中,各个结构单元由多个分开的部件组成,它们概要地表示在图4及图5上。
详细地说,在图4所示的方式中,一个由带有一个硬塑料膜2及一个软塑料膜3的塑料块1组成的分离室前部分与一个由带有一个玻璃膜10及一个软塑料膜11的金属块8组成的分离室后部分彼此通过一个间隔支架5设置。在塑料块1中设有多个-在图示实施例中为四个-电极空间4。在金属块8中设有冷却管9。此外设有多个介质输入口7及多个分级位置6。图5表示被分级的试样的转移12。
因此,图4中分离室前部分的结构单元由一个基本组件、即一个实心的胶质玻璃块1组成,在其中设有至多8个电极空间4和用于介质输入及分级部分的专门方法模件的孔。在该基本组件1上设有一个由硬塑料材料作的薄膜2,其中在对于相应的应用所需的塑料块1的电极空间区域中它具有用于电流传输的孔,及所需的塑料块1的电极空间不会封闭。这也适合于在介质输入口7及分级部分6的区域中为硬塑料膜2的实施例。硬塑料膜2向着分离空间的面可直接地化学改性或在所示方式中由塑料膜3覆盖,后者的表面直接地限定了分离空间并这样地被化学改性,即试样的电渗及吸附作用减到最小程度。
通过所述的基本组件即塑料决1与根据应用可改性的两个塑料膜2,3的组合可以满足所述的所有要求,这些要求涉及所需分离级的数目、分离空间的几何形状及分离过程的相应阶段的具体电泳边界条件。
在图6及7中各详细地表示出分离室前部分及分离室后部分的结构。如图7中所示,分离室后部分由多个层即金属块8、玻璃膜10及软塑料膜11组成。这些层可用不同方式组合,以便优化用于相关应用的分离装置。
分离室后部分的基本组件因此是实心的金属块8,它与一个外部冷却部分连接可实现在电泳分离期间所产生的热的有效散出。金属块8向着分离空间的表面被由玻璃或陶瓷薄膜作的电绝缘薄膜10覆盖,其中电绝缘薄膜被塑料薄膜11覆盖,后者的表面直接地限定分离空间并被化学改性以实现分离过程的优化。通常向着分离空间的塑料膜、即膜3及11在其材料及化学改性的类型上是相同或相似的,但它们在一定的方法组合的情况下也可以不同。
图8,9及10表示可使用在根据本发明的方法中的不同分级模件。图8中的分级组件在该标准实施方式中包括三个用于分级的出口,其中在特别的实施形式中可设置五个或七个分级出口,如在图9及图10中分别所示的。在这些例中,分离介质的流动方向由箭头13给出,及表示出用于横向流的输入口位置14,用于试样物质的n个分级位置15,及用于剩余介质的n+1个出口16。
在工作中,用一个配给泵的相同输送速率输送的两个分开的输送通道各在电极附近的分级出口区域中与分离空间连接,其中视输送泵的转向而定,通过一个连接部分向电极附近的分离空间输送介质,及同时通过第二连接部分从分离空间中以相同容积速率排出介质。通过在电极附近的分离空间中介质的同时输入及排出将改变试样分级位置的区域中的流断面,如在图11,12及13中所示的。
图11表示两个分析物17,18,一个用于横向流的泵21,用于横向流的输入导管20及用于流断面的掩模19。在图11中表示无横向流的流断面。图12表示当开通横向流时的流断面及图13表示当开通横向流但泵21反向转动时的流断面。
以下将描述一个制备的长时间试验,即在两个典型的变换应用中分级模件的工作:
在一种任意的被分离物质的制备离析的情况下,这样地选择两通道泵的输送速率,即可通过为此设置的试样出口中的一个收集待离析的物质。两通道泵的输送速率在制备离析的期间保持不变。如果在试样分级软管中流出的分析物仅用很小的时延被定量地检测,则用于分离方法控制的检测可被如下地利用,即具有最多生成量及纯度的分析物可被离析出来。
但如果当电泳分离期间泵的输送速率连续地变化时,先后相继分离的物质通过试样分离位置被收集。通过输送速率的改变及通过两通道泵转向的改变,所有被分离的试样将相继地通过分级位置被洗提,及接着随着时间控制或峰值控制的收集容器更换输送到检测系统及部分收集器。
当试样边带的局部迁移在试样分级位置的方向上必需达到大于20mm时,增加试样出口的数目是合适的,其中在分离空间的宽度的值大的情况下该数目可任意的增加,以便实现试样洗提的最佳质量。
Claims (10)
1.用于从分析物中无载体的电泳分离试样物质的电泳装置,所述电泳分离包括:
一个用于试样物质的粗分级的第一分离阶段,在该第一分离阶段中具有一介质入口和一分级出口,
至少一个第二分离阶段,用于试样物质的细分级;
其中,至少一个分离阶段的分级出口与另一个分离阶段的介质入口相连,其特征在于:该电泳装置至少具有一个分离室,在该分离室中构成多个分开的分离空间,所述分离空间具有分开的电极、分开的分级出口及分开的试样入口,所述分离空间属于一个或多个分离阶段。
2.根据权利要求1的电泳装置,其特征在于:每一个分离阶段包括多个分离空间,所述分离空间分别构成在一个公共的分离室中。
3.根据权利要求1或2的电泳装置,其特征在于:所述分离阶段被设计成分级能够相互并列地同时进行。
4.根据权利要求1或2的电泳装置,其特征在于:所述分离阶段被设计成相互并列地同时进行的分级能够与串联的分级相组合地进行。
5.根据权利要求1或2的电泳装置,其特征在于:对于相邻的分离空间设置公共的电极。
6.根据权利要求1或2的电泳装置,其特征在于:所述分级出口具有不同数目的分级位置。
7.根据权利要求1或2的电泳装置,其特征在于:与所述分离空间在电极附近的分级位置的区域中相连接的是一个配给泵的两个分开的输送通道,以便输入介质及排出相同容积的介质。
8.根据权利要求1或2的电泳装置,其特征在于:所述分离室由一个前部分及一个后部分构成,这些部分各由多个分开的室部件组成。
9.根据权利要求8的电泳装置,其特征在于:后部分由一个实心金属块组成。
10.根据权利要求9的电泳装置,其特征在于:在金属块中设有冷却管。
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DE10063097.9 | 2000-12-18 | ||
DE10063097A DE10063097B4 (de) | 2000-12-18 | 2000-12-18 | Elektrophoresevorrichtung |
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EP (2) | EP1845370B1 (zh) |
JP (1) | JP4143719B2 (zh) |
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CN (1) | CN1232817C (zh) |
AT (2) | ATE424556T1 (zh) |
AU (2) | AU2002234556B2 (zh) |
CA (1) | CA2431921C (zh) |
DE (3) | DE10063097B4 (zh) |
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2000
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EP1346211B1 (de) | 2007-08-01 |
JP4143719B2 (ja) | 2008-09-03 |
ES2287172T3 (es) | 2007-12-16 |
KR20030062430A (ko) | 2003-07-25 |
EP1346211A2 (de) | 2003-09-24 |
CA2431921C (en) | 2010-11-02 |
DE10063097B4 (de) | 2007-04-19 |
US7491304B2 (en) | 2009-02-17 |
CA2431921A1 (en) | 2002-06-27 |
DE10063097A1 (de) | 2002-07-04 |
DE50112805D1 (de) | 2007-09-13 |
WO2002050524A3 (de) | 2002-12-27 |
AU2002234556B2 (en) | 2007-06-21 |
EP1845370A1 (de) | 2007-10-17 |
ATE424556T1 (de) | 2009-03-15 |
DE50114751D1 (de) | 2009-04-16 |
US20040045826A1 (en) | 2004-03-11 |
CN1481500A (zh) | 2004-03-10 |
WO2002050524A2 (de) | 2002-06-27 |
ATE368851T1 (de) | 2007-08-15 |
EP1845370B1 (de) | 2009-03-04 |
JP2004516472A (ja) | 2004-06-03 |
AU3455602A (en) | 2002-07-01 |
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