Microsecond analysis of transient molecules using bi-directional capillary electrophoresis.
Microsecond analysis of transient molecules using bi-directional capillary electrophoresis.
复制标题
使用双向毛细管电泳对瞬态分子进行微秒分析。
DOI：
10.1021/ac901283y
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发表时间：
2009
影响因子：
7.4
通讯作者：
Shear,JasonB
中科院分区：
文献类型：
--
作者：
Ritschdorff,EricT;Plenert,MatthewL;Shear,JasonB
我们证明了通过诱导新生的，带相反电荷的光化学产物从它们的产生点向相反方向迁移来最大限度地减少瞬态化学物质的电泳分析时间的可行性。在这种方法中，单独的探针位点位于电泳通道内，位于由高数值孔径光学器件形成的光反应位点的高场和低场，带正电荷（并且在某些情况下为中性）的组分朝向一个探针位点迁移，带负电荷的物质在相反的方向上朝向第二探针位点迁移。作为概念验证，羟基吲哚、5-羟色胺（血清素）、5-羟基色氨酸和5-羟基吲哚-2-羧酸的荧光光产物在几何修饰的毛细管内形成，并通过电泳和电泳运输到几微米远的探针位点。虽然可以在单个通道中检测所有组分，或者使用双通道成像方法来独立地检测阳性和阴性组分，但是我们发现最快速的分析方法涉及其中激光以高频（1 kHz）交替地指向相对的探针位点的方案，该策略允许在没有串扰的情况下检测阳性和阴性物质，即使当组件具有重叠的检测时间时。通过对两个序列的交叉指状电泳迹线求和，在每个时间通道上执行重复信号平均。这种方法允许光产物被检测，而不受带相反电荷物质的干扰，使得混合物中的正物质和负物质能够在约100 ° C下被精确地分析。6 μs，比以前使用单向电泳的可行时间快几倍。
We demonstrate the feasibility for minimizing electrophoretic analysis times of transient chemical species by inducing nascent, oppositely charged photochemical products to migrate in opposite directions from their point of creation. In this approach, separate probe sites are positioned within an electrophoretic channel both upfield and downfield from a photoreaction site formed by high-numerical-aperture optics, with positively charged (and in some cases neutral) components migrating toward one probe site and negatively charged species migrating in the opposite direction, toward the second probe site. As a proof-of-concept, fluorescent photoproducts of the hydroxyindoles, 5-hydroxytryptamine (serotonin), 5-hydroxytrptophan, and 5-hydroxyindole-2-carboxylic acid, are formed within a geometrically modified capillary and are transported electrophoretically and electroosmotically to probe sites several micrometers away. Although it is possible to detect all components in a single channel, or to use a two-channel imaging approach to independently detect positive and negative components, we have found the most rapid analysis approach involves a protocol in which laser light is alternately directed to opposing probe sites at high frequency (1 kHz), a strategy that allows positive and negative species to be detected with no cross-talk, even when components have overlapping detection times. Fluorescence-signal-averaging is performed on each temporal channel via summation of the two sequences of interdigitized electrophoretic traces. This approach allows photoproducts to be detected free from interferences from oppositely charged species, enabling positive and negative species in a mixture to be analyzed electrophoretically in ca. 6 μs, a period several-fold faster than was previously feasible using unidirectional electrophoresis.