Two-step perpendicular free-solution isoelectric focusing in a microchamber array chip
Two-step perpendicular free-solution isoelectric focusing in a microchamber array chip
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DOI：
10.1039/c004176a
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发表时间：
2010-01-01
期刊：
影响因子：
6.1
通讯作者：
Shimura, Kiyohito
中科院分区：
文献类型：
--
作者：
Ishibashi, Ryo;Kitamori, Takehiko;Shimura, Kiyohito
基于两步垂直等电聚焦（IEF）原理，设计了一种用于小规模蛋白质分离的无溶液微流控芯片装置。微芯片由两个单独的玻璃板组成，每个板的正面蚀刻有100个缺口的正方形阵列。通过改变两个板的相对位置，可以形成10个通道作为交错凹口的范围，并且可以切换到垂直方向，或者可以通过凹口的完美重叠产生100个每个140 nL的腔室。在第一IEF中，载体两性电解质的整个pH范围被分级成10个连续的pH段，并且每个段通过与第一IEF成直角的第二IEF进一步分级成10个级分。该芯片能够在较低的电压和较短的时间内进行IEF分离，与在一个方向上操作的线性装置相比。在最大施加电压500 V下，在10 min的总分离时间内，在不同的室中回收具有小等电点（pI）差异（Δ pI = 0.3）的双标记肽。
A free-solution microfluidic chip device was fabricated for small-scale protein fractionation based on the principle of two-step perpendicular isoelectric focusing (IEF) previously reported in a gel slab. The microchip was composed of two separate glass plates with a square array of 100 indentations etched on the facing side of each plate. By changing the relative position of the two plates, ten channels can be formed as ranges of the staggered indentations and can be switched to the perpendicular direction, or 100 chambers of 140 nL each can be produced by perfect overlapping of the indentations. The entire pH range of the carrier ampholyte was fractionated into 10 successive pH segments in the first IEF, and each of the segments was further fractionated into 10 fractions by the second IEF at a right angle to the first one. This chip enabled IEF fractionation at a lower voltage and in a shorter time, compared with a linear apparatus operated in one direction. Fluorescence-labeled peptides with a small isoelectric point (pI) difference (Delta pI = 0.3) were recovered in different chambers in a total separation time of 10 min at a maximum applied voltage of 500 V.