Microfluidic high-resolution free-flow isoelectric focusing
Microfluidic high-resolution free-flow isoelectric focusing
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DOI：
10.1021/ac071419b
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发表时间：
2007-11-01
影响因子：
7.4
通讯作者：
Schasfoort, Richard B. M.
中科院分区：
文献类型：
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作者：
Kohlheyer, Dietrich;Eijkel, Jan C. T.;Schasfoort, Richard B. M.
描述了一种用于蛋白质分离的微流控自由流动等电聚焦玻璃芯片。自由流动等电聚焦证明了一组荧光标准品，涵盖了广泛的等电点从pH 3至10，以及蛋白质HSA。相对于早期开发的装置，开发了改进的微流体FFE芯片。改进包括使用多鞘流和引入预分离的两性电解质。预分离的两性电解质通常用于大规模的常规自由流动等电聚焦仪器，但尚未用于微机械装置。此外，通道深度进一步减小。这些调整导致了更高的分离分辨率和峰值容量，这是以前发表的自由流动等电聚焦芯片无法实现的。在1.2和2 mm宽之间产生pH 2.5至11.5的几乎线性的pH梯度。在2.5 s的停留时间和20 V mm（-1）的电场下，7个等电聚焦标记物成功且清晰地分离。使用pI标记物的实验证明，该装置完全能够分离分析物，其等电点的最小差异为Delta（pI）= 0.4。此外，结果表明，甚至可以实现更好的分辨率。等电点的理论最小差异为（pI）= 0.23，导致1.8 mm内的峰容量为29个峰。这是先前发表结果的峰容量的8倍。pI标记物的聚焦导致浓度增加20倍或更高。在分辨率方面的进一步改进似乎是可能的，为此，我们设想电渗流的影响必须进一步降低。微流体自由流动等电聚焦装置的性能将使新的应用成为可能，因为该装置可能用于临床分析，其中通常低样品体积可用并且快速分离时间是必不可少的。
A microfluidic free-flow isoelectric focusing glass chip for separation of proteins is described. Free-flow isoelectric focusing is demonstrated with a set of fluorescent standards covering a wide range of isoelectric points from pH 3 to 10 as well as the protein HSA. With respect to an earlier developed device, an improved microfluidic FFE chip was developed. The improvements included the usage of multiple sheath flows and the introduction of preseparated ampholytes. Preseparated ampholytes are commonly used in large-scale conventional free-flow isoelectric focusing instruments but have not been used in micromachined devices yet. Furthermore, the channel depth was further decreased. These adaptations led to a higher separation resolution and peak capacity, which were not achieved with previously published free-flow isoelectric focusing chips. An almost linear pH gradient ranging from pH 2.5 to 11.5 between 1.2 and 2 mm wide was generated. Seven isoelectric focusing markers were successfully and clearly separated within a residence time of 2.5 s and an electrical field of 20 V mm(-1). Experiments with pI markers proved that the device is fully capable of separating analytes with a minimum difference in isoelectric point of Delta(pI) = 0.4. Furthermore, the results indicate that even a better resolution can be achieved. The theoretical minimum difference in isoelectric point is (pI) = 0.23 resulting in a peak capacity of 29 peaks within 1.8 mm. This is an 8-fold increase in peak capacity to previously published results. The focusing of pI markers led to an increase in concentration by factor 20 and higher. Further improvement in terms of resolution seems possible, for which we envisage that the influence of electroosmotic flow has to be further reduced. The performance of the microfluidic free-flow isoelectric focusing device will enable new applications, as this device might be used in clinical analysis where often low sample volumes are available and fast separation times are essential.