Label-free microfluidic free-flow isoelectric focusing, pH gradient sensing and near real-time isoelectric point determination of biomolecules and blood plasma fractions.
Label-free microfluidic free-flow isoelectric focusing, pH gradient sensing and near real-time isoelectric point determination of biomolecules and blood plasma fractions.
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DOI：
10.1039/c5an01345c
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发表时间：
2015-10
期刊：
影响因子：
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通讯作者：
E. Poehler;C. Herzog;Carsten Lotter;Simon A. Pfeiffer;D. Aigner;T. Mayr;S. Nagl
中科院分区：
文献类型：
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作者：
E. Poehler;C. Herzog;Carsten Lotter;Simon A. Pfeiffer;D. Aigner;T. Mayr;S. Nagl
我们展示了制造，表征和应用的微流控芯片能够连续电泳分离，通过自由流动等电聚焦（FFIEF）。通过在整个分离床下集成近红外（NIR）荧光pH传感器层，在几秒钟内实现了pH梯度的在线观察和生物分子等电点（pI）的测定。使用用于在266 nm激发下对生物分子的固有荧光成像的光学设置，可以避免标记步骤，并且可以分离、收集和分析天然生物分子的pI。该微芯片成功地用于蛋白质α-乳白蛋白和β-乳球蛋白、血浆蛋白以及抗生素氨苄青霉素和氧氟沙星等电聚焦过程中的分离监测和pH梯度的同时观察。所获得的pI与文献数据吻合良好，证明了该系统的适用性。在微芯片末端从不同级分连续分离15分钟后获得的分离的抗生素的质谱验证了通过微流体等电聚焦的分离，并表明进一步的芯片上或芯片外处理步骤的可能性。
We demonstrate the fabrication, characterization and application of microfluidic chips capable of continuous electrophoretic separation via free flow isoelectric focussing (FFIEF). By integration of a near-infrared (NIR) fluorescent pH sensor layer under the whole separation bed, on-line observation of the pH gradient and determination of biomolecular isoelectric points (pI) was achieved within a few seconds. Using an optical setup for imaging of the intrinsic fluorescence of biomolecules at 266 nm excitation, labelling steps could be avoided and the native biomolecules could be separated, collected and analysed for their pI. The fabricated microchip was successfully used for the monitoring of the separation and simultaneous observation of the pH gradient during the isoelectric focussing of the proteins α-lactalbumin and β-lactoglobulin, blood plasma proteins and the antibiotics ampicillin and ofloxacin. The obtained pIs are in good agreement with literature data, demonstrating the applicability of the system. Mass spectra from the separated antibiotics taken after 15 minutes of continuous separation from different fractions at the end of the microchip validated the separation via microfluidic isoelectric focussing and indicate the possibility of further on- or off-chip processing steps.