Free-flow electrophoresis (FFE) is an all-liquid-phase electrophoresis technique without any supporting media, which has both analytical and preparative functions. Compared to other electrophoresis techniques, FFE has been used for the separation of peptides, proteins, cells, and microorganisms due to its advantages of mild separation environment, high recovery, and sustainable separation. Both the online detection of the characteristic parameters for each component solution and the real-time control of the progress of the separation experiment are of considerable importance for the study of FFE separation. Since the existing FFE devices do not have the online detection function, there are obvious deficiencies in their practicability. The absence of online detection function not only made it impossible to track the progress of the separation experiment in real time, but also made it difficult to detect the properties of the component solutions, which still require offline testing after separation. In this study, a multi-channel capacitively coupled contactless conductivity detection (MC-C4D) device has been developed to solve this problem, and an automatic measurement software has also been developed. The MC-C4D device used a parallel time-sharing contactless conductivity detection technique. It consisted of several contactless conductivity detection modules arranged in parallel, which in turn consisted of a number of contactless conductivity cells that were switched on/off by analog multiplexers for detecting the conductivity of the solution flowing through the cells in real time. The number of cells was equal to the number of components of the FFE. The components were connected to each of the FFE flow channels, such that the MC-C4D device could be used to measure the conductivity of the solution flowing through each channel in parallel online. To verify the performance of the MC-C4D device, calibration was conducted by using potassium chloride (KCl) standard solutions on MC-C4D device. The experimental data showed that the detection range of MC-C4D was 0.015-2.5 mS/cm, and the limit of detection (LOD) was 0.002 mS/cm. The intra-day relative standard deviation (RSD, n=3) was 2.31%, the measurement relative error (RE) was 3.03%, and the measurement difference between channels was 1.60%. All these data validated that the device had the advantages of wide detection range, low LOD, good repeatability, high accuracy, and low variation between channels. The MC-C 4D device was also applied to reciprocating free-flow isoelectric focusing (RFFIEF) electrophoresis for real-time online detection of the conductivity of each component solution during protein focusing. At the start of isoelectric focusing, when the ions had not reached equilibrium loading in the electric field and the pH gradient had not yet been fully developed, there was little difference in conductivity between the different channels and the channel conductivity curve was relatively flat. As the experiment progressed, the proteins gradually started to enrich the anodic end. As the proteins accumulated towards the isoelectric point, their own net charge gradually decreased, and thus, the conductivity of the solution in the channels near the anodic region also decreased. Under sufficient isoelectric focusing, protein enrichment was evident. In the focusing region, the conductivity of the solution in the corresponding channel decreased further. There was also an increase in the conductivity of the solution in the corresponding channel due to the accumulation of ions near the electrode ends. These results showed that the MC-C4D device not only enabled real-time online detection of the conductivity of each component solution in FFE, but also aided in mastering the progress of separation experiment in RFFIEF, thus improving the practicality of the FFE device. Thus, the MC-C4D device, which had the advantages of good performance, small size, simple circuit system, easy installation and commissioning, and low cost, could play an important role in multi-channel measurement, online inspection, and process monitoring.
现有自由流电泳(FFE)装置因不具备在线检测功能,其实用性仍然存在明显不足。针对这一问题,该工作发展了一种多通道电容耦合式非接触电导检测(MC-C4D)装置并开发了自动测量软件。MC-C4D装置采用了并行分时的非接触电导检测技术,即由多个同样的非接触电导检测模块并行排列,而单个电导检测模块又由多个非接触电导检测池组成,采用模拟开关切换这些检测池,能够分时检测流经相应检测池溶液的电导率。多个电导检测模块的检测池总数等于FFE的组分数,它们分别串行接入到FFE各流路中,这样MC-C4D装置就可在线并行分时在线测量各组分溶液的电导率。为验证所设计MC-C4D装置的检测性能,采用配制的氯化钾标准溶液作为检测对象对MC-C4D装置进行了标定和测试。实验数据表明,MC-C4D装置电导率检测范围为0.015~2.5 mS/cm,检出限(LOD)为0.002 mS/cm,日内相对标准偏差(RSD, n=3)为2.31%,测量相对误差(RE)为3.03%和通道间测量相对偏差为1.60%,这些参数表明该装置检测范围较大,LOD低,重复性好,准确性高,通道间测量相对偏差小。另外,将MC-C 4D装置应用于往复式自由流等电聚焦电泳(RFFIEF)在蛋白质聚焦过程中对各组分溶液电导率进行实时在线检测,结果表明,所开发的MC-C4D装置不仅可实现对FFE各组分溶液电导率的实时在线检测,而且还可在RFFIEF实验中辅助掌握分离的实验进度,提高FFE装置的实用性。
Keywords: capacitively coupled contactless conductivity detection (C4D); free-flow electrophoresis (FFE); multi-channel; online detection; protein separation.