自由流电泳进样技术及其应用的研究
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摘要
自由流电泳(Free-Flow Electrophoresis, FFE)是一种兼具制备和分析功能的纯液相电泳，具有反应过程可控、条件温和、回收率较高、分辨率良好、生物活性保持完好且应用广泛等优点。FFE发展至今，主要的研究重点和难点是如何在缩窄样品带宽，改善分辨率的同时增加通量。目前的解决方法主要是：(1)减小样品的初始带宽；(2)改变FFE的运行模式(如间断式FFE的引入)；(3)使用聚焦的方式(如IEF)。但是相关的研究却存在一些问题：(1)减小样品的初始带宽会导致通量的损失，使FFE失去制备的意义；(2)至今没有阐明间断式FFE模式与连续式FFE模式相比能否在相同通量条件下具有更好的分辨率；(3)聚焦不仅需要大量的两性电解质，使得分离制备成本大幅上涨，而且两性物质(如蛋白质)在聚焦位置(即等电点处)溶解度大幅下降，使样品易损失。基于前两个问题，本文做了一些样品带宽控制方面的初步研究。
在开展实验之前，我们对已经搭建的重力自平衡-自由流电泳装置做了一系列改进，包括：(1)更换自平衡收集器的材质；(2)电极室缩短；(3)减小收集导管死体积；(4)加厚分离腔上下板；(5)在分离腔上板设置冷却挡板。经过改进后的整个装置占地空间由原来的80cm×100cm×160cm缩小为现在的70cm×85cm×45cm。
本文中，我们开展了如下一系列实验工作：
(1)研究如何利用样品/背景流量之比控制样品带宽、分辨率和载量。这里我们选用了两种进样方式；其一为保持背景缓冲液流量不变，改变样品进样流量；其二为保持样品进样流量不变，改变背景缓冲液流量。利用含有结晶紫的商品化甲基绿染料作为可视化样本，我们发现通过调节样品和背景缓冲液流量的比率可以很好的控制混合染料(甲基绿与结晶紫)的带宽、分辨率和通量。这些结果对于今后FFE装置的设计有着极为重要的意义。
(2)在相同通量的情况下，定量比较间断式FFZE和连续式FFZE在分辨率提高方面的差异。实验中，我们仍然采用了含有结晶紫的商品化甲基绿染料作为可视化样本，同时严格控制了4个实验参数，即相同的模式染料载量(3.49、1.75、1.17、0.88mg/h)，相同的运行时间(5、10、15、20min)，相同的样品/背景流量之比(=10.64×10~(-3))、相同的电压(=200V)。实验结果表明在连续式模式中带宽受流体动力学影响发生明显拓宽，而间断式模式可以显著消除流体动力学引起的拓宽，极大增加染料的分辨率。最后，间断式FFZE被成功应用于两种模式抗生素(PCA和Plt，二者共表达于一种新型菌株Pseudomonas aeruginosa M18的发酵液中)的完全分离。这一结果可能会改变今后FFE的运行模式。
(3)利用重力自平衡-自由流电泳装置分离制备Pseudomonas sp. M18发酵液中提取的低浓度吩嗪-1-羧酸(PCA)。从Pseudomonas sp. M18发酵液中提取大约0.3mM的低浓度PCA作为FFE分离纯化的初始样本。实验考察了3个影响分离效果的因素，即背景缓冲液pH和浓度，以及冷却循环系统。从实验结果我们可以发现不管有没有冷却循环系统，背景缓冲液pH和浓度对于PCA的分离影响都非常有限。相较而言，冷却循环系统对分离效果却有着非常大的影响。如果没有冷却循环系统，PCA的带宽控制将会非常困难，如果有则会变得容易。通过实验我们得到最佳实验条件为：10mM pH5.5磷酸缓冲液作为背景缓冲液，30mM pH5.5磷酸缓冲液作为电极缓冲液，5.46ml/min背景缓冲液流量，10min保留时间，500V电压。此时，PCA可以连续不断的获得较高产量的制备，所得回收率可达85%，通量约为7ml/h (115μl/min的样品流量)。实验结果表明FFE技术可以成为研究天然生物农药小分子的重要替代手段。
通过一系列的实验研究，我们发现：(1)可以通过控制样品/背景流量之比来控制样品的分辨率和载量；(2)可以应用间断式FFZE获得样品分辨率的大幅提高，而不损失样品通量；(3)可以将我们自制的FFE应用于一些难溶于水、难电离的物质。这些结果将有利于今后我们实验室FFE的进一步研制及应用的拓展。
Free-Flow Electrophoresis (FFE) is a pure-liquid electrophoresis for bothpreparative and analytical purposes with the advantages of controllable process,gentle condition, high recovery, well resolution, complete preservation of activity, andwide applications. Until now, the development of FFE is to focus on how to narrowthe bandwidth, improve the resolution and increase the throughput. The currentsolutions are to (i) decrease the initial sample bandwidth;(ii) change the FFE runningmode (e.g. the introduction of interval FFE);(iii) use the focusing method (such asIEF). However, there are some problems remained.(i) To decrease the initial samplebandwidth can lower the sample throughput in no accordance with the preparativepurpose of FFE.(ii) The resolution of interval FFE mode has not been reported to bemore superior to that of continuous one at the same throughput.(iii) The focusingmethod does not only need a lot of ampholytes, making the cost higher, but also loseamphoteric substances (such as proteins) at their pIs. Based on the previous twoproblems, this paper has done some prelimilary works on the bandwidth controlling.
Before experiment, we made a series of modifications on the previousconstructed FFE device with gratis gravity, including (i) the material of self-balancecollector (SBC) were changed,(ii) the lengths of electrode cavities were shortened;(iii) the dead volume of collection tubes were reduced;(iv) the up and down plateswere thickened; and (v) the cooling barriers were set on the up plate. Thus, the size ofwhole device (70cm×85cm×45cm) was greatly reduced in contrast to theprevious version of FFE apparatus (80cm×100cm×160cm).
After the device modifications, we carried out the following experiments:
(1) It was studied on how to use the flux ratio between the sample andbackground solutions to control the sample bandwidth, resolution and throughput.Two injection methods were described herein. The first method was the one in whichsample injection flux were variable, whereas the background flux was a constantvalue. The second was the one in which the background flux were flexible, while the sample flux was stable. With the help of methyl green and crystal violet as twoviewable model compounds, we found that the bandwidth, resolution and throughputof mixed dyes could be under better control by the adjustment of flux ratios betweensample and background flux. The results were of significance to the future designs onour newly-developed FFE device with gratis gravity.
(2) Under the same throughput, the resolution promotion of interval FFZE andcontinuous FFZE were compared quantitatively. In this paper, a commercial dye withmethyl green and crystal violet was well chosen as the model sample. Thecomparative experiments were conducted under the same sample loading of the modeldye (viz.3.49,1.75,1.17and0.88mg/h), the same running time (viz.5,10,15and20min), the same flux ratio between sample and background buffer (=10.64×10~(-3)) andthe same voltage (=200V). Under the given conditions, the relevant experimentsdemonstrated that the band broadening was evidently caused by hydrodynamic factorin continuous FFZE mode, and the interval mode ccould clearly eliminate thehydrodynamic broadening existing in the continuous mode, greatly resulting in theresolution increase of dye separation in FFZE. Finally, the interval FFZE wassuccessfully used for the complete separation of two model antibiotics (hereinpyoluteorin and phenazine-1-carboxylic acid co-existing in fermentation broth of anew strain Pseudomonas aeruginosa M18). These results might change FFE runningmode in the future.
(3) The low concentration PCA (=0.3mM) extracted from fermentation brothof Pseudomonas sp. M18was selected to be isolated and prepared with a newly facileFFE device with gratis gravity. Three factors of pH value and concentration ofbackground buffer, and the cooling circle of FFE device were investigated for thepurification of PCA in the device. It was found that the pH value and concentration ofbackground buffer had mild influences on the separation of PCA whether withcooling circle or not. However, the cooling circle had a much greater impact on theseparation of PCA. The controlling of the band zone of PCA in FFE chamber wouldbe difficult if without cooling circle, while the controlling would become easy if withcooling circle. Under the optimal conditions (10mM pH5.5phosphate as background buffer,30mM pH5.5phosphate buffer as electrode solution,5.46mL/minbackground flux,10min residence time of injected sample, and500V), PCA couldbe continuously prepared from its impurities with relative high purity. The flux ofsample injection was115μL/min, viz.7mL sample throughput per hour, and therecovery was up to85%. All of the experiments indicated that FFE was a goodalternative tool for the study on natural biological control agents.
According to a series of experiments, we concluded that (i) the sample resolutionand throughput could be controlled by the flux ratio of sample and backgroundsolutions;(ii) the application of interval FFZE could increase largely the sampleresolution without the loss of the sample throughput;(iii) the substances difficult todissolve and ionize could be separated in our homemade device. All these resultswould be beneficial to the deep investigations and extension of applications in ourFFE in the future.
在开展实验之前，我们对已经搭建的重力自平衡-自由流电泳装置做了一系列改进，包括：(1)更换自平衡收集器的材质；(2)电极室缩短；(3)减小收集导管死体积；(4)加厚分离腔上下板；(5)在分离腔上板设置冷却挡板。经过改进后的整个装置占地空间由原来的80cm×100cm×160cm缩小为现在的70cm×85cm×45cm。
本文中，我们开展了如下一系列实验工作：
(1)研究如何利用样品/背景流量之比控制样品带宽、分辨率和载量。这里我们选用了两种进样方式；其一为保持背景缓冲液流量不变，改变样品进样流量；其二为保持样品进样流量不变，改变背景缓冲液流量。利用含有结晶紫的商品化甲基绿染料作为可视化样本，我们发现通过调节样品和背景缓冲液流量的比率可以很好的控制混合染料(甲基绿与结晶紫)的带宽、分辨率和通量。这些结果对于今后FFE装置的设计有着极为重要的意义。
(2)在相同通量的情况下，定量比较间断式FFZE和连续式FFZE在分辨率提高方面的差异。实验中，我们仍然采用了含有结晶紫的商品化甲基绿染料作为可视化样本，同时严格控制了4个实验参数，即相同的模式染料载量(3.49、1.75、1.17、0.88mg/h)，相同的运行时间(5、10、15、20min)，相同的样品/背景流量之比(=10.64×10~(-3))、相同的电压(=200V)。实验结果表明在连续式模式中带宽受流体动力学影响发生明显拓宽，而间断式模式可以显著消除流体动力学引起的拓宽，极大增加染料的分辨率。最后，间断式FFZE被成功应用于两种模式抗生素(PCA和Plt，二者共表达于一种新型菌株Pseudomonas aeruginosa M18的发酵液中)的完全分离。这一结果可能会改变今后FFE的运行模式。
(3)利用重力自平衡-自由流电泳装置分离制备Pseudomonas sp. M18发酵液中提取的低浓度吩嗪-1-羧酸(PCA)。从Pseudomonas sp. M18发酵液中提取大约0.3mM的低浓度PCA作为FFE分离纯化的初始样本。实验考察了3个影响分离效果的因素，即背景缓冲液pH和浓度，以及冷却循环系统。从实验结果我们可以发现不管有没有冷却循环系统，背景缓冲液pH和浓度对于PCA的分离影响都非常有限。相较而言，冷却循环系统对分离效果却有着非常大的影响。如果没有冷却循环系统，PCA的带宽控制将会非常困难，如果有则会变得容易。通过实验我们得到最佳实验条件为：10mM pH5.5磷酸缓冲液作为背景缓冲液，30mM pH5.5磷酸缓冲液作为电极缓冲液，5.46ml/min背景缓冲液流量，10min保留时间，500V电压。此时，PCA可以连续不断的获得较高产量的制备，所得回收率可达85%，通量约为7ml/h (115μl/min的样品流量)。实验结果表明FFE技术可以成为研究天然生物农药小分子的重要替代手段。
通过一系列的实验研究，我们发现：(1)可以通过控制样品/背景流量之比来控制样品的分辨率和载量；(2)可以应用间断式FFZE获得样品分辨率的大幅提高，而不损失样品通量；(3)可以将我们自制的FFE应用于一些难溶于水、难电离的物质。这些结果将有利于今后我们实验室FFE的进一步研制及应用的拓展。
Free-Flow Electrophoresis (FFE) is a pure-liquid electrophoresis for bothpreparative and analytical purposes with the advantages of controllable process,gentle condition, high recovery, well resolution, complete preservation of activity, andwide applications. Until now, the development of FFE is to focus on how to narrowthe bandwidth, improve the resolution and increase the throughput. The currentsolutions are to (i) decrease the initial sample bandwidth;(ii) change the FFE runningmode (e.g. the introduction of interval FFE);(iii) use the focusing method (such asIEF). However, there are some problems remained.(i) To decrease the initial samplebandwidth can lower the sample throughput in no accordance with the preparativepurpose of FFE.(ii) The resolution of interval FFE mode has not been reported to bemore superior to that of continuous one at the same throughput.(iii) The focusingmethod does not only need a lot of ampholytes, making the cost higher, but also loseamphoteric substances (such as proteins) at their pIs. Based on the previous twoproblems, this paper has done some prelimilary works on the bandwidth controlling.
Before experiment, we made a series of modifications on the previousconstructed FFE device with gratis gravity, including (i) the material of self-balancecollector (SBC) were changed,(ii) the lengths of electrode cavities were shortened;(iii) the dead volume of collection tubes were reduced;(iv) the up and down plateswere thickened; and (v) the cooling barriers were set on the up plate. Thus, the size ofwhole device (70cm×85cm×45cm) was greatly reduced in contrast to theprevious version of FFE apparatus (80cm×100cm×160cm).
After the device modifications, we carried out the following experiments:
(1) It was studied on how to use the flux ratio between the sample andbackground solutions to control the sample bandwidth, resolution and throughput.Two injection methods were described herein. The first method was the one in whichsample injection flux were variable, whereas the background flux was a constantvalue. The second was the one in which the background flux were flexible, while the sample flux was stable. With the help of methyl green and crystal violet as twoviewable model compounds, we found that the bandwidth, resolution and throughputof mixed dyes could be under better control by the adjustment of flux ratios betweensample and background flux. The results were of significance to the future designs onour newly-developed FFE device with gratis gravity.
(2) Under the same throughput, the resolution promotion of interval FFZE andcontinuous FFZE were compared quantitatively. In this paper, a commercial dye withmethyl green and crystal violet was well chosen as the model sample. Thecomparative experiments were conducted under the same sample loading of the modeldye (viz.3.49,1.75,1.17and0.88mg/h), the same running time (viz.5,10,15and20min), the same flux ratio between sample and background buffer (=10.64×10~(-3)) andthe same voltage (=200V). Under the given conditions, the relevant experimentsdemonstrated that the band broadening was evidently caused by hydrodynamic factorin continuous FFZE mode, and the interval mode ccould clearly eliminate thehydrodynamic broadening existing in the continuous mode, greatly resulting in theresolution increase of dye separation in FFZE. Finally, the interval FFZE wassuccessfully used for the complete separation of two model antibiotics (hereinpyoluteorin and phenazine-1-carboxylic acid co-existing in fermentation broth of anew strain Pseudomonas aeruginosa M18). These results might change FFE runningmode in the future.
(3) The low concentration PCA (=0.3mM) extracted from fermentation brothof Pseudomonas sp. M18was selected to be isolated and prepared with a newly facileFFE device with gratis gravity. Three factors of pH value and concentration ofbackground buffer, and the cooling circle of FFE device were investigated for thepurification of PCA in the device. It was found that the pH value and concentration ofbackground buffer had mild influences on the separation of PCA whether withcooling circle or not. However, the cooling circle had a much greater impact on theseparation of PCA. The controlling of the band zone of PCA in FFE chamber wouldbe difficult if without cooling circle, while the controlling would become easy if withcooling circle. Under the optimal conditions (10mM pH5.5phosphate as background buffer,30mM pH5.5phosphate buffer as electrode solution,5.46mL/minbackground flux,10min residence time of injected sample, and500V), PCA couldbe continuously prepared from its impurities with relative high purity. The flux ofsample injection was115μL/min, viz.7mL sample throughput per hour, and therecovery was up to85%. All of the experiments indicated that FFE was a goodalternative tool for the study on natural biological control agents.
According to a series of experiments, we concluded that (i) the sample resolutionand throughput could be controlled by the flux ratio of sample and backgroundsolutions;(ii) the application of interval FFZE could increase largely the sampleresolution without the loss of the sample throughput;(iii) the substances difficult todissolve and ionize could be separated in our homemade device. All these resultswould be beneficial to the deep investigations and extension of applications in ourFFE in the future.
引文
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