移动化学反应界面的应用：尿液指纹分析和等电聚焦电泳不稳定性机制的再研究
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摘要
移动化学反应界面(Moving Chemical Reaction Boundary, MCRB)理论可以用于对两性电解质进行在线富集,已有的实验也证明,MCRB理论可以对高盐样品进行有效的物质富集,而不受其中高浓度盐分的影响,这对于众多生物样品如尿液等来说是十分适用的。另一方面,由MCRB理论推导出的判别式可以判断界面移动方向,为等电聚焦动力学的研究提供了新的视角。
本文的主要研究内容是移动化学反应界面理论的应用研究。一方面,利用移动化学反应界面理论并结合毛细管电泳快速、高效、简便、经济和易于自动化的特点,进行了尿液指纹分析的研究。快速、灵敏的尿液指纹分析对发现新的生物标志物和进行临床检测具有重要意义。根据MCRB理论,以Gly-HCl作为样品缓冲液、Gly-NaOH作背景电解质的缓冲液,分别对缓冲液的pH值、浓度、工作电压等实验条件进行了优化。实验结果证明该方法大大提高了尿液指纹分析的灵敏度和数据的容量,与常规方法只能获得10个峰相比,MCRB方法大大改善了尿液指纹分析的灵敏度:观测到了超过90个的富集峰,达到了20倍以上的富集效果。表明MCRB介导的富集方法对尿液分析具有巨大潜在价值。
另一方面,对大量文献材料中报道的有关等电聚焦中pH梯度不稳定性的实验(包括计算机模拟实验),采用MCRB理论衍生得到的判别式Rr进行分析和预测,并与实际的结果相对照。结果表明,在阴极漂移中,Rr均大于零,即阳极液中氢离子的迁移数大于阴极液中氢氧根离子的迁移数;而在阳极漂移中,Rr均小于零,即阴极液中氢氧根离子的迁移数大于阳极液中质子的迁移数。实验结果与MCRB的理论是完全一致的,从而揭示出MCRB理论在揭示等电聚焦中pH梯度不稳定现象的动力学方面的正确性和价值。这一结果对提高等电聚焦电泳的稳定性意义重大,而等电聚焦正是双向电泳和蛋白质组学研究中的重要工具。
本文的最后一部分,选用硼砂作为缓冲液,以α-萘胺及NaBH3CN作为衍生化试剂,探索了采用场放大进样方法,用毛细管电泳对葡萄糖和果糖进行分离和富集的实验条件,最优的实验条件为60 mM,pH 11.0硼砂缓冲液,1.0 psi压力进样90s,和常规方法相比,葡萄糖的检测灵敏度提高了20多倍,果糖提高了近60倍。
The Moving Chemical Reaction Boundary (MCRB) theory can be utilized for the on-line stacking of zwitterions. It had also been manifested that the MCRB technique can achieve effective stacking in a high-salt matrix, which is applicable to biofluids such as urine. On the other hand, the judgment expressions derived from MCRB theory can be used to determine the moving direction of the boundary, which provides new clues for the study of Isoelectric Focusing (IEF) dynamics.
Rapid and sensitive profiling of urine holds evident significance for discovery of new biomarkers and for clinical diagnosis. A simple, rapid and sensitive moving chemical reaction boundary (MCRB)-induced stacking technique was described herein for the first time for the analysis of urine profiling by CE. The carefully designed MCRB was formed with acidic Gly-HCl sample buffer and alkaline Gly-NaOH running buffer. The experimental conditions such as the pHs, concentrations of the buffers and the applied voltage were optimized. Compared with the conventional CZE with only ten observable peaks and poor sensitivity, the MCRB could significantly improve the sensitivity of urine profiling: More than 90 stacking peaks had been observed and more than 20 fold sensitivity enhancement achieved. The results indicated that the MCRB-induced stacking is a useful and potential tool for urine profiling.
The MCRB theory was also applied to re-investigate the instability of natural pH gradient of ampholytes in classic IEF. By using simple judgment expressions, the Rr value was used for the comparative analyses of the transference numbers of hydrogen ions in the acidic anolyte and hydroxyl ions in the alkaline catholyte in numerous IEF experiments cited from references. The re-investigations showed that (1) in the runs of cathodic drifting of pH gradient, all the values of Rr were evidently or slightly higher than the key important value of zero, namely the transference number of hydrogen ions in the anolyte was greater than that of hydroxyl ions in the catholyte; (2) conversely, in the anodic drifting, Rr < 0, namely the transference number of hydroxyl ions in the catholyte was over that of hydrogen ions in the anolyte. The paper supplied a novel viewpoint for the study of the instability of pH gradient in classic IEF.
In the last part, the optimal experimental conditions for the simutaneous stacking of glucose and fructose were investigated. Sodium borate was chosen as the buffer andα- naphthylamine and NaBH3CN as the derivation agents. The optimal conditions were 60 mM pH 11 sodium borate buffer, 1.0 psi pressure injection for 90s. Compared with the normal CZE method, the detection sensitivity of glucose was enhanced by more than 20 times, and that of fructose nearly 60 times.
本文的主要研究内容是移动化学反应界面理论的应用研究。一方面,利用移动化学反应界面理论并结合毛细管电泳快速、高效、简便、经济和易于自动化的特点,进行了尿液指纹分析的研究。快速、灵敏的尿液指纹分析对发现新的生物标志物和进行临床检测具有重要意义。根据MCRB理论,以Gly-HCl作为样品缓冲液、Gly-NaOH作背景电解质的缓冲液,分别对缓冲液的pH值、浓度、工作电压等实验条件进行了优化。实验结果证明该方法大大提高了尿液指纹分析的灵敏度和数据的容量,与常规方法只能获得10个峰相比,MCRB方法大大改善了尿液指纹分析的灵敏度:观测到了超过90个的富集峰,达到了20倍以上的富集效果。表明MCRB介导的富集方法对尿液分析具有巨大潜在价值。
另一方面,对大量文献材料中报道的有关等电聚焦中pH梯度不稳定性的实验(包括计算机模拟实验),采用MCRB理论衍生得到的判别式Rr进行分析和预测,并与实际的结果相对照。结果表明,在阴极漂移中,Rr均大于零,即阳极液中氢离子的迁移数大于阴极液中氢氧根离子的迁移数;而在阳极漂移中,Rr均小于零,即阴极液中氢氧根离子的迁移数大于阳极液中质子的迁移数。实验结果与MCRB的理论是完全一致的,从而揭示出MCRB理论在揭示等电聚焦中pH梯度不稳定现象的动力学方面的正确性和价值。这一结果对提高等电聚焦电泳的稳定性意义重大,而等电聚焦正是双向电泳和蛋白质组学研究中的重要工具。
本文的最后一部分,选用硼砂作为缓冲液,以α-萘胺及NaBH3CN作为衍生化试剂,探索了采用场放大进样方法,用毛细管电泳对葡萄糖和果糖进行分离和富集的实验条件,最优的实验条件为60 mM,pH 11.0硼砂缓冲液,1.0 psi压力进样90s,和常规方法相比,葡萄糖的检测灵敏度提高了20多倍,果糖提高了近60倍。
The Moving Chemical Reaction Boundary (MCRB) theory can be utilized for the on-line stacking of zwitterions. It had also been manifested that the MCRB technique can achieve effective stacking in a high-salt matrix, which is applicable to biofluids such as urine. On the other hand, the judgment expressions derived from MCRB theory can be used to determine the moving direction of the boundary, which provides new clues for the study of Isoelectric Focusing (IEF) dynamics.
Rapid and sensitive profiling of urine holds evident significance for discovery of new biomarkers and for clinical diagnosis. A simple, rapid and sensitive moving chemical reaction boundary (MCRB)-induced stacking technique was described herein for the first time for the analysis of urine profiling by CE. The carefully designed MCRB was formed with acidic Gly-HCl sample buffer and alkaline Gly-NaOH running buffer. The experimental conditions such as the pHs, concentrations of the buffers and the applied voltage were optimized. Compared with the conventional CZE with only ten observable peaks and poor sensitivity, the MCRB could significantly improve the sensitivity of urine profiling: More than 90 stacking peaks had been observed and more than 20 fold sensitivity enhancement achieved. The results indicated that the MCRB-induced stacking is a useful and potential tool for urine profiling.
The MCRB theory was also applied to re-investigate the instability of natural pH gradient of ampholytes in classic IEF. By using simple judgment expressions, the Rr value was used for the comparative analyses of the transference numbers of hydrogen ions in the acidic anolyte and hydroxyl ions in the alkaline catholyte in numerous IEF experiments cited from references. The re-investigations showed that (1) in the runs of cathodic drifting of pH gradient, all the values of Rr were evidently or slightly higher than the key important value of zero, namely the transference number of hydrogen ions in the anolyte was greater than that of hydroxyl ions in the catholyte; (2) conversely, in the anodic drifting, Rr < 0, namely the transference number of hydroxyl ions in the catholyte was over that of hydrogen ions in the anolyte. The paper supplied a novel viewpoint for the study of the instability of pH gradient in classic IEF.
In the last part, the optimal experimental conditions for the simutaneous stacking of glucose and fructose were investigated. Sodium borate was chosen as the buffer andα- naphthylamine and NaBH3CN as the derivation agents. The optimal conditions were 60 mM pH 11 sodium borate buffer, 1.0 psi pressure injection for 90s. Compared with the normal CZE method, the detection sensitivity of glucose was enhanced by more than 20 times, and that of fructose nearly 60 times.
引文
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