药物与细胞膜相互作用的ppKCE动力学参数

本文在区段-区段动力学毛细管电泳（ppKCE）的理论基础上,以兔红细胞膜、人红细胞膜为受体,首次同时测得了表征药物与细胞膜间结合快慢的正向结合速率常数kon、反向解离速率常数koff、以及配体与受体间相互作用的结合常数Kb。实验中考察了迁移时间及峰高的稳定性,pH值对药物与细胞膜相互作用动力学参数的影响。该方法为评价药物疗效、毒性及药物动力学研究提供一种新的方法。     

Capillary electrophoresis integrated immobilized enzyme reactor for kinetic and inhibition assays of β‐secretase as the Alzheimer's disease drug target

Capillary electrophoresis integrated immobilized enzyme reactors are becoming an increasingly popular alternative for enzyme kinetic and inhibition assays thanks to their unique set of features including cost effectiveness, repeated use of the enzyme, minuscule sample consumption, rapid analysis time and easy automation. In this work we present the development and application of a capillary electrophoresis integrated immobilized enzyme reactor based on magnetic particles for kinetic and inhibition studies of β‐secretase, a key enzyme in the development of Alzheimer's disease and a promising drug target. We document the optimization of the immobilization procedure, characterization of immobilized β‐secretase, optimization of a mutually compatible incubation protocol and separation method as well as the production of the capillary electrophoresis integrated immobilized enzyme reactor. The applicability of the capillary electrophoresis integrated immobilized enzyme reactor was demonstrated by kinetic assay with an unlabelled substrate and by inhibition assays using three structurally different reference inhibitors. The resulting kinetic and inhibition parameters clearly support the applicability of the herein presented method as well as document the fundamental phenomena which need to be taken in account when comparing the results to other methods.     

动力学毛细管电泳法求解蛋白质与单链脱氧核糖核苷酸结合的解离常数及可靠性分析

平衡混合物的非平衡毛细管电泳(NECEEM)可用于测定复合物的解离常数(K_d)。该文以单链脱氧核糖核苷酸结合蛋白(SSB)和单链脱氧核糖核苷酸(ssDNA)相互作用为模型,分析了相互作用强弱的电泳图谱特征,测定了不同条件下复合物的K_d,建立了基于积分偏差的误差分析方法。结果表明,SSB与ssDNA相互作用强弱所致的电泳图差异、SSB与ssDNA的浓度比、毛细管分离温度均影响K_d的计算。此外,基于25℃时SSB与5′-GGTTGGTGTGGTTGG-3′(15mer)人凝血酶适配体作用模型的数值分析结果表明,在现有实验条件下,手动积分峰面积时产生的面积偏差对K_d计算结果有影响,但在10%的偏差区间内,所求K_d值的最大相对偏差小于7%,所求K_d值的误差可忽略。该文证实了基于NECEEM求解动力学相关参数的方法可靠。     

A semipermanent coating for preventing protein adsorption at physiological pH in kinetic capillary electrophoresis

Protein adsorption to the inner capillary wall hinders the use of kinetic capillary electrophoresis (KCE) when studying noncovalent protein-ligand interactions. Permanent and dynamic capillary coatings have been previously reported to alleviate much of the problems associated with protein adsorption. The characteristic limitations associated with permanent and dynamic coatings motivated us to look at a third type of coating - semipermanent. Here, we demonstrate that a semipermanent capillary coating, designed by Lucy and co-workers, comprised of dioctadecyldimethylammonium bromide (DODAB) and polyoxyethylene (POE) stearate, greatly reduces protein adsorption at physiological pH - a necessary requirement for KCE. The coating (i) does not inhibit protein-DNA complex formation, (ii) prevents the adsorption of the analytes, and (iii) supports an electoosmotic flow required for many applications of KCE. The coating was tested in three physiological buffers using a well-known DNA aptamer and four proteins that severely bind to bare silica capillaries as standards. For every protein, a condition was found under which the semipermanent coating effectively suppresses protein adhesion. While no coating can completely prevent the adsorption of all proteins, our findings suggest that the DODAB/POE stearate coating can have a broad impact on CE at large, as it prevents the absorption of several well studied, highly adhesive proteins at physiological pH.     

毛细管电泳与芯片毛细管电泳的双检测技术

综述了毛细管电泳和芯片毛细管电泳的3种双检测技术,包括荧光-散射等光学双检测技术、安培-非接触电导等电化学双检测技术和荧光-非接触电导等光电联用双检测技术.介绍了3种双检测方法的仪器的检测原理及应用,并展望了双检测技术的发展前景.引用文献54篇.     

毛细管电泳在纳米粒子分离和性质表征中的应用进展

毛细管电泳方法被成功用于分离性质不同的纳米粒子,并且可对纳米粒子的表面zeta电势、等电点及表面电荷等表面性质进行表征.本文综述了毛细管电泳法在纳米粒子的分离分析以及其表面性质研究中的应用进展.     

免疫亲和毛细管电泳的研究进展

免疫亲和毛细管电泳方法结合了免疫分析的高特异性和毛细管电泳分离的高效、快速、样品用量少等优点,是复杂样品中特定组分分析的重要方法之一。激光诱导荧光检测器的使用以及毛细管电泳分离前免疫预富集过程的引入,可以进一步提高分析测定的灵敏度,使其能够用于痕量物质的高灵敏测定。本文结合作者所在课题组的工作,对免疫亲和毛细管电泳的两种主要模式,即均相的毛细管电泳免疫分析(CEIA)和非均相的免疫亲和毛细管电泳(IACE)的研究进展进行了综述。     

Comparison of hydrodynamically closed two‐dimensional capillary electrophoresis coupled with ultraviolet detection and hydrodynamically open capillary electrophoresis hyphenated with mass spectrometry in the bioanalysis of varenicline

Two capillary electrophoresis methods for monitoring renally excreted varenicline, a highly effective drug prescribed for smoking cessation, in human urine were developed and compared. A method combining capillary electrophoresis with mass spectrometry was proposed for the fast analysis of varenicline (analysis time up to 7 min). Here, mass spectrometry was a prerequisite for achieving high sensitivity and selectivity of the analysis suitable for the quantification of a 15 ng/mL level of varenicline in un‐pretreated urine matrices. An alternative approach, two‐dimensional (column‐coupled) capillary electrophoresis with enhanced sample load capacity and ultraviolet detection, was proposed as a low‐cost alternative to capillary electrophoresis with mass spectrometry. The isotachophoresis on‐line sample treatment included simple elimination of the major matrix constituents and stacking of the sample in a large volume so that threefold lower quantitation limits could be easily achieved in comparison to the capillary electrophoresis with mass spectrometry. On the other hand, longer analysis time (ca. 4.5‐fold) and more complex electrolyte system in the coupled zone electrophoresis step (including two additives enhancing separation selectivity, i.e. isopropanol and cyclodextrin) were prerequisites for the complete separation of varenicline from the sample matrix. Anyway, both the developed methods were validated according to the Food and Drug Administration guidelines showing favorable performance parameters, suitable for their routine biomedical use.     

亲和毛细管电泳技术及其应用

对近几年新发展起来的亲和毛细管电泳技术（ACE）的原理、分类及方法作了简要介绍,着重介绍了亲和毛细管区带电泳、毛细管亲和凝胶电泳、胶束电动色谱中的亲和电泳、亲和毛细管等电聚焦、亲和探针毛细管电泳等过程和方法。对ACE在分子生物学、生物化学中的应用及该技术在亲和常数测定、核酸片段识别、竞争免疫分析、药物先导化合物的筛选等方面的应用也作了介绍。     

毛细管电泳多次进样法快速测定尿肌酐

采用毛细管电泳多次进样技术快速测定了尿肌酐。采用３６ｃｍ×５０μｍｉ．ｄ涂层柱,０．１ｍｍｏｌ／ＬｐＨ２．５的磷酸缓冲液,在２００ｎｍ处检测。连续进样５个样品总分析时间为１２ｍｉｎ,比单次进样节省２０ｍｉｎ左右。日内、日间变异系数均小于７．０％,用吡啶作内标,在肌酐浓度为５～８０ｍｇ／Ｌ范围内,肌酐浓度与肌酐同吡啶的峰高比值的线性关系很好（ｒ＝０．９９９６）。测定１２个样品同生化分析仪测定的结果相关性好（ｒ＝０．９７７３）,且分析时间节省了约３０ｍｉｎ。     

毛细管电泳在农药残留检测上的应用

对近20年来毛细管电泳分析方法在农药残留分析中的应用进行了论述.按照农药的用途分类,分别评述了应用毛细管电泳分析检测杀虫剂和杀菌剂以及除草剂残留的研究进展,并对毛细管电泳在农药残留分析中的应用进行了展望.     

Continuous on-line concentration based on dynamic pH junction for trimethoprim and sulfamethoxazole by microfluidic capillary electrophoresis combined with flow injection analysis system

A novel, rapid and continuous on-line concentration approach based on dynamic pH junction for the analysis of trimethoprim (TMP) and sulfamethoxazole (SMZ) by microfluidic capillary electrophoresis (CE) combined with flow injection analysis is developed in this paper. Stacking is due to decreases in the velocity of analytes when migrating from the low-pH sample zone (sample was dissolved in 50 mM HCl) to a relatively high-pH buffer (30 mM phosphate buffer, pH 8.5) filled in the capillary. This results in 2.9-4.7-fold improvement in concentration sensitivity relative to conventional capillary electrophoresis methods. The separation could be achieved within 2 min and sample throughput rate can reach up to 38 h(-1).     

使用含离子涂层柱的毛细管电泳和毛细管电色谱的研究进展

 毛细管电色谱是近年发展起来的高效、高选择性的微分离技术。与一般的毛细管电泳和使用ODS反相填料的毛细管电色谱相比 ,含离子涂层柱的毛细管电泳和毛细管电色谱能提供较大且可控的电渗流 ,便于拓宽分离对象 ,优化分离条件。对使用含离子涂层柱的毛细管电泳和电色谱的特点、发展和应用状况进行了综述。     

Affinity Capillary Electrophoresis: From Binding Measurement to Combinatorial Library Screening

Affinity capillary electrophoresis is a new procedure for receptor-ligand binding studies. Since its introduction in the early 1990s, this method has proved valuable in chiral separation of racemates, the measurement of binding constants, the estimation of kinetic rate constants, the determination of stoichiometries, the investigation of electrostatic interactions, the estimation of effective charges and molecular weights of biomolecules, the characterization of enzymatic catalysis, and, most recently, combinatorial library screening in solutions. This technique demands small amounts of samples, involves no radiolabeled materials or chemically immobilized ligands, and does not require changes in spectroscopic characteristics upon binding. This paper reviews the most recent applications of affinity capillary electrophoresis in binding measurement and combinatorial library screening.     

毛细管电泳法分离四环素及其杂质

用毛细管电泳方法分离四环素和它的主要杂质４－差向四环素，脱水四环素，４－差向脱水四环素和２－乙酰基－２－脱酰胺四环素，相对次要的杂质氯四环素和脱甲四环素也能被分离。并系统研究了缓冲液ｐＨ，缓冲液浓度以及毛细管温度和电压对分离的影响。紫外检测波长是２７０ｎｍ，测定的相对标准偏差为１．９％。所得结果与ＨＰＬＣ方法进行了比较     

Kinetic CE: foundation for homogeneous kinetic affinity methods

Kinetic capillary electrophoresis (KCE) is defined as capillary electrophoresis of species that interact during electrophoresis. KCE can serve as a conceptual platform for development of homogeneous kinetic affinity methods for affinity measurements (measurements of binding parameters and quantitative measurements) and affinity purification (purification of known molecules and search of unknown molecules). A number of different KCE methods can be designed by varying initial and boundary conditions - the way interacting species enter and exit the capillary. KCE methods will find multiple practical applications in the designing of biomedical diagnostics and the development of drug candidates. Here, the concept of KCE, its up-to-date applications, and future prospective are reviewed.     

新多肽配体GE11与表皮生长因子受体的结合能力分析

应用亲和毛细管电泳（ACE）分析方法,对表皮生长因子受体（EGFR）和新多肽配体GE11之间的结合能力进行分析。结果表明,EGFR与多肽配体GE11之间存在特异性相互作用,考察EGFR在不同浓度GE11溶液中的迁移情况,采用非线性、双倒数、Y-倒数和X-倒数4个数据处理方法得到较好的数据拟合,并测得结合常数。该文为筛选多肽配体以及测定受体与多肽配体之间的结合常数提供了简便的方法,将有力推动肿瘤靶向药物输送的研究。     

毛细管电泳-柱端电化学检测池的设计

在射壁式检测器的基规上,设计了一种用于毛细管电泳-电化学检测装置的检测池,巧妙地利用因表面张力现象而留驻波滴曲面的放大作用,直接调整毛细管与电极之间的相对位置,无需显微镜-微调节器装置,具有空间体积小,操作更简便的优点。     

非水毛细管电泳进展

 毛细管电泳通常是在以水为溶剂的缓冲溶液中进行的,事实上以纯有机溶剂替代水介质同样可以完成特殊样品的电泳分离,且存在诸多优点。以所建立的非水毛细管电泳方法为核心,总结了该方法中有机溶剂、电解质的选择原则及溶质-添加剂相互作用模式,并综述了它在无机离子、中性物质、有机酸等化合物分离分析中的应用。71篇。     

Development of fully automated quantitative capillary electrophoresis with high accuracy and repeatability

A quantitative capillary electrophoresis (qCE) was developed by utilizing a rotary type of nano‐volume injector, an autosampler, and a thermostat with cooling capacity. The accuracy and precision were greatly improved compared with conventional capillary electrophoresis. The 10 nL volume accuracy was guaranteed by the carefully designed nano‐injector with an accurate internal loop. The system repeatability (precision) in terms of RSD <0.5% for migration time and 1% for peak area were achieved by using DMSO as a test sample. We believe that this fully automated qCE system has the potential to be employed broadly in quality control and quality assurance in the pharmaceutical industry. Copyright © 2015 John Wiley & Sons, Ltd.