流动注射-毛细管电泳联用及应用进展

流动注射是一种高效进样及在线溶液处理手段。毛细管电泳是一种高分离效率、高选择性的分析技术,但传统的毛细管电泳间歇式进样方式效率低且难用于过程分析,将流动注射进样技术与毛细管电泳结合,既弥补了毛细管电泳的进样缺陷,又可兼具两者的优点。有关两种技术的联用一直都在探索之中。文中对近年流动注射一毛细管电泳联用及应用研究进行了综述。     

毛细管电泳进样技术新进展

评述了毛细管电泳进样技术新成果。对直接在线进样，二维分离体系中毛细管电泳分离的增样，相关毛细管电泳增样，超微量样品及单个分子的进样，近端进样，双向进样和高温下的进样装置的应用状况作了介绍。     

基于短毛细管的高速毛细管电泳系统的研究进展

概述了基于短毛细管的高速毛细管电泳系统的研究进展。重点介绍了适用于基于短毛细管的高速毛细管电泳系统的各种进样方法及其在生物分离分析领域的应用,包括光门进样、流动门进样、电动进样、自发进样、流体动力进样和扩散进样等方法。     

毛细管电泳流动注射-负压进样装置的研究

设计了一种用于毛细管电泳系统的流动注射-负压进样装置。样品由蠕动泵输送到进样阀后再由缓冲液带到分离毛细管入口,由毛细管出口端施加的负压引入。进样时间由自制精密控时电路控制,经进样条件的优化,能获得良好的重现性。实验中两种阳离子峰面积和迁移时间的RSD(n=8)≤2.7%,优于传统重力进样,而且操作简便;与非接触电导检测器组装成流动注射-毛细管电泳系统,可实现快速、高效的在线分析。初步应用于无机阳离子的分离,取得了满意的结果。     

毛细管电泳技术在线酶分析研究进展

生物酶是生物体内活细胞产生的生物催化剂,控制着生物体的新陈代谢、营养和能量转换等反应过程.建立生物酶活性及催化反应或抑制反应动力学的快速、准确和有效的测定方法,对于理解生物反应过程、药物研发以及疾病诊治等具有重要意义.毛细管电泳技术由于其具有分离效率高、分析速度快、操作简单和样品消耗少等优点,在酶分析研究中越来越受到关注,正逐渐发展为生物酶快速分析的技术平台.根据酶的存在形态,毛细管电泳在线酶分析可分为均相模式(包括电泳媒介微分析法和在线连续监测酶分析)和异相模式(固定化酶反应器),本文综述了近10年来这2种不同模式及其在酶抑制剂筛选、酶动力学研究和底物定量检测等方面的应用.     

芯片电泳推拉式压力进样方法

芯片电泳作为微流控分析系统的典型代表,广泛涉及材料、微加工方法、微液流控制、分离模式和检测方法等诸多方面.与传统分析系统一样,样品制备和引入也是微全分析系统实现样品到结果首先面临的问题.电进样一直是芯片电泳系统的主流进样方法.而传统毛细管电泳系统中与电进样同样经常使用的压力进样方法则很少用于芯片电泳系统.     

气动毛细管微滴进样-化学发光法测定酚磺乙胺

传统的化学发光(CL)分析有分立式取样和流动注射式两种方式,各有优缺点,目前国内外的仪器都采用手动进样或电动进样方法,速度和进样量均很难精确控制,对微升级的样品尤为困难,另外也无法实现在线分析。本文提出的气动毛细管微滴进样化学发光(AFCM-CL)检测法是以气体压力作为     

毛细管电泳法在酶抑制动力学研究中的应用进展

毛细管电泳法因其分离高效、分析快速和进样微量且柱体不易受污染等特点,被广泛用于酶抑制剂的相关研究中,如酶抑制剂筛选、抑制活性评估及抑制类型判断等方面。本文介绍了近年来毛细管电泳法在酶抑制动力学研究中的发展,包括用于酶抑制动力学研究的测定模式和在酶抑制动力学研究的应用两个部分。     

微流量液相色谱在线大体积进样的方法和专用装置

本发明涉及微分离系统的进样方法,具体地说是一种微流量液相色谱在线大体积进样的方法和专用装置。在微流量液相色谱分离系统的流路中,添加多流路分流切换装置,使得系统在进样和分离时,流动相可以从不同流路分流。样品利用在固定相上的保留,实现色谱系统的大体积进样。     

毛细管电泳直接分析与水不互溶溶液中痕量物质的新方法

本文采用在线反萃取-场放大进样方法实现了毛细管电泳与溶剂微萃取的直接联用.     

基于DNA定向固定化技术构建毛细管固定化酶微反应器的研究进展

生物酶影响着物质代谢和质能转换等生命活动,生物体内某些酶的活性变化会导致疾病的发生。发展新型的酶分析方法对深刻理解生物代谢过程、疾病诊断和药物研发等具有重要意义。毛细管电泳（CE）具有分离效率高、分析速度快、操作简单和样品消耗少以及可与多种检测手段联用等优点,在酶分析研究中越来越受到关注。CE酶分析主要包括离线和在线两种模式,其中,固定化酶微反应器与毛细管电泳联用（CE-IMER）的在线酶分析已经成为主要的酶分析方法之一。CE-IMER充分结合了固定化酶和CE的优势,将游离酶固定在毛细管内,不仅可以显著提高酶的稳定性和重复使用性,而且可以实现纳升规模溶液的自动化酶分析,进而显著降低酶分析成本。目前已有大量方法制备IMER用于CE酶分析,然而如何构建性能良好、可再生使用、酶固载量大、自动化程度高的CE-IMER一直是该领域重点研究的问题。DNA定向固定化技术（DDI）可以充分利用DNA分子的碱基互补配对（A-T,C-G）,在温和的生理条件下特异性固定生物大分子。由于短链双螺旋DNA分子具有较强的机械刚性和物理化学稳定性,通过DDI将酶固定在载体表面,有利于降低传质阻力,提高酶与底物的接触能力,进而促进酶促分析过程。该文主要综述了利用DDI构建新型IMER在CE酶分析中的应用现状,并对其未来发展进行了展望。     

Controllable and high-performance immobilized enzyme reactor: DNA-directed immobilization of multienzyme in polyamidoamine dendrimer-functionalized capillaries

In recent years, CE-integrated immobilized enzyme reactors (IMERs) for single-enzyme immobilization have attracted considerable attention. However, there has been little research on multienzyme immobilization in CE. Here, we introduce a method for fabricating a CE-integrated IMER, using DNA-directed immobilization to fix glucose oxidase and horseradish peroxidase in the capillary, which had been functionalized with polyamidoamine dendrimer (PAMAM). Owing to the reversibility of DNA hybridization, the reactor is capable of dynamic immobilization. Moreover, by introducing the PAMAM, the loading capacity of the IMER is greatly enhanced, and the PAMAM can spontaneously form complexes with DNA and then contribute to the efficiency and stability of the reactor. After 25 days storage, the prepared IMER ultimately retained approximately 70% of its initial activity. We also used the IMER to detect glucose, and the favorable linearity was obtained over the concentration range of 0.78–12.5 mM, with an LOD of 0.39 mM, demonstrating that the CE-integrated IMER can be applied to actual samples. We believe that this strategy can be extended to other multienzyme immobilization systems, and CE-integrated IMERs are potentially useful in a wide range of biochemical research applications.     

Advances in Capillary Electrophoresis-Based Enzyme Assays

Capillary electrophoresis (CE) has become a flexible and accurate, high-efficiency analytical separation technique in many areas requiring only minute amounts of sample and chemicals. Thus, CE has also been recognized as a suitable technique to study enzymatic reactions including the determination of Michaelis–Menten kinetic data or the identification and characterization of inhibitors. The most often applied CE-based enzyme assay modes can be divided into two categories: (1) pre-capillary assays where incubations are performed offline followed by CE analysis of substrate(s) and/or product(s) and (2) in-capillary assays in which the enzymatic reaction and analyte separation are performed in the same capillary. In case of the in-capillary assays, the enzyme may be immobilized or in solution. The latter is also referred to as electrophoretically mediated microanalysis (EMMA), while in the case of immobilized enzyme the term immobilized enzyme reactor (IMER) is used. The present review summarizes the literature on CE-based enzyme assays published between January 2010 and April 2015. Immobilized enzyme reactors as well as microfluidic devices applied to the study of enzymatic activity will also be briefly addressed.

     

Development of off-line and on-line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substrates

Fast and convenient CE assays were developed for the screening of adenosine kinase (AK) inhibitors and substrates. In the first method, the enzymatic reaction was performed in a test tube and the samples were subsequently injected into the capillary by pressure and detected by their UV absorbance at 260 nm. An MEKC method using borate buffer (pH 9.5) containing 100 mM SDS (method A) was suitable for separating alternative substrates (nucleosides). For the CE determination of AMP formed as a product of the AK reaction, a phosphate buffer (pH 7.5 or 8.5) was used and a constant current (95 microA) was applied (method B). The methods employing a fused-silica capillary and normal polarity mode provided good resolution of substrates and products of the enzymatic reaction and a short analysis time of less than 10 min. To further optimize and miniaturize the AK assays, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product employing electrophoretically mediated microanalysis (EMMA, method C). After hydrodynamic injection of a plug of reaction buffer (20 mM Tris-HCl, 0.2 mM MgCl2, pH 7.4), followed by a plug containing the enzyme, and subsequent injection of a plug of reaction buffer containing 1 mM ATP, 100 microM adenosine, and 20 microM UMP as an internal standard (I.S.), as well as various concentrations of an inhibitor, the reaction was initiated by the application of 5 kV separation voltage (negative polarity) for 0.20 min to let the plugs interpenetrate. The voltage was turned off for 5 min (zero-potential amplification) and again turned on at a constant current of -60 microA to elute the products within 7 min. The method employing a polyacrylamide-coated capillary of 20 cm effective length and reverse polarity mode provided good resolution of substrates and products. Dose-response curves and calculated K(i) values for standard antagonists obtained by CE were in excellent agreement with data obtained by the standard radioactive assay.     

Advances in screening enzyme inhibitors by capillary electrophoresis

Capillary electrophoresis (CE) has attracted lots of attention due to its simplicity, low sample consumption, low solvent volume, high resolution, and high speed. Based on these advantages, it has been widely used in enzyme inhibitor screening. There are two main operation modes on enzyme inhibitor screening: off‐line (precapillary enzyme assays) in which process CE was used as an analytical tool; online (in‐capillary enzyme assays) which combined the sample injection, mix, reaction, separation, and detection within a single run. Additionally, diverse of new materials were introduced to immobilize enzyme, which has been coupled with CE for the study of enzyme activity and its inhibitor screening. This review gives an overview of the developments and applications for the CE‐based enzyme inhibitor screening.     

Sequential capillary electrophoresis analysis using optically gated sample injection and UV/vis detection

We present sequential CE analysis of amino acids and l ‐asparaginase‐catalyzed enzyme reaction, by combing the on‐line derivatization, optically gated (OG) injection and commercial‐available UV‐Vis detection. Various experimental conditions for sequential OG‐UV/vis CE analysis were investigated and optimized by analyzing a standard mixture of amino acids. High reproducibility of the sequential CE analysis was demonstrated with RSD values (n = 20) of 2.23, 2.57, and 0.70% for peak heights, peak areas, and migration times, respectively, and the LOD of 5.0 μM (for asparagine) and 2.0 μM (for aspartic acid) were obtained. With the application of the OG‐UV/vis CE analysis, sequential online CE enzyme assay of l ‐asparaginase‐catalyzed enzyme reaction was carried out by automatically and continuously monitoring the substrate consumption and the product formation every 12 s from the beginning to the end of the reaction. The Michaelis constants for the reaction were obtained and were found to be in good agreement with the results of traditional off‐line enzyme assays. The study demonstrated the feasibility and reliability of integrating the OG injection with UV/vis detection for sequential online CE analysis, which could be of potential value for online monitoring various chemical reaction and bioprocesses.     

Electrophoretically mediated microanalysis

This review describes the existing developments in the use of the capillary electrophoretic microanalytical technique for the in-line study of enzyme reaction, electrophoretically mediated microanalysis (EMMA). The article is divided into a number of parts. After an introduction, the different modes, basic principle, procedure, and some mathematical treatments of EMMA methodology are discussed and illustrated. The applications of EMMA for enzyme assay and for non-enzymatic determination are summarized into two tables. In addition to classical capillary electrophoresis (CE) instrument EMMA, special emphasis is given to a relatively new technique: EMMA on CE microchip. Finally, conclusions are drawn.     

Dual injection capillary electrophoresis: foundations and applications

The state of the art of capillary electrophoresis (CE) approaches based on dual injection is here reported. Dual injection strategies have been proposed with three main objectives: (i) to provide information about reaction kinetics and/or related parameters, (ii) to perform in-capillary derivatization for improving separation and/or determination, (iii) to develop electrophoretic methods for the simultaneous analysis of anionic and cationic compounds. For the first two purposes, dual injection, which involves sample and reagent, can be realized either from the same end of the capillary (electrophoretically mediated microanalysis, EMMA) or from the two ends of the capillary (electroinjection analysis, EIA). The third objective, with dual injection of sample from the two ends of the capillary, takes advantage of moving cationic and anionic compounds with opposite directions. The foundations of each alternative, conditions necessary for working with them, restrictions, applications as well as perspectives are reviewed in order to establish the advantages, shortcomings, and convenience or no of their use in comparison to conventional CE.     

Capillary electrophoretic approach to screen for enzyme inhibitors in natural extracts

This paper demonstrates development of electrophoretically mediated micro analysis (EMMA), for screening protein tyrosine phosphatase (PTP) inhibitors in natural extracts. It is demonstrated that capillary electrophoresis (CE) separation of the substrate and the product allows for using the assay in an on-column format to monitor the reaction without typically used fluorogenic substrates. Michaelis-Menten kinetics parameters calculated based on the EMMA results (Km = 1.2-1.5 microM) were in a good agreement (Km = 1.0-1.5 microM) obtained using an off-line CE functional assay (CE FA). EMMA of PTP titrated with different concentrations of ligand demonstrated the peak-shift phenomenon normally seen in affinity capillary electrophoresis. This feature of EMMA gives an indication of the binding affinity of the ligand in addition to its functional activity, providing another dimension in characterization of the protein-inhibitor interaction. It was demonstrated that simultaneous screening of the primary PTP target and a secondary, counter target (PTP-C) using the EMMA format can be used to prioritize hits based on their specificity.     

The First Online Capillary Electrophoresis-Microscale Thermophoresis (CE-MST) Method for the Analysis of Dynamic Equilibria—The Determination of the Acidity Constant of Fluorescein Isothiocyanate

This article presents the first successful application of a capillary electrophoresis-microscale thermophoresis tandem technique (CE-MST) for determining the values of equilibrium constant, realized by connecting online the CE and MST instruments using a fused-silica capillary. The acid-base dissociation of fluorescein isothiocyanate, expressed by the acidity constant value (pK_a), was used as a model. The measurement procedure consisted of introducing a mixture containing the analyte and a deliberately added interferent into the CE capillary, electrophoretic separation of the analyte from the interferent, the detection of the analyte with a CE-integrated detector, detection with a MST detector, and then stopping the flow temporarily by turning off the voltage source to conduct the thermophoretic measurement. The analysis of migration times, peak areas and MST responses obtained concurrently for the same sample allowed us to determine the pK_a value using three independent methods integrated within one instrumentation. The analyte was effectively separated from the interferent, and the acidity values turned out to be consistent with each other. An attempt was also made to replace the standard commercial CE instrument with a home-made portable CE setup. As a result, the similar pK_a value was obtained, at the same time proving the possibility of increasing cost efficiency and reducing energy consumption. Overall, the CE-MST technique has a number of limitations, but its unique analytical capabilities may be beneficial for some applications, especially when sample separation is needed prior to the thermophoretic measurement.