毛细管电泳序列分析技术用于在线酶分析研究进展

Advances in on-line enzyme assays by sequence analysis-based capillary electrophoresis

毛细管电泳技术具有操作简单、样品消耗量少、分离效率高和分析速度快等优势,不仅是一种高效的分离分析技术,而且已经发展成为在线酶分析和酶抑制研究的强有力工具。酶反应全程的实时在线监测,可以实现酶反应动力学过程的高时间分辨精确检测,以更准确地获得反应机制和反应速率常数,有助于更好地了解酶反应机制,从而更全面深入地认识酶在生物代谢中的功能。此外,准确、快速的在线酶抑制剂高通量筛选方法的发展,对加快酶抑制类药物的研发以及疾病的临床诊断亦具有重要意义。电泳媒介微分析法(EMMA)和固定化酶微反应器(IMER)是毛细管电泳酶分析技术中常用的在线分析方法。这两种在线酶分析法的进样方式通常为流体动力学进样和电动进样,无法实现酶反应过程中的无干扰序列进样分析。近年来,基于快速序列进样的毛细管电泳序列分析技术已经发展成为在线酶分析的另一种强有力手段,以实现高时间分辨和高通量的酶分析在线检测。该文从快速序列进样的角度,综述了近年来毛细管电泳序列分析技术在线酶分析的研究进展,并着重介绍了各种序列进样方法及其在酶反应和酶抑制反应中的应用,包括光快门进样、流动门进样、毛细管对接的二维扩散进样、流动注射进样、液滴微流控进样等。

Due to unique advantages such as short analysis time,high separation efficiency and sensitivity,easy automation,extremely low sample and reagent volume requirements,and the ability to utilize several detection methods,capillary electrophoresis(CE)is used as a high-efficiency separation technique,and has been developed as a powerful tool for on-line enzyme assays.On-line enzyme assays based on CE have been applied to almost all aspects of enzyme assays over the past two decades,including the evaluation of enzyme activities and kinetics,identification and characterization of enzyme inhibitors and activators,detection of enzyme substrates,investigation of enzyme-mediated metabolic pathways,and proteome analysis.One potential use of enzyme assays is in tracing enzymatic reactions from beginning to the end at high temporal resolution.Measurements of enzyme reactions at high temporal resolution can result in more accurate estimates of reaction mechanisms and reaction rate constants,which is vitally important for improving understanding of the functions of enzymes in metabolism and for identifying the potential use of enzymes in clinical diagnostics.Furthermore,high-throughput online enzyme analysis is of great importance for the analysis of enzyme reactions and enzyme inhibition reactions.The development of accurate,rapid and high-throughput enzyme inhibition screening methods is especially important for accelerating the development of new drugs.Electrophoretically mediated microanalysis(EMMA)and CE-integrated immobilized enzyme microreactor(IMER)are the two most used techniques for online CE enzyme assays.The EMMA technique utilizes different electrophoretic mobilities of enzymes and substrates to initiate reactions within the capillary and to separate the components of the reaction mixture for the final in-capillary quantification.In a CE-integrated IMER,the enzyme is bound to the capillary surface or to a suitable carrier attached to the capillary through physical adsorption,cross-linking,covalent bonding or other methods.The enzyme reactor is usually located at one end of the capillary;the enzyme-catalyzed reaction occurs when the substrates pass through the enzyme reactor and the substrates/products of the enzymatic reaction are separated and online detected by CE at the downstream end of the capillary.In both either techniques,the samples are usually introduced into the capillary by electrokinetic injection or by hydrodynamic injection.Because both injection methods require that the capillary inlet be physically moved from the sample container to the running buffer for CE analysis after each sample injection,it is unlikely that EMMA or microreactor techniques can be successfully used to perform sequential online analysis.Therefore,a CE sequence analysis technique based on rapid sequential injection has been developed as another powerful method for online enzyme analysis.Compared with the widely used electrokinetic and hydrodynamic injection methods used in traditional CE online enzyme analysis methods,rapid sequence injection methods can achieve sequential injection without any physical disturbance of the capillary inlet,allowing for the successful performance of online enzyme assays with high temporal resolution and at high throughput.A rapid,sequential,and automatic sample introduction system is an important part of online enzyme analysis based on CE sequence analysis.Several sequential injection methods such as optical-gating injection,flow-gated injection,two-dimension diffusion injection,flow injection and droplet microfluidics combined with CE have been developed to successfully perform online enzyme assays with high temporal resolution and high throughput.In this paper,we will review recently developed CE online enzyme assays and inhibition studies based on rapid sequential injection.We review the progress and applications of various sequential sample injection approaches that have been developed for sequential on-line CE analysis of enzyme reactions at high temporal resolution and high-throughput screening of enzyme inhibitors,including optical-gating injection,flow gated injection,two-dimension diffusion injection,flow injection and droplet microfluidics.