Advances in CE-mediated microanalysis: an update

This review, as a continuation of two earlier reports, gives an overview of the recent developments, over the period from 2005 until now, in the use of electrophoretically mediated microanalysis (EMMA) methodology for the on-line study of enzymatic reaction and derivatization. The article is divided into two parts: (i) on-line enzymatic reaction by EMMA and (ii) on-line derivatization by EMMA. Following a brief introduction, a literature overview on enzymatic reaction is provided. The second part starts with an introduction of the purpose of derivatization and the nomenclature used in the area of in-capillary derivatization based on EMMA. The development of more integrated analytical platform that combines in-capillary derivatization and sample preconcentration is discussed. Reported derivatization procedures are summarized.     

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.

     

Advances in capillary electrophoretically mediated microanalysis: an update

This review, as a continuation of an earlier report, gives an overview of recent developments, over the period from 2003 until now, in the use of capillary electrophoretic techniques for the in-line study of enzymatic reactions, derivatization, and chemical reactions. The article is divided into two parts: (i) in-line enzymatic reactions and (ii) in-line derivatization and chemical reactions. The first part introduces electrophoretically mediated microanalysis (EMMA) and discusses and illustrates the different modes of EMMA. A literature overview on enzymatic reactions is provided. The second part starts with an introduction of the procedures and the nomenclature used in the area of in-line derivatization and chemical reactions based on EMMA. Reported derivatization and chemical reaction procedures are discussed and summarized.     

Chromatographic separation for domoic acid using a fragment imprinted polymer

A method for real-time visualisation of reactions performed in-capillary by the technique of electrophoretically mediated microanalysis (EMMA) is described, using a two dimensional imaging detection system. The UV absorbance detector is based on a complementary metal oxide semiconductor (CMOS) active pixel sensor. Imaging of analyte peaks absorbing at 200 nm and migrating over length of 14 mm in the capillary dimension allowed measurement of velocities and lengths of reactant and product zones. By contrast with use of single point detection, velocities of species generated by reaction anywhere within the capillary are readily measured with CMOS imaging: this is of particular benefit for EMMA experiments where reaction occurs during zone overlap. For the oxidation of glutathione by hydrogen peroxide, reaction times were varied over the range 0.5-20 s by changing voltages for electrokinetic injection and zone migration, and reactant and product peak areas were obtained for kinetic analysis of the reaction. The use of EMMA conditions with CMOS imaging allows the whole process of reaction, separation and quantification to be carried out in nanolitre volumes on-capillary in a single run on a time scale of less than 5 min.     

毛细管电泳序列分析技术用于在线酶分析研究进展

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。     

Electrophoretically mediated microanalysis for in-capillaryelectrical cell lysis and fast enzyme quantification by capillary electrophoresis

In this study, a novel capillary electrophoresis (CE)-based enzymatic assay was developed to evaluate enzymatic activity in whole cells. β-Galactosidase expression was used as an example, as it is a biomarker for assessing replicative senescence in mammalian cells. It catalyzes the hydrolysis of para-nitrophenyl-β-d-galactopyranoside (PNPG) into para-nitrophenol (PNP). The CE-based assay consisted of four main steps: (1) hydrodynamic injection of whole intact cells into the capillary, (2) in-capillary lysis of these cells by using pulses of electric field (electroporation), (3) in-capillary hydrolysis of PNPG by the β-galactosidase—released from the lysed cells—by the electrophoretically mediated microanalysis (EMMA) approach, and (4) on-line detection and quantification of the PNP formed. The developed method was applied to Escherichia coli as well as to human keratinocyte cells at different replicative stages. Results obtained by CE were in excellent agreement with those obtained from off-line cell lysates which proves the efficiency of the in-capillary approach developed. This work shows for the first time that cell membranes can be disrupted in-capillary by electroporation and that the released enzyme can be subsequently quantified in the same capillary. Enzyme quantification in cells after their in-capillary lysis has never been conducted by CE. The developed CE approach is automated, economic, eco-friendly, and simple to conduct. It has attractive applications in bacteria or human cells for early disease diagnostics or insights for development in biology.

Kinetic study of gamma-glutamyltransferase activity by electrophoretically mediated microanalysis combined with micellar electrokinetic capillary chromatography

The use of capillary electrophoresis for the determination of gamma-glutamyltransferase (GGT) activity with gamma-glutamyl-p-nitroanilide (Glu-p-NA) as a substrate was investigated. The reaction velocity was quantified spectrophotometrically by the corrected peak area of the product p-nitroaniline (pNA) at 380 nm. Micelles composed of sodium deoxycholic acid were used in the background electrolyte in order to obtain a baseline separation between the substrate and the product. The presence of the micelles did not influence the enzymatic reaction. The electrophoretic system was used, not only for the separation and quantitation of the different reaction compounds but also for the in-capillary mixing of the enzyme and substrate plugs. This methodology is known as electrophoretically mediated microanalysis (EMMA). With the developed in-capillary activity assay an average Michaelis constant (K(M)) for GGT was calculated to be 2.09 mM (RSD = 7.3%, n = 3), a value consistent with previously reported values.     

Advances in capillary electrophoretically mediated microanalysis

Recent developments in the use of capillary electrophoretic techniques for the in-line study of enzyme reactions and derivatization protocols are reviewed. The article is divided into two parts: (i) in-line enzyme reactions and (ii) in-line derivatization. The first part introduces electrophoretically mediated microanalysis (EMMA) and discusses and illustrates the different modes of EMMA. A literature overview is provided, starting from 1996, and the investigated enzymes are classified into two tables based on the mode of engagement (i.e., continuous or transient) of the developed EMMA-based assay. The second part starts with an introduction of the procedures and the nomenclature used in the area of in-line derivatization protocols based on EMMA. Reported derivatization procedures are discussed and classified in tables, according to the functional group that is derivatized.     

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.     

Electrophoretically mediated microanalysis assay for sirtuin enzymes

An electrophoretically mediated microanalysis (EMMA) assay for the human sirtuin SIRT1 has been developed using 9-fluorenylmethoxycarbonyl (Fmoc)-labeled peptides, i.e. Fmoc-KK(Ac)-NH(2), Fmoc-KK(Ac)L-NH(2) and Fmoc-RHKK(Ac)-NH(2), as substrates. The partial filling mode was applied due to the incompatibility between the incubation buffer, pH 8.0, and the BGE that had a pH of 2.7 or 2.3 depending on the analytes. Incubation and subsequent analyte separation were carried out in a 37/30 cm, 50 μm id fused-silica capillary at 37°C. An injection sequence of incubation buffer, enzyme, substrate, enzyme and incubation buffer was selected because the electrophoretic mobility of SIRT1 was not known. The assay was optimized with regard to the length of the injected plugs, the mixing voltage and mixing time as well as the activity (concentration) of SIRT1. The EMMA assay was subsequently applied to the determination of the Michaelis-Menten constants, K(m), and the maximum velocity, V(max), as well as the determination of the inhibitory constants, IC(50), of inhibitors. Data obtained with the in-capillary assay were in accordance with the literature data or an offline SIRT1 assay.     

Study of enzyme kinetics of phenol sulfotransferase by electrophoretically mediated microanalysis

Electrophoretically mediated microanalysis (EMMA) was applied for the study of the kinetic parameters of the enzymatic reaction of phenol sulfotransferase SULT1A1 isoenzyme with 4-nitrophenol as a substrate. The SULT1A1 activity was determined by the quantitation of the product, 4-nitrophenyl sulfate, at 274 nm by using different injection and separation steps. This new approach solved the problem of the presence of the very strong inhibitor, adenosine 3',5'-bisphosphate (PAP), in the co-substrate solution (adenosine 3'-phosphate 5'-phosphosulfate, PAPS) which is unstable at room temperature. The inhibitor PAP was electrophoretically separated from the co-substrate PAPS before the injection of enzyme and substrate inside the capillary (and thus before their in-capillary encountering). With the developed in-capillary SULT1A1 activity assay an average Michaelis constant (Km) for 4-nitrophenol was calculated to be 0.84 microM, a value which is consistent with a previously reported value. Strong substrate inhibition (above a 4-nitrophenol concentration of 2.5 microM) was observed, and this is also in accordance with literature values.     

Applications of in-capillary reaction micellar electrokinetic chromatography in the food industry

This review describes the quantitative analysis of in-capillary reactions by using capillary electrophoresis (CE) in the food industry. An electrophoretic analysis of products of an enzyme reaction of a substrate by in-capillary reaction was useful for the activity measurement of glucoamylase in sake rice koji. p-Nitrophenyl-beta-D-maltoside was employed as a substrate and p-nitrophenyl-beta-D-glucopyranoside was the product of the enzyme reaction. The glucoamylase activity of sake rice koji samples gave a good linear relationship with the peak area observed in the in-capillary enzyme reaction method. Also, in-capillary micellar electrokinetic chromatography (MEKC) was used for analyzing the Monascus pigment-mediated degradation of mutagenic 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole. During the electrophoresis, the mutagen and the pigment, due to their different migration velocities, mix for a certain period of time to interact, and then they are separated and quantitated. The in-capillary reaction MEKC method can be applied to the routine quality control of enzyme activities in the food industry and the evaluation of mutagenic compounds in food materials.     

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.     

Micellar electrokinetic chromatography: current developments and future

This review highlights recent methodological and instrumental advances in micellar electrokinetic chromatography (MEKC). Enhancements in sensitivity and selectivity of the technique through the use of on-line preconcentration approaches (stacking and sweeping) and nonconventional pseudostationary phases, namely nonionic and zwitterionic surfactants, mixed micelles and polymers, are discussed in detail. Laser-induced fluorescence and mass spectrometry, as alternatives to UV-absorption detection, have been covered to evaluate their advantages and limitations when applied to analysis in an MEKC format. Some thoughts on future directions in this area such as in-capillary reactions, coated capillaries and MEKC on microchips are also presented.     

Speciation of chromium by in-capillary derivatization and electrophoretically mediated microanalysis

An electrophoretic method for chromium speciation analysis--as Cr(III) and Cr(VI)--based on in-capillary derivatization with 1,5-diphenylcarbazide (DPC) is here proposed. As Cr(III) does not react with DPC, it was oxidized also in-capillary to Cr(VI) by Ce(IV). For this purpose, a capillary electrophoresis (CE) mode called electrophoretically mediated microanalysis (EMMA) based on sequential injection of sample and reagents--namely, DPC, sample and Ce(IV)--was employed. The conditions of both reactions--Cr(III) oxidation and Cr(VI)-DPC derivatization--were optimized in order to quantify separately the Cr(VI)-DPC complex from the original Cr(VI) in the sample and that from oxidation of Cr(III) to Cr(VI). The electrophoretic conditions were independently optimized for variables influencing the resolution and those affecting sensitivity. The method thus developed was applied to the determination of Cr(III) and Cr(VI) in glass material, for which different sample preparation methods--namely, EPA method 3060A, ultrasound-assisted leaching and microwave-assisted digestion--were tested. Microwave-assisted digestion was found to be the best sample preparation alternative in terms of efficiency of the step--99.6 and 98.3% for Cr(VI) and Cr(III), respectively--and procedure time--20 min. The complete method was validated with the certified reference material BAM-S004.     

Investigating the effects of conductivity on zone overlap with EMMA: computer simulation and experiment

In this paper, we demonstrate, using both experiment and simulation, how sample zone conductivity can affect plug-plug mixing in small molecule applications of electrophoretically mediated microanalysis (EMMA). The effectiveness of in-line mixing, which is driven by potential, can vary widely with experimental conditions. Using two small molecule systems, the effects of local conductivity differences between analyte plugs, reagent plugs and the BGE on EMMA analyses are examined. Simul 5.0, a dynamic simulation program for CE systems, is used to understand the ionic boundaries and profiles that give rise to the experimentally obtained data for EMMA analyses for (i) creatinine determination via the Jaffe reaction, a reaction involving a neutral and an anion, and (ii) the redox reaction between gallate and 2,6-dichloroindophenol, two anions. Low sample conductivity, which is widely used in CE analyses, can be detrimental for in-line reactions involving a neutral reactant, as rapid migration of the ionic component across a low conductivity neutral zone results in poor reagent plug overlap and low reaction efficiency. Conversely, with two similarly charged reagents, a low conductivity sample plug is advantageous, as it allows field-amplified stacking of the reagents into a tight reaction zone. In addition, the complexity of simultaneously overlapping three reagent zones is considered, and experimental results validate the predictions made by the simulation. The simulations, however, do not appear to predict all of the observed experimental behavior. Overall, by combining experiment with simulation, an enhanced appreciation for the local field effects in EMMA is realized, and general guidelines for an advantageous sample matrix can be established for categories of EMMA analyses.     

Evaluation of an in-capillary approach for performing quantitative cytochrome P450 activity studies

An automated in-capillary assay requiring very small quantities of reagents was developed for performing in vitro cytochrome P450 (CYP450) drug metabolism studies. The approach is based on the following: (i) hydrodynamic introduction of nanoliter volumes of substrate and enzyme solutions in the sandwich mode, within a capillary; (ii) mixing the reagents by diffusion across the interfaces between the injected solutions; (iii) collection of the capillary content at the end of the in-capillary assay; and (iv) off-line analysis of the incubation mixture by ultrahigh pressure liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS). After optimizing the injection sequence of the reagents, the in-capillary approach was applied to the quantitative determination of the kinetics of drug metabolism reactions catalyzed by three CYP450 isozymes involved in human drug metabolism: CYP1A2, CYP2D6, and CYP3A4. It was demonstrated that this in-capillary method was able to provide similar kinetic parameters for CYP450 activity (e.g., Michaelis constants and turnover values) as the classical in vitro method, with a drastic reduction of reagent consumption.     

Strategies for in-capillary derivatization of amino acids in capillary electrophoresis using 1,2-naphthoquinone-4-sulfonate as a labeling reagent.

This paper examines the potentiality of in-capillary derivatization for improving the sensitivity of the spectrophotometric detection of amino acids in capillary zone electrophoresis. 1,2-Naphthoquinone-4-sulfonate was selected as the labeling agent of amino acids. The underivatized sample and the reagent solution segments are injected by pressure into the capillary prior to applying the running voltage. The corresponding derivatization reaction occurs inside the capillary once the potential is applied, as it induces mixing of the sample with the reagent. Several introduction modes consisting of tandem or sandwich configuration have been evaluated. These techniques result in a straightforward and automated way of carrying out a derivatization. Furthermore, in-capillary procedures may become much more attractive than conventional pre-capillary derivatization in terms of sensitivity and reproducibility. The optimum operation mode found consists of a sandwich system where the sample is injected in between two reagent segments. The method was applied to the determination of amino acids in feed samples. Results show a good concordance with those given by a standard amino acid analyzer.     

Multi-compound electrophoretic assays for tyramine oxidase with a UV area detector imaging multiple windows on a looped capillary

Active pixel sensor UV area imaging and capacitively coupled contactless conductivity detection have been applied in an electrophoretically mediated microanalysis (EMMA) assay for substrate specificity of tyramine oxidase (Arthrobacter sp.). Use of the UV area imaging detector to monitor four windows in a capillary with three loops provided intrinsic self-referencing for all species and identified tyramine and 2-phenethylamine as the only reactive components in a multi-compound mixture. Continuous engagement EMMA experiments showed significant benefits by comparison with plug-plug EMMA, improving sensitivity by extending enzyme-substrate interaction times and allowing measurement of time-dependent reaction in the substrate zones passing the four windows.     

Field-amplified sample injection and in-capillary derivatization for sensitivity improvement of the electrophoretic determination of histamine

The feasibility of the combination of field-amplified sample injection (FASI) and in-capillary derivatization was explored for improving sensitivity of histamine in capillary electrophoresis (CE). Naphthalene-2,3-dicarboxaldehyde (NDA) was used as derivatization reagent. The reagent and sample was introduced by tandem mode. The derivatization was accomplished by at-inlet mode with standing time of 1.5 min. The combination of FASI and in-capillary derivatization was successfully achieved with about 400-fold concentration sensitivity enhancement compared to pre-capillary derivatization at the same set-up. The detection limit of concentration for histamine reached 1.25 x 10(-11) M by CE and fluorescence detection with S/N = 3. Parameters affecting FASI and in-capillary derivatization process including sample matrix, buffer concentration and reagent injection amount, were investigated.