In-capillary determination of creatinine with electrophoretically mediated microanalysis: Characterization of the effects of reagent zone and buffer conditions

Previous work has demonstrated proof-of-concept for carrying out the clinically useful Jaffe reaction between creatinine and picrate within a capillary tube using electrophoretically mediated microanalysis (EMMA). Here, it is shown that careful control of reagent plug length as well as concentration and pH of the background electrolyte (BGE) can result in a marked improvement in the sensitivity of this assay. Increasing the length of the picrate reagent zone is shown to give rise to as much as a 3–4-fold enhancement, and increasing the concentration and/or pH of the borate buffer also results in an additional, albeit modest, improvement in sensitivity. Interestingly, borate BGE concentrations approaching 100 mM give rise to an unexplained drop in reaction efficiency, an effect which can be avoided by utilizing lower borate concentration with higher pH. The improvements appear to primarily minimize electrodispersion of the picrate reagent, allowing higher picrate concentration in the reaction zone. The same conditions also appear to minimize the electrodispersion of the in-line product as well. With optimized EMMA parameters, the sensitivity of the in-line Jaffe chemistry can be enhanced to an extent that there is no need for the two capillary “high sensitivity” detection system required in previous work. Using optimized conditions, three different human serum samples spanning the expected clinical range of creatinine concentrations were successfully analyzed. Overall, this work illustrates the importance of systematically characterizing the conditions under which EMMA analyses are carried out.     

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.     

Determination of kanamycin by electrophoretically mediated microanalysis with in-capillary derivatization and UV detection

An electrophoretically mediated microanalysis (EMMA) approach, used to perform on-line chemistry between two small molecules, has been characterized and optimized. The plug-plug type EMMA method involved electrophoretic mixing and subsequent reaction of nanoliter plugs of kanamycin-containing samples and 1,2-phthalic dicarboxaldehyde and mercaptoacetic acid within the confines of the capillary column, which acts as a microreactor. Analyses were performed by pressure-injecting a plug of kanamycin sandwiched in two reagent plugs. A potential of 375 Vcm(-1) was then applied to electrophoretically mix the two reactants, and an incubation time of up to 5 min allowed the reaction to proceed prior to the application of a separation potential of 588 Vcm(-1). UV detection was at 335 nm. The background electrolyte was 30 mM sodium tetraborate at pH 10.0, containing 16% of methanol. The method was validated in terms of linearity, limits of quantitation and detection, and precision. The method allows determination of kanamycin in bulk samples as a fully automated procedure.     

Flow-reversal flow injection analysis-II Determination of glucose with a double-pump system

A new type of flow-injection procedure is proposed in which the samples are reversely pumped to the detector. In this procedure the injected samples are pumped into the reaction loop of a 6-way valve, then the valve is rotated to reverse the flow and the sample/reagent plug is pumped to the detector by another pump. The dispersion of the sample zone is low and the consumption of the reagent is very small. Therefore, its analytical potential for analysis with expensive reagents or long reaction times is high. The procedure has been applied to the determination of glucose in serum with an enzyme kit.     

Predefine resolution of enantiomers in partial filling capillary electrophoresis and two discontinuous function plugs coupling in‐capillary

In common partial filling CE (PF‐CE), the capillary contains the selectors plug between the injection and detector end to avoid the selector going into the detector zone. To expand this method, we propose a mode of two discontinuous function plugs coupling in‐capillary, named as plug–plug PF‐CE (ppPF‐CE). Initially, we present the method to predefine the effective length of chiral selector to meet the requirement of enantiomers' resolution, which could avoid some experimental procedures. With α‐CD as a chiral selector, a satisfactory resolution of enantiomers d,l ‐tryptophan and d,l ‐tyrosine was obtained with a partial filling α‐CD plug of optimal length and concentration. Subsequently, a second plug containing hydroxypropyl methylcellulose, organic solvents (acetonitrile and methanol), anionic and cationic surfactants (SDS and CTAB), and different concentrations of sodium phosphate buffer was inserted after the selector plug. Effects of plug–plug filling on enantiomers' migration and resolution are discussed. The ppPF‐CE might be a new flexible mode for CE application.     

Coupling of sequential injection analysis and capillary electrophoresis - Laser-induced fluorescence via a valve interface for on-line derivatization and analysis of amino acids and peptides

The on-line coupling of sequential injection analysis (SIA) and capillary electrophoresis (CE) via an in-line injection valve is presented. The SIA system is used for automated derivatization of amino acids and peptides. Dichlorotriazinylaminofluorescein serves as the derivatization agent, thus enabling sensitive laser-induced fluorescence detection of the derivatized analytes. The SIA procedure includes the following steps: (a) introduction of reagent and sample zones in a holding coil, (b) sample and reagent mixing in a reaction coil, (c) stop-flow step for increase of the reaction time, and (d) delivery of derivatized sample into the loop of the micro-valve interface. A small portion of the analyte zone is introduced electrokinetically in the separation capillary via the valve interface and CE analysis is performed. Factors affecting the CE separation, such as pH, the borate and sodium dodecyl sulphate concentration of the background electrolyte have been optimized. The derivatization conditions have been studied to obtain a high reaction yield in a relative short time. The transfer of a part of the reaction plug into the loop of the valve interface has been optimized. Using des-Tyr(1)-[Met]-enkephalinamide as test compound, it is demonstrated that after automated derivatization, on-line electrophoretic analysis could be achieved. Glycine has been selected as the internal standard in order to correct for variations in reaction time and filling of the injection loop. For the enkephalin, good reproducibility (RSD<4.5% calculated by the ratio of the peak areas) and linearity (0.5-5 microg mL(-1), R(2)>or=0.994) are obtained with a detection limit of 30 ng mL(-1) (S/N=3).     

Electrophoretically mediated microanalysis with small molecules: the Jaffé method for creatinine carried out in a capillary tube

An eletrophoretically mediated microanalysis (EMMA) approach, used to perform online chemistry between two small molecules, has been characterized and optimized. The "plug-plug" type EMMA method involved electrophoretic mixing and subsequent reaction of nanoliter plugs of creatinine-containing samples and alkaline picrate (Jaffe reaction) within the confines of the capillary column, which acts as a microreactor. Analyses were performed by pressure injecting a plug of picrate followed by a plug of the creatinine-containing sample. A potential was then applied to electrophoretically mix the two reactants, and an incubation time of up to 6 min allowed the reaction to proceed prior to the application of a 27 kV separation potential with absorbance detection at 485 nm. The use of a 50 microm inner diameter(ID) extended light path capillary (150 microm pathlength) was found to be adequate for determining elevated levels of creatinine in human blood sera, but could not be used to quantify normal levels. Quantification of both normal and elevated levels of creatinine in sera was possible with a 75 microm ID high-sensitivity cell (1200 microm pathlength). Calibration plots using the latter for creatinine in human blood sera spanned the expected clinical range and were linear between 40 microM and 1.2 mM (r2 = 0.996) with an estimated limit of detection of 17 microM (signal-to-noise ratio S/N = 3). A quantitative comparison of results obtained with the reported EMMA method and accepted clinical methodology correlated very well (slope = 1.001).     

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.     

Simultaneous determination of multicomponents by flow injection analysis: determination of copper and zinc in serum by using zincon as colouring reagent

A flow injection system is described for the simultaneous determination of copper and zinc with a single detector. Two sample plugs are injected into the same carrier stream sequentially. One is for zinc determination and the other is for the sum of copper and zinc. For zinc determination, copper masking reagent is simultaneously injected into a parallel carrier stream and merged with the sample plug by using the merging zone technique. Zincon is used as the colour reagent for the spectrophotometric determination of copper and zinc. The results for the analysis of serum by the proposed method correspond well with those obtained by an AAS method. The rate of analysis is about 45 samples/hr.     

Physicochemical characterization of variously packed porous plugs of hydroxyapatite: streaming potential coupled with conductivity measurements

A homemade instrument was used for the measurement of the streaming potential, conductivity, and permeability of plugs packed in different densities with hydroxyapatite (HAP) particles at 25 degrees C and pH = 7.0 +/- 0.2. KCl solutions with ionic strength values in the range of 0.3-300 mM, equilibrated with HAP for 3 days, were forced to flow through the plugs. It was found that the particle volume fraction of the plug obtained from conductivity measurements was slightly higher than that obtained by weighing the solid. This suggested that, in addition to the volume of the solid itself, the volume of liquid trapped in the cavities of the particles does not contribute to the conductivity of the plug. The pH change recorded in the solution passed through the plug was attributed to the protonation/deprotonation of the HAP surface groups. Denser packing of the HAP crystallites resulted to higher surface conductivities. It was suggested that this trend was due to the easier interparticle ion transport in close-packed plugs. Considering zeta-potential, the values computed by neglecting surface conductivity were significantly underestimated, especially at low ionic strength values and at dense packing. More realistic values for the HAP zeta-potential were obtained taking into account the surface conductivity. These values were practically independent of the material packing during the plug preparation. Finally, the total surface conductivity was found to be limited behind the slipping plane of the electric double layer developed at the interface of HAP in contact with electrolyte solution.     

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.     

Amberlyst-15® in ionic liquid: an efficient and recyclable reagent for the benzylation and hydroalkylation of β-dicarbonyl compounds

Benzylation and hydroalkylation of 1,3-dicarbonyl compounds using Amberlyst-15 immobilized in ionic liquid [Bmim][PF₆] as an efficient reusable reagent was studied. The reagent was compared with other solid acid reagents along with role of the ionic liquid. The effect of various reaction parameters like type of reagent, solvent, substrate molar ratio, reaction time, and temperature were studied. Present protocol is advantageous due to the ease in handling of reagent, simple work-up procedure, economical and environmentally benign process. The products were obtained in good to excellent yield and applicable to wide variety of substrates.     

Capillary electrophoretic analysis of carbohydrates derivatized by in-capillary condensation with 1-phenyl-3-methyl-5-pyrazolone

Our previous papers on capillary electrophoresis (CE) have shown that samples can be derivatized in a capillary and the derivatives can be analyzed immediately after derivatization, provided that the derivatization reaction is so rapid as to complete in seconds. The present paper presents extended application of in-capillary derivatization to a much slower reaction such as the condensation of reducing carbohydrates with 1-phenyl-3-methyl-5-pyrazolone (PMP) which requires 30 min at 70 degrees C in pre-column derivatization by manual operation. It was necessary to first drive the introduced plugs of sample and reagent solutions to put them together at the entrance of the heated portion of a capillary, then to allow the superimposed plugs to react for a relevant period. We showed how to determine the introduction times of the sample and the reagent solutions as well as intermediate running buffer, the voltages to be applied for plug driving and product analysis, and the duration of voltage application, all of which are important for effective in-capillary derivatization. An example of the analysis of maltooligosaccharides by this technique is presented. It was shown that maltooligosaccharides were quantitatively derivatized with PMP in 35 min at 57 degrees C, and the derivatives could be analyzed in ca. 15 min by CE immediately after derivatization. Separation was satisfactory in 200 mM borate buffer, pH 8.2 containing sodium dodecyl sulfate to a concentration of 200 mM. Although the theoretical plate number, and accordingly the resolution, were significantly lower than the corresponding values in pre-capillary derivatization, reasonable reproducibility was ensured for both migration time (RSD 3.5% on average) and peak area (RSD less than 3%) under the optimized conditions. It is notable that sample amount could be lowered to the 10 fmol level, in contrast to the 10 pmol level in pre-capillary derivatization. In addition, since the technique employed here (the modified at-inlet technique of in-capillary derivatization) is easily automated, the established system will be highly beneficial for routine analysis of carbohydrates. Analysis by this technique was also shown to be useful for kinetic study of the derivatization reaction.     

Sample preconcentration by field amplification stacking for microchip-based capillary electrophoresis

A microchip structure for field amplification stacking (FAS) was developed, which allowed the formation of comparatively long, volumetrically defined sample plugs with a minimal electrophoretic bias. Up to 20-fold signal gains were achieved by injection and separation of 400 microm long plugs in a 7.5 cm long channel. We studied fluidic effects arising when solutions with mismatched ionic strengths are electrokinetically handled on microchips. In particular, the generation of pressure-driven Poiseuille flow effects in the capillary system due to different electroosmotic flow velocities in adjacent solution zones could clearly be observed by video imaging. The formation of a sample plug, stacking of the analyte and subsequent release into the separation column showed that careful control of electric fields in the side channels of the injection element is essential. To further improve the signal gain, a new chip layout was developed for full-column stacking with subsequent sample matrix removal by polarity switching. The design features a coupled-column structure with separate stacking and capillary electrophoresis (CE) channels, showing signal enhancements of up to 65-fold for a 69 mm long stacking channel.     

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。     

Computer simulation on a continuous moving chelation boundary in ethylenediaminetetraacetic acid-based sample sweeping in capillary electrophoresis

This paper introduces a mathematic mode of moving chelation boundary (MCB) for computer simulation of a continuous EDTA-based sample sweeping in capillary electrophoresis (CE). Besides the equations of MCB used herein, the mode also includes electro-neutrality equation, constant current density, jump boundary condition of MCB, Kohlrausch’ regulating function expressed in MCB formulation, product of water, ionic apparent mobility, ionic strength and conductivity of electrolyte as well as simple equilibrium reaction, etc. The simulation software is developed based on the mode. With the software the relevant simulation is carried out, and the corresponding experiments on a MCB are performed. The results on the simulation and experiments demonstrate that (1) the software can simulate a dynamic process, characteristic peak shape and relevant electrophoregram of a MCB; (2) the simulator can quantitatively compute velocities of MCB and complex boundary (CB), all of ionic concentrations (especially the concentration of complex) and sweeping efficiency; (3) these simulation results mentioned above are generally in accordance with the experiments. The simulation software holds evident significances for the study on a MCB and conditional optimization in such an EDTA-based sample sweeping of metal ion in CE.     

Effects of injector geometry and sample matrix on injection and sample loading in integrated capillary electrophoresis devices

The double-T injector design employed in many microchip capillary electrophoresis devices allows for the formation of very small (50-500 pL) sample plugs for subsequent analysis on-chip. In this study, we show that sample plugs formed at the channel junction can be geometrically defined. The channel width and injector symmetry prove to be of great importance to good performance. A unique pushback of solvent into the side channels can be induced when the side channels have a very low resistance to flow, and this helps to better define the injected sample plug. Samples and running buffers of differing ionic strength (e.g., 10 mM KCl buffer and 20 mM KCl sample) can yield widely variable results in terms of plug shape and amount injected (variations of 1.5 to 10x). Applying bias voltages to all the intersecting channels aids in controlling the plug shape. However, when the ionic strengths of buffer and sample are not matched, the actual amount injected (up to 10x variations) can be inconsistent with the appearance of the plug formed in the injector (up to only 30 % variations). Operating at constant pH and ionic strength produced the most consistent results. This report examines the effects of altering the injector geometry and solution ionic strengths, and presents the results of using bias voltages to control plug formation. The observed results should provide a benchmark for modeling of the fluid dynamics in channel intersections.     

Quantitative study on selective stacking of zwitterions in large-volume sample matrix by moving reaction boundary in capillary electrophoresis

The paper advanced the theoretical procedures for quantitative design on selective stacking of zwitterions in full capillary sample matrix by a cathodic-direction moving reaction boundary (MRB) in capillary electrophoresis (CE) under control of electroosmotic flow (EOF). With the procedures, we conducted the theoretical computations on the selective stacking of two test analytes of L-histidine (His) and L-tryptophan (Trp) by the MRB created with 30 mM pH 3.0 formic acid-NaOH buffer and 2-80 mM sodium formate. The results revealed the following three predictions. At first, the MRB cannot stack His and Trp plugs if less than 12.5 mM sodium formate is used to form the MRB and prepare the sample matrix. Second, the MRB can stack His and/or Trp sample plugs completely if higher than 50 mM sodium formate is chosen to form the MRB. Third, the MRB can only focus His plug completely, but stack Trp plug partially if 20-50 mM sodium formate is used; this implied the complete MRB-induced selective stacking to His rather than Trp. All the three predictions were quantitatively proved by the experiments. With great dilution of sample matrix and control of EOF, controllable, simultaneous and MRB-induced selective stacking and separation of zwitterions were achieved. The theoretical results hold evident significances to the quantitative design of selective stacking conditions and the increase of detection sensitivity of zwitterions in CE. In addition, the control of EOF by cetyltrimethylammonium bromide (CTAB) can evidently improve the stacking efficiency to both His and Trp.     

Off-line and in-line determination of catechol- O-methyltransferase activity in a capillary electrophoretic system

The use of capillary electrophoresis for the determination of catechol-O-methyltransferase (COMT) activity with dihydroxybenzoic acid as a substrate was investigated. Both an off-line and in-line capillary electrophoresis determination of COMT activity was developed and the two approaches are discussed. In the presented methods, substrate and reaction products are monitored at the same time. The initial velocity of the reaction is quantified spectrophotometrically by the corrected peak area of the products at 200 nm. In the off-line setup, capillary zone electrophoresis is used to separate and quantify the different reaction compounds. Each electrophoretic run required only 37 nL of the enzymatic reaction solution. Based on the off-line assay, an in-line determination of COMT activity was developed by a methodology known as electrophoretically mediated microanalysis (EMMA). All the different steps (i.e. mixing, incubation, separation and in-line quantitation) are combined in the capillary, which is used as a microreactor for the enzymatic reaction. Full automation of the assay is achieved with this microscale approach.     

"Drop-and-Sip" fluid handling technique for the reagent-release capillary (RRC)-based capillary-assembled microchip (CAs-CHIP): sample delivery optimization and reagent release behavior in RRC.

The experimental conditions of the sample delivery inside the reagent-release capillary-based capillary-assembled microchip (RRC-based CAs-CHIP) were optimized and the reagent release procedure in the RRC is discussed. Recently, our group introduced the basic concept of the "drop-and-sip" fluid handling technique (Anal. Chem., 2007, 79, 908). A microliter volume of sample solution is dropped on the inlet hole and is sipped into another hole, producing a sample plug flow in the main poly(dimethyl siloxane) (PDMS) channel, concurrently filling each sensing capillary that faces the main PDMS channel. However, the detailed evaluation of the successful sample delivery condition and the reagent release behavior in the RRC has not been fully discussed. Under our experimental conditions, ca. 0.6 - 2.4 s of sample plug-RRC contact time allowed the successful sample introduction into the RRC by capillary force without any reagent leakage or disturbance of the sample plug flow. On the other hand, reagent release behavior inside the RRC is governed by both convective and diffusive mass transport, which leads to a faster mixing time of the sample with reagents immobilized inside the RRC compared to that expected from the simple diffusion alone.