Isotachophoresis of proteins in a networked microfluidic chip: experiment and 2-D simulation

This paper reports both the experimental application and 2-D simulation of ITP of proteins in a networked microfluidic chip. Experiments demonstrate that a mixture of three fluorescent proteins can be concentrated and stacked into adjacent zones of pure protein under a constant voltage of 100 V over a 2 cm long microchannel. Measurements of the isotachophoretic velocity of the moving zones demonstrates that, during ITP under a constant voltage, the zone velocity decreases as more of the channel is occupied by the terminating electrolyte. A 2-D ITP model based on the Nernst-Planck equations illustrates the stacking and separation features of ITP using simulations of three virtual proteins. The self-sharpening behavior of ITP zones dispersed by a T-junction is clearly demonstrated both by experiment and by simulation. Comparison of 2-D simulations of ITP and zone electrophoresis (ZE) confirms that ZE lacks the ability to resharpen protein zones after they pass through a T-junction.     

Isotachophoresis of allergenic extracts

One aspect of the isotachophoretic determination of protein patterns in biological samples of interest is the characterization of allergens. This group of (glyco) proteins, causing allergic reactions, is used both for diagnosis and in the treatment of allergy. The aim of this investigation was to obtain a maximum amount of information, within one run, on the (glyco)protein composition of a number of allergenic extracts (e.g., from pollen or house dust mites). Commercially available extracts were dialysed prior to analysis to remove disturbing buffer constituents. A high-pH system was chosen in order to obtain a maximum amount of information from the samples (1-2 microliter). The leading electrolyte was 0.01 M C1-, buffered with Tris (pH 8.2), containing 0.2% w/v hydroxyethylcellulose, and the terminating electrolyte was beta-alanine, buffered to pH 10 with Ba(OH)2. The total analysis time was 15-20 min using a PTFE capillary (0.2 mm I.D.). The pre-separation current was 30 microA and the current during detection was 15 microA. UV absorption was measured at 280 nm. For optimal discrimination of the compounds of interest, an ampholyte mixture was used for spacing. The analytical procedure yielded highly reproducible UV patterns. Significant differences between various allergenic extracts were observed. It was concluded that isotachophoresis is a powerful method for the physico-chemical characterization of individual allergenic extracts, e.g., with respect to manufacturing and quality control.     

Isotachophoresis of proteins in uncoated open-tubular fused-silica capillaries with a simple approach for column conditioning.

The isotachophoretic determination of proteins in uncoated open-tubular fused-silica capillaries of 50 and 75 microns I.C. with on-column multi-wavelength detection is reported. Small amounts of hydroxypropylmethylcellulose added to the leader provide an efficient method of dynamic column conditioning which allows the high-resolution isotachophoretic determination of most proteins to be performed in the presence of an electro-osmotic flow. Different approaches for cationic and anionic analyses are discussed and illustrated with selected examples.     

柱偶联装置在毛细管等速电泳中的应用

根据自制的ＯＣＥＰ－１型离子分析仪的特点，设计了一套新的住偶联装置。提高了分离容量，可以用来检测复杂混合物中的低浓度组分。用等速电泳法测定了高浓度Ｃｌ＾－，Ａｃ＾－存在时的Ｆ＾－。这个装置对于处理离子性组分，无论在研究工作还是实际分析工作中，都是很有用的。     

Use of Counterflow in Bidirectional Capillary Isotachophoresis

Electrochemical cell using counterflow in bidirectional capillary isotachophoresis is designed. With this cell, steady-state concentrations of several solutions in cationic and anionic capillary isotachophoresis are determined simultaneously. Transport numbers of ionic components are calculated and compared with literature data.     

Isotachophoresis for rapid transformation of Escherichia coli

A frequent limitation of electroporation (EP) and chemical transformation (CT) are the need of tedious and time-consuming procedures for inducing transformation competence, the substantial number of cells required, and the low transformation yields typically achieved. Here, we show a new and rapid electrokinetic method for transformation of small number of noncompetent Escherichia coli TOP10 cells (2–3 × 10⁵) at room temperature. Escherichia coli TOP10 cells and plasmid DNA are sequentially injected into a 50 μm ID capillary and focused into 11.5 nL by isotachophoresis (ITP) induced by application of high DC voltage (–16 kV). Through ITP, a large excess of plasmid DNA is brought in contact with the cell surface, with the contact time adjusted by application of a counter-pressure (1.3 psi) opposing the ITP movement. The transformation rate was more than 1000-fold higher compared to EP and CT at survival rates greater than 60%.     

A New Potential Gradient Detection System for Isotachophoresis

Abstract

Concerning the potential gradient detector for isotachophoresis, the authors solved the problem of bubble generation on the sensing electrodes which frequently resuls in suspension of isotachophoresis, which had been the biggest impediment to the practical development of this type of detector.

In order to obtain stable measurements of zone boundaries without descernible generation of bubbles even at 250 μA of migration current, several modifications were made. The projecting part of the sensing electrodes were scraped off resulting in reduction of the electric current between two sensing electrodes, the cross-sectional area of the cell was expanded resulting in reduction of the current density in the sensing cell, and finally, the sensing cell, which is situated at a high voltage to ground, was isolated from ground potential by converting the electrical signal provided by the sensing cell into an optical signal, resulting in a reduction of the current to ground.

The detection limit was 2×10^?9 gram equivalent for adipic acid. A boundary between zones was detectable when the mobility difference between the zones was about 1% in the case of a sample having a diffusion coefficient of about 10⁵ cm²/sec.     

Inorganic Arsenic and Selenium Determination Using Miniaturised Isotachophoresis

A new method allowing the simultaneous determination of arsenic(V), selenium(IV) and selenium(VI) using miniaturised isotachophoresis has been developed. The method uses 0.02 M nitric acid buffered to pH 5.5 with histidine as the leading electrolyte. Using a miniaturised poly(methyl methacrylate) chip device with an integrated conductivity detector, separations of model samples and an industrial process stream sample were achieved. Limits of detection were calculated to be 0.85 mg L⁻¹ for arsenic(V), 0.95 mg L⁻¹ for selenium(IV) and 1.0 mg L⁻¹ for selenium(VI). A method for the analysis of arsenic(III), using a glycolic acid based leading electrolyte to eliminate carbonate interference is also presented.     

Separation of Proteins and Other Compounds by Capillary Isotachophoresis

Electrophoresis, as the name implies, consists essentially in moving charged particles under the influence of an electric field. Since the first practical experiments by Tiselius¹, most of the variations in electrophoretic techniques have been based on the principle of zone electrophoresis. Only in the last decade did electrophoretic separation techniques, based on other principles, become available; isoelectric focusing and isotachophoresis. It is becoming more and more clear that neither of these techniques can provide the ideal solution to every protein separation problem but that each of them has its special field of application where it can give the researcher a maximum of information.     

Measurement of Histamine in Stinging Insect Venoms by Isotachophoresis

Abstract

An isotachophoretic histamine assay has been developed to determine the histamine content in hymenoptera stinging insect venoms. The amount of histamine was lowest in bee venom (0.9%) while the histamine content of vespid venom extracts varied between 2.7–5.2%. Histamine, determined quantitatively by isotachophoresis, correlated well with the histamine values achieved with reversed-phase high performance liquid chromatography.     

Protein Separation and Enzyme Purification by Preparative Capillary Isotachophoresis

The use of preparative capillary isotachophoresis (CITP), operating in a discontinuous fractionation mode, for separation and fast purification of the enzyme uridine diphosphate galactopyranose mutase (UGM) from the cell extract of Escherichia coli overproducing the recombinant enzyme is presented in this feasibility study. UGM is required to produce galactofuranose for the cell wall biosynthesis of many pathogenic microorganisms and represents a very attractive candidate for the development of new antimicrobial drugs. CITP separations were carried out under slightly alkaline pH conditions (8.7), in which UGM enzyme is negatively charged. Significantly simplified proteinous matrix isolated in several fractions by employed preparative CITP procedure with the aid of properly selected discrete spacers was subsequently confirmed by SDS PAGE with Coomassie staining. It was shown that preparative CITP is very effective tool for fast purification of the target enzyme from other proteinous matrix constituents when purification and isolation step lasted 20 min. The enzymatic activity of UGM was confirmed in the sample after the preparative CITP purification step, which is a crucial requirement for further biochemical applications.     

Isotachophoresis as an on-line concentration pretreatment technique in capillary electrophoresis

One of the major disadvantages of capillary electrophoresis (CE) is its limited loadability. Therefore, the on-line coupling of isotachophoresis (ITP) and CE was studied with regard to its potential for the improvement of the minimum concentration that can be measured by CE. Based on the concentrating and separating power of ITP, detection limits could be lowered by at least two orders of magnitude. Especially for biological samples containing proteins, it appeared that in non-treated capillaries the electromigration characteristics are hardly influenced when isotachophoretic pretreatment is applied. The potential of ITP-CE coupling is illustrated by the analysis of o-phthaldialdehyde and fluorescein isothiocyanate derivatives of a number of amino acids.     

Selected Imidazolium Ionic Liquids as a Terminating Electrolyte in Isotachophoresis

Results of application of selected imidazolium ionic liquids [especially EMIM (1-ethyl-3-methyl-imidazolium) cation] as a new type of terminating electrolyte (TE) in isotachophoresis (ITP) with conductometric detection are presented. In experiments seven different types of leading electrolyte (LE) with new terminating electrolyte were studied. After selection of parameters influenced on the analysis resolution, buffers were successfully used in control of qualitative and quantitative analysis (sodium, potassium, calcium, and magnesium) of different type of waters. The new ITP method was satisfying and basic validation parameters were assigned.     

等速电泳法同时检测无机和有机阴离子

本文介绍了利用自制的ＯＣＥＰ－１型离子分析仪,建立了以Ｃｌ－为前导,正丁酸很为尾随,８－羟基喹啉为缓冲配对离子的等速电泳分离系统的新分离分析方法,对ＮＯ＿３,ＮＯ＿２,Ｆ￣－,ＨＣＯＯ￣－及ＣＨ３ＣＯＯ￣－进行分离和同时检测,获得满意的结果。     

等速电泳分析人血红蛋白

应用等速电泳（ＩＴＰ）分析了２０例健康者、１０例糖尿病患者及３例异常血红蛋白病患者的血红蛋白组分。健康者血红蛋白的ＩＴＰ分析图可分为５个部分,即ＨｂＡ_（１ａ）,ＨｂＡ_（１ｂ）＋ＨｂＡ_（１ｃ）＋ＨｂＦ,ＨｂＡ_０,ＨｂＡ_２和ＨｂＵ,并计算出各组分的均值±标准差和各组分的相对值。病理组的ＩＴＰ分析图和各组分的测定值均与健康者明显不同。     

油田水中短链有机酸的等速电泳法研究

本文采用水相蒸发除去大量的氯离子,用等速电泳法对油田水中短链有机酸进行了定性定量分析。结果表明,油田地层水中有机酸成分主要有甲酸、乙酸、丙酸、丁酸、苯甲酸和乳酸等,本法回收率和相对标准偏差分别在96％～105％和2.4％～7.6％之间。     

Isotachophoresis separations of enantiomers on a planar chip with coupled separation channels

The use of a poly(methylmethacrylate) chip, provided with a pair of on-line coupled separation channels and on-column conductivity detectors, to isotachophoresis (ITP) separations of optical isomers was investigated. Single-column ITP, ITP in the tandem-coupled columns, and concentration-cascade ITP in the tandem-coupled columns were employed in this investigation using tryptophan enantiomers as model analytes. Although providing a high production rate (about 2 pmol of a pure tryptophan enantiomer separated per second), single-column ITP was found suitable only to the analysis of samples containing the enantiomers at close concentrations. A 94-mm separation path in ITP with the tandem-coupled separation channels made possible a complete resolution of a 1.5 nmol amount of the racemic mixture of the enantiomers. However, this led only to a moderate extension of the concentration range within which the enantiomers could be simultaneously quantified. The best results in this respect were achieved by using a concentration-cascade of the leading anions in the tandem-coupled separation channels. Here, a high production rate, favored in the first separation channel, was followed by the ITP migration of the enantiomers in the second channel under the electrolyte conditions enhancing their detectabilities. In dependence on the migration configuration of the enantiomers, this technique made possible their simultaneous determinations when their ratios in the loaded sample were 35:1 or less (D-tryptophan a major constituent) and 70:1 or less (L-tryptophan a major constituent).     

等速电泳法测定人齿菌斑培养液中乳酸含量

应用等速电泳法分离并测定了人齿菌斑培养液中的乳酸，测定回收率在９４．２％－１０２．２％之间，相对标准偏差小于４．５％，比较了抗龋者与易感龋者的菌斑在相同培养条件下的乳酸产量，并对不同饥饿时间下菌斑所产的乳酸进行了测定与比较。