Transient Isotachophoresis Research Articles

Focusing of anionic micelles using sample‐induced transient isotachophoresis: Computer simulation and experimental verification in MEKC

The specific phenomenon of high-salt micelle focusing used as a preconcentration method for uncharged solutes in MEKC has been interpreted in terms of complementary transient isotachophoresis (tITP) process. High matrix chloride can focus anionic SDS micelles not only due to a field-enhanced effect but also by creating the tITP state in which chloride acts as a leading ion. The latter micelle-enrichment event was simulated by SIMUL software in order to understand tITP conditions under which micelle stacking can be expected to occur. Specifically, effects of chloride matrix concentration and the nature of BGE co-ion, performing a role of terminating ion, were explored. A greater extent of micelle stacking was observed at a higher chloride concentration in borate than in phosphate buffer system. This proof-of-principle simulation result was confirmed in real experiments using metal-based chemotherapeutic drugs. It was concluded that analyte enrichment requires prolonged interaction with the micelle, which is feasible at a proper ratio between sample matrix and BGE co-ion concentrations.

Highly sensitive transient isotachophoresis sample stacking coupling with capillary electrophoresis-amperometric detection for analysis of doping substances

A simple and effective method of capillary electrophoresis-amperometric detection (CE-AD) coupled with transient isotachophoresis (tITP) was developed for the trace determination of doping substances. Compared with the conventional capillary electrophoresis method, the maximum enhancement factor in terms of peak heights was up to 5500-fold when the tITP technique was adopted. Under the optimum conditions, the detection limit (S/N=3) for methylephedrine (MDP), celiprolol (CEL), sotalol (SOT) and indapamide (IDP) were 4.2 x 10(-14), 6.3 x 10(-13), 5.8 x 10(-14) and 9.5 x 10(-13)molL(-1), respectively. The RSDs of four analytes were 1.0-2.3% for migration time and 2.6-3.8% for peak current, respectively. The proposed method was successfully applied to determine the contents of SOT and IDP in real urine sample, and the excretion curve of IDP within 48h was also investigated. The recoveries of the four doping in urine ranged from 90.0 to 102%.

Precise Prediction of DNA Sizes with Transient Isotachophoresis-Capillary Gel Electrophoresis Analysis on a Microchip

Abstract The migration time in transient isotachophoresis (tITP) separation is affected by sample salinity due to the dependence of ITP time on sample-zone conductivity. The sample-to-sample variation of migration time in microchip tITP-capillary gel electrophoresis (CGE) analysis is an undesired factor for precise DNA sizing. In this study, a DNA-sizing method based on relative migration-time proportion (RMP) was developed to eliminate the effect of sample salinity on sizing precision. RMP is defined as the ratio of the migration-time difference between the target fragment and the lower marker to that of the migration-time difference between the upper marker and the lower marker. The RMP values were tested to be reproducible in microchip tITP-CGE separations irrespective of sample salinity. Size of a target DNA was predicted by placing its RMP value in the equation derived from RMPs of standard DNA ladder vs . DNA sizes. The precision and reproducibility of the sizing method were validated by testing multiple standard PCR amplicons. Experimental results showed that the RMP method was simple and reliable, thus well suited to precise DNA sizing with microchip tITP-CGE technique.

On‐chip coupling of free‐solution transient ITP and CGE for highly efficient separation of dsDNA with variable sample loading amounts

We developed an on-chip DNA analysis method, in which free-solution transient isotachophoresis (FstITP) were coupled with CGE. Using chloride ions in the sample matrix and HEPES in the background electrolyte, respectively, as the leading and terminating ions, dsDNAs were isotachophoretically preconcentrated in free-solution and then separated in sieving polymer. The coupling of FstITP and CGE enabled higher separation efficiency due to higher preconcentration rate in free-solution. The FstITP-CGE analysis offered adjustable signal intensities by varying sample injection time. With the maximum sample loading volume, the LOD of the FstITP-CGE analysis was determined to be 0.24 ng/mL by confocal laser-induced fluorescence detection. The FstITP-CGE method is simple, robust and flexible, thus well suited to the analysis of highly saline DNA samples at different concentration level.

Microsystem for Isolation of Fetal DNA from Maternal Plasma by Preparative Size Separation

Routine prenatal diagnosis of chromosomal anomalies is based on invasive procedures, which carry a risk of approximately 1%-2% for loss of pregnancy. An alternative to these inherently invasive techniques is to isolate fetal DNA circulating in the pregnant mother's plasma. Free fetal DNA circulates in maternal plasma primarily as fragments of lengths <500 bp, with a majority being <300 bp. Separating these fragments by size facilitates an increase in the ratio of fetal to maternal DNA. We describe our development of a microsystem for the enrichment and isolation of cell-free fetal DNA from maternal plasma. The first step involves a high-volume extraction from large samples of maternal plasma. The resulting 80-microL eluate is introduced into a polymeric microsystem within which DNA is trapped and preconcentrated. This step is followed by a transient isotachophoresis step in which the sample stacks within a neighboring channel for subsequent size separation and is recovered via an outlet at the end of the channel. Recovered fractions of fetal DNA were concentrated 4-8 times over those in preconcentration samples. With plasma samples from pregnant women, we detected the fetal SRY gene (sex determining region Y) exclusively in the fragment fraction of <500 bp, whereas a LEP gene (leptin) fragment was detected in both the shorter and longer recovery fractions. The microdevice we have described has the potential to open new perspectives in noninvasive prenatal diagnosis by facilitating the isolation of fetal DNA from maternal plasma in an integrated, inexpensive, and easy-to-use microsystem.

In-line preconcentration of oxidized and reduced glutathione in capillary zone electrophoresis using transient isotachophoresis under strong counter-electroosmotic flow

Transient isotachophoresis (tITP) can improve the sensitivity of capillary electrophoresis (CE). In general, it was carried out under the condition of suppressed electroosmotic flow (EOF). However, some special conditions, such as extreme low pH background electrolyte and coating were needed to achieve the requirements of suppressed EOF. In this work, an approach of tITP under the strong counter-EOF in open system (counter-EOF-tITP) is presented for the rapid and sensitive preconcentrating the reduced glutathione (GSH) and the oxidized glutathione (GSSG) without modifying the capillary and the commercial CE instrument. The parameters of the experimental system, such as the concentration of leading electrolyte, the injected amount of terminating electrolyte and the injected pressure of sample were investigated in detail to understand the mechanism of counter-EOF-tITP. The sensitivity enhancement factors were of 320 for GSH and 280 for GSSG. In addition, the detection limit of 23.4 and 18.0 μg L −1 for GSH and GSSG was achieved, respectively. The method's applicability was demonstrated by determining GSH and GSSG in tomato and human serum.

Imaging and Quantification of Isotachophoresis Zones Using Nonfocusing Fluorescent Tracers

We present a novel method for visualizing isotachophoresis (ITP) zones. We introduce negligibly small concentrations of a fluorophore that is not focused by isotachophoresis. This nonfocusing tracer (NFT) migrates through multiple isotachophoresis zones. As it enters each zone, the NFT concentration adapts to the local electric field in each zone. ITP zones can then be visualized with a point detector or camera. The method can be used to detect, identify, and quantify unknown analyte zones and can visualize complex and even transient electrophoresis processes. This visualization technique is particularly suited to microfluidic and laboratory-on-a-chip applications, as typical fluorescence microscopes and charge-coupled device (CCD) cameras can provide high-resolution spatiotemporal data. We present a theoretical description, a methodology for identifying analytes, and experimental validation. We also visualize and analyze a complex, transient DNA ITP preconcentration and separation.

High-sensitive analysis of DNA fragments by capillary gel electrophoresis using transient isotachophoresis preconcentration and fluorescence detection

In this report aimed on further development of a high-sensitivity capillary gel electrophoresis (CGE) method for analysis of DNA fragments, we firstly explored online transient isotachophoresis (tITP) preconcentration combined with fluorescence detection (FD). The fluorescence signal (excitation: 488 nm; emission: 590 nm) was generated using the intercalating dye of ethidium bromide (EB). It was found when the leading electrolyte (LE) was injected behind the sample zone, such a special tITP mode has significant advantages to solve the bubble formation issue and to improve the analytical performance stability. Two standard DNA samples, a 50 bp DNA step ladder and the φX174/HaeIII digest, were used to evaluate the qualitative and quantitative abilities of the tITP-FD approach. A highly diluted sample (10,000-fold in the water, e.g. the φX174/HaeIII digest diluted from 500 μg/ml to the 50 ng/ml level) was enriched and detected; the LOD was down to 0.09 ng/ml for the 72 bp fragment, apparently improved more than 1000-fold in comparison with UV detection. Although the RSD of peak areas ( n = 3) was around 15.5% for the sample was electrokinetically injected, good linearity of peak area response showed that the proposed method is suitable for quantitative analysis.

Estratégias de pré-concentração em eletroforese capilar (CE): parte 1. Manipulação da velocidade eletroforética do analito

Capillary electrophoresis has become a well-established and routine-based separation technique. It is based on the differences between charged analyte mobility in aqueous or organic electrolytes. Its major limitation is the sensitivity due to small sample injection volumes and the narrow diameter of the capillaries, especially when UV detection is used. There are a number of ways to increase the concentration sensitivity. This report shows some on-line preconcentration strategies to perform it in free solution capillary electrophoresis that are based on manipulation of the analyte electrophoretic velocity during the sample introduction (stacking, field amplification and transient isotachophoresis).

Magnetic Beads Based Immunoaffinity Capillary Electrophoresis of Total Serum IgE with Laser-Induced Fluorescence Detection

A magnetic beads based immunoaffinity capillary electrophoresis method for total Immunoglobulin E quantification in serum has been developed. The method combines speed, automation ability, and minimal sample consumption. Only 1 microL of serum is required while the whole immunoaffinity capillary electrophoresis method is performed in less than 50 min. The concomitant use of online immunocapture, transient isotachophoresis, and laser-induced fluorescence detection provides a sensitivity in the low picomolar range and a highly linear fluorescence response over 4 orders of magnitude (IgE concentration ranging from 2.4 to 2400 ng/mL). After validation with a reference material, the method has been successfully applied to the quantification of total IgEs in patient sera. The results compared well with classical ImmunoCap data.

On‐line preconcentration and quantitative analysis of peptide hormone of brain and intestine using on‐column transient isotachophoresis coupled with capillary electrophoresis/electrospray ionization mass spectrometry

A new approach was described to achieve very sensitive analysis of peptide hormone of brain and intestine by capillary electrophoresis coupled with a transient isotachophoresis (tITP) preconcentration method. The system used was electrospray ionization mass spectrometry (ESI-MS) as detector and equipped with a sheath flow configuration. The effects of sample matrix, pH and concentration of leading electrolyte (LE), sample injection time, and ESI-MS instrumental parameters on the efficiency of the sample stacking were investigated in detail. Under the optimized conditions, lower than micromole (0.01 microM) concentrations of the peptides were easily detected. Compared to traditional hydrodynamic injection methods, about 40-230-fold increase in detection sensitivity was obtained by this technique. A distinguishing feature of the described approach is that the background electrolyte can serve as terminating electrolyte (TE), which simplifies the process of the experiments. The method was further evaluated by the analysis of low concentration active peptide mixtures spiked in hypothalamus tissue of the rat.

Focusing and stabilization of bis‐intercalating dye–DNA complexes for high‐sensitive CE‐LIF DNA analysis

Multiple labeling of nucleic acids by intercalative dyes is a promising method for ultrasensitive nucleic acid assays. The properties of the fast dissociation and instability of dye-DNA complexes may prevent from their wide applications in CE-LIF nucleic acid analysis. Here, we describe an optimum CE focusing method by using appropriately paired sample and separation buffers, Tris-glycine buffer and Tris-glycine-acetic acid buffer. The developed method was applied in both uncoated and polyacrylamide coated fused-silica capillary-based CE-LIF analysis while the sample and separation buffers were conversely used. The complexes of intercalative dye benzoxazolium-4-pyridinium dimer and dsDNA were greatly focused (separation efficiency: 1.8 million theoretical plates per meter) by transient isotachophoresis mechanism in uncoated capillary, and moderately focused by transient isotachophoresis in combination of field amplified sample stacking and further stabilized by the paired buffer in polyacrylamide coated capillary. Based on the developed focusing strategy, an ultrasensitive DNA assay was developed for quantitation of calf thymus dsDNA (from 0.02 to 2.14 pM). By the use of an excitation laser power as low as 1 mW, the detection limits of calf thymus dsDNA (3.5 kb) are 7.9 fM in concentration and 2.4x10(-22) mol (150 molecules) in mass. We further demonstrate that the non-gel sieving CE-LIF analysis of DNA fragments can be enhanced by the same strategy. Since the presented strategy can be applied to uncoated and coated capillaries and does not require special device, it is also reasonable to extend to the applications in chip-based CE DNA analysis.

Use of a heterogeneous buffer combination in microchip electrophoresis for high‐resolution separation by on‐line concentration of DNA samples

We have developed a novel high-resolution separation technique of DNA fragments in a heterogeneous combination of a sample buffer and a separation buffer. The use of a heterogeneous buffer combination is a simple method for on-line concentration of DNA fragments, in which a sample buffer is simply exchanged with one including taurine anions. The mobility of taurine anions, co-ions for DNA, is lower than the that of acetate anions in a separation buffer. The difference in the mobility invokes transient isotachophoresis. The current technique allows DNA fragments to be effectively concentrated and the separation length of microchips to be shorter than that of conventional ones by a factor of three without deterioration in separation resolution and any modification of a chip design. Fragments of 100-bp DNA ladders (100-1000 bp) were separated with high resolution (0.72-10.7) within 60 s with a 10 mm separation length on a polymethyl methacrylate chip. Furthermore, fragments of 10-bp DNA ladders (10-330 bp) were separated with high resolution (0.69-2.00) with a 10 mm separation length within 50 s without band broadening. The current achievements will make it possible to fabricate compact devices for microchip electrophoresis.

Sensitive determination of anions in saliva using capillary electrophoresis after transient isotachophoretic preconcentration

A transient isotachophoresis–capillary electrophoresis (tITP–CE) system for the determination of minor inorganic anions in saliva is described. The complete separation and quantification of bromide, iodide, nitrate, nitrite, and thiocyanate has been achieved with only centrifugation and dilution of the saliva sample. In-line tITP preconcentration conditions, created by introduction of the plugs of 5 mM dithionic acid (leading electrolyte) and 10 mM formic acid (terminating electrolyte) before and after the sample zone, respectively, allowed the limits of direct UV absorption detection (at 200 nm) to be up to 50-fold improved as compared with CE without tITP. As a result, nitrate and thiocyanate were still detectable at 4.6 and 3.8 μg l −1, respectively, in 1000 times diluted saliva. The daily variations of anionic concentrations in saliva samples taken from a smoking health volunteer were discussed based on the results of tITP–CE analysis. It was confirmed that the thiocyanate concentration in saliva noticeably increased after smoking. This is apparently the first report on simultaneous quantification of more than four anionic salivary constituents using CE.

Determination of iodate in table salt by transient isotachophoresis–capillary zone electrophoresis

A transient isotachophoresis–capillary zone electrophoresis (tITP–CZE) method was first time developed for determining iodate in table salt. Sensitivity enhancement was accomplished by coupling on-capillary tITP with CZE. The interference of sample matrix was overcome by using electrophoretic buffer containing high concentration of sodium chloride. The optimal terminating electrolyte for tITP was 1500 mmol/L phosphate and the separation buffer of capillary zone electrophoresis was 10 g/L sodium chloride (pH 8.0) containing 20 mmol/L cetyltrimethylammonium chloride (CTAC). Calibration graphs based on peak height and peak area showed good linearity. Use of chromate as the internal standard significantly improved the precision of the quantitative results. The wavelength of UV detection for iodate was set at 218 nm with the detection limits of 3.5 μg/L for iodate. The quantitative results of iodate in table salt measured by the tITP–CZE method were compared with those measured by the redox titration and there was no significant difference between the two means. The method developed was sensitive, fast and simple.

Capillary Electrophoresis-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for the Identification of Cationic Metabolites via a pH-Mediated Stacking-Transient Isotachophoretic Method

Capillary electrophoresis-mass spectrometry (CE-MS) is still widely regarded as an emerging tool in the field of metabolomics and metabolite profiling. A major reason for this is a reported lack of sensitivity of CE-MS when compared to gas chromatography-mass spectrometry GC/MS and liquid chromatography-mass spectrometry. The problems caused by the lack of sensitivity are exacerbated when CE is coupled to Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), due to the relatively low data acquisition rate of FT-ICR MS. Here, we demonstrate the use of an online CE sample preconcentration method that uses a combination of pH-mediated stacking and transient isotachophoresis, coupled with FT-ICR MS to improve the overall detection of cationic metabolites in the bacterium Desulfovibrio vulgaris Hildenborough. This method showed a significant increase in signal-to-noise ratio when compared to CE normal sample stacking, while providing good separation efficiency, reproducibility, and linearity. Detection limits for selected amino acids were between 0.1 and 2 microM. Furthermore, FT-ICR MS detection consistently demonstrated good mass resolution and sub-ppm mass accuracy.

最新のキャピラリー電気泳動とチップ電気泳動 キャピラリー電気泳動法およびマイクロチップ電気泳動法のＥｌｅｃｔｒｏｋｉｎｅｔｉｃ ｓｕｐｅｒｃｈａｒｇｉｎｇ：スタッキング機構と小イオンから生体高分子までの高感度分析への応用

This review summarizes the mechanism and applications of electrokinetic supercharging (EKS), an online preconcentration technique utilizing electrokinetic injection (EKI) coupled with transient isotachophoresis (tITP) in capillary electrophoresis and microchip electrophoresis. When the sample components (targets) are electrokinetically injected in the EKS procedure, the electrolyte filled in the separation capillary must (at least partly) contain a leading electrolyte (LE) that contains a co-ion with greater mobility than that of the targets to fulfill the condition of the ITP preconcentration. To complete preconcentration, further introduction of terminating electrolyte (TE) is useful. Obviously, the concentration factors of EKS strongly depend on essential EKI and tITP stages, thus two sections of this review are concentrated on the introduction of EKI and tITP. For EKI, several factors affecting the injected amount are discussed and concluded here. For tITP, different introduction modes for the sample, the optimization of the LE and TE to enhance preconcentration are also mentioned. So far, several high-sensitivity applications to metal ions, drugs, peptides, DNA fragments, and SDS-proteins have illustrated that EKS was an effective and promising stacking technique.

Online Preconcentration by Transient Isotachophoresis in Linear Polymer on a Poly(methyl methacrylate) Microchip for Separation of Human Serum Albumin Immunoassay Mixtures

Online preconcentration of human serum albumin (HSA) and its immunocomplex with a monoclonal antibody by on-chip transient isotachophoresis is reported. An 800-fold signal enhancement was achieved following the preconcentration on standard cross-channel microchips made of poly (methyl methacrylate). Sample injection, preconcentration, and separation were done continuously and controlled solely by a sequential voltage switching program. The preconcentration was followed by on-chip nondenaturing gel electrophoresis in methylcellulose solution. The method was applied to microchip electrophoresis immunoassay of HSA. Baseline separation of HSA and its immunocomplex was achieved in 25 s in the first 1 cm of the microchannel. In a direct immunoassay, the minimum detectable concentration of fluorescent labeled HSA by laser-induced fluorescence detection was 7.5 pM.

Ketopinic acid and diisoproylideneketogulonic acid as chiral ion-pair selectors in capillary electrophoresis: Enantiomeric impurity analysis of S-timolol and 1 R,2 S-ephedrine

1 S,4 R-(+)-ketopinic acid [(+)-KPA] has been introduced as a chiral selector for the separation of pharmacologically active amines by non-aqueous capillary electrophoresis (NACE). (+)-KPA gave enantioresolution for most of the compounds previously separated by 2 R,3 S,4 R,5 S-(−)-2,3:4,6-di- O-isopropylidene-2-keto- l-gulonic acid [(−)-DIKGA], but with a reversed migration order. A complete enantioresolution (Rs = 4.2) was obtained for timolol, a compound that could not be resolved using (−)-DIKGA as the selector. Thus, (+)-KPA was evaluated for the enantiomeric purity determination of S-timolol. A method based on pre-concentration by transient isotachophoresis (tITP) provided a limit of detection (LOD) of 0.2% R-timolol in S-timolol samples. Because of the lack of enantioresolution of ephedrine when (+)-KPA was used as the selector, a method with (−)-DIKGA has been developed and validated for determination of the enantiomeric purity of the 1 R,2 S enantiomer. The method gave good precision and accuracy with an LOD (S/N = 3) of 0.033% for the enantiomeric impurity 1 S,2 R-ephedrine.

Direct sample injection for capillary electrophoretic determination of organic acids in cerebrospinal fluid

Organic acids in cerebrospinal fluid (CSF) are potential diagnostic markers for neurological diseases and metabolic disorders. A capillary electrophoretic (CE) method for the direct analysis, i.e., without any sample preparation, of six organic acids in CSF was developed. A capillary coating consisting of a triple layer of charged polymers (polybrene-dextran sulfate-polybrene) was used in combination with a negative separation voltage, providing fast and efficient analysis of acidic compounds. Separation conditions, such as background electrolyte (BGE) concentration and pH were optimized, and the influence of albumin and sodium chloride was systematically studied using a set of test compounds. With injection volumes of ca. 44 nL, plate numbers of up to ca. 150,000 were obtained with a BGE of 200 mM sodium phosphate (pH 6.0). It appeared that high sodium chloride concentrations in the sample hardly affected the peak width and shape of the organic acids, most probably due to transient isotachophoresis effects occurring in the sample zone. Adverse effects of CSF proteins, which frequently compromise the CE performance, could be effectively minimized by the triple layer coating in combination with rinses of 0.1 M hydrochloric acid. Overall, the developed CE system allowed direct injections of CSF samples, yielding good separation efficiencies and stable migration times (RSDs<2%) for organic acids. Validation of the method with artificial and real CSF samples showed good linear responses (r>0.99), and LODs for the organic acids were in the range of 2-8 microg/mL when applying UV detection. RSDs for migration times and peak areas were <2% and <7%, respectively. The applicability of the CE system is shown for the determination of organic acids in CSF samples.