Metabolomics, which can be defined as the measurement of the level of all intracellular metabolites, has become a powerful new tool for gaining insight into cellular and physiological responses. Here we have developed a comprehensive and quantitative analysis method based on capillary electrophoresis mass spectrometry (CE-MS). In this marriage of techniques, CE confers rapid analysis and efficient resolution, and MS provides excellent selectivity and sensitivity. Previous work in our laboratory demonstrated that CE-MS techniques were quite useful for the metabolome analysis. We applied this approach to profile liver metabolites following acetaminophen-induced hepatotoxicity and revealed ophthalmate as a sensitive indicator of reduced glutathione (GSH) depletion. Our results specifically indicate that serum ophthalmate is a sensitive indicator of hepatic GSH depletion, and may be a new biomarker for oxidative stress.
Various kinds of functional polymers have been developed for capillary and microchip electrophoresis. A versatile alternative to entangled and random-coiled polymers, Pluronic F127, is a typical functional polymer for a DNA separation matrix in microchip electrophoresis. This temperature-sensitive and viscosity-tunable polymer provided excellent resolutions over a wide range of DNA sizes based on a different separation mechanism compared with conventional polymers such as cellulose-derivatives. In this review, we will describe different type of viscosity-tunable polymers as well as our recent work using Pluronic F127.
Possibilities of micro gel isoelectric focusing were discussed considering; 1) the advantages of gel electrophoresis, 2) the advantages of gel isoelectric focusing using carrier-ampholytes, and 3) the advantages of micro gel electrophoresis system for the rapid separation of functional proteins under non-denaturing conditions. The importance to develop liquid handling systems in sub-microliter level was stressed for the future separation systems of high-molecular-mass protein complexes, in which the 2-D gel size would be around 1 cm × 1 cm and all the separation processes should be automated.
“Capillary-Assembled Microchip (CAs-CHIP)” is fabricated by embedding the chemically-functionalized square capillaries into lattice polydimethylsiloxane channel having same channel dimensions as the outer dimensions of square capillaries. This new approach of chip fabrication allowed us not only the integration of various chemical functions, but also that of multiphase flow process onto a single microchip. Here we propose two types of different applications of CAs-CHIP for electrophoretic separations. First one is the usage as a pretreatment attachment for capillary electrophoresis separation, and the other is the usage as an “injection cross” structure for microchip capillary electrochromatography using an octadecylsilane-modified square capillary. Preliminary results of these two applications suggested the wide applicability of CAs-CHIP in the field of electrophoretic separations.
Carbohydrate analysis absolutely depends on high-resolution separation, because oligosaccharides derived from glycoconjugates are usually composed of an extremely complex mixture of carbohydrates including isomers. Capillary electrophoresis (CE) is one of the most powerful techniques in terms of resolving power, and a combination of CE and laser-induced fluorescence (LIF) detection enables to detect carbohydrates at fmol (10−15 mol) to amol (10−18 mol). CE is currently applied to the analysis of various carbohydrates derived from glycoconjugates. This review describes the recent development in high sensitive analysis of carbohydrates using LIF-CE, and the techniques for profiling of carbohydrates using capillary affinity electrophoresis (CAE).
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.
Recently, nanostructures have become one of the anticipated nanotechnologies for genomics and proteomics. Although microchip electrophoresis has become a powerful tool for analyzing biomolecules, more improvement of analytical method is highly required for medical diagnosis. In this paper, we introduce a new method using a nanostructure as a sieving matrix during microchip electrophoresis. Nanostructure prepared from bacterial cellulose (Nata de Coco) is proposed. This method has dramatically improved the separation efficiency of biomolecules and it is highly expected for the medical diagnosis and biological analysis in future.
Sample solutions are often introduced to microchip electrophoretic systems by electrokinetic injection, which often causes selective injection of the components with higher electrophoretic mobility, and impairs the reliability of quantitative analysis. One promising alternative to overcome this limitation is on-line sample trapping enabling the concentration of all analytes in sample reservoir to the separation channel. Here we describe the methodology of concentration techniques of field amplified stacking, micellar sweeping, isotachophoresis, perm-selectivity, and solid phase extraction mainly concerned on microchip electrophoresis. Then we also introduce our recent research on highly effective preconcentration of anionic samples using sulfonate-type polyacrylamide gel fabricated by in-situ photopolymerization.
For RNA size separation in a small sample volume (<10 nL), a strong denaturant to cleave the intramolecular hydrogen bonds that maintain the high-order structures of RNA and optimization for a small sample volume are required. We suggested, “in-capillary denaturing gel electrophoresis” as the RNA separation based on capillary gel electrophoresis, that realizes the denaturation and separation simultaneously in a capillary tube. We found that carboxylic acids were strong denaturants for in-capillary denaturing gel electrophoresis, and the performance of RNA separation was dramatically improved with a running buffer containing acetic acid. Based on the decrease of DNA melting temperature, we estimated that the denaturing ability of 2.0 M acetic acid was stronger than that of either 2.5 M formaldehyde or 7.0 M urea. The baseline separation of RNA with a size of 100−10,000 nt was achieved in only 25 min by in-capillary denaturing gel electrophoresis containing 2.0 M acetic acid. The resolution and number of plates of RNA separation were higher and larger than those obtained in a conventional capillary gel electrophoresis with sample preparation with 7.0 M urea.
Recently the development and the practical application of monoclonal antibodies for clinical use are rapidly expanding in pharmaceutical business trend. Because of the heterogeneities of antibodies as glycoproteins, a new approach to test the products is required. Capillary electrophoresis (CE) is one of the important candidates that meet this demand. CE separation is based on the various modes involving capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE) and capillary isoelectric focusing (CIEF). All of them are used for the analysis and test of monoclonal antibodies for clinical use. Especially with CGE, a non-glycosylated heavy chain is separated from a glycosylated one and detected. CIEF is an important technology to analyze the heterogeneity of antibodies mainly based on the N-linked oligosaccharide composition. N-linked oligosaccharide structure is also important as it effects on the bio-activity of the antibodies like antibody-dependent cellular cytotoxity. The fluorescence and charge insertion into oligosaccharides by chemical labeling is a valuable strategy to make CE useful for the analysis of them which released by enzyme digestion of antibodies. CZE is also useful not only for peptide mapping but also as the base of the CE-mass spectrometry approach to analyze the structure of peptides and N-linked oligosaccharide chains. These applications are discussed with examples.
Nitric Oxide (NO) has been identified as an intrinsic mediator to play key roles in physiological and pathological conditions in human body. Recently, the relationships between NO and diseases are attracted much attention. Due to its short half-life, the amount of NO is generally estimated from the concentrations of NO metabolites, i.e. nitrite (NO2−) and nitrate (NO3−). The colorimetric kit (Griess method) is widely used for determination of NO2− and NO3−; however, this method is relatively complicated and time-consuming because of the intricate chemical reactions. The other methods, such as high performance liquid chromatography, are also complicated and time-consuming.Recently, in order to obtain high resolution and high speed separation of NO metabolites, some studies using capillary electrophoresis (CE) have been reported. Since, microchip CE (MCE) has an advantage in high-throughput assay, we have developed a high-throughput NO assay using MCE with UV detection.In this paper, we described the development of a novel running buffer for determination of NO2− and NO3− in human fluids, the achievement of complete MCE separation by controlling the applied voltages and the investigation of on-chip preconcentration using transient isotachophoresis. As a result, a novel running buffer based on the ionic composition of human serum was developed for CE, and then the MCE separation of NO2− and NO3− in a spiked human serum was achieved within 6.5 seconds.
Recent development involves practical and universal usage of microfabricated devices (microfluidic chip) for analysis of biological molecules. Agilent 2100 Bioanalyzer, launched in 1999, is a unique tool capable of handling nucleic acids, proteins and cells on one platform, and for obtaining accurate, reproducible and high-resolution analytical results. For example, RNA is the starting point for several applications, like RT-PCR and microarray, and knowing that the starting RNA is of sufficient quality is vital for success of these applications. Bioanalyzer not only acquires migration profiles of RNA samples, but also automatically assesses their integrity in an objective manner. And besides, it has so many benefits over conventional bioanalytical methods, the elimination of time consuming procedure and the reduction of the sample contamination. In this report, we show its basic feature and new applications for small RNA and highly sensitive analysis of proteins.
We developed a novel on-line sample preconcentration technique named transient-trapping (tr-trapping) to improve the concentration sensitivity by using a partially injected short micellar plug in microchip micellar electrokinetic chromatography (MCMEKC). Our proposed theoretical model indicated that a trap-and-release mechanism enables a short micellar zone which was partially injected into the separation channel to work as an effective concentration and separation field. To perform tr-trapping in MCMEKC, we fabricated a 5 way-cross microchip to realize the successive injection of a shorter micellar plug and a longer sample plug. When the injection times of micelle (tinj,M) and sample solution (tinj,S) were 1.0 and 2.0 s, respectively, both the preconcentration and separation of two dyes were completely finished within only 4.0 s. At tinj,S of 8.0 s, a 400-fold improvement of the detectability was achieved in comparison with conventional MCMEKC. The resolution obtained with tr-trapping-MCMEKC was also better than that with conventional MCMEKC in spite of the 160-fold shorter length of the injected micellar zone at tinj,M of 1.0 s. These results clearly demonstrate that the tr-trapping technique in MCMEKC provides a rapid separation, high efficiency, and high sensitivity even in the short separation channel on the microchip.
A Surfactant gradient method was developed for miceller electrokinetic chromatography (MEKC). In the present method, electroosmotic flow was suppressed almost completely and thus, micelles in a running solution of the inlet vial were introduced in turn into the capillary during operations. Under such situations, we can perform surfactant gradient MEKC separations only by changing the composition of surfactants in the inlet vial during a single run. Here, we represented MEKC separations of unsubstituted benzoate and nine substituted benzoates as model organic anions using mixed systems of CTAC as a cationic surfactant and Tween 20 or Brij 35 as non-ionic surfactants possessing polyoxyethylene chains (POE-NSs). In a pure CTAC system, the synergistic influences of attractive electrostatic and hydrophobic interactions gave rise to quite large retention factors of many of the benzoate anions, resulting in their co-elution. Addition of an adequate amount of the POE-NSs to the pure CTAC system decreased the electrostatic interaction significantly to give remarkably improved separation of the analytes, but long analysis time was required. Surfactant gradient methods decreasing the concentration of POE-NSs in the mixed systems were useful to decrease analysis time and to improve separation simultaneously in both stepwise manners and continuous manners. The fundamentals of the present method were discussed in detail. We also mentioned its availability as a platform to construct highly functional separation systems.
This paper describes the concept of MCE-202 “MultiNA”, a new microchip electrophoresis system for DNA / RNA analysis. MCE-202 “MultiNA” realizes high-throughput and cost-effective analysis with originally developed reusable microchips and reagent kits. Application data for Norovirus detection and meat identification are also presented.
Capillary electrophoresis (CE) is a highly efficient separation method and used for separation of many compounds. We developed a fast and sensitive analytical method for β-amyloid (Aβ) aggregates by the combination of CE-laser-induced fluorescence and a fluorescence reagent, thioflavine T. The developed method is also applicable to a high-throughput screening of Aβ aggregation inhibitors, which are expected to be an effective therapeutic agent candidates of Alzheimer’s disease.