Microchip Electrophoresis
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Amino acids are essential compounds for living organisms, and their determination in biological fluids is crucial for the clinical analysis and diagnosis of many diseases. However, the detection of most amino acids is hindered by the lack of a strong chromophore/fluorophore or electrochemically active group in their chemical structures. The highly sensitive determination of amino acids often requires derivatization. Capillary electrophoresis is a separation technique with excellent characteristics for the analysis of amino acids in biological fluids. Moreover, it offers the possibility of precapillary, on-capillary, or postcapillary derivatization. Each derivatization approach has specific demands in terms of the chemistry involved in the derivatization, which is discussed in this review. The family of homocyclic o-dicarboxaldehyde compounds, namely o-phthalaldehyde, naphthalene-2,3-dicarboxaldehyde, and anthracene-2,3-dicarboxaldehyde, are powerful derivatization reagents for the determination of amino acids and related compounds. In the presence of suitable nucleophiles they react with the primary amino group to form both fluorescent and electroactive derivatives. Moreover, the reaction rate enables all of the derivatization approaches mentioned above. This review focuses on articles that deal with using these reagents for the derivatization of amino acids and related compounds for ultraviolet-visible spectrometry, fluorescence, or electrochemical detection. Applications in capillary and microchip electrophoresis are summarized and discussed.
The review presents an evaluation of the development of on-line, at-line and in-line sample treatment coupled with capillary and microchip electrophoresis over the last 10 years. In the first part, it describes different types of flow-gating interfaces (FGI) such as cross-FGI, coaxial-FGI, sheet-flow-FGI, and air-assisted-FGI and their fabrication using molding into polydimethylsiloxane and commercially available fittings. The second part deals with the coupling of capillary and microchip electrophoresis with microdialysis, solid-phase, liquid-phase, and membrane based extraction techniques. It mainly focuses on modern techniques such as extraction across supported liquid membrane, electroextraction, single drop microextraction, head space microextraction, and microdialysis with high spatial and temporal resolution. Finally, the design of sequential electrophoretic analysers and fabrication of SPE microcartridges with monolithic and molecularly imprinted polymeric sorbents are discussed. Applications include the monitoring of metabolites, neurotransmitters, peptides and proteins in body fluids and tissues to study processes in living organisms, as well as the monitoring of nutrients, minerals and waste compounds in food, natural and wastewater.
- MeSH
- elektroforéza kapilární metody MeSH
- elektroforéza mikročipová * metody MeSH
- mikrodialýza MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
This review presents recent developments and applications of capillary and microchip electromigration methods in proteomics and peptidomics. Sample preparation methods as well as instrumental innovations in the coupling of these advanced electromigration methods with mass spectrometry detection employed in proteomic and peptidomic analyses are presented. Interesting applications of various capillary electromigration methods in bottom-up as well as top-down proteomics, including investigation of post-translational modifications of proteins are described. In addition, several examples of the use of capillary electromigration methods combined with mass spectrometry detection in clinical proteomics and peptidomics are demonstrated.
The emergence of drug-resistant bacteria and new or changing infectious pathogens is an important public health problem as well as a serious socioeconomic concern. Immunomagnetic separation-based methods create new possibilities for rapidly recognizing many of these pathogens. Nanomaterial-based techniques including fluorescent labeling by quantum dots as well as immunoextraction by magnetic particles are excellent tools for such purposes. Moreover, the combination with capillary electrophoresis in miniaturized microchip arrangement brings numerous benefits such as fast and rapid analysis, low sample consumption, very sensitive electrochemical and fluorescent detection, portable miniaturized instrumentation, and rapid and inexpensive device fabrication. Here the use of superparamagnetic particle-based fully automated instrumentation to isolate pathogen Staphylococcus aureus and its Zn(II)-containing proteins (Zn-proteins) is reported using a robotic pipetting system speeding up the sample preparation and enabling to analyze 48 real samples within 6 h. Cell lysis and Zn-protein extractions were obtained from a minimum of 100 cells with the sufficient yield for SDS-PAGE (several tens ng of proteins).
- MeSH
- bakteriální proteiny izolace a purifikace MeSH
- elektroforéza kapilární metody MeSH
- elektroforéza mikročipová metody MeSH
- imunomagnetická separace metody MeSH
- kvantové tečky * MeSH
- Staphylococcus aureus chemie izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
This review article summarises aspects of the determination of amino acids using capillary and chip electrophoresis in combination with contactless conductivity detection from their historical beginnings to the present time. Discussion is included of the theory of conductivity detection in electromigration techniques, the design of contactless conductivity cells for detection in capillaries and on microchips, including the use of computer programs for simulation of the conductivity response and the process of the electrophoretic separation of amino acids. Emphasis is placed on optimisation of the background electrolyte composition, chiral separation, multidimensional separation, stacking techniques and the use of multidetection systems. There is also a description of clinical applications, the determination of amino acids in foodstuffs, waters, soils and composts with emphasis on modern techniques of sample treatment, such as microdialysis, liquid membrane extraction and many other techniques.
CE with capacitively coupled contactless detection (C4D) was used to determine 3-methylhistidine (3-MH) and 1-methylhistidine (1-MH). The C4D response to 3-MH was studied in a BGE consisting of 500 mM acetic acid and ammonia at varying concentration and the results were compared with the theory. Complete separation of a model mixture of 3-MH, 1-MH, and histidine (His) was attained in two optimized BGEs, one containing 500 mM HAc, 20 mM NH4OH, and 0.1 % m/v hydroxyethylcellulose (HEC), pH 3.4 (I) and the other consisting of 100 mM morpholinoethanesulfonic acid (MES), 25 mM LiOH, and 0.1 % m/v HEC, pH 5.5 (II). These optimized BGEs were tested in CE/C4D analyses of urine. Promising results were obtained for separation and determination of 3-MH, 1-MH, and His on a silicon microchip, using aluminum strips as the C4D electrodes; the three analytes were baseline-separated within less than 30 s with a separation channel effective length of 38 mm. The LOD were satisfactory and amounted to 26.4 microM for 3-MH and 18.3 microM for 1-MH.
Analyses of very complex samples involving capillary and chip electrophoresis often require dual (multiple) detection to attain sufficient selectivity of determination. The present work reviews and critically evaluates selected combinations of electrochemical detection techniques among themselves and with absorption spectrometric, fluorescence and luminescence techniques. Amperometry, contact and contactless conductometry, UV photometry and fluorescence measurements are paid special attention. Some information is also given on combinations of spectrometric techniques with mass spectrometry. The properties of the detection systems are critically discussed, examples are illustrated in figures and some details on the characteristics of dual detectors and their applications are tabulated.
Point-of-care systems based on microchip capillary electrophoresis require single-use, disposable microchips prefilled with all necessary solutions so an untrained operator only needs to apply the sample and perform the analysis. While microchip fabrication can be (and has been) standardized, some manufacturing differences between microchips are unavoidable. To improve analyte precision without increasing device costs or introducing additional error sources, we recently proposed the use of integrated internal standards (ISTDs): ions added to the BGE in small concentrations which form system peaks in the electropherogram that can be used as a measurement reference. Here, we further expand this initial proof-of-principle test to study a clinically-relevant application of K ion concentrations in human blood; however, using a mock blood solution instead of real samples to avoid interference from other obstacles (e.g. cell lysis). Cs as an integrated ISTD improves repeatability of K ion migration times from 6.97% to 0.89% and the linear calibration correlation coefficient (R2 ) for K quantification from 0.851 to 0.967. Peak area repeatability improves from 11.6-13.3% to 4.75-5.04% at each K concentration above the LOQ. These results further validate the feasibility of using integrated ISTDs to improve imprecision in disposable microchip CE devices by demonstrating their application for physiological samples.
Nearly all processes in living organisms are controlled and regulated by the synergy of many biomolecule interactions involving proteins, peptides, nucleic acids, nucleotides, saccharides, and small molecular weight ligands. There is growing interest in understanding them, not only for the purposes of interactomics as an essential part of system biology, but also in their further elucidation in disease pathology, diagnostics, and treatment. The necessity of detailed investigation of these interactions leads to the requirement of laboratory methods characterized by high efficiency and sensitivity. As a result, many instrumental approaches differing in their fundamental principles have been developed, including those based on capillary electrophoresis. Although capillary electrophoresis offers numerous advantages for such studies, it still has one serious limitation, its poor concentration sensitivity with the most commonly used detection method-ultraviolet-visible spectrometry. However, coupling capillary electrophoresis with a more sensitive detector fulfils the above-mentioned requirement. In this review, capillary electrophoresis combined with fluorescence, mass spectrometry, and several nontraditional detection techniques in affinity interaction studies are summarized and discussed, together with the possibility of conducting these measurements in microchip format.
- MeSH
- chromatografie afinitní MeSH
- design vybavení MeSH
- elektroforéza kapilární * MeSH
- elektroforéza mikročipová MeSH
- fluorescenční spektrometrie MeSH
- hmotnostní spektrometrie MeSH
- krevní proteiny analýza MeSH
- lidé MeSH
- peptidy analýza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Automated chip capillary electrophoresis was used in detection and isolation of lactoferrin and a human matrix metalloproteinase (MMP-9) as well as in investigation of interactions and cleavage of collagen with MMP-9. The method is a sensitive and simple technique superior to SDS-PAGE. It is useful in proteomic research.
