Choice of different dyes to label tyrosine and nitrotyrosine

In this work, we will present some attempts to analyze tyrosine and nitrotyrosine using capillary electrophoresis and either UV-Visible detection or laser-induced fluorescence (LIF) detection. An argon ion (488 nm) laser is used for fluorescein isothiocyanate (FITC) and 7-fluoro-4-nitro-2,1,3-benzoxadiazole (NBD-F). A near infrared (780 nm) laser is used for NIR 780 derivatives. The UV-Visible limit of detection is 2.5 microM whereas it is in the range of 30 nM for LIF detection.     

Recent developments in capillary isoelectric focusing

The developments in capillary isoelectric focusing (cIEF) over the period 2003-2007 are reviewed. With the focus on technological aspects, cIEF papers published in the fields of methodology, new techniques, detection, multidimensional systems, miniaturization and applications are summarized. The methodology section covers recent research in ampholytes composition, detergents and other additives, carrier ampholyte free cIEF, coatings and other capillary modifications. In the section on new systems adjustments to the technique (e.g. dynamic IEF), different applications of cIEF (e.g. as injection system) and new devices are reported. Systems focusing on whole column imaging, fluorescence and chemiluminescence detection and coupling to mass spectrometers are discussed in the section on detection. Interfacing cIEF with MS via RPLC systems and hyphenation of cIEF with capillary electrochromatography and other capillary electrophoresis modes are also summarized. Papers focusing on miniaturization are reviewed in the section on microfluidic devices. The section on applications will show analysis of biopharmaceutical compounds and isolated proteins for metabolomic studies. For the analysis of complex biological matrices, generally multidimensional systems are needed, which are mentioned throughout this review.     

Sample stacking revisited: a personal perspective

One of the major challenges in capillary electrophoresis and other miniaturization separation techniques is to maintain high detection sensitivity in the increasingly smaller dimension. Numerous on-column sample preconcentrating procedures, based either on electrokinetic focusing or chromatographic effects, have been developed. This review will discuss some practical approaches to sample stacking from a personal perspective. Several recent developments in sample stacking on microfluidic devices are reviewed.     

Advances in amperometric and conductometric detection in capillary and chip-based electrophoresis

Amperometric and conductometric detection are currently the two major electrochemical detection modes in capillary and chip electrophoresis. The ease of miniaturization and integration of electrochemical detection elements offers a high potential for the development of portable analytical devices based on electromigrative separations. The challenges and basic concepts of both detection principles in the context of capillary/chip electrophoresis are shortly introduced and milestones of the methodical developments are summarized from a historical perspective. Recent advances and applications are discussed with more detail. Particular attention is paid to new trends in this area of research such as measurements in short capillaries and the enormous progress and increased popularity of contactless conductivity detection. Correspondence: Frank-Michael Matysik, Institute of Analytical Chemistry, University of Leipzig, Linnéstr. 3, D-04103 Leipzig, Germany     

Applications of capillary electrophoresis to the determination of antibiotics in food and environmental samples

In this paper we review applications of capillary electrophoresis (CE) to the determination of antibiotic residues in food derived from animals and in environmental samples. Although many CE methods have been used to determine antibiotics in the pharmaceutical field (drug quality control or therapeutic monitoring in biological samples), food and environmental applications have been increasing in recent years. Due to the maximum residue limits established by the EU, in Directive 2377/90/EEC, for foodstuffs of animal origin and considering the low levels that can be found in environmental or waste waters or soils, different strategies to increase sensitivity have been developed, including off-line preconcentration, on-line stacking modes to use higher sample volumes, or in-line solid-phase extraction. Also, several detection techniques, such as fluorescence, laser-induced fluorescence, electrochemical detection, or mass spectrometry have been used; the last of these also enables unequivocal identification of the residues, required by Commission Decision 2002/657/EC. All these aspects will be discussed in this paper, in relation to the main groups of antibiotics used in veterinary and human medicine, for which applications in food and environmental samples have been developed by using CE as an efficient alternative to liquid chromatography.     

Light-emitting diode induced fluorescence (LED-IF) detection design for a pen-shaped cartridge based single capillary electrophoresis system

CGE is a well-established separation technique for the analysis of biologically important molecules such as nucleic acids. The inherent high resolving power, rapid analysis times, excellent detection sensitivity, and quantification capabilities makes this method favorable compared to conventional manual polyacrylamide and agarose slab gel electrophoresis techniques. In this paper we introduce a novel single-channel capillary gel electrophoresis system with LED-induced fluorescence detection also utilizing a compact pen-shaped capillary cartridge design for automatic analysis of samples from a 96-well plate. To evaluate the suitability of the system, 1000 genomic DNA(gDNA) samples were analyzed in gel filled capillaries and detected by the microball ended excitation and emission optical fiber based LED-induced fluorescence detection system. Excellent migration time reproducibility of RSD <0.75% was obtained over the course of 1000 runs. The system rapidly distinguished between intact and degraded gDNA samples, therefore provided important information if they could be used for downstream quantitative PCR processing where high-quality intact gDNA was key. We envision that this novel system design will rapidly find new applications in both research and clinical diagnostic laboratories as a highly sensitive and easy to use bio-analytical approach.     

Capillary electrochromatography-laser-induced fluorescence method for separation and detection of dansylated dialkylamine tags in encoded combinatorial libraries

LC-fluorescence and LC-MS methods have been previously reported for use in decoding bead-based combinatorial libraries. We present the use of capillary electrochromatography (CEC) for highly selective decoding in combination with laser-induced fluorescence (LIF) detection for high sensitivity. The results are compared to prior data obtained using HPLC with fluorescence detection. The use of CEC shows promise for miniaturization and multiplexing for future applications, and the use of LIF detection can allow for detection at sub-pmol amounts.     

Hyphenated chromatographic methods for biomaterials

The improvement in hyphenated analytical techniques has significantly widened their applications to the analysis of biomaterials. In this article, we discuss recent advances in applications of hyphenated chromatographic techniques including capillary electrophoresis to the analyses of biological samples. As tools of separation, gas chromatography, high-performance liquid chromatography and capillary electrophoresis are considered with special emphasis on applications utilizing the hyphenation of these methods to mass spectrometry. Moreover, applications using other detection methods such as Fourier transform infrared spectroscopy hyphenated to gas chromatography and photodiode array detector combined with high-performance liquid chromatography or capillary electrophoresis are also discussed. Owing to their high sensitivity, luminescence-based detection systems such as laser-induced fluorescence and chemiluminescence are also included in this review.     

A data treatment method for detecting fluorescence anisotropy peaks in capillary electropherograms

A data treatment method is presented to detect fluorescence anisotropy (FA) peaks in capillary electrophoresis electropherograms. The data treatment method converts plots of fluorescence anisotropy vs. time that contain no peaks that are distinguishable from the noise of the anisotropy background into plots that show distinct fluorescence anisotropy peaks. The method was demonstrated using laser-induced fluorescence anisotropy data from individual Aβ (1–42) aggregates separated using capillary electrophoresis. Applying this data treatment method enabled the detection of anisotropy peaks for individual Aβ aggregate fluorescence peaks that were not observed prior to the data treatment method. The data treatment method is not specifically designed for Aβ aggregate analysis or capillary electrophoresis, and it should be applicable to other applications and other separation methods with FA detection.     

Label-free fluorescence detection in capillary and microchip electrophoresis

Herein, we summarize the current status of native fluorescence detection in microchannel electrophoresis, with a strong focus on chip-based systems. Fluorescence detection is a powerful technique with unsurpassed sensitivity down to the single-molecule level. Accordingly fluorescence detection is attractive in combination with miniaturised separation techniques. A drawback is, however, the need to derivatize most analytes prior to analysis. This can often be circumvented by utilising excitation light in the UV spectral range in order to excite intrinsic fluorescence. As sensitive absorbance detection is challenging in chip-based systems, deep-UV fluorescence detection is currently one of the most general optical detection techniques in microchip electrophoresis, which is especially attractive for the detection of unlabelled proteins. This review gives an overview of research on native fluorescence detection in capillary (CE) and microchip electrophoresis (MCE) between 1998 and 2008. It discusses material aspects of native fluorescence detection and the instrumentation used, with particular focus on the detector design. Newer developments, featured techniques, and their prospects in the future are also included. In the last section, applications in bioanalysis, drug determination, and environmental analysis are reviewed with regard to limits of detection.     

Fundamentals and practice for ultrasensitive laser-induced fluorescence detection in microanalytical systems

Laser-induced fluorescence is an extremely sensitive method for detection in chemical separations. In addition, it is well-suited to detection in small volumes, and as such is widely used for capillary electrophoresis and microchip-based separations. This review explores the detailed instrumental conditions required for sub-zeptomole, sub-picomolar detection limits. The key to achieving the best sensitivity is to use an excitation and emission volume that is matched to the separation system and that, simultaneously, will keep scattering and luminescence background to a minimum. We discuss how this is accomplished with confocal detection, 90 degrees on-capillary detection, and sheath-flow detection. It is shown that each of these methods have their advantages and disadvantages, but that all can be used to produce extremely sensitive detectors for capillary- or microchip-based separations. Analysis of these capabilities allows prediction of the optimal means of achieving ultrasensitive detection on microchips.     

Micro flow modules with combined fluid flow channel and optical detection waveguide - hyper Rayleigh scattering as a case study

Micro flow modules with optical detection have been fabricated in a way which enables optical waveguiding inside and a defined interaction length along the fluid channel. Because of the usually lower refractive index of the solution compared with that of the substrate, so-called "leaky" optical wave-guiding must be employed. The combination of the fluid flow channel function with that of the optical waveguide has advantages for all miniaturized optical detection cells. It has been shown for hyper Rayleigh scattering (HRS) that improvement of the analytical principle is inherent in the miniaturization. The detection limit can be enhanced by at least a factor of 20. The applied HRS measurement procedure also enables simultaneous detection of two photon absorption (TPA) fluorescence. The severe boundary conditions of capillary electrophoresis were used as micro flow module design constraints to enable the transfer of the approach to other types of analysis.     

Challenges and applications of isotachophoresis coupled to mass spectrometry: A review

Electrophoretic separations are of growing interest to tackle complex analytical challenges. Nevertheless, capillary electrophoresis, as the most common mode, still suffers from insufficient detection limits due to low capillary loadability. ITP is of growing interest as preconcentration method for capillary electrophoresis and is also interesting to be applied as an independent analytical method. While mass spectrometric detection is common for capillary electrophoresis, the combination of ITP with MS is still a niche technique. In this work, we want to give an overview on isotachophoretic effects in CE-MS and ITP-MS methods, as well as coupling techniques of ITP with CE-MS. The challenges and possibilities associated with mass spectrometric detection in ITP and its coupling to capillary electrophoresis are critically discussed.     

基于PMMA毛细管电泳芯片的多位点突变检测研究

利用基于激光诱导荧光(LIF)检测的芯片毛细管电泳平台,批量制作了低成本聚甲基丙烯酸甲酯(PMMA)芯片,通过修饰管道,优化有效分离距离、分离介质等条件,可在90s内完成DNA片段的分离检测,实现单碱基分离,并在此平台上成功地对遗传性耳聋三个常见突变位点实现分型检测,为这种低成本的PMMA芯片应用于分型相关的临床诊断领域奠定了基础。     

Applications of capillary electrophoresis with electrochemical detection in pharmaceutical and biomedical analyses

As a high efficiency separation technique, capillary electrophoresis has been widely used in various fields of analytical science. This review discusses the applications of electrochemical detection systems combined with capillary electrophoresis in pharmaceutical and biomedical analysis. These detection methods mainly involve amperometric detection but also include conductivity detection and potentiometric detection. Its applications in the field are divided into six parts, including catechol compounds, thiols, amino acids and peptides, carbohydrates, general pharmaceuticals, and other related compounds. A relatively detailed discussion is described for each compound under the current studied. On this basis, we have suggested several conceivable directions for capillary electrophoresis with electrochemical detection in the future.     

Near-infrared spectroscopy for liquids of microliter volume using capillaries with wall transmission

In the present study we propose a capillary tube method for measuring near-infrared (NIR) spectra of microliter liquid and solution samples. This method enables one to measure NIR spectra of liquids and solutions of only 2.5 microl. As an example of the capillary tube method, Fourier-transform NIR spectra of benzene were measured using a capillary tube with a diameter of 1 mm. Positions and intensities of the NIR bands in the spectra obtained by the capillary tube were almost identical to those measured using a quartz cuvette cell. Moreover, capillary NIR spectra of human blood serum obtained from a patient with backbone neoplasm were also measured. Subtraction of the NIR spectrum of water from those of human blood serum develops unambiguously some NIR bands due to blood components such as proteins and glucose. Our results suggest that capillary NIR spectroscopy will open new areas of NIR applications for small amounts of liquid and solution samples.     

人的基因组及DNA序列分析──过去、现去及将来（上）

人的基因组研究已成为生命科学前沿领域中最热门的课题之一。ＤＮＡ序列分析是基因组研究的关键技术．本文对人的基因组分析及其对ＤＮＡ序列分析的要求进行了论述．对ＤＮＡ序列分析方法如板凝胶电泳自放射显影法、板凝胶电泳激光荧光法、毛细管电泳激光荧光法、阵列毛细管凝胶电泳激光荧光法。超薄层板在胶电泳激光荧光法作了详细评论．并对正在开发的不用凝胶电泳分离的直接测序新技术和新方法，如质谱法、原子探针法（扫描隧道显微镜、原子力显微镜）、杂交法、流动单分子荧光检测法等进行了评论。     

Capillary array scanner for time-resolved detection and identification of fluorescently labelled DNA fragments

The first capillary array scanner for time-resolved fluorescence detection in parallel capillary electrophoresis based on semiconductor technology is described. The system consists essentially of a confocal fluorescence microscope and a x,y-microscope scanning stage. Fluorescence of the labelled probe molecules was excited using a short-pulse diode laser emitting at 640 nm with a repetition rate of 50 MHz. Using a single filter system the fluorescence decays of different labels were detected by an avalanche photodiode in combination with a PC plug-in card for time-correlated single-photon counting (TCSPC). The time-resolved fluorescence signals were analyzed and identified by a maximum likelihood estimator (MLE). The x,y-microscope scanning stage allows for discontinuous, bidirectional scanning of up to 16 capillaries in an array, resulting in longer fluorescence collection times per capillary compared to scanners working in a continuous mode. Synchronization of the alignment and measurement process were developed to allow for data acquisition without overhead. Detection limits in the subzeptomol range for different dye molecules separated in parallel capillaries have been achieved. In addition, we report on parallel time-resolved detection and separation of more than 400 bases of single base extension DNA fragments in capillary array electrophoresis. Using only semiconductor technology the presented technique represents a low-cost alternative for high throughput DNA sequencing in parallel capillaries.     

毛细管电泳化学发光检测系统新进展

分析技术的微型化、集成化和自动化是当前分析化学发展的一个新趋势。本文综述了毛细管电泳化学发光检测系统微型化、集成化和自动化的最新研究进展。