Électrophorèse capillaire et hémoglobinopathies

For 20 years, capillary electrophoresis has become a major tool in clinical laboratories. In protein analysis, the great diversity of separation principles has allowed it to replace classical electrophoresis and to compete with liquid chromatography. Since the very beginning, this technique has been applied to haemoglobin analysis, firstly to separate abnormal forms, then to quantify minor fractions. This article reviews the different capillary electrophoresis methods that have been developed for the separation and the quantification of hemoglobin fractions or of its basic elements.     

High performance microfluidic capillary electrophoresis devices

This paper presents a novel microfluidic capillary electrophoresis (CE) device featuring a double-T-form injection system and an expansion chamber located at the inlet of the separation channel. This study addresses the principal material transport mechanisms depending on parameters such as the expansion ratio, the expansion length, the fluid flow. Its design utilizes a double-L injection technique and combines the expansion chamber to minimize the sample leakage effect and to deliver a high-quality sample plug into the separation channel so that the detection performance of the device is enhanced. Experimental and numerical testing of the proposed microfluidic device that integrates an expansion chamber located at the inlet of the separation channel confirms its ability to increase the separation efficiency by improving the sample plug shape and orientation. The novel microfluidic capillary electrophoresis device presented in this paper has demonstrated a sound potential for future use in high-quality, high-throughput chemical analysis applications and throughout the micro-total-analysis systems field.     

Separation and detection of DNA polynucleotides using capillary electrophoresis. Application to detection of polymerase chain reaction-amplified human immunodeficiency virus and HLA DNA.

The polymerase chain reaction (PCR) is a technique for rapid amplification of target DNA sequences. During the past several years, a large number of research applications of PCR have appeared, many of which may prove to be useful clinically. We report the use of capillary electrophoresis, a fully automated technique, as an alternative to polyacrylamide gel electrophoresis for the detection of PCR-amplified viral and cellular DNA. We describe conditions for rapid separation, detection, and discrimination of PCR products from the human immunodeficiency virus type 1 gag gene and the HLA-DQ-alpha gene amplified from the human immunodeficiency virus provirus-containing U1.1 cell line. The sensitivity achieved with the use of capillary electrophoresis analysis was roughly equivalent to that of ethidium bromide staining of polyacrylamide gel electrophoresis gels. Further refinement of capillary electrophoresis for automated detection and quantitation of PCR-amplified products should expedite more widespread application of PCR analysis in the clinical laboratory.     

毛细管区带电泳分离珠蛋白肽链

目的介绍一种可快速分离临床样品珠蛋白肽链的新技术。方法用未涂层毛细管，酸性尿素、ＴｒｉｔｏｎＸ－１００、巯基乙醇、磷酸盐电泳缓冲液，采用毛细管区带电泳法，对ＨｂＡ标准品、６０名正常人、１３例β地中海贫血患者及１例ＨｂＨ病患者，进行毛细管电泳分离其珠蛋白肽链。结果用纳升级样品，在４０分钟内即能一次性分离出α、β、Ｇγ、δ和Ａγ５种珠蛋白肽链；电泳图基线平稳，目标峰清晰，电泳重复性良好；时间变异系数在３％以内，峰面积变异系数在４％以内。另外对６０名正常人及１４例地中海贫血患者珠蛋白肽链含量进行了分析。结论毛细管区带电泳技术能成功地分离珠蛋白肽链，该技术具有简便、快速、高效、样品用量少等优点，适用于对临床样品进行血红蛋白病的快速诊断。     

Electromigration methods for amino acids, biogenic amines and aromatic amines

Methods of electromigration in laboratory apparatus of small-bore size have recently undergone development at a remarkably rapid pace, leading to a variety of new analytical techniques. One such technique is called "capillary electrophoresis" (CE), which is further classified on the basis of electromigration mode, viz., "capillary zone electrophoresis" (CZE), which, in turn, has several variations. This review aims to give a short overview of the various electromigration methods for amino compounds by using CE. Firstly, this review briefly summarizes the detection methods employed for detection of monoamines and polyamines by CE for both native and derivative forms. Next, current CE methods are described, and their applications to detection of amino acids, biogenic amines, aromatic amines, including heteroaromatic amines and their enantiomers, are introduced from representative papers. Finally, new methods for single-cell analysis and microchip CE techniques are focused on.     

Analysis of small-scale biological compartments by capillary electrophoresis

Two characteristics of capillary electrophoresis make the technique attractive for the separation of the components of microscale compartments within living organisms: small sample volume requirements and direct compatibility with biofluids. Indeed, capillary electrophoresis has been used for analysis down to a sub-cellular level. There are also potentially many applications of capillary electrophoresis to biological compartments on a super-cellular scale, which are nevertheless so small that they make analysis by conventional separations techniques difficult or impractical. The analytical challenges in small-scale bioanalysis are first to develop a suitable method for collection of sample and its introduction into the separation capillary, and secondly, to achieve the required separation. Examples reviewed here will primarily focus on the analysis of tear fluid or airway surface liquid, cases in which the amount of sample that can be collected range from around 10 microl to around 100 nl.     

Neutral amino acids monitoring in phenylketonuric plasma microdialysates using micellar electrokinetic chromatography and laser-induced fluorescence detection

Neutral and non-polar amino acids such as phenylalanine (Phe), valine (Val), tyrosine (Tyr), threonine (Thre) and GABA are hard to resolve by capillary zone electrophoresis (CZE). Their separation is possible by adding a surfactant to the mobile phase. This method is called micellar electrokinetic chromatography (MEKC). We used MEKC with laser-induced fluorescence detection (LIFD) to separate and quantitate these amino acids in plasma microdialysates of patients with phenylketonuria (PKU). This disease is an inborn enzymatic defect with decreased conversion of Phe to Tyr that causes severe neurological damage and mental deterioration, which is diagnosed by measuring plasma Phe and Phe/Tyr ratio. The amino acids tested had linear concentration-signal relation. PKU patients had significantly higher Phe, lower Tyr, 21 times higher Phe/Tyr ratio and decreased values of Val and Thre than controls. These results show that microdialysis of biological fluids coupled with MEKC-LIFD is a convenient technique to measure neutral amino acids in clinical disorders such as PKU.     

Analysis of trace amino acid neurotransmitters in hypothalamus of rats after exhausting exercise using microdialysis

A simple but effective coupling of microdialysis and capillary electrophoresis with laser induced fluorescence detection technique was applied to analysis of amino acid neurotransmitters in the hypothalamus of rats after acute exhausting exercise. The separation of amino acids was achieved using an uncoated fused-silica capillary (57 cm x 75 microm I.D.) with a buffer of 10 mM disodium tetraborate at pH 10 and an applied voltage of 12.5 kV. The detection limit was 10(-10) M for each amino acid. It is sufficiently sensitive and rapid for the determination of amino acids in a 5-microl Microdialysate. In comparison to pre-exercise, a significant increase in the levels of six hypothalamic amino acids (arginine, glycine, lysine, glutamic acid, alanine, gamma-amino-n-butyric acid) was found after exercise. These results demonstrate that the increase of metabolic amino acids in the hypothalamus of rats can be induced by exhausting exercise and suggests that amino acid neurotransmitters may play functional roles in the central effects of exercise.     

Determination of immunoreactive gonadotropin-releasing hormone in serum and urine by on-line immunoaffinity capillary electrophoresis coupled to mass spectrometry

The need for urgent diagnoses has propelled the development of automated analyses that can be performed in a short time at reasonable cost. One such method is immunoaffinity capillary electrophoresis. This emerging hybrid technology employs two powerful techniques coupled on-line for the direct and rapid determination of analytes present in biological fluids. The first technique, immunoaffinity, is used for the selective extraction of a molecule present in a complex matrix, utilizing a microscale-format chamber affinity device. An analyte (affinity target) present in serum or urine is captured by an immobilized molecular recognition antibody molecule (affinity ligand) bound to a solid support constituent (glass beads or an appropriate porous structure) of a microchamber affinity device. The second technique, capillary electrophoresis, is used for the high-resolution analytical separation of the purified and concentrated affinity target material after elution from the microchamber affinity device. In this work, immunoaffinity capillary electrophoresis was developed for the identification and characterization of a single constituent of a complex matrix. Immunoreactive gonadotropin-releasing hormone was determined in serum and urine specimens derived from a normal individual and from a patient suffering from benign prostatic hyperplasia. Furthermore, the on-line immuno-separation system was coupled in tandem to mass spectrometry to obtain molecular mass information of the affinity isolated and CE separated neuropeptide. This hybrid immuno-analytical technology is simple, rapid, selective and sensitive. In addition, an attempt was also made to characterize other urinary constituents by CE-MS that may lead to marker activity in the urine of the diseased subject. The hyphenation of analytical techniques has proved valuable in enhancing their individual features. The future of bioanalysis using miniaturized affinity systems is discussed in this paper.     

High-throughput single-strand conformation polymorphism analysis by capillary electrophoresis

Mutation detection plays a great role in genetic and medical research and clinical diagnosis of inherited diseases and particular cancers. Single-strand conformation polymorphism (SSCP) analysis is one of the most popular methods for detection of mutations. Recently, automated capillary electrophoresis (CE) systems have been used in SSCP analysis instead of conventional slab gel electrophoresis. SSCP analysis in combination with CE is a rapid, simple, sensitive and high-throughput mutation screening tool, and has been successfully applied for mutation detection involving human tumor suppressor genes, oncogenes and disease-causing genes. The new technique has a great potential for mutation screening of large numbers of samples in clinical diagnosis. This review discusses basic issues about the methodology of SSCP analysis based on CE and summarizes several key applications.     

Separation of DNA fragments and single strand conformation polymorphism analysis in bare capillaries using poly(acrylamide-dimethylacrylamide) as a separation medium

A short chain poly(acrylamide-dimethylacrylamide) (PADMA) was synthesized in aqueous phase using isopropanol as a chain transfer agent, and was characterized according to the chemical composition and molecular mass. This polymer can form a stable dynamic coating on the inner surface of the capillary, thereby suppressing the electroosmotic flow and DNA-capillary wall interaction. The sieving medium has low viscosity and capillary filling with this medium and medium replacement were conveniently carried out by commercial capillary electrophoresis instruments. The effects of components and concentration of copolymers on the separation of DNA fragments were investigated. Highly efficient separation of DNA fragments, successful single strand conformation polymorphism (SSCP) analysis and good reproducibility of the migration time were obtained in bare capillaries using these copolymers as sieving media. Our preliminary results demonstrate that PADMA will become an alternative matrix for DNA separation by capillary electrophoresis.     

Capillary electrophoresis: principle and applications

Capillary Zone Electrophoresis is a new separation technique which is performed in capillaries (ID smaller than 100 microns). The separations are carried out according to several modes which are mainly the Free Solution Capillary Electrophoresis, the Micellar Electrokinetic Capillary Chromatography and the Gel Capillary Electrophoresis. Many detection systems can be used. This technique has many advantages: high separative power, short time of analysis, low cost of operation and very low sample consumption. Capillary electrophoresis and HPLC appear to be complementary because they are based on different physical and chemical properties. Capillary electrophoresis has a great potential of applications for both chemical and biochemical analysis. This new and very efficient analytical technique is of growing interest for biochemical analysis and its use is likely to increase rapidly.     

Two-dimensional gel electrophoresis as an aid in the analysis of monoclonal gammopathies

Two-dimensional gel electrophoresis is the most powerful protein separation technique currently available. In the authors' laboratory it has proved useful in the analysis of specimens from patients with monoclonal gammopathies when those specimens were otherwise difficult to diagnose. Examples of problematic specimens include biclonal gammopathies, heavy chain diseases, and monoclonal gammopathies that show a small or invisible spike on serum protein electrophoresis on cellulose acetate. In addition, two-dimensional electrophoresis is being used to investigate the pathophysiologic features of myeloma kidney disease, especially regarding potential interactions of Bence Jones proteins and kidney proteins.     

Applications of capillary electrophoresis in DNA mutation analysis of genetic disorders.

AIM: To facilitate DNA mutation analysis by use of capillary electrophoresis. METHODS: The usefulness and applications of capillary electrophoresis in DNA fragment sizing and sequencing were evaluated. RESULTS: DNA mutation testing in disorders such as cystic fibrosis, Huntington disease, alpha thalassaemia, and hereditary fructose intolerance were undertaken effectively. However, sizing the (CAG)n repeat in the case of Huntington disease was a potential problem when using capillary electrophoresis. Separation polymers used in capillary electrophoresis are still in the developmental phase, with improved ones being released regularly. CONCLUSIONS: In the DNA diagnostic setting, capillary electrophoresis is a valuable development because it expands the scope for automation and has useful analytical properties. The potential to perform complex multiplexing within one electrophoresis run facilitates DNA diagnosis. The different mobility of DNA fragments in capillary electrophoresis compared with conventional gel electrophoresis will require, in some circumstances, additional care when results are being interpreted or reported. Capillary electrophoresis is a cheap alternative for combined automated sequencing and fragment analysis that utilises multicolour fluorescence capability. However, in its present form, it is not useful for large scale sequencing.     

Coupling of optical characterization with particle and network synthesis for biomedical applications

Polymeric microspheres containing a magnetic core have been used in cancer therapy for biophysical targeting of antitumor agents and in magnetic resonance imaging as contrasting agents. For the Human Genome Project, deoxyribose nucleic acid (DNA) capillary electrophoresis has become the most widely used analytical technique where a key component is the design of an effective separation medium. The synthesis and optical characterization of polymeric coated superparamagnetic nanoparticles and of (self-assembled) polymer networks by means of a range of physical techniques, including laser light scattering and laser-induced fluorescence detection, are presented. (1) Polymeric microspheres with a superparamagnetic core. A water-in-oil microemulsion approach has been used successfully to synthesize the superparamagnetic core and the polymeric microsphere in one continuous step. The synthesis permits us to control the magnetic nanoparticle size and the thickness of the hydrogel, ranging from 80 to 320 nm. Magnetite concentration in the microspheres, calculated by vibrating-sample magnetometry, was found to be up to 3.3 wt %. The internal structure of the microspheres, as observed by atomic force microscopy, confirmed a core-shell model. (2) Development of new separation media for DNA capillary electrophoresis. Block copolymers in selective solvents can self-assemble to form supramolecular structures in solution. The nanostructures can be characterized in the dilute concentration regime by means of laser light scattering. At semidilute concentrations, the mesh size, the supramolecular structure, and the surface morphology can be investigated by means of small angle x-ray scattering and atomic force microscopy. The structural knowledge and the information on chain dynamics can then be correlated with electrophoresis using laser-induced fluorescence detection to provide a deeper understanding for the development of new separation media.     

Poliovirus separation from cell extracts using capillary electrophoresis: potential use in vaccine production and control?

Rapid assessment of the concentration of virus particles in a given sample remains a challenge. Modern separation methods, such as capillary electrophoresis, were proposed recently to study viruses and viral infection or to separate and characterize viral vaccines in a time-efficient manner. Even though capillary electrophoresis is much more rapid than traditional virological methods and has the advantages of automation, increased precision and reliability, it has the drawback of reduced sensitivity for low concentrations. A sensitivity improvement is then necessary in many cases for a successful application. However, to date, only highly purified viral samples were examined using capillary electrophoresis. The injection of larger sample volumes, followed by intra-capillary concentration, was used in this study for cell extracts. Poliovirus was successfully detected rapidly, without any laborious staining procedures and incubation times. The method is simple, fast, automatic, requires only minute amounts of samples and reagents, and no expensive dyes or biological reagents. Additionally, the method showed a potential for monitoring the viral load during growth and purification, with obvious prospects for the optimization of the variable and time-consuming virus propagation procedures. The results of this study provide a potential basis for the development of routine methods for viral particles analysis, irrespective of their infective properties. In the future, the capillary electrophoresis test could help study the relationship between the intact poliovirus particles and the D-antigenic properties of a viral suspension, or could represent a supplementary or alternative test for virus concentration and D-antigen assays during vaccine production.     

Capillary electrophoresis in the pharmaceutical industry: applications in discovery and chemical development

Over the last 10 to 15 years capillary electrophoresis (CE) has become an extensively used separation technique in the pharmaceutical industry. The attraction of the various modes of operation of CE to analysts is their complementarity to other more established methodology, in particular high-performance liquid chromatography. CE methods have been developed not only for the resolution of drug substances that vary widely in their structure, size and stereochemistry, but also for the determination of the physico-chemical constants of analytes, such as pKa and isoelectric point (pI) values, binding and complexation constants, and octanol-water partition coefficients.     

Chromatographic and electrophoretic approaches in ink analysis

Inks are manufactured from a wide variety of substances that exhibit very different chemical behaviors. Inks designed for use in different writing instruments or printing methods have quite dissimilar components. Since the 1950s chromatographic and electrophoretic methods have played important roles in the analysis of inks, where compositional information may have bearing on the investigation of counterfeiting, fraud, forgery, and other crimes. Techniques such as paper chromatography and electrophoresis, thin-layer chromatography, high-performance liquid chromatography, gas chromatography, gel electrophoresis, and the relatively new technique of capillary electrophoresis have all been explored as possible avenues for the separation of components of inks. This paper reviews the components of different types of inks and applications of the above separation methods are reviewed.     

Séparation électrophorétique des isoenzymes de la phosphatase alcaline par le Kit Hydragel 15 Iso-PAL®

We evaluated a new electrophoresis Kit (Hydragel 15 Iso-PAL®, Sebia) for the separation of alkaline phosphatase (ALP) isoforms. This agarose gel technique uses wheat germ lectin affinity for sialic acids present on ALP isoenzymes. Similar results were obtained between serum and plasma (lithium heparinate), particularly for the bone fraction (< 5% difference). In healthy subjects, bone and liver H1 fractions are the two major components of ALP activity (up to 90%) with a ratio close to 1/1. This technique displays a practical advantage for laboratories, due to the absence of sample pre-treatment, the use of a single gel and automation on the Hydrasys® system (2 h analysis time for 7 patients). This method also offers a clinical advantage for bone and hepatobiliary diseases, allowing a whole panorama of ALP activity.     

Subcutaneous microdialysis: a simple technique for monitoring the extracellular biochemical environment. Combination with capillary electrophoresis and laser-induced fluorescence detection

Microdialysis is a simple technique that allows monitoring endogenous or exogenous substances in any extracellular compartment. It has many useful experimental and clinical applications. The sampling of the extracellular fluid of the subcutaneous compartment is especially useful for metabolic evaluation in critically ill patients, pharmacokinetic studies and blood glucose monitoring. We built a subcutaneous microdialysis probe, with a cellulose hollow fiber (13,000 molecular weight cut off, 200 microns outside diameter) glued to stainless steel tubing (26 ga. outside diameter). It was implanted in the subcutaneous tissue of a critically ill child or anesthetized mice to obtain amino acids patterns by means of capillary electrophoresis with laser induced fluorescence detection (CE-LIFD). The probe was also implanted in ambulatory volunteers to monitor glucose. The results confirmed that subcutaneous microdialysis is a very simple, inexpensive and not aggressive method with advantages over repeated venipuncture sampling and endovenous microdialysis sampling. The present report shows that subcutaneous microdialysis with the proper analytical technique can be used to monitor the chemical composition of the interstitial compartment in very different preclinical or clinical conditions.