Brief introduction of capillary electrophoresis

Directory 1 Pinyin 2 English Reference 3 Basic Principles of High Performance Capillary Electrophoresis 4. 1 Capillary Zone Electrophoresis (CZE Capillary Zone Electrophoresis) 4.2 Capillary Gel Electrophoresis (CGE) 4.3 Micellar Electrokinetic Capillary Chromatography (MECC Micellar Electrokinetic Capillary Chromatography) 4.4 Isoelectric Focusing (IEF) 4.5 Isokinetic Electrophoresis (ITP). 4.6 Capillary electrochromatography (CEC) 4.7 Affinity Capillary Electrophoresis (ACE) 4.8 Electrochromatography (characteristics of recognizing monoclonal protein by electrochromatography 5.3 Hemoglobin component analysis 5.4 Myoglobin analysis 5.5 Lipoprotein analysis 5.6 Glycosylated hemoglobin (HbAlc) analysis 5.7 Isozyme separation 5.8 Immune complex analysis 5.9 DNA fragment and chromosome analysis 5. 1 Application of 0 in therapeutic drug monitoring 5. 1 1 detection of other small molecules/ions 6 prospect 1 pinyin máo xěguěn diàn y

2 English reference capillary electrophoresis

Capillary electrophoresis (CE), also known as high performance capillary electrophoresis (HPCE), is one of the fastest developing analytical methods in recent years. 198 1 year, Jensen and lukacs first proposed to separate in a capillary column with an inner diameter of 75μm and established modern capillary electrophoresis. 1984 Terabe et al. established micellar capillary electrokinetic chromatography. 1987 Hjerten established capillary isoelectric focusing, Cohen and Karger proposed capillary gel electrophoresis. From 1988 to 1989, the first batch of commercial capillary electrophoresis instruments appeared. In a few short years, CE has developed rapidly because it has met the requirements of separation and analysis of peptides, protein (including enzymes and antibodies), nucleotides and even deoxyribonucleic acid (DNA) in various fields of life science represented by bioengineering.

The basic principle of high performance capillary electrophoresis is that particles move in the opposite direction of charge at a uniform speed under the action of electric field. It is an efficient separation technology of macromolecules and micromolecules in a hollow capillary with a tiny inner diameter (inner diameter 10~200um). Both ends of the capillary are immersed in the buffer solution, and the electrodes connected with the high-voltage power supply are respectively inserted. This voltage causes the analysis sample to migrate along the capillary. According to the difference of charge and volume between separated substances, various molecules are separated under high pressure. In capillary electrophoresis with free zone, electrophoresis movement (charged molecules move to electrodes with opposite polarity) and electroosmotic flow (electrolyte movement caused by inner wall charge of capillary and applied potential energy) lead to separation.

The size of electroosmotic flow depends on electric field strength, PH value of electrolyte, composition of buffer solution, ionic strength, internal friction and capillary surface. These factors can improve the separation effect alone or in combination. Ultraviolet can be detected directly through the small window on the capillary, or by laser-induced fluorescence, diode array, electrochemistry and mass spectrometry. The method of sample introduction is to press the sample into the capillary by air pressure or voltage. There are many separation modes in high performance capillary electrophoresis (HPCE), and their separation mechanisms are different and complementary.

4 Separation mode of high performance capillary electrophoresis 4. 1 separation principle of capillary zone electrophoresis (CZE): Capillary zone electrophoresis is called free solution capillary electrophoresis, but capillary electrophoresis is the simplest form. The separation mechanism is based on the difference of the ratio of net charge to mass of each substance. Different ions move at different speeds in the medium according to their different surface charge densities, which leads to separation. It requires uniform buffer solution and constant electric field intensity in capillary, which is the most widely used separation mode at present. It is suitable for the analysis of protein, amino acids, peptides and ions. The capillary tube does not need to be filled with any substance except electrolyte, and the operation is simple and the degree of automation is high.

4.2 Separation principle of capillary gel electrophoresis (CGE): Gel electrophoresis is a kind of zonal electrophoresis, in which the gel is moved into the capillary and used as a support for separation. Gel is a solid dispersion system. It is porous, similar to the function of molecular sieve. The separated substances are separated one by one according to the size of their respective molecules by the gel force loaded into the capillary, and the macromolecules are separated first. It is suitable for the analysis of biological macromolecules. The molecular weight of protein can be determined by separation based on the size of molecular species (SDSPAGE), and an oligonucleotide with different bases can be identified. It can separate DNA fragments, such as microsatellite (STR) analysis, and can change the gel concentration.

4.3 Separation principle of micellar electrokinetic capillary chromatography (MECC): It is the intersection of electrophoresis technology and chromatography technology. When ionic surfactant is added to the buffer and its concentration is large enough, the monomers of this surfactant combine to form spheres (micelles). At present, sodium dodecyl sulfate (SDS) micelle is the most commonly used. MECC exists between two phases, and they have different retention times because of their retention ability in micelles. With the increase of CZE, the buffer solution forms a positive charge on the tube wall, which leads to the strong maximum seepage moving to the negative electrode. Surface SDS micelles tend to migrate to the positive electrode because of the negative shell, and the electroosmosis speed is generally greater than the migration speed of micelles to the positive electrode, forcing micelles to eventually move to the negative electrode at a lower speed. The separation between neutral particles is based on their own hydrophobicity, and the interaction between particles with different hydrophobicity and micelles is different. High hydrophobic force and long residence time.

MECC is the only HPCE mode that can separate neutral ions from charged components.

4.4 Principle of isoelectric focusing (IEF) separation: The migration of amphoteric electrolyte in the separation medium forms a pH gradient in the capillary, and peptides and protein with different isoelectric points migrate to their different isoelectric points and stop according to this gradient, thus creating a very narrow focusing area, which is separated by using the tiny difference of isoelectric points. After focusing different protein at different positions, the buffer solution of the cathode becomes salt, and finally, high pressure is added to reduce the gradient, so that the components pass through the detector one by one, and accurate isoelectric points are obtained.

4.5 Isokinetic Electrophoresis (ITP) separation principle: An electrophoretic method of focusing separation, in which the separated components move forward together with the electrolyte. Like IEF, the electroosmotic flow of ITP in capillary is zero, and the buffer system consists of two electrolytes with different fluidity. In the separation process, the trailing electrolyte (whose mobility is lower than that of the separated component) is first introduced into the capillary. Under the action of strong electric field, the separated components are focused and separated in the gap between the two electrolytes.

The choice of treated or untreated silicon capillary is correct, and 0.25% hydroxypropyl methyl fiber can inhibit electroosmosis. The leading electrolyte is 5nM phosphoric acid and the trailing electrolyte is valine. At the beginning of separation, because the electrolyte with high mobility completely fills the capillary, the current will increase rapidly, and when entering the separation process, the current will decrease as the electrolyte with low mobility enters the capillary.

4.6 Separation principle of capillary electrochromatography (CEC): It is a chromatographic process in which many stationary phases of high performance liquid chromatography are filled into capillary tubes, and the interaction between samples and stationary phases is the separation mechanism, and the current mobile phase is the driving force.

4.7 Separation principle of affinity capillary electrophoresis (ACE): In the electrophoresis process, there is biological specific affinity, that is, the specific affinity between the receptor (receptor) and the ligand (ligand) forms the receptor ligand complex. By changing the electrophoresis patterns of receptors and ligands before and after affinity interaction, we can get the information of the effect products of the changes of affinity size and structure of receptors and ligands.

4.8 Electrokinetic chromatography (EKC) separation principle: it is an electrophoresis mode named after electrokinetic phenomenon, involving the principles of electroosmosis, electrophoresis and chromatography, and is mainly used for the separation of chiral compounds.

4.9 separation principle of nonaqueous capillary electrophoresis (NaCE): it is a mode of electrophoretic separation of analytes in organic solvents. The use of non-aqueous medium can increase the selectivity of the method and is beneficial to the separation of water-insoluble substances.

5. Clinical Application of High Performance Capillary Electrophoresis 5. 1 Serum Protein ce separation effect analysis, and can accurately calculate the relative concentration of each protein, avoiding the errors caused by various factors in the process of gel electrophoresis dyeing and decoloration. The results of HPCE method have good repeatability, high reliability and easy storage and retrieval. Serum prealbumin concentration can indicate nutritional status and is an important index to judge malignant tumor, inflammation, liver cirrhosis and Hodgkin's disease. Most electrophoresis methods are difficult to distinguish, but HPCE method is easy to separate and quantify. The detection wavelength is 2 14 or 200 nm. Capillary electrophoresis improved the resolution of albumin. This greatly improves the sensitivity of detecting double albuminemia. CE provides sufficient resolution in α 1 region to distinguish α 1 acidic glycoprotein from α 1 antitrypsin. In α2 region, α2 macroglobulin is not easy to distinguish from haptoglobin, but it has high resolution in β globulin region. CE method can be used to analyze polyclonal immunoglobulins such as nephrotic syndrome, chronic inflammation, autoimmune diseases and liver cirrhosis, showing clear characteristics. Serum protein electrophoresis is an important screening test for monoclonal protein (MP) anemia, such as multiple myeloma and macroglobulinemia, which is highly sensitive to the detection of typical monoclonal light chains and monoclonal proteins. Oligoclonal protein component is an oligogammaglobulinemia with uncertain clinical significance or reflecting the existence of infectious diseases. It is also a part of some clinical disease processes, such as B-cell lymphoma, use of chemical immunosuppressants, autoimmune or immune complex diseases.

5.2 Identification of the characteristics of monoclonal protein by immunoadsorption. The agarose spheres coated with specific anti-IgG IgM antibodies were incubated with serum samples, and CE was detected before and after incubation. A special peak was eliminated with the agarose spheres coated with specific antibodies to indicate which monoclonal component it was, so as to identify the type, subtype and light chain type of immunoglobulin.

5.3 Analysis of Hemoglobin Components More than a dozen Hb variant chains can be separated by isoelectric focusing capillary electrophoresis (CIEF) and zone electrophoresis (CZE). Some authors used CZE method to separate blood samples from normal people and thalassemia patients in alkaline phosphate buffer (PBS) with PH 1 1.8. The separation speed was very fast (< 8 min), and the electrophoresis patterns of the two were obviously different. After fetal red blood cells are treated, their hemoglobin can be separated, and α, β, γ globulin chains and C globulin chains can be separated. For example, if a buffer with a low pH of pH3.2 is used, although the analysis time is prolonged, the resolution effect of variants is better. Obviously, CE technology plays an important role in the differential diagnosis of hemoglobinopathy.

5.4 Analysis of Myoglobin Abnormal increase of myoglobin in blood and urine is frequent in patients with acute myocardial infarction. It is difficult to determine low concentration of myoglobin by immunoturbidimetry. CZE can quickly separate low concentration myoglobin from urine and distinguish it from hemoglobin within 8 minutes.

5.5 Lipoprotein analysis can separate plasma lipoproteins into 14 subcomponents. If surfactant is added to the separation buffer, two main components, high density lipoprotein (HDL) and low density lipoprotein (LDL), can be quantified in a short time, and LDL can be further separated into three subcomponents, namely LDL, medium density lipoprotein (ILD) and very low density lipoprotein (VLDL), and the proportion of each component can be determined.

5.6 HbAlc analysis CE can separate the glycosyl configurations of several glycoproteins, and can identify isomers such as hba 1c a 1 and Alc, which is of great significance for the monitoring of diabetes.

5.7 Separation of Isozymes Many isozymes were successfully separated by HPLC. The principle is that the sample is separated by electrophoresis in the capillary, and after the isoenzyme separation band is formed, the power supply is cut off, and then the liquid buffer containing the substrate is added, so that the enzyme can catalyze the substrate to develop color and form the isoenzyme band with correct detection, and then the power supply is turned on again to continue electrophoresis, so that the colored bands formed by isoenzymes pass through the detector in turn. By measuring the optical density beyond the maximum absorption, we can analyze and measure the separated isoenzymes, such as detecting A and B isoenzymes of βbN acetylglucosaminidase, amylases P (pancreas) and S (saliva), creatine phosphokinase, alkaline phosphatase, proteolytic enzyme, γ -glutamine transpeptidase, kallikrein, angiotensin reductase, cathepsin B and 5' nucleotidase.

5.8 Analysis of immune complexes CE can quickly separate immune complexes from bound antigens and antibodies, and the detection limit of L- functional FCE for monoclonal antibodies can reach mg, which can be used to identify low-concentration immune complexes in mixed liquids.

5.9 DNA fragment and chromosome analysis HPCE separation of DNA molecules requires adding macromolecular crosslinking agents such as polyacrylamide, polyethylene glycol, methyl cellulose and other substances as molecular sieves in the buffer, which can effectively separate narrow-range DNA. Some authors used HPCE to adapt to X-linked recessive genetic diseases and successfully analyzed the gene polymorphism of DNA restriction fragments. Studies have shown that HPCE can be used for prenatal diagnosis of carriers and fetuses. At present, CE can isolate three bpDNA fragments. If larger molecular DNA fragments are analyzed, of course, there are still many technologies to be improved in HPCE analysis of DNA fragments, among which the most prominent problem is to choose the appropriate crosslinking agent to improve the resolution of existing methods and become an effective tool for genome analysis.

5. Application of10 in therapeutic drug monitoring CE can simply and quickly analyze various forms of drug components in biological samples, and it is also widely used in pharmacological research, forensic examination and clinical toxicology. For example, after solid-phase extraction, HPCE separation and laser-activated fluorescence detector determination, the detection limit of methotrexate can reach 0.1~1nmol/l; The anti-leukemia drug spore pyrimidine βD Ala was used as glycoside, and the sample was extracted with simple organic solvent. The detection limit is 8 μ mol/l; Mesh electrokinetic capillary electrophoresis (MECC) was used to monitor antihypertensive drugs (nitrofurantoin was used as D and ne), and SDS and γ -cyclodextrin were added to the separation solution. After extraction with organic solvent, the minimum detection limit is10ug/L. For example, amoxicillin, an antibiotic drug, can directly participate in the injection of plasma samples without sample pretreatment, but adding SDS to the buffer can reduce the adsorption of protein on the wall. Cef antibiotics (cef work x work me) can be decomposed into five metabolites by gastrointestinal bacteria after oral administration, and the final products are excreted with urine. Detection of drug concentration in blood and urine can be used as a clinical observation index. The blood and urine drug concentrations of some drugs can be used as clinical observation indicators; Some MECC used in drug analysis can be directly separated without special treatment, such as smooth muscle spasmolysis agent, which can alleviate the pain of patients with urinary calculi. The detection limit of MECC can reach 200 μ g/L. Antiasthmatic drugs (theophylline) are often used to treat asthma and asphyxia in premature infants. Serum, saliva and urine samples were determined by direct injection with multiple wavelengths. The linear range is 0~200umol/L, and the accuracy is good in the range of 5~ 1 10. Hypnotic and sedative drugs are widely used in clinic, which are easy to produce drug dependence, and the poisoning dose is close to the therapeutic dose. If the drug concentration needs to be monitored, the minimum detection limit can reach ng/L, and antiepileptic drugs can also be used. It can also analyze the composition of toxic substances in human body, such as morphine and its main metabolites, heroin, cocaine, morphine and other sedatives. In the treatment and monitoring of diabetes, we can prevent hypoglycemia caused by improper use of drugs by detecting the concentration of hypoglycemic drugs.

5. The detection of11other small molecules/ions can separate weak anions such as blood contrast agent and oxalate content in blood and urine samples within 3~4m to detect more than a dozen porphyrin substances and vitamin C isomers in urine samples. 10 organic acid markers, such as dimethyl malonate, glutaric acid, 3- methylglutaric acid, N- acetylaspartic acid, 2- aminoacetic acid, propionic acid, lactic acid, isovaleric acid, uric acid and 2- oxoisovaleric acid, can be used for screening of hereditary organic aciduria in newborns.

6 Prospect The limitation of traditional electrophoresis technology is that when the voltage at both ends reaches a certain value, it will generate self-heating (Joule heat) in the electrolyte ion flow, resulting in the gradient of radial viscosity and velocity, resulting in wider band and lower efficiency. The essence of HPCE is that it is carried out in a capillary tube (10~200mm) with high heat dissipation efficiency. The operation is simple, the analysis speed is fast, the sample injection is less than other separation methods, the sensitivity is high, the cost is relatively low, and the capillary tube and self-made buffer can be reused. It has developed rapidly since 1980s, and it can be divided into two categories: clinical type and scientific research type. Protein analysis and identification are the most widely used clinical types. This technology is very mature, and it has been used as a routine test abroad in the 1990s, and it is also being started in China. High performance capillary electrophoresis (HPCE) is a recognized frontier in analytical chemistry and biomedicine, and will be accepted by clinical medical laboratories. It can be used not only for the analysis of routine items, but also for the inspection of other special items, and also plays an important role in clinical applications in many other fields.