Capillary electrophoresis high-speed capillary electrophoresis separation system

Capillary zone electrophoresis is the most basic protein separation mode of chip capillary electrophoresis. This is a simple and rapid separation method based on the different migration rates of different protein molecules in the electric field. Many protein samples have been successfully separated by zone electrophoresis.

Colyer et al. used capillary electrophoresis chip to separate human serum protein samples in zone electrophoresis mode, and could distinguish four protein bands (IgG, transferrin, a- 1- antitrypsin and albumin bands, which were used to simulate 7, P, dl and albumin bands in serum protein samples respectively). Among them, the fluorescence labeling of protein was carried out after separation. Because of the low sensitivity of serum protein labeled with fluorescent dye TNS(2- toluidine black. 1- ene -5- sulfonate), it is impossible to separate the five bands of actual human serum protein samples. Xiao et al. adopted the zone electrophoresis mode, and used 50 mmoVL phosphate buffer (pH 2. 15) as the working buffer, and realized the rapid separation of cytochrome c and lysozyme in 35 seconds on a polydimethylsiloxane () chip with a channel width of 30um. Dodge et al. designed a PDMS chip integrating 8 microvalves and/kloc-0 micropumps, which realized the effective control of liquid flow through microvalves and micropumps. They first separated bovine serum albumin and myoglobin by zone electrophoresis, then introduced the separated protein components into a micro-mixer for enzymolysis through a valve, and finally analyzed the products by mass spectrometry. This work shows that the chip technology can be used for the pretreatment of complex protein samples before mass spectrometry analysis. Zhuang et al. used 75 mmol/L borate buffer (pH 10.3) as the electrophoresis buffer system on the chip to separate immunoglobulin, O/ 1 antitrypsin, bovine serum albumin and iron transfer protein. The urine samples of patients with pregnancy-induced hypertension syndrome, rheumatic heart disease and multiple myeloma were analyzed by electrophoresis, and the results were consistent with Helena electrophoresis system in the United States within 2 minutes.

An important problem to be solved in the separation of protein by chip capillary electrophoresis is the adsorption of macromolecular protein on the channel surface. The tiny adsorption between the protein and the inner wall of the chip channel will reduce the separation efficiency of protein, resulting in wide and trailing peak deformation, which will affect the repeatability of separation. In capillary zone electrophoresis separation mode, two methods are usually used to inhibit the adsorption of protein, namely, permanent modification of the inner wall of the channel and dynamic modification by adding additives to the buffer.

Wu et al. effectively separated two basic proteins (lysozyme and ribonuclease) and two typical acidic proteins (bovine serum albumin and oral lactoglobulin) by using multi-layer 88% hydrolyzed polyvinyl alcohol (PVA) modified chip. The coating can inhibit the generation of electroosmotic flow and the adsorption of protein in the range of pH 3 ~ 1 1, and the effect is stable, and the separation effect is still good after 70 consecutive runs. The research team then processed epoxy modified polymer coating on the surface of PDMS chip channel by self-assembly method to inhibit the adsorption of protein, and successfully separated lysozyme and ribonuclease A A..Chiem et al. added inorganic electrolyte NaCl and neutral surfactant Tween 20 to the running buffer to inhibit the adsorption of protein, and separated and analyzed monoclonal antibodies by chip capillary zone electrophoresis. Gel electrophoresis is a widely used separation technique in protein omics and protein separation. It is a separation mode in which polymers such as gel are used as separation media, and the network structure is used for separation according to the different molecular volumes of the tested components. Using gel electrophoresis mode to separate the protein on the chip is more conducive to achieving high-speed and high-efficiency separation operation. Yao et al. used sodium dodecyl sulfonate (SDS) gel electrophoresis separation mode to compare the performance of chip SDS capillary gel electrophoresis with that of conventional capillary gel electrophoresis system. The results show that the separation efficiency of the former is obviously better than that of the latter, and the separation time is obviously lower than that of the latter.

Like conventional capillary gel electrophoresis, the commonly used screening media in chip capillary gel electrophoresis are divided into gel and non-gel polymer solutions. Crosslinked polyacrylamide gel is a widely used gel screening medium. Herr et al. transplanted the traditional SDS- PAGE method to the chip for the first time, and prepared 6% cross-linked polyacrylamide gel in the channel of the chip as the screening medium through photopolymerization. Five kinds of protein with relative molecular mass (m) between 5500 and 39000 were separated within 30s, and the separation distance was only. At the late stage of'1',the research team prepared 22% cross-linked polyacrylamide membranes in microchannels for preconcentration of protein samples, which effectively enriched protein molecules with a relative molecular weight of 12000 ~ 205000, and used 8% cross-linked polyacrylamide gel as the screening medium for separation.

Agirregabiria et al. used SU-8 optical gel to make microchannels on polymethyl methacrylate (PM-MA) chip, and used polyacrylamide gel with concentration of 12% as screening medium to separate protein. Subsequently, the research team integrated metal electrodes on the chip and successfully separated two kinds of protein with relative molecular weights of 20 000 and 97 000, respectively. However, cross-linked polyamide-amide gel has some problems, such as complex preparation and difficult use. Compared with it, linear polyacrylamide (PLA), polyvinyl alcohol (PEG), polyethylene oxide (PEO) and other non-gel screening media have the advantages of simple preparation, convenient use, and can be injected into the channel after polymerization without polymerization in the channel, so they are suitable for use in complex channel systems, so they have been widely used in chip capillary gel electrophoresis. Yao et al. used SDS 14 200 gel buffer (Beckman Coulter product) to separate six kinds of protein and Giordano with relative molecular weights of 9000 ~ 1 16000 on a glass chip within 35 s, and added nano-orange dye to the samples and buffer to dynamically label protein, and optimized the separation buffer system. Finally, 5% PEO(M, = 100 000) was selected as the screening medium. The detection limit of this system for bovine serum albumin is 500ng/mL, and the separation and analysis of actual human serum samples have been completed.

In chip capillary gel electrophoresis, the adsorption of protein on the inner wall of the channel is still an important problem to be solved. Bousse et al. physically coated the inner wall of the microchannel of the glass chip with polydimethyl acrylamide (PDMA) to reduce the electroosmotic flow to 0.5×10 ~ mzv s, and the protein standard sample of Bio—Rad Company was separated by SDS gel electrophoresis within 40 s, and the separation efficiency reached 107 plate/m. Nagata et al. used PEG coating on the PMMA chip. Using 5% linear polyacrylamide as screening medium, the trypsin inhibitor was separated at high speed. The separation time of bovine serum albumin and galactosidase was only 8 s in a channel with a separation length of 3 mm The principle of protein separation by chip isoelectric focusing is basically the same as that by conventional capillary isoelectric focusing, and the separation is based on the difference of protein isoelectric point (pI). Hofmann et al. first applied capillary tube to protein analysis.

Li et al. separated bovine serum albumin and enhanced green fluorescent protein on the chip and polycarbonate chip by isoelectric focusing. Das, wait. 26 3 Using polymer chip, the separation length and voltage conditions were optimized in isoelectric focusing electrophoresis mode. Finally, in the channel with the length of 1.9 cm, green fluorescent protein and R phycoerythrin were separated in 1.5 min, and the separation voltage was 500 V. Cui et al. successfully separated phycoerythrin, isoelectric focusing and phycoerythrin on the chip. The author also reported that the channel distance needed to complete protein separation can be changed by changing the concentration of methylcellulose in the sample and separation medium. Tsai et al. suppressed protein adsorption by modifying glass chip channel with hexamethyldisiloxane plasma polymerization membrane, and separated phycocyanin (PI: 4.65), hemoglobin (pI: 7.0) and cytochrome C (PI: 9.6) in isoelectric focusing separation mode. When protein was separated by chip isoelectric focusing, Huang et al. adopted the method of adding hydroxymethyl cellulose as an additive in amphoteric electrolyte solution to inhibit protein adsorption. The successful application of chip capillary electrophoresis promotes the development of high-speed and efficient chip two-dimensional electrophoresis technology. For multi-component complex protein samples, the traditional one-dimensional separation method can not meet the requirements, so two-dimensional separation technology is needed to improve the separation efficiency and increase the peak capacity. Compared with the traditional capillary electrophoresis system, the two-dimensional electrophoresis separation on the chip can realize the direct intersection or communication of channels by designing the channel structure of the chip, without making a complicated two-dimensional capillary electrophoresis interface, thus avoiding the phenomenon of band expansion caused by the existence of dead volume of the interface.

In the research of protein separation by chip two-dimensional electrophoresis, the first-dimensional separation mode mostly adopts isoelectric focusing mode. Chen et al. made a two-dimensional capillary electrophoresis chip, and used one-dimensional isoelectric focusing and two-dimensional gel electrophoresis to separate and analyze the fluorescent labeled bovine serum albumin and carbonic anhydrase, as well as the egg white labeled with Dichotos red. Li et al. designed a 4t- sheet for two-dimensional separation of polymer cores by isoelectric focusing and gel electrophoresis. After isoelectric focusing separation in the first dimension, protein samples can be separated in multiple parallel channels by gel electrophoresis in the second dimension. The whole separation process was completed within 10 min and the peak capacity reached 1 700. Herr et al: "1 A two-dimensional chip system of isoelectric focusing zone-free electrophoresis with cross-channel configuration has been developed. The chip channel is 200 barrel meters wide and 20 barrel meters deep. The sample to be measured is separated by isoelectric focusing in the transverse channel, and the separated sample zone enters the longitudinal zone electrophoresis channel under the drive of electric field for two-dimensional separation. The separation performance was evaluated by fluorescence microscope imaging, and the separation peak capacity reached 1 300 within 5 min. Wang et al. made a microvalve on the chip to prevent the mixing of separation buffers between one-dimensional isoelectric focusing and two-dimensional gel electrophoresis systems, and effectively separated four standard proteins within 20 rain. It has also been reported that protein two-dimensional electrophoresis combined with SDS gel electrophoresis and micellar electrokinetic capillary electrophoresis was used to separate PMMA chips. The system completed the separation of 10 protein in 12 min, and the peak capacity was about 1000.

In addition, there is a two-dimensional separation system based on a chip, which is mainly used for the separation and analysis of protein enzymatic hydrolysate. Usually, the first dimension separation adopts micellar electrokinetic capillary electrophoresis or capillary electrochromatography mode, and the second dimension separation adopts zone electrophoresis mode. 1 A two-dimensional separation system combining micellar electrokinetic capillary electrophoresis (the first dimension) and zone electrophoresis (the second dimension) was established on a glass chip for the first time, and it was applied to the separation of trypsin degradation products such as cytochrome c, ribonuclease and D-L albumin. After that, the research team improved the system, lengthened the length of the first-dimensional electrophoresis channel and used a small-diameter corner channel to reduce diffusion. Bovine serum albumin hydrolysate was separated in about 65438±05min, and the peak capacity reached 4 200. 200 1 they also developed a chip two-dimensional electrophoresis system combining open-tube electrochromatography and zone electrophoresis. In the electrochromatographic separation part, an annular channel with octadecyl trimethoxysilane coating is used, and in the zone electrophoresis part, a straight channel with the length of 1.2 μm is used. The first casein trypsin hydrolysate was separated within 65438 03 minutes.

Compared with the one-dimensional separation chip, the two-dimensional chip separation system has higher separation efficiency and peak capacity, which is expected to play a greater role in the separation of complex protein samples. Microfluidic chip capillary electrophoresis system has outstanding advantages in the separation and analysis of protein, especially in clinical inspection and field monitoring. At the same time, it is also of great significance to the development of integration, miniaturization and portability of analytical instruments. According to literature reports, Renzi et al. developed a handheld microfluidic chip electrophoresis separation device for protein. The device consists of an electrophoresis chip, a small laser-induced fluorescence detection system and a high-voltage power supply, and its volume is only1.5cm×1.5cm×1.5cm×19.0cm. It can be used for field analysis, bedside medical diagnosis and forensic analysis. In recent years, there have been reports on the detection of clinical urinary protein and lipoprotein by chip capillary electrophoresis in China. Recently, Pandey et al. used Caliper and Agilent P200 protein chips to detect microalbuminuria, and integrated and automated the electrophoresis separation and fluorescence detection of protein, thus realizing its application in clinical laboratories.

At present, many researchers are devoted to the research of microfluidic chip capillary electrophoresis combined with mass spectrometry to further improve the separation and analysis ability of complex samples. The above system has a broad application prospect in protein separation analysis and protein formation research. Especially for the multi-dimensional separation and analysis of complex protein samples, chip capillary electrophoresis is fast and efficient, and can be used as one of the one-dimensional separation methods to significantly improve the analytical flux of protein. It is believed that with the deepening of research and the continuous development of related technologies, microfluidic chip capillary electrophoresis protein separation technology will become more and more mature and play an important role in biochemical analysis, clinical diagnosis and protein group research.