Use of nanomaterials in capillary and microchip electrophoresis

This review gives an overview of different separation strategies with nanomaterials and their use in capillary electrophoresis (CE) and capillary electrochromatography, as well as in microchip electrophoresis, including metal and metal oxide nanoparticles, carbon nanotubes, fullerene and polymer nanoparticles, as well as silica nanoparticles. The paper highlights the new developments and innovative applications of nanoparticles as pseudostationary phases or immobilized on the capillary surface for CE separation. The separation and characterization of target nanoparticles with different sizes by CE are reviewed likewise.     

Characterization of proteins in the human serum proteome.

This paper presents a multidimensional profile of the human serum proteome, produced by a two-dimensional protein fractionation system based on liquid chromatography followed by characterization with capillary electrophoresis (CE). The first-dimension separation was done by chromatofocusing over a pH range from 8.5 to 4.0, where proteins were separated by their isoelectric points (pI). In this dimension, fractions were collected based on pH. The first-dimension pI fractions were then resolved in the second dimension by high-resolution, reversed-phase chromatography with a gradient of trifluoroacetic acid (TFA) in acetonitrile and TFA in water. A selected protein fraction collected from the second dimension by time was characterized by CE for molecular-weight estimation and for presence of isoforms. Molecular-weight estimation was done by sodium dodecyl sulfate capillary gel electrophoresis, where proteins were separated in the range of 10,000-225,000 Da. Detection of isoforms was done by capillary isoelectric focusing over a pH range of 3-10. A selected second-dimension fraction that contained the putative serum iron-binding protein transferrin was analyzed by these two CE techniques for molecular-weight determination and the presence of isoforms. The combination of two-dimensional protein fractionation and CE characterization represents an advanced tool for proteomics.     

New procedure for the determination of nisin in milk

A new procedure is described for the assay of nisin in milk by using capillary zonal electrophoresis. Since nisin assay in milk and in other matrices is hampered by its adsorption to both the protein and lipid milk fractions, a simple extraction procedure was devised to remove caseins and lipids, which interfere with nisin detection. An excellent linear response to nisin concentration was observed in the range from 10 to 100 g/ml with a mean coefficient of variation of 3.8%. © Rapid Science Ltd. 1998     

Monitoring solution‐phase combinatorial library synthesis by capillary electrophoresis

Capillary electrophoresis has been applied to monitor model reactions in solution‐phase combinatorial chemistry. In particular, the simultaneous alkylation reactions of secondary amines with a series of benzyl halides has been investigated. Reactant and product concentrations were monitored using capillary electrophoresis in a non‐aqueous buffer system. The simplified sample preparation was a key feature making this an attractive method of analysis. The results demonstrate that capillary electrophoresis is a useful tool for monitoring reactions to determine initial rates, rate constants, and extinction correlation coefficients for quantitative analysis in combinatorial chemistry, and is a broadly applicable technique for the analysis of a variety of organic and bioorganic transformations. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng (Comb Chem) 61:169–177, 1998/1999.     

Effect of sialic acid residues of human α1-acid glycoprotein on stereoselectivity in basic drug-protein binding

The function of sialic acid groups at the terminal of sugar chains of human α₁-acid glycoprotein (AGP) was investigated with respect to chiral discrimination between optical isomers of basic drugs, using high-performance capillary electrophoresis/frontal analysis (HPCE/FA), a novel analytical method developed for the determination of unbound drug concentration with ultramicrosample volume (100–200 nl). Native human AGP and desialylated AGP were used as test proteins, and propranolol (PRO) and verapamil (VER) were used as model drugs. The unbound concentration of (S)-VER was 1.31 times higher than that of (R)-VER in native AGP solution. This selectivity was not affected by desialylation. Further, enzymatic elimination of galactose residues, which neighbored sialic acid groups, did not change the binding of either isomer of VER. On the other hand, the unbound concentration of (R)-PRO was 1.27 times higher than that of (S)-PRO in native AGP solution. Desialylation caused the unbound concentration of (S)-PRO to rise to the same level of (R)-PRO, resulting in loss of enantioselectivity. Thus, it follows that sialic acid groups of AGP, as a whole, are not responsible for chiral recognition between enantiomers of VER but are involved in enantioselectivity toward the isomers of PRO. Chirality 9:291–296, 1997. © 1997 Wiley-Liss, Inc.     

山黧豆活性成分β-ODAP的检测方法研究进展

β-N-草酰-L-α,β-二氨基丙酸(β-N-oxalyl-L-α,β-diaminopropionic acid,β-ODAP)是山黧豆中的一种重要非蛋白氨基酸,与三七中的三七素为同一种次生代谢物,具有止血、兴奋神经、抗菌等多种生物活性。自20世纪60年代和80年代在山黧豆等豆类和三七等植物中分别发现β-ODAP以来,科研工作者建立了多种检测方法。该文对近年来国内外有关检测β-ODAP的传统分析法、高效液相色谱法(HPLC)、色谱-质谱法(GC/LC-MS)、毛细管电泳法及酶学分析法等的基本原理及应用研究进展进行了综述,这些检测方法各有所长,具体选择依实验条件和实验目的而定。其中,柱前衍生化高效液相色谱法是目前应用最广泛的方法,高成本同位素内标的LC-MS则被誉为“金标准”。但特异、快速和低成本的检测方法一直是人们的追求目标,因而进一步完善酶传感器技术是β-ODAP检测领域在将来的主要研究方向。     

无胶筛分毛细管电泳中核酸的迁移行为及分离的研究

无胶筛分毛细管电泳分析小于1kb的核酸,其迁移率与碱基数的对数成线性关系,长度大于1kb核酸的迁移率不是仅由其分子大小决定。据此可推测小于1kb核酸片段的大小。采用不更换聚合物法分析核酸,迁移时间的变异系数小于1.3％,适于大量样本的快速测定。考虑温度对核酸迁移行为的影响时,观察到22℃时,柱效最高。电进样与压力进样相比,分析大于300bp核酸的柱效提高,但不适于定量分析。     

Electrochemiluminescence detection with integrated indium tin oxide electrode on electrophoretic microchip for direct bioanalysis of lincomycin in the urine

In this article, an antibiotic, lincomycin was determined in the urine sample by microchip capillary electrophoresis (CE) with integrated indium tin oxide (ITO) working electrode based on electrochemiluminescence (ECL) detection. This microchip CE-ECL system can be used for the rapid analysis of lincomycin within 40s. Under the optimized conditions, the linear range was obtained from 5 to 100 microM with correlation coefficient of 0.998. The limit of detection (LOD) of 3.1 microM was obtained for lincomycin in the standard solution. We also applied this method to analyzing lincomycin in the urine matrix. The limit of detection of 9.0 microM was obtained. This method can determine lincomycin in the urine sample without pretreatment, which demonstrated that it is a promising method of detection of lincomycin in clinical and pharmaceutical area.     

Monitoring differential synthesis of RNA in individual cells by capillary electrophoresis

A capillary electrophoresis (CE)-based technique is reported here to monitor differential RNA synthesis in individual Chinese hamster ovary cells at distinct stages of the cell proliferation cycle. Cell synchronization was achieved by the shake-off method, in which mitotic (M) cells were dislodged, and cells at G(1), S, and G(2) phases were harvested 2.5, 10, and 13 h, respectively, after synchronizing the mitotic cells. Thirty-two cells (eight from each phase) were analyzed by injecting each cell into the capillary, lysing it with dilute surfactant, separating the RNA by capillary electrophoresis, and detecting the peaks with laser-induced fluorescence. The results from single cells show that the total amount of RNA increased at each successive stage (from G(1) to M), while the relative synthetic rates of different RNA fractions varied with progression through the cycle. There was a threefold increase in the synthetic rate of total RNA from S to G(2), compared with G(1) to S. In addition, differential accumulation of specific RNA fractions was observed, with the low-molecular-mass fraction exhibiting a much higher synthetic rate from G(2) to M, relative to the rates of the larger ribosomal RNA (rRNA) fractions. Comparison of the large rRNA fractions with one another reveals that at S phase more 28S rRNA was accumulated than 18S rRNA, and at G(1) and M phases, the synthetic rate of 28S rRNA was slowed compared with that of 18S. Minimal sample preparation, combined with the separation power of CE and single-cell detection sensitivity of laser-induced fluorescence, results in a simple method for assessing differential accumulation of RNA from distinct individual cells.     

Comparative genomic analysis of isolates belonging to the six species of the genus Thermus using pulsed-field gel electrophoresis and ribotyping

Fifty isolates belonging to the six validly described species of the genus Thermus (T. aquaticus, T. filiformis, T. thermophilus, T. scotoductus, T. brockianus, and T. oshimai) isolated from hot springs of different geographical areas were compared using macrorestriction analysis of genomic DNA and ribotyping. With the exception of presumed clones, the macrorestriction patterns of isolates obtained with EcoRI or NdeI were distinct. However, isolates belonging to the same species exhibited similar profiles particularly when they were isolated from the same hot spring. The estimated genomic size of strains of the Thermus spp. varied between approximately 1.8 and 2.5 Mbp. Ribotyping with BamHI and HindIII produced 30 and 35 distinct ribotypes, respectively. In spite of the variability of the hybridization patterns produced, the ribotypes obtained for isolates belonging to the same species also shared, in general, several fragments of identical size, and these fragments were similar when isolates originated from the same spring. Received: 7 October 1996 / Accepted: 10 March 1997     

Rapid monitoring of autolysis process of proteases by capillary electrophoresis

A protease, MCP-01, produced by a deep-sea psychrotrophic strain of Pseudoaltermonas sp. SM9913 was purified and its autolysis reaction at 20 °C–50 °C was monitored by capillary electrophoresis. Capillary electrophoresis provides a rapid assay because the degree and state of autolysis of protease MCP-01 could be observed within 6 min. The autolysis rate increased as the temperature rose in the tested range. After 30 min incubation at 30 °C, 77% of MCP-01 autolyzed into peptides. However, its activity for the hydrolysis of casein was reduced by only 4%. The rate of loss of activity of MCP-01 was thus slower than that of autolysis of MCP-01 at 30 °C. Similar results were obtained when MCP-01 was incubated at 20 °C, 40 °C and 50 °C. Large peptides produced by autolysis of MCP-01 therefore still have catalytic activity. When these large peptides autolyzed further into smaller peptides, the enzyme conformation that retained its catalytic activity was destroyed and activity was lost.     

Escherichia coli ß-galactosidase is heterogeneous with respect to a requirement for magnesium

Commercially obtained E. coli ß-galactosidase was stored at 25 °C in buffer containing 1 mM MgCl₂ and in buffer containing no added MgCl₂. Samples were removed at set times and the activity of individual enzyme molecules assayed. When stored in the presence of 1 mM magnesium, the number of active molecules did not change over a 2.5-h period. When stored in the absence of added MgCl₂, over half the enzyme molecules became inactive within the first hour. However, those molecules which retained activity remained active for the duration of the experiment. This indicates that there may exist two populations of E. coli ß-galactosidase, one which requires storage in the presence of the higher concentration of Mg²⁺ in order to remain active. There was no observed correlation between this requirement for magnesium and reaction rate. Additionally, the presence of the 1 mM MgCl₂ was found to decrease the average activity of the ß-galactosidase molecules under the conditions employed.     

Estimation of receptor-ligand interactions by the use of a two-marker system in affinity capillary electrophoresis

The study of receptor-ligand interactions by affinity capillary electrophoresis (ACE) requires an accurate form of analysis. Here, we examine the use of two noninteracting standards (markers) in the analysis of binding constant data in ACE studies. This concept is demonstrated using two model systems: carbonic anhydrase B (CAB, EC 4.2.1.1) and arylsulfonamides, and vancomycin (Van) from Streptomyces orientalis and the dipeptide N-acetyl-d-Ala-d-Ala. In this procedure a plug of receptor and noninteracting standards is injected, and analysis of the change in the relative migration time ratio of the receptor, relative to the noninteracting standards, as a function of the concentration of the ligand yields a value for the binding constant. The findings described here demonstrate that data from ACE studies can best be analyzed using two noninteracting standards, yielding values comparable to those estimated using other binding and ACE techniques.     

safum: statistical analysis of SSCP fingerprints using PCA projections,dendrograms and diversity estimators

The program safum provides a smart interface to import, visualize and compare fingerprinting profiles, especially on capillary electrophoresis single strand conformation polymorphism data, in conjunction with basic statistical analysis tools. It includes principal component analysis, two‐ or three‐dimensional representations, dendrograms based on Euclidean distance, and easily exportable files for subsequent applications. safum is useful for the analysis of spatial or temporal sequences of microbial community fingerprints obtained with an ABI prism sequencer.     

聚乙二醇修饰牛血清白蛋白的反应与分析

采用N,N′-羰基二咪唑活化法活化单甲氧基聚乙二醇5000分子一端的羟基,对活化后的单甲氧基聚乙二醇分子进行了元素分析。用该活化产物对牛血清白蛋白的赖氨酸侧链氨基进行化学修饰。应用毛细管电泳对聚乙二醇修饰后的产物进行了分析,并与高效液相色谱分析结果作了对照研究,表明毛细管电泳对修饰后的牛血清白蛋白有更好的分析效果。     

毛细管电泳-激光诱导荧光分析血清多胺的研究

为进一步探讨多胺的生物学作用,建立了毛细管电泳-激光诱导荧光(λ_ex=488 nm,λ_em=513 nm)分析血清多胺方法.在碱性介质中,多胺与荧光素异硫氰酸酯进行衍生化反应,硼酸盐(pH 8.6)作为运行缓冲液,运行电压20 kV,腐胺、精胺、精脒和1,6-己二胺（内标）在8 min内达到基线分离.考察了方法的线性范围、稳定性、检测限和方法的回收率等,方法具有样品处理简单,灵敏度高,速度快等特点.用于正常对照大鼠和肿瘤大鼠血清多胺的测定.结果提示：实验组肿瘤大鼠血清精胺和精脒水平显著高于正常对照大鼠和实验组未出现肿瘤大鼠血清精胺和精脒水平（P＜0.05）,正常对照组大鼠和实验组未出现肿瘤大鼠血清精胺和精脒水平间无显著性差异（P＞0.05）,各组间血清腐胺水平均无显著性差异（P＞0.05）.     

Enantioseparations with the macrocyclic antibiotic ristocetin a using a countercurrent process in CE

The chiral selectivity of ristocetin A was examined in a countercurrent process in CE using a coated column to suppress electroosmotic flow. Excellent enantioseparations of several nonsteroidal antiinflammatories, dansylamino acids, dinitrophenyl‐derivatized amino acids, and other optically active compounds were achieved. The chiral selectivity of ristocetin A also was examined as a function of antibiotic concentration and pH. Enantioresolution was found to significantly improve with a slight increase in migration time upon increasing chiral selector concentration. Enantioselectivities were found to be greatly influenced by pH of the running buffer. Chirality 11:663–668, 1999. © 1999 Wiley‐Liss, Inc.     

Determination of ions, amino acids/amines in the haemolymph of fairy shrimps by capillary electrophoresis with indirect UV and laser-induced fluorescence detection

We identified and quantified the major inorganic ions present in a haemolymph of four species of fairy shrimp, to determine its ionic strength, using capillary electrophoresis, non-destructive micro-technique for the analysis of small amounts of body fluids. This is the first speciation and quantitative analysis of inorganic ions in the haemolymph of fairy shrimps.     

无胶筛分毛细管电泳分离DNA片段的现状与展望

毛细管电泳已DNA片段分离分析的重要手段。本简述了毛细管电泳中采用无胶筛分介质分离DNA片段的机理研究,介绍了筛分介质近年的研究发展状况,依据分离介质的化学组成,分单聚物、共聚物和混聚物等3个部分进行了评述,并对其发展前景进行了展望。