Capillary electrophoresis-based immunoassay.

Capillary electrophoresis-based immunoassay (CEIA) is a developing analytical technique with a number of advantages over conventional immunoassay, such as reduced sample consumption, simpler procedure, easy simultaneous determination of multiple analytes, and short analysis time. However, there are still a number of technical issues that researchers on CEIA have to solve before the assay can be more widely used. These issues include method to improve the concentration sensitivity of the assay, requirement for robust separation strategy for different analytes, and method to increase the throughput of the assay. The approaches to solve these issues are reviewed. Several studies have been devoted to develop general separation strategies for CEIA, and to enhance the sensitivity of detection. The recent development of microchip-based CEIA is encouraging and is likely to address more drawbacks of CEIA, particularly on the throughput issue.

Effect of various modes of pressurization on the separation in capillary electrochromatography.

In capillary electrophoresis and capillary electrochromatography, the driving factor of the separation is electroosmotic flow (EOF). Pressurized capillary electrochromatography, in which the separation is controlled by EOF as well as the pressure, becomes more and more attractive. We studied the influence of various pressures on capillary electrochromatographic separation. The results reveal that in pressurized capillary electrochromatography, which was performed by EOF combined with the forward and reverse pressure, the main driving factor is still the EOF. It was also found that, when reverse pressure was applied in capillary electrochromatography, the repeatability of the capillary electrochromatographic separation was increased dramatically.

Capillary electrophoretic determination of apoptosis of HeLa cells induced by trichosanthin.

Apoptosis is a distinct mode of cell death that is responsible for deletion of cells in normal tissues; it also occurs in specific pathologic contexts. The observation of apoptosis is very important in the research of cancer and cancer therapy. The traditional observation method of apoptosis was agarose gel electrophoresis, which is depending on the determination of ladder-liking DNA fragments extracted from apoptotic cells. It is time-consuming and low-sensitive. Recently, the sieving capillary electrophoresis has been used to detect apoptosis too. However, the problem of DNA fragments contamination is still existing. Here, we have developed a capillary electrophoresis method that could detect apoptosis of whole cell directly and do not need to extract DNA fragments from cells. Apoptosis of adherent cell HeLa cell of carcinoma induced by cyclophosphamide was used as the model to establish the method. The effluence of medicine concentration on apoptosis of cells was studied in detail. It was also found that the method could detect the change of cells in the early period of apoptosis. The induction of apoptosis of HeLa cell by trichosanthin was determined with the method, and the result of flow cytometry was also proved that trichosanthin could result in apoptosis of HeLa cells.

Single-strand conformation polymorphism analysis to detect the p53 mutation in colon tumor samples by capillary electrophoresis.

The polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique is developed for the detection of point mutations in DNA samples, and is very useful in the research of tumors. The traditional SSCP was carried out with slab gel electrophoresis (SGE), but this is time-consuming and labor-intensive, particularly for clinical diagnoses. We have developed a capillary electrophoresis (CE) method for SSCP detection with a linear polyacrylamide gel solution as the sieving matrix. Twenty colon tumor samples were detected with SSCP-CE and the point mutation in exon 7 of the p53 gene was found in six of the samples. Based on the sequencing results, the accuracy of SSCP-CE was better than that of SSCP-SGE. We hope this rapid and convenient method could be applied in the clinical diagnosis of tumors soon.

Pressurized gradient capillary electrochromatographic separation of eighteen amino acid derivatives.

A pressurized gradient capillary electrochromatography (pCEC) instrument was developed to separate 18 amino acid derivatives. A reversed-phase C18 column (3 microm, 130 mm x 75 microm I.D.) and an acetate buffer (50 mmol/l NaAc, pH 6.4) with an ion-pair reagent (1% N,N-dimethylformamide) were used to separate derivatized amino acids from a standard solution (2 microg/ml), and the wavelength of the UV-Vis detector was 360 nm. The pressure on the capillary column was kept at approx. 70 Pa and 3 kV positive voltage was added on the outlet end of column. The effect of voltage on the eluting order of amino acids and the resolution of separation were studied, and it was found that when the voltage was higher than 3 kV, the adsorption of amino acids in the porous C18 column occurred. The effect of salt concentration, injection volume, and column length on the separation of amino acids was determined. The amino acid sample was separated by pCEC, and RSDs of the migration times of each amino acid were all less than 2.5%.

Variations of the bilirubin uridine-diphosphoglucuronosyl transferase 1A1 gene in healthy Taiwanese.

The activity of uridine-diphosphoglucuronosyl transferase 1 (UGT1) may influence the concentration of serum bilirubin. Because UGT1 is too labile to be measured with classical biochemical methods, we analysed the whole UGT1A1 gene in 290 healthy Taiwanese adults by using the polymerase chain reaction method, and investigated the relationship between UGT1A1 genotypes and serum bilirubin levels. The results showed that slightly more than 50% of the subjects had one or more variant sites in UGT1A1 gene. The most common variant was A(TA)6TAA/A(TA)7TAA (6/7) in the promoter area, followed by heterozygous variation within the coding region, compound heterozygous and homozygous variations. Among the four variant sites within the coding region, 211 G to A was the predominate one, 1091 C to T was a novel variation, and 686 C to A was associated with 6/7. Subjects with 6/7 or heterozygous variation within the coding region or compound heterozygous (plus one homozygous) variation had significantly higher bilirubin levels than those with wild UGT1A1 gene. When the 290 subjects were stratified into six groups according to their serum bilirubin concentrations, the bilirubin levels were correlated well to the frequencies of variant UGT1A1 gene. Our results show that there is a strong association between UGT1A1 gene and bilirubin levels in healthy Taiwanese adults. The occurrence of A(TA)7TAA allele was relatively rare and the variation rate within the coding region was much higher in Taiwanese compared to that in Caucasians.

Fabrication and photoelectric properties of self-assembled bilayer lipid membranes on conducting glass.

Supported bilayer lipid membranes (s-BLMs with and without the doping of fullerene C60) self-assembled on indium-tin oxide (ITO) glass were fabricated and characterized by cyclic voltammetry and electrochemical impedance spectroscopy using a three-electrode system. The photoelectric properties of the ITO supported planar lipid bilayers were studied. Light intensity of irradiation, bias voltage, and concentration of donors have been found to be limiting factors of the transmembrane photocurrent. The facilitation effect of C60 doping in s-BLMs on the photoinduced electron transfer across s-BLM is discussed. This novel self-assembled ITO/s-BLM system may provide a simple and mechanically stable model for the study of the photoelectric and photodynamic properties of biomembranes.

[Analysis of phenylethanoid glycosides in the extract of herba Cistanchis by LC/ESI-MS/MS].

AIM: To analyze the phenylethanoid glycosides in Cistanche deserticola Y. D. Ma and its alternatives. METHODS: An HPLC/MS/MS method has been developed for the analysis of seven kinds of phenylethanoid glycosides in Cistanche deserticola Y. D. Ma, C. salsa (C. A. Mey) G. Beck and C. tubulosa (Schenk) R. Wight. The [M - H]- ions were observed for five standards and Cistanche extracts. The glycosidic linkages, the core, and the attached sugar (s) of the phenylethanoid glycosides can be determined from the collision-induced dissociation spectra of the molecular. RESULTS: Seven kinds of phenylethanoid glycosides (echinacoside, acteoside, cisacteoside, isoacteoside, 2'-acetylacteoside, cistanoside A, osmanthuside B) in Cistanche deserticola Y. D. Ma, six kinds (echinacoside, acteoside, cisacteoside, isoacteoside, 2'-acetylacteoside and cistanoside A) in C. salsa (C. A. Mey) G. Beck and five kinds (echinacoside, acteoside, cisacteoside, isoacteoside and 2'-acetylacteoside) in C. tubulosa (Schenk) R. Wight were detected. CONCLUSION: The difference of the relative distribution of these phenylethanoid glycosides in each extract was found out. Phenylethanoid glycosides are the specific constituents in Cistanchis, which can be used to distinguish different species in Genus Cistanchis.

Enzymatic activity measurement of phospholipid hydroperoxide glutathione peroxidase by capillary electrophoresis.

Based on the separation of 1-palmitoyl-2-(13-hydroperoxy-cis-9,trans-11-octadecadienoyl)-L-3- phosphatidylcholine (PC-OOH) and 1-palmitoyl-2-(13-hydroxy-cis-9,trans-11-octadecadienoyl)-L-3- phosphatidylcholine (PC-OH) and the quantitative determination of PC-OH, the enzymatic activity of phospholipid hydroperoxide glutathione peroxidase (PHGPx) can be measured by capillary electrophoresis. The separation was carried out in a fused-silica capillary (30 cm x 100 microns id) at 15 kV positive voltage. Sodium borate (100 mM; pH = 8.4) was used as the running buffer, and the photodiode array detector wavelength was 232 nm. The determination can be completed in 5 min. The detection limit was 5 pmol; and the relative standard deviation (RSD) of the peak area was less than 1% with an average recovery of 98.6%. Compared with traditional methods such as HPLC and spectrophotometry, it is faster and more convenient. Using capillary electrophoresis, the enzymatic activities of PHGPx expressed by the rice PHGPx gene in E. coli. M15 was determined as 1.25 x 10(-5) mumol min-1, and the specific activity of partially purified trans-gene PHGPx was 3.1 x 10(-2) mumol min-1 per mg. The stability of the trans-gene PHGPx was also studied.

[Development of integrated capillary electrophoresis chips].

Capillary electrophoresis in its different operation modes has been demonstrated that it is suitable to be integrated into a planar microdevice which is called integrated capillary electrophoresis chip (ICE chip) or microchip. The use of microfabrication and micromachining allows the integration of complex channel nets and functional units such as pre- and post-column reaction chambers on a single chip, providing good sensitivity and high separation efficiency with low reagent consumption and short analysis time. In the last 10 years an important number of publications have been reported in this area. In the present paper, the development and applications are systematically reviewed with 39 references.

Microemulsion electrokinetic chromatography of proteins.

Microemulsion electrokinetic chromatography was used to separate a test mixture of proteins effectively. The separation was carried out in a 42.5 cm (to the detector) x 50 microns I.D. fused-silica capillary using a microemulsion system consisting of 80 mM heptane, 120 mM SDS, 900 mM butanol in 2.5 mM borate buffer, pH 8.5-9.5. Optimum separation conditions were investigated with respect to the running voltage, temperature, pH and the composition of microemulsion. Results were compared with those obtained in micellar electrokinetic chromatography and capillary zone electrophoresis. The examined method is practical and successfully applied to the assay of genetically engineering pharmaceuticals, recombinant human granulocyte macrophage colony stimulating factor injection and recombinant human granulocyte colony stimulating factor injection.

Detection of the genotype of apolipoprotein H purified from the serum of a Chinese subject by capillary isoelectric focusing electrophoresis.

Capillary isoelectric focusing electrophoresis (cIEF) has been developed to detect the genotype of apolipoprotein H, which was purified from the serum of a Chinese subject. Depending upon the observation of splitting peaks in cIEF, it is possible to determine if the protein was expressed from a heterozygote gene.

Application of capillary electrophoresis, liquid chromatography, electrospray-mass spectrometry and matrix-assisted laser desorption/ionization - time of flight - mass spectrometry to the characterization of recombinant human erythropoietin.

High performance capillary electrophoresis (HPCE), high performance liquid chromatography (HPLC), matrix-assisted laser desorption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS), on-line CE-electrospray ionization-mass spectrometry (CE-ESI-MS) and on-line LC-ESI-MS have been employed to characterize a heterogeneous glycoprotein, recombinant human erythropoietin (rHuEPO) expressed from Chinese hamster ovary (CHO) cells. The analysis was demonstrated through two specific levels of detail: the intact protein and tryptic digests of the protein. Six glycoforms of rHuEPO were separated by HPCE; seventeen tryptic fragments in a total of 21 nonglycosylated and glycosylated peptides were characterized; the O-linked glycopeptides were analyzed directly by CE-ESI-MS and LC-ESI-MS. In particular, four glycans of O-acetylation of sialic acid were identified in the O-linked glycosylated fragments. The molecular weight of rHuEPO was accurately determined by MALDI-TOF-MS.

[Identification of semen Cuscutae by HPCE].

A method for identification of semen Cuscutae, a traditional Chinese herb, was developed. The proteins in semen Cuscutae were extracted under acid or basic conditions and separated by high performance capillary electrophoresis. The electrophoretograms of acidic or basic extractants from Cuscuta chinensis Lam., Cuscuta australis R. Br. and Cuscuta japonica Choisy showed significant differences, which can be used to identify the three different semen Cuscutae. The results of the identification for 13 pharmacognosical samples agreed well with those of scanning electronic microscopy and tissue microanalysis.

[Determination of three components in aspirin compound tablets by use of UV-PLS method].

The optimum experimental condition for simultaneous UV-spectrophotometric determination of the contents of aspirin, phenacetin and caffeine in aspirin compound tablets (APC) and the basic principle and application of partial least squares method(PLS) in simultaneous multicomponent determination have been studied. Confidence intervals of the three components are 100.1 +/- 0.23% (aspirin), 100.0 +/- 0.25% (phenacetin) and 100.1 +/- 0.33% (caffeine) (confidence 95%). No information has ever been available in the literature for the application of PLS in pharmaceutical analysis. Compared with other traditional computing methods, PLS is a more perfect multicomponent determination method. It is especially applicable to analyzing samples in batches. It is faster and produces more accurate and reliable results. PLS provides a new method for in-line UV-visible spectrophotometric automation.