Capillary electrophoresis of urinary normal and modified nucleosides of cancer patients

This paper gives a capillary electrophoretic method for the separation of 15 urinary normal and modified nucleosides from cancer patients in less than 40 min. A 500 mmx50 mu m uncoated capillary column (437.5 mm to window) was used. The effects of the voltage and the sodium dodecyl sulfate (SDS) concentration in the buffer on the separation were studied. With reproducibilities of migration times better than 1.2% (R.S.D.) and determined concentrations better than 5-25%, depending on the concentrations of nucleosides in the urine, the analytical characteristics of the method were food. Using this developed method, the concentrations of 13 normal and modified nucleosides, extracted on a phenyl boronic acid affinity chromatography column, in 25 urines from patients of 14 kinds of cancer were determined. The levels (nmol/mol creatinine) of modified nucleosides in urines from cancer patients were increased as compared with those in normal urines. (C) 1998 Elsevier Science B.V.

Quantitative evaluation of the interaction between pUC19DNA and ovalbumin by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) was used to study the interaction between pUC19DNA (pUC19) and ovalbumin (Ova). Samples containing pUC19 and Ova at various ratios were incubated at room temperature and were then separated by CZE with tris-acetate buffer at pH 7.2. Reduction in ultraviolet (UV) absorbance of pUC19 was due to the decrease of free pUC19 after binding to Ova. The binding constant of the interaction calculated from the Scatchard plot was (1.46+/-0.15) x 10(5) M-1. The use of polyacrylamide-coated capillary showed better effects than that of uncoated capillary. The results show that it is important to keep a constant ionic strength in the samples in order to obtain accurate quantitative data in binding assays by CZE.

Interaction between netropsin and double-stranded DNA in capillary zone electrophoresis and affinity capillary electrophoresis

Capillary zone electrophoresis (CZE) and affinity capillary electrophoresis (ACE) were applied to study the interaction between netropsin and a 14mer double-stranded DNA (dsDNA). The use of a polyacrylamide coated capillary can suppress the electroosmotic flow (EOF) and the adsorption of DNA onto the wall. Better analysis of the DNA was achieved in a coated capillary upon Tris-acetate. In CZE, the peak width broadened due to the affinity interaction between dsDNA and netropsin. In ACE, o-toluic acid, a negatively charged molecule was used as the indicator to monitor the changes of EOF when netropsin was added to the running buffer. The 14mer dsDNA showed different mobilities upon various concentrations of netropsin due to the affinity interaction between the dsDNA and netropsin. The binding constants of this interaction were (1.07 +/- 0.10) . 10(5) M-1 calculated from CZE and (4.75 +/- 0.30) . 10(4) M-1 from ACE using a Scatchard plot. The binding stoichiometry was 1:1 calculated from CZE which was superior to ACE in this study. (C) 2002 Elsevier Science B.V. All rights reserved.

Profiling of Complex Microbial Populations by Denaturing Gradient Gel Electrophoresis Analysis of Polymerase Chain Reaction-Amplified Genes Coding for 16S rRNA

We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.

Soil microbial community analysis using denaturing gradient gel electrophoresis

The most biological diversity on this planet is probably harbored in soils. Understanding the diversity and function of the microbiological component of soil poses great challenges that are being overcome by the application of molecular biological approaches. This review covers one of many approaches being used: separation of polymerase chain reaction (PCR) amplicons using denaturing gradient gel electrophoresis (DGGE). Extraction of nucleic acids directly from soils allows the examination of a community without the limitation posed by cultivation. Polymerase chain reaction provides a means to increase the numbers of a target for its detection on gels. Using the rRNA genes as a target for PCR provides phylogenetic information on populations comprising communities. Fingerprints produced by this method have allowed spatial and temporal comparisons of soil communities within and between locations or among treatments. Numerous samples can be compared because of the rapid high throughput nature of this method. Scientists now have the means to begin addressing complex ecological questions about the spatial, temporal, and nutritional interactions faced by microbes in the soil environment.

Separation and detection of chlorpromazine hydrochloride and promethazine hydrochloride using electrochemiluminescence followed by capillary electrophoresis

Phenothiazine drugs, chlorpromazine hydrochloride (CPZ) and promethazine hydrochloride (PMZ), were determined with Ru(bpy)(3)(2+) electrochemiluminescene by the capillary electrophoresis (CE-ECL). It was found that both CPZ and PMZ could produce an intermediate that acted as coreactants to react with Ru(bpy)(3)(2+) to produce excited states which were capable of emitting light. This CE-ECL detection method had high sensitivity, good selectivity and reproducibility for CPZ and PMZ determination.

Analysis of amphetamines in urine with liquid-liquid extraction by capillary electrophoresis with simultaneous electrochemical and electrochemiluminescence detection

Amphetamines including methamphetamine, 3,4-methylenedioxyamphetamine and 3,4-methylenedioxymethamphetamine were separated and detected by CE using simultaneous electrochemical (EC) and electrochemiluminescence (ECL) detection (CE-EC/ ECL). Factors that influenced the separation and detection performance, such as the detection potential, the pH value and concentration of the running buffer, the separation voltage and the pH of the detection buffer, were investigated.

Quality analysis of herbal medicine products prepared from Herba sarcandrae by capillary electrophoresis with electrochemical detection

A capillary electrophoresis with electrochemical detection(CE-ED) method was developed for the quality analysis of herbal medicine products prepared from the same herb of Herba Sarcandrae: Fufang Caoshanhu tablets, Qingrexiaoyanning capsules, and Xuekang oral liquids. Under the optimal analysis conditions, the low detection limit[1.0x10(-7) mol/L(S/N=3)] and the wide linear range(1.0x10(-7)-1.0x10(-4) mol/L) were obtained for quality standard compound of isofraxidin. The precisions of the peak current and the migration time(as RSDs) for the real sample analysis were 2.0%-2.6%, and 1.2%-1.8% for isofraxidin, respectively.

Kinetic Study of Prolidase Activity in Erythrocytes against Different Substrates Using Capillary Electrophoresis with Electrochemiluminescence Detection

Capillary electrophoresis (CE) with electrochemiluminescence (ECL) detection was used to explore the kinetics ofthe enzymatic reaction. The different effects ofreaction conditions including the concentration of Mn2l, incubation temperature and pH on PFOlidase (PLD, EC 3.4.13.9) activity in erythrocyte lysates against three different substrates, Gly-Pro, Val-Pro and Leu-Pro were investigated. Also, the effects of colchicine which can prevent or delay cancer ofliver on the PLD activity were studied.

Sensitive, Label-Free Protein Assay Using 1-ethyl-3-methylimidazolium Tetrafluoroborate Supported Microchip Electrophoresis with Laser-Induced Fluorescence Detection

Based on the dimer-monomer equilibrium movement of the fluorescent dye Pyronin Y (PY), a rapid, simple, highly sensitive, label-free method for protein detection was developed by microchip electrophoresis with LIF detection. PY formed a nonfluorescent dimer induced by the premicellar aggregation of an anionic surfactant, SDS, however, the fluorescence intensity of the system increased dramatically when proteins such as BSA, bovine hemoglobin, cytochrome c, and trypsin were added to the solution due to the transition of dimer to fluorescent monomer. Furthermore, 1-ethyl-3-methylimidazolium tetrafluoroborate (EMImBF(4)) instead of PBS was applied as running buffers in microchip electrophoresis.

Analysis of quinolizidine alkaloids in Sophora flavescens Ait. by capillary electrophoresis with tris(2,2 '-bipyridyl) ruthenium (II)-based electrochemiluminescence detection

A capillary electrophoresis method coupled with electrochemiluminescence detection for the analysis of quinolizidine alkaloids was established, especially, oxymatrine (OMT) which could not be measured by previous electrochemiluminescence methods was detected sensitively herein. Complete separation of sophoridine (SR), matrine (MT) and OMT was achieved within 13 min using a background electrolyte of 50mM phosphate buffer at pH 8.4 and a separation voltage of 15 kV. The calibration curves showed a linear range from 2.8 x 10(-8) to 4.4 x 10(-7) M for SR, 2.7 x 10(-8) to 4.4 x 10(-7) M for MT.

Simultaneous determination of pethidine and methadone by capillary electrophoresis with electrochemiluminescence detection of tris(2,2 '-bipyridyl)ruthenium(II)

In this paper, we described a simple and rapid method, capillary electrophoresis with electrochemiluminescence (CE-ECL) detection using tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)(3)(2+)), to simultaneously detect pethidine and methadone. Analytes were injected to separation capillary of 67.5 cm length (25 mu m i.d., 360 mu m o.d.) by electrokinetic injection for 10 s at 10 kV.

Determination of isocyanates by capillary electrophoresis with tris(2,2 '-bipyridine) ruthenium(II) electrochemiluminescence

CE with tris(2,2'-bipyridyl) ruthenium(II) (Ru(bpy)(3)(2+)) electrochemiluminescence (ECL) detection for the quantitative determination of isocyanates was first reported. Hexamethylene diisocyanate (HDI) and hexyl isocyanate (HI) were used as the model analytes. Commercially available N,N-diethyl-N'-methylethylenediamine was used as the derivatization reagent. It has both a secondary amine group and a tertiary amine group. The secondary amine group can quantitatively react with isocyanate group, and the tertiary amine group can react with Ru(bpy)(3)(2+) to produce strong ECL signal for sensitive detection. The derivatization reaction was almost instantaneous and is much faster than other reported derivative reactions using other derivative reagents.

Ionic liquids used in and analyzed by capillary and microchip electrophoresis

This review, covering reports published from 2001 to December 2008, shows how ionic liquids (ILs) have made significant contributions in the improvement of capillary and microchip electrophoresis (CE and mu CE) for the separation and detection of analytes such as phenols and aromatic acids, metal ions, medicines, enantiomers. biological materials, etc. Furthermore, CE methods applied in the sensitive and accurate determination of physico-chemical properties of ILs have been summarized. Accordingly, research vacancies and future development trends in these areas are discussed.