Capillary Electrophoretic Analysis Research Articles

Integrated portable genetic analysis microsystem for pathogen/infectious disease detection.

An integrated portable genetic analysis microsystem including PCR amplification and capillary electrophoretic (CE) analysis coupled with a compact instrument for electrical control and laser-excited fluorescence detection has been developed. The microdevice contains microfabricated heaters, temperature sensors, and membrane valves to provide controlled sample positioning and immobilization in 200-nL PCR chambers. The instrument incorporates a solid-state laser and confocal fluorescence detection optics, electronics for sensing and powering the PCR reactor, and high-voltage power supplies for conducting CE separations. The fluorescein-labeled PCR products are amplified and electrophoretically analyzed in a gel-filled microchannel in <10 min. We demonstrate the utility of this instrument by performing pathogen detection and genotyping directly from whole Escherichia coli and Staphylococcus aureus cells. The E. coli detection assay consists of a triplex PCR amplification targeting genes that encode 16S ribosomal RNA, the fliC flagellar antigen, and the sltI shigatoxin. Serial dilution demonstrates a limit of detection of 2-3 bacterial cells. The S. aureus assay uses a femA marker to identify cells as S. aureus and a mecA marker to probe for methicillin resistance. This integrated portable genomic analysis microsystem demonstrates the feasibility of performing rapid high-quality detection of pathogens and their antimicrobial drug resistance.

Differential metabolic effects on mitochondria by silica hydride using capillary electrophoresis.

Working as an extension of a newly developed method for a capillary electrophoretic analysis of purine nucleotides, nucleosides, bases, and catabolism, an assay of the differential metabolic properties by a novel organosiliceous anionic hydride compound, silica hydride, was evaluated with Chinese hamster ovary mitochondria using a 50-microm poly(acryloylaminopropanol)-coated, fused-silica capillary. The results of this organellar differential analysis indicate a correlation of increased redox pair of NADH to NAD(+) ratios by two times and an increase in ATP levels in the assayed mitochondria by six times. Glucose levels in the organelles were half of the original values. This study validates the electrophoretic method utilizing live organelle fractions for differential metabolic analysis and additionally illustrates some of the emerging novel properties of silica hydride. As confirmation of the results obtained in this assay, additional methods of standard protocol were used to monitor the mitochondrial metabolic activity.

Determination of bromide and potassium in saline groundwaters by capillary electrophoresis without prior dilution

Two capillary electrophoretic analysis methods are presented; one optimized for the analysis of Br − and the other for determination of K + in groundwaters of high conductivity. The water sampling was performed from the planned final disposal facility area for spent nuclear fuel at Olkiluoto in the municipality of Eurajoki, Finland. Bromide was analysed in an acidic electrolyte solution containing 5 mM formic acid and 42 mM NaCl (pH 3.5) and using direct UV detection (200 nm). Sample stacking was needed for the preconcentration. Potassium was analysed at pH 4.5 using imidazole-18-crown-6 ether solution. The accuracies of the Br − and K + methods were tested using laboratory-made reference sample mixtures with high salt concentration. In the Br − analyses, the limits of detection and determination were 0.1 and 1 mg L −1, respectively. The developed CE analysis for K + in saline water was repeatable (RSD% 14.5–18.0) and the detection and the determination limits were 0.5 and 2.0 mg L −1, respectively. The interlaboratory results showed that CE measurements of Br − and K + were compatible with those made with traditional solvent chemistry techniques.

Variations in content and structure of glycosaminoglycans of the vitreous gel from different mammalian species.

The vitreous of all species is composed of essentially the same type of extracellular matrix macromolecules organized to a transparent gel. In this study, the composition and fi ne chemical structure of the glycosaminoglycans (GAGs) in the vitreous gel from sheep and goat were determined and compared with those of human and pig vitreous gels. The results showed that, in all examined species; hyaluronan (HA) was the predominant GAG, whereas chondroitin sulphate (CS) was the minor one. In the vitreous gel of the most relative species, i.e. sheep and goat, higher amounts of both of HA and CS were estimated as compared with pig and human tissues. The distribution of hydrodynamic sizes of HA and CS was significantly differed among different species. All HA preparations consisted of molecules with great variability in hydrodynamic sizes. The relative proportions of the large HA molecules (size >1.8 x 10(6) kDa) were significantly higher in sheep and goat as compared with human and pig vitreous gel. The length of CS chains was also of larger size in sheep and goat (50 and 58 kDa, respectively) than the respective chains in human and pig vitreous gel (38 and 28 kDa, respectively). The sulphation patterns of CS preparations were determined following enzymic treatments, HPLC and capillary electrophoretic analyses. The human vitreous-derived CS chains showed quite different sulphation profile than that of CS isolated from other species, since 4-sulphated disaccharides were identified as the dominant moiety. In conclusion, significant compositional and structural variations between the vitreous matrixes of different species at the GAG level were identified. The functional significance of these species-dependent variations is discussed.

OPTIMIZING STABILITY OF ORANGE JUICE FORTIFIED WITH WHEY PROTEIN AT LOW pH VALUES

Abstract The influence of temperature, heating time and pH on the stability of whey protein‐fortified Valencia orange juice was determined by uronic acid content, degree of esterification (DE), % transmission measurements (%T) and capillary electrophoretic analysis of the juice‐protein supernatants. Uronic acid content and charge of pectins showed no significant change in heat‐treated samples with added proteins. The %T decreased with decreasing pH and increasing temperature and heating time for α‐lactalbumin (α‐lac), β‐lactoglobulin (β‐lg) and whey protein isolate (WPI). The lowest transmission values were shown at pH 3.0 and 85C. Capillary electropherograms confirmed more extensive juice‐protein interactions in WPI and β‐lg added juices than in those containing α‐lac, especially at low pH, resulting in more stable juice‐protein mixtures.

Quantitative chiral analysis in capillary electrophoresis from unresolved peaks using derivative electropherograms, experimental design, and artificial neural networks.

Quantitative capillary electrophoretic analysis of chiral compounds might be difficult or even impossible when baseline separation is not reached. In this work, the use of n-th derivative of the electropherogram was studied and examined on model and experimental data. The electropherograms should be first smoothed using Savitzky-Golay method and the quantitative analysis is then possible using either a graphical method or multivariate calibration applying a combination of experimental design (ED) and artificial neural networks (ANNs). The best results were obtained for the first derivative, higher derivatives are not suitable because of noise accumulation. The method was applied to real experimental data to quantify chiral amino acids from unresolved peaks, but it is applicable for quantitative analysis of any other chiral analytes from poorly resolved peaks. Precision of analysis from partially resolved peaks reached was about +/- 3.2% relative standard deviation.

Capillary electrophoretic analysis of the derivatives and isomers of benzoate and phthalate

A capillary electrophoretic method for the analysis of 12 commonly found derivatives and isomers of benzoate and phthalate, including p-toluic acid, p-acetamido and p-hydroxy derivatives of benzoic acid, salicylic acid and its acetyl ester, 2- and 4-isomers of carboxybenzaldehyde, meta-, para-, and ortho-isomers of phthalic acid, and monomethyl terephthalic acid was developed. Capillary electrophoresis (CE) was performed in the free zone electrophoresis mode. Performing CE in 10 m M phosphate buffer, pH 7.0 could separate most of the benzoic acid derivatives except the structural or positional isomers. The positional isomers of phthalic acids could be completely separated with co-addition of α- and β-cyclodextrins. Addition of poly(ethylene glycol) 600 (4%) could further resolve some structural isomers. The CE method developed here is rapid, i.e. complete separation could be achieved in less than 8 min for the nine monoanionic benzoate derivatives and in less than 14 min for the three dianionic phthalate isomers. The new method has good precision and linearity and can be readily applied to real samples for quantitative analysis. It is sensitive and can detect sub-ppm (w/w) level of impurity in real terephthalic samples.

Capillary electrophoretic profiling and pattern recognition analysis of urinary nucleosides from thyroid cancer patients

Metabolic profiling analysis by capillary electrophoresis (CE) was combined with pattern recognition methods to see some correlation between urinary nucleoside levels and thyroid cancer. A total of 15 nucleosides were identified in urines from 12 female thyroid cancer patients and 12 healthy females studied. From the scatter plot evaluation, inosine showed the highest estimated diagnostic power with ca. 97.725% confidence level, followed by N 2-methylguanosine. Star symbol graphs showed differences in levels of both minor and major nucleosides between cancer and normal groups more efficiently, compared with histogram. The stepwise discriminant analysis (SDA) selected N 2-methylguanosine, N 2, N 2-dimethylguanosine and 1-methylguanosine as the most discriminating variables between thyroid cancer and normal groups. The canonical discriminant analysis (CDA) correctly classified all urine specimens studied into two separate clusters of cancer and normal groups in a canonical plot. The principal component analysis (PCA) distinguished cancer patients from normal controls in a principal component plot. The cluster analysis (CA) yielded a dendrogram displaying group separation without any single wrong linkage.

Analysis of underivatized amino acids in protein hydrolysates for cosmetic application by capillary electrophoresis

AbstractIn this work, the use of two chromophores, 3,5‐dinitrobenzoate and phthalate, and the additives α‐cyclodextrin and methanol was investigated with the goal of developing three electrolyte systems for the capillary electrophoretic analysis of free underivatized amino acids in commercially available protein hydrolysates of diverse origin: keratin, silk fiber, milk, soybean, fish tissue, and cattle leather. Sixteen amino acids could be effectively separated by two distinct methods. Several amino acids were identified in the studied hydrolysates and confirmed by spiking techniques. As expected, those products which originated from acidic hydrolysis were richer in free amino acids than those which originated from enzymatic hydrolysis. The quantitative determination of Asp, Glu, Cys, Ser, and Pro in Queratan, a keratin hydrolysate, was also performed and some method validation parameters were established. The coefficient of variation for migration time was better than 1% for all amino acids except Asp, Leu, Lys, and Pro. Overall, peak height repeatability was slightly better than peak area. Calibration curves based on peak heights present a good fit, with coefficients of correlation better than 0.99. The LOD for the amino acids identified in Queratan ranged from 1.0 to 7.0 mg L–1.

English

&nbsp; European (Vitis vinifera) and American (Vitis labrusca) grape species succumb to a bacterial disease known as Pierce&rsquo;s Disease (PD). In contrast, muscadine grape genotypes (Vitis rotundifolia) are tolerant/resistant to PD. This is due to the unique biochemical composition of muscadine xylem.&nbsp; However, because of low protein concentration, conventional methods such as low-pressure chromatography and PAGE are unsuitable for grape xylem protein characterization.&nbsp; In addition, these procedures are tedious, time-consuming and require large amount of sample.&nbsp; This study reports a procedure for isolating and separating proteins from muscadine and bunch grape xylem tissue. The procedure consists of separation of xylem from cortex and phloem, removal of pigments and other gummy substances from xylem with ethanol: ethylacetate (2:1) and subsequent Capillary Electrophoretic (CE) analysis of xylem protein extracts to achieve desired resolution.&nbsp; Number of peaks, peak height and areas, retention time and baseline position were used to compare resolution and study the effect of sample and separation buffer.&nbsp;&nbsp;Xylem tissue proteins extracted with 0.05% sodium borate buffer (pH 8.3) and subjected to CE using 1.2% sodium borate (pH 8.3) as a separation buffer were found to yield most satisfactory resolution of grape xylem proteins.&nbsp; The data obtained by CE were consistent and reproducible, and hence, is well suited to obtain excellent resolution of xylem tissue protein for identifying differences in protein composition among the grape genotypes. Key words:&nbsp; Capillary electrophoresis, grape,&nbsp;Vitis, xylem, Pierce&rsquo;s disease, protein.

Capillary electrophoretic separation of dicarboxylic acids in atmospheric aerosol particles

2,3-Pyrazinedicarboxylic acid (PZDA), 2,6-pyridinedicarboxylic acid (PDA) and 2,3-pyridinedicarboxylic acid (QUIN) solutions were studied as background electrolytes (BGEs) in the capillary electrophoretic analysis of dicarboxylic acids in aerosol particles with indirect UV detection. The BGEs were selected on the basis of similarity in structure with the analytes so that mobilities would be compatible. Optimised pH values for PZDA, PDA and QUIN solutions were 10.6, 11.0 and 10.2, respectively. Myristyltrimethylammonium hydroxide and myristyltrimethylammonium bromide were added to reverse the electroosmotic flow in the solutions in the direction of anode to enable fast anion detection. Separation was obtained for nine dicarboxylic acids (C 2–C 10) differing in the number of CH 2 groups in their skeleton. The electrophoretic mobilities were determined to lie in the range 3.0×10 −4–7.0×10 −4 cm 2 V −1 s −1. The relative standard deviations (RSDs) for the absolute migration times of the analytes were mostly less than 0.5% ( n=6) in PZDA solution. In PDA solution the within-day and day-to-day RSD values for migration were less than 1% and between 2 and 4%, respectively. Peak heights and areas mostly deviated between 1 and 15% in both PZDA and PDA solutions. Detection limits ranged between 1 and 5 mg/l. Methods were applied to the analysis of dicarboxylic acids isolated from aerosol particles.

Optimization of cellular nucleotide extraction and sample preparation for nucleotide pool analyses using capillary electrophoresis

Cell extraction and further sample preparation for nucleotide pool analysis using capillary electrophoresis was faster and simpler using volatile extraction solvents (e.g. organic solvents and de-ionized water) compared to the commonly applied acids dissolved in water (e.g. perchloric acid and trichloracetic acid). Temperature had to be controlled during the whole sample preparation process to prevent degradation, and extracts had to be cleaned from proteins and other large molecules prior to capillary electrophoretic analysis to improve reproducibility. Capillary electrophoresis using borate and cyclodextrins in the background electrolyte was used for determining 11 cellular nucleotides simultaneously. In order to optimize the assay, 0–100% acetonitrile, 0–100% ethanol, and 0–100% methanol in de-ionized water were applied to extract nucleotides from mouse lymphoma cells, and nucleotide yields, recovery, and reproducibility were compared. The assay met the commonly accepted validation limits for biological fluids, if 20–80% acetonitrile in water and 40–60% ethanol in water were used as extraction solvents.

Analysis of carbendazim and thiabendazole in lemons by CE-DAD

A capillary electrophoretic method for the determination of two benzimidazole fungicides, carbendazim and thiabendazole, in lemons has been developed. The two fungicides were separated in a law-pH phosphate buffer containing acetonitrile. Reproducibility tests measuring both migration times and peak areas gave low relative standard-deviation values. Calibration graphs were linear and the detection limits, defined as a signal-to-noise ratio of 3:1, were 2.3 and 2.0 μg mL−1 for thiabendazole and carbendazim, respectively. Analysis of carbendazim and thiabendazole residues in lemons was performed after lemon homogenization, extraction with ethyl acetate and purification through a series of separatory-funnel, acid-base partitions. This lemon clean-up procedure ensured no interference in the capillary electrophoretic analysis from lemon endogenous ingredients.

Capillary electrophoretic analysis of genomic DNA methylation levels.

Changes in DNA methylation have been found in the large majority of tumors. This phenomenon includes both genome-wide hypomethylation and gene- specific hypermethylation. However, the clinical relevance of either mechanism has remained contentious. In order to determine DNA methylation levels from a large number of clinical samples, we have established a method for accurate high-throughput quantification of 5-methylcytosine in genomic DNA. Our protocol requires a small amount (<1 micro g) of DNA that is enzymatically hydrolyzed to single nucleotides. Single nucleotides are then derivatized with a fluorescent marker and separated by capillary electrophoresis. After calibration of the method, we have determined cytosine methylation levels from tumor samples of 81 patients that had been diagnosed with chronic lymphocytic leukemia (CLL). These patients showed a high variability in their methylation levels with a general trend towards hypomethylation. Because of its high accuracy and throughput our method will be useful in determining the role of genomic DNA methylation levels in tumorigenesis.

Appearance of allelic drop-out in STR-multiplex amplified samples after capillary electrophoresis can be avoided by removal of residual primers

In this study, the effect of sample purification on total signal intensities of genRES® MPX-2 (nine-locus multiplex) amplified DNA prior to capillary electrophoretic analysis has been investigated. Sample purification with the QIAQUICK PCR purification kit led to an increase of the relative fluorescent signal intensity by a factor of 3.8±0.8. In addition, increase of injection time showed a linear increase of signal intensity between 3 and 10 s. Higher injection times may influence electrophoretic fragment separation and, therefore, lead to broad signals which may not be sized correctly.

Laser-induced fluorescence as a powerful detection tool for capillary electrophoretic analysis of heparin/heparan sulfate disaccharides.

In quest for high sensitivities necessary for determining the disaccharide composition of heparin/heparan sulfate present in trace amounts in biologic samples, an ultrahighly sensitive capillary electrophoresis (CE) method using laser-induced fluorescence (LIF) detection was developed. Heparin/heparan sulfate-derived Delta-disaccharides were derivatized with the fluorophore 2-aminoacridone and resolved by a reversed-polarity CE method. Estimation of the limit of detection in concentration term and limit of quantitation showed that LIF detection of AMAC-derivatives of Delta-disaccharides resulted in 27-744 times higher sensitivity as compared to those detected by UV at 255 nm. These data suggest that CE-LIF is a powerful tool to quantify minute amounts of heparin/heparan sulfate disaccharides.

Derivatization trends in capillary electrophoresis: an update.

This survey as a sequel of two earlier reports gives an overview of recent developments, starting from 1999, in the use of derivatization protocols in capillary electrophoretic (CE) analysis. Derivatization is mainly used for enhancement of the detection sensitivity in CE, for which a combination of fluorescence labeling and laser-induced fluorescence detection is favorable. Moreover, especially in the field of saccharide assay, derivatization to introduce charge into the molecule, is common. Derivatization procedures are classified in tables, focused on precapillary, on-line, on-capillary and postcapillary arrangements and divided in sections concerning the functional group that is derivatized. The most frequently reported groups are amines and the reducing end of (oligo)saccharides, but thiols, carbonyl and carboxyl groups, steroids and inorganic ions have also been reported about. Other reasons for derivatization are to enhance chiral separation, introduction of a suitable charge into the molecule or to improve mass spectrometric detection. The use of derivatization techniques for special cases, such as the analysis of neurotransmitters, insulin antibodies and mitochondria has also been incorporated as well as a study on the adsorption of proteins onto capillary walls during CE in which derivatization plays a role.

Detection of Chemically-Induced Damage in Layered DNA Films with Co(bpy)33+ by Square-Wave Voltammetry

Damage of double stranded (ds) DNA by metabolites of lipophilic pollutants and drugs constitutes a major toxicity pathway. We evaluated films of DNA and poly(dimethyldiallylammonium chloride) (PDDA) grown layer-by-layer on pyrolytic graphite electrodes for the detection of DNA damage by the model metabolite styrene oxide, which forms chemical adducts with DNA bases. The method features electroactive probe Co(bpy)33+, which binds to films with DNA as the outer layer [(PDDA/ds-DNA)2] in greater amounts when the ds-DNA is intact than when it is damaged. Square-wave voltammetry peaks of Co(bpy)33+ decreased with incubation time of (PDDA/DNA)2 electrodes in styrene oxide solutions, while no changes in the peaks were found for control incubations with non-reactive toluene. Comparison with capillary electrophoretic analyses suggested that this voltammetric method can detect roughly 0.05% damage. The presence of hemoprotein in the films did not interfere with the analysis. The method appears promising for the rapid detection of relative rates of DNA damage by human liver metabolites.

Chemical synthesis of beta-defensins and LEAP-1/hepcidin.

A large and steadily growing subfamily of antimicrobially active peptides of animals and plants is formed by the defensins, which are highly disulfide-bonded, cationic peptides with a molecular mass of about 4 kDa. The synthesis of the human beta-defensins 1 and 2 (hBD-1, hBD-2) as well as of the novel murine beta-defensins 7 and 8 (mBD-7 and mBD-8) is reported. The peptides were synthesized by solid-phase peptide synthesis using fluorenylmethoxycarbonyl chemistry. The linear products were oxidized in the presence of the cysteine/cystine redox system to the biologically active molecules. The correct disulfide connectivity of the resulting cyclic products was partly verified by mass spectrometry and sequence analysis of the fragments obtained after tryptic cleavage. In addition, the recently discovered antimicrobially active human peptide LEAP-1/hepcidin, which contains four disulfide bonds, was successfully synthesized and subsequently oxidized. For Liver-expressed anti microbial peptide (LEAP)-1/hepcidin and hBD-1, the identity of native and synthetic peptides was demonstrated by high-pressure liquid chromatography and capillary electrophoretic analysis. The general synthetic procedure is suitable to rapidly perform the total chemical synthesis of novel fully bioactive defensins, which are expected to be identified soon, as well as of structurally modified analogs.

Purification of STR-multiplex-amplified microsamples can enhance signal intensity in capillary electrophoresis.

In this study, the effect of sample purification on total signal intensities of samples amplified with genRES MPX-2 (nine-locus multiplex) prior to capillary electrophoretic analysis has been investigated. Sample purification with the Qiaquick PCR purification kit led to an increase of the relative fluorescent signal intensity by a factor of 3.8 +/- 0.8. In contrast, the application of larger sample volumes led to a decrease of signal intensities from 20% to 80%, depending on whether the samples were purified or not. In addition, increase of injection time showed a linear increase of signal intensity between 3 s and 10 s. Increasing the number of PCR cycles from 30 to 33 also led to a significant increase of signal intensities. Nevertheless, this increase greatly depended on the fragment lengths and was sometimes accompanied by the appearance of non-specific signals. In combination, optimisation of sample preparation and increase of injection time may intensify signals up to 12-fold, thereby increasing the overall sensitivity of the assay. This may be of special interest for forensic analysis of microspecimens containing limited amounts of DNA.