bluuebunny/crossref_metadata_2025_split huggingface dataset:
Microchip capillary electrophoresis with a boron‐doped diamond electrochemical detector for analysis of aromatic amines
Authors: Shin Dongchan , Tryk Donald A. , Fujishima Akira , Muck Alexander , Chen Gang , Wang Joseph 
Published: August 2004

AbstractThe attractive features of a boron‐doped diamond (BDD) thin‐film detector for microchip capillary electrophoretic (CE) separations of dye‐related amino‐substituted aromatic compounds are described. The diamond electrode was employed in the end‐column amperometric detection of 4‐aminophenol (4‐AP), 1,2‐phenylenediamine (1,2‐PDA), 2‐aminonaphthalene (2‐AN), 2‐chloroaniline (2‐CA), and o‐aminobenzoic acid (o‐ABA), and its attractive behavior was compared to commonly used screen‐printed carbon and glassy‐carbon electrodes. These conventional electrode materials exhibit a significant degree of passivation and low sensitivity to the above‐mentioned environmental pollutants. The diamond‐based electrochemical detection system displayed a favorable analytical performance, including lower noise levels, higher peak resolution with enhanced sensitivity, and improved resistance against electrode passivation. Factors influencing the on‐chip analysis were assessed and optimized. The diamond detector displayed detection limits of 2.0 and 1.3 μM for 4‐AP and 2‐AN, respectively, and a wide linear response for these compounds over the 2–50 μM range. The enhanced stability was demonstrated by relative standard deviation (RSD) values of 1.4% and 4.7% for 100 μM 1,2‐PDA and 200 μM 2‐CA, respectively, for repetitive detections (n = 7). Besides, the simultaneously observed current decrease was 2.4 and 9.1% for 1,2‐PDA and 2‐CA, respectively (compared to 21.8 and 41.0% at the screen‐printed carbon electrode and 28.3 and 34.1% at the glassy carbon electrode, respectively). The favorable properties of the diamond electrode indicate great promise for environmental applications in CE and other microchip devices.
Determination of SARS‐coronavirus by a microfluidic chip system
Authors: Zhou Xiaomian , Liu Dayu , Zhong Runtao , Dai Zhongpeng , Wu Dapeng , Wang Hui , Du Yuguang , Xia Zhinan , Zhang Liping , Mei Xiaodai , Lin Bingcheng 
Published: August 2004

AbstractWe have developed a new experimental system based on a microfluidic chip to determine severe acute respiratory syndrome coronavirus (SARS‐CoV). The system includes a laser‐induced fluorescence microfluidic chip analyzer, a glass microchip for both polymerase chain reaction (PCR) and capillary electrophoresis, a chip thermal cycler based on dual Peltier thermoelectric elements, a reverse transcription‐polymerase chain reaction (RT‐PCR) SARS diagnostic kit, and a DNA electrophoretic sizing kit. The system allows efficient cDNA amplification of SARS‐CoV followed by electrophoretic sizing and detection on the same chip. To enhance the reliability of RT‐PCR on SARS‐CoV detection, duplex PCR was developed on the microchip. The assay was carried out on a home‐made microfluidic chip system. The positive and the negative control were cDNA fragments of SARS‐CoV and parainfluenza virus, respectively. The test results showed that 17 positive samples were obtained among 18 samples of nasopharyngeal swabs from clinically diagnosed SARS patients. However, 12 positive results from the same 18 samples were obtained by the conventional RT‐PCR with agarose gel electrophoresis detection. The SARS virus species can be analyzed with high positive rate and rapidity on the microfluidic chip system.
Determination of pore/protein size via electrophoresis and slit sieve model
Authors: Sarbolouki Mohammad N. , Mahnam Karim , Rafiee‐Pour Hossain‐Ali 
Published: August 2004

AbstractWe used electrophoresis for three purposes: (i) estimation of the mean pore size of polyacrylamide gels via measuring electrophoretic mobility of globular proteins of known sizes in combination with simple sieve (cylindrical and slit) models; (ii) determination of the average size of protein molecules (native or denatured) by the use of the same models; (iii) monitoring the changes in molecular dimensions of proteins in the course of their denaturation. Both models yield results that are in good agreement with those found via the more elaborate techniques (considering the principal differences involved). The approach provides a direct and convenient way of monitoring the variations in protein sizes during the course of their denaturation in gels having a gradient of denaturants, and possibly the number of conformational states involved in the process, a facet that is quite unique and useful. The simpler slit model seems to yield better results in the latter case and is moreover supported by the recently reported data on electrophoresis of DNA molecules through the 1 μm slits of a microbrush matrix made of micropillars arranged in a hexagonal lattice.
Capillary isoelectric focusing – reproducibility and protein adsorption
Authors: Graf Michael , Wätzig Hermann 
Published: August 2004

AbstractCapillary isoelectric focusing (CIEF) is an important tool for the quality assurance of biotechnologically maintained drugs and for proteome analysis. The critical performance parameters of this technique are the precisions of isoelectric point (pI) values and peak areas. Compared to capillary zone electrophoresis (CZE), where precise results can be obtained (e.g., 0.5% relative standard deviation (RSD) for peak areas, n = 60), only few data are available for CIEF experiments. So far, reproducible data of pI values (RSD = 0.5%) have been acquired, but peak areas show inferior results (about 3–15% RSD). Nonstable capillary coatings and protein adsorption have been discussed as possible reasons. Recent work of Righetti et al. [25, 27] has proven that the use of coated capillaries can reduce the adsorption of proteins by 50% but cannot prevent it. In our CIEF experiments irregular and poorly reproducible peak patterns have been observed. In a long‐time experiment of 106 repeated runs, an overall RSD of 10% was obtained for peak areas, RSD of 2% only in series of about 10 consecutive replicates. Especially at higher concentrations the reproducibility deteriorates. This seems to be the result of a self‐amplifying process, induced by adsorbed protein molecules, leading to further agglomerations. CZE control experiments in linear polyacrylamide (LPA)‐coated capillaries proved a strong pH dependency of these effects within a small range. Compared to bare fused‐silica surfaces, adsorption effects are reduced but not inhibited. An enhancement of reproducibility in CIEF experiments can be achieved only by controlling the interactions of proteins and capillary walls.
Separation and determination of aconitine alkaloids in traditional Chinese herbs by nonaqueous capillary electrophoresis
Authors: Li Yuqin , Qi Shengda , Chen Xingguo , Hu Zhide 
Published: August 2004

AbstractAn easy, rapid, and simple nonaqueous capillary electrophoresis (NACE) method was developed for the identification and determination of three aconitine alkaloids, hypaconitine (HN), aconitine (AN), and mesaconitine (MN) within 6 min. The most suitable running buffer was composed of 60 mM ammonium acetate, 0.5% acetic acid, and 15% acetonitrile (ACN) in methanol with a fused‐silica capillary column (50 cm×75 μm ID). In the concentration range 12.5–1000 mg/L the calibration curves reveal linear relationships between the peak area for each analyte and its concentration (correlation coefficients: 0.9997 for HN, 0.9999 for AN, and 0.9995 for MN). The relative standard deviations of the migration time and peak area of the three alkaloids were 0.13, 0.57, 0.33 and 2.87, 1.06, 3.49%, respectively. The method was successfully applied to determine the three alkaloids in two commonly used traditional Chinese herbal medicines, the recoveries of the three constituents ranging between 94.7–101.9% for HN, 98.3–102.3% for AN, and 98.1–104.6% for MN.
Cerebrospinal fluid amyloid β peptide patterns in Alzheimer's disease patients and nondemented controls depend on sample pretreatment: Indication of carrier‐mediated epitope masking of amyloid β peptides
Authors: Bibl Mirko , Esselmann Hermann , Otto Markus , Lewczuk Piotr , Cepek Lukas , Rüther Eckart , Kornhuber Johannes , Wiltfang Jens 
Published: August 2004

AbstractA quantitative urea‐based amyloid β (Aβ)‐sodium dodecyl sulfate‐polyacrylamide gel electrophoresis with Western immunoblot (Aβ‐SDS‐PAGE/immunoblot) reveals highly conserved and disease‐specific Aβ peptide patterns (Aβ 1‐37, 1‐38, 1‐39, 1‐40, 1‐42) in Alzheimer's disease (AD) patients and nondemented controls. For further standardization of this method, we analyzed cerebrospinal fluid (CSF) of eight probable AD patients and seven nondemented controls using different preanalytical procedures for Aβ‐SDS‐PAGE/immunoblot and Aβ1‐42‐enzyme linked immunosorbent assay (ELISA). Both diagnostic groups were discriminated significantly by absolute levels of Aβ1‐42 and ratios of Aβ1‐42/40, 1‐42/38, 1‐42/39. Preanalytical freezing of CSF led to a highly significant loss of all Aβ peptide species. This effect was most pronounced for Aβ1‐42 and completely prevented by SDS‐heat denaturation prior to freezing. Prolonged storage of SDS‐heat denatured CSF led to a selective loss of Aβ1‐42 and impaired the discrimination of diagnostic groups as measured by Aβ‐SDS‐PAGE/immunoblot. Neither freezing nor storage significantly affected absolute Aβ1‐42 levels as determined by Aβ1‐42‐ELISA, but both impaired the discrimation of diagnostic groups. Hence, we suggest immediate analysis of samples for Aβ1‐42‐ELISA, analysis after a short freezing interval for Aβ‐SDS‐PAGE/immunoblot, and avoidance of prolonged storage intervals. Remarkably, Aβ‐SDS‐PAGE/immunoblot measured threefold higher levels of Aβ1‐42 in CSF than Aβ1‐42‐ELISA. In summary, our results indicate carrier‐mediated epitope masking of Aβ1‐42.
Separation and recovery of intact gold‐virus complex by agarose electrophoresis and electroelution: Application to the purification of cowpea mosaic virus and colloidal gold complex
Authors: Soto Carissa M. , Szuchmacher Blum Amy , Wilson Charmaine D. , Lazorcik Jason , Kim Moon , Gnade Bruce , Ratna Banahalli R. 
Published: August 2004

AbstractColloidal gold has been coupled to a mutant cowpea mosaic virus (CPMV), which contains 60 cysteine residues on the surface. A purification process was developed to separate the gold‐containing viral nanoblocks (VNBs) from the free gold. Agarose electrophoresis was utilized to separate the mixture followed by electroelution of the desired sample to recover the intact virus. Mobility of Au‐VNB and free colloidal gold was facilitated by the addition of thioctic acid (TA). 30% of the gold‐containing virus was recovered after electroelution as determined by absorbance measurements. Histogram analysis of transmission electron microscopy (TEM) images demonstrated the efficient separation of gold‐containing virus from free gold. TEM and scanning electron microscopy (SEM) images indicated that the virus was recovered intact. Monodisperse spherical particles of nominal size of 45 nm were observed under SEM.
Determination of amino acids in rat vitreous perfusates by capillary electrophoresis
Authors: Thongkhao‐On Kongthong , Kottegoda Sumith , Pulido Jose S. , Shippy Scott A. 
Published: August 2004

AbstractIn vivo determinations of amino acids are important for improving our understanding of physiological states of biological tissue function and dysfunction. However, the chemically complex matrix of different biological fluids complicates the assay of this important class of molecules. We introduce a method for characterizing the amino acid composition of submicroliter volumes of vitreous humor perfusates. Low‐flow push‐pull perfusion sampling is compatible with collecting small volume samples in a complicated matrix that are potentially difficult to separate. An efficient, sensitive, and rapid analysis of amino acids from in vivo perfusates of the vitreous is presented with 3‐(4‐carboxybenzoyl)‐2‐quinoline‐carboxaldehyde (CBQCA) derivatitation and capillary electrophoresis (CE) separation with laser‐induced fluorescence detection (LIF). Derivatization with CBQCA for up to 2 h provided high sensitivity and low detection limits at the nM level. Seventeen amino acids including D‐serine (D‐Ser) and D‐aspartate (D‐Asp) were resolved in less than 10 min. Importantly, D‐Ser is separated from its enantiomeric pair. Characterization of vitreal amino acids with this assay technique will be useful for understanding ocular diseases and physiological mechanisms in vision.
A statistical comparison of silver and SYPRO Ruby staining for proteomic analysis
Authors: White Ian R. , Pickford Russell , Wood John , Skehel J. Mark , Gangadharan Bevan , Cutler Paul 
Published: August 2004

AbstractSilver staining has been the method most commonly employed for high sensitivity staining of proteins following two‐dimensional gel electrophoresis. Whilst this method offers detection in the nanogram range it does have major drawbacks including a lack of linearity, nonstoichiometric staining of proteins, a lack of compatibility with the microchemical preparation of proteins for identification by mass spectrometric techniques, and a highly subjective assessment of the staining endpoint. SYPRO Ruby is a relatively new, ruthenium complex‐based stain which is reported to offer advantages over silver, particularly in overcoming the limitations cited above. We describe a series of experiments where several protein staining procedures commonly employed are compared. To enable optimization of the in situ digestion procedure, a statistical approach has been undertaken. The effects of a variety of staining, digestion, and analysis protocols on the downstream processing of a test radiolabeled protein were studied. The data confirms that as well as offering sensitivity similar to silver, SYPRO Ruby staining is reproducible, linear, and offers a higher level of compatibility with the identification of proteins by mass spectrometry.
High reproducibility of large‐gel two‐dimensional electrophoresis
Authors: Challapalli Kiran Kumar , Zabel Claus , Schuchhardt Johannes , Kaindl Angela M. , Klose Joachim , Herzel Hanspeter 
Published: August 2004

AbstractTwo‐dimensional gel electrophoresis (2‐DE) facilitates the separation of thousands of proteins from highly complex protein mixtures and has become a central method in proteomics in recent years. In the present study, we examined the technical variability of large 2‐DE gels with respect to sample preparation, electrophoresis procedure, data acquisition, and biological variation by analyzing a disease (Huntington's disease) and control state with a commercially available software package, PROTEOMWEAVER™. Scatter plots and correlation coefficients were obtained to quantify both technical and biological variation. Even 2‐DE gels run separately in both dimensions yielded correlation coefficients around 0.88 and deviations from the mean close to 20% for low‐intensity spots. This indicates a high technical reproducibility of the 2‐DE procedure developed in our laboratory. Variability within a biological condition was low and comparable to technical variation (at least 0.87). Two‐dimensional (2‐D) gels obtained from samples of different biological conditions (health vs. disease) achieved a variability similar to intracondition and technical variability. These findings highlight the importance of multiple gel and spot‐by‐spot comparisons to identify biological significant changes. Minor errors introduced by technical and biological variation allow a comparison of all gels within a study which facilitates the tackling of complex biological problems.
Separation of proteins on surface‐modified poly(dimethylsiloxane) microfluidic devices
Authors: Dou Yue‐Hua , Bao Ning , Xu Jing‐Juan , Meng Fei , Chen Hong‐Yuan 
Published: August 2004

AbstractSeparation and detection of proteins have been realized on nonionic surfactant‐modified poly(dimethylsiloxane) (PDMS) microfabricated devices with end‐column amperometric detection. The hydrophobic PDMS channels are turned into hydrophilic ones after being modified with Brij35 and facilitate the separation of proteins. The coating can remarkably reduce the adsorption of large protein molecules and is stable in the range of pH 6–12. The detection of proteins in such channels needs less rinsing time and thus efficiency is raised. Even large molecules of proteins can also be detected with better reproducibility and enhanced plate numbers. The relative standard deviation (RSD) of the migration time for glucose oxidase (GOD) is 2.2% (n = 19). Separation of GOD and myoglobin has been developed in modified channels. Predominant operational variables, such as the coating conditions, the concentration of surfactant and buffer, are studied in detail.
Capillary and microchip electrophoresis of basic drugs with contactless conductivity detection
Authors: Tanyanyiwa Jatisai , Hauser Peter C. 
Published: August 2004

AbstractThe extension of contactless conductivity detection in electrophoresis to the determination of basic drugs is demonstrated using β‐adrenergic blocking agents (β‐blockers) and other physiologically active amines as examples. The high‐voltage approach to conductivity detection was employed for conventional capillaries as well as microchip devices. Acidic buffers were used in all cases. A buffer consisting of 100 mM acetic acid and 1 mM histidine was deemed most optimal for the separation of six β‐blockers and best results for the analysis of the other amines were achieved with a 20 mM lactic acid buffer at low pH‐value. The detection limits ranged from 0.06 to 5 μM. To demonstrate potential practical applications, a main component assay was conducted for three pharmaceutical formulations. On‐chip, five pharmaceutical amines could be baseline‐resolved in a 8 cm long microchannel in 90 s, albeit a reduced sensitivity and peak capacity compared to conventional capillary electrophoresis.
Nonlinear electroosmosis in hierarchical monolithic structures
Authors: Nischang Ivo , Tallarek Ulrich 
Published: August 2004

AbstractWe studied the dependence of electroosmotic flow (EOF) velocity and separation efficiency for neutral analytes in 100 μm ID capillary monoliths on a variation of the mobile phase ionic strength and applied electrical field strength, i.e., we covered a range for the concentration of Tris buffer from 10−5 to 10−2 M and applied electrical field strengths up to 105 V/m. The silica‐based monoliths are hierarchically structured having intraskeleton mesopores and interskeleton macropores. While a linear dependence of the average EOF velocity on applied field strength could be observed with 5×10−3 M Tris (turning slightly nonlinear at a higher concentration due to thermal effects), this dependence becomes systematically nonlinear as the Tris concentration is reduced towards 10−4 M. Increased velocities by more than 50% compared to those expected from linear behavior are realized at 105 V/m. Concomitantly, as the Tris concentration is reduced from 10−3 to 10−4 M, we notice an improvement in plate heights by a factor of more than 2 (they approach 2 μm for ethylbenzoate). We complementary analyzed the onset of the nonlinear EOF dynamics in a hierarchical monolith and the significantly reduced axial dispersion in view of nonequilibrium electrokinetic effects which may develop in porous media due to the presence of ion‐permselective regions, e.g., the mesoporous monolith skeleton. In this respect, a decreasing mobile phase ionic strength favors the formation of nonequilibrium concentration polarization in strong electrical fields, and a coupling of the electrostatics and hydrodynamics then may explain nonlinear EOF velocities and increasing separation efficiencies depending on the Tris concentration and applied field strength.
In situ monitoring of kinetics of metabolic conversion of ATP to ADP catalyzed by MgATPases of muscle Gastrocnemius skinned fibers using micellar electrokinetic chromatography
Authors: Kulp Maria , Kaljurand Mihkel , Käämbre Tuuli , Sikk Peeter , Saks Valdur 
Published: August 2004

AbstractA method for the in situ measurement of the kinetics of ATP metabolic transformation using capillary electrophoresis (CE) has been developed. The depletion of ATP and formation of ADP were monitored in situ by using saponin‐permeabilized muscle fibers. The method of micellar electrokinetic chromatography, employing reversed electroosmotic flow by cationic surfactant and reversed‐polarity mode, provided an efficient and reproducible separation of nucleotides and enabled kinetic analysis of the reaction to be performed in a large range of nucleotide concentrations that approaches physiological concentrations of ATP in the muscle cells, without the need for precipitation of proteins prior to sample application. The analytes were detected at a nM level with a reproducibility of about 7%. This reproducibility enabled the comparison of different competing kinetic models of ATP conversion to ADP and the results show that the MgATPase activity in the fast‐twitch gastrocnemius muscle followed biphasic kinetics that corresponds to the allosteric character of regulation of the enzyme(s) activity at physiological ATP concentrations. The results also confirmed that the combination of minimal sample volume requirements, rapid measurement and reproducibility makes the micellar CE a valuable tool for the analysis of biological fluids and understanding the processes of biological interest.
Improved resolution power of electrophoretic fractionation of DNA using a voltage gradient “up and down” application
Authors: Bellavia Daniele , Sanfilippo Rosalia , Sisino Giorgia , Palmeri Laura , Barbieri Rainer 
Published: August 2004

AbstractThe improved resolution power of electrophoretic fractionation of DNA in a wide range of molecular masses is demonstrated using an “up and down” application of voltage gradient gel electrophoresis (VGGE). This application also allows separation of different DNA fragments which are poorly fractionated in conventional electrophoresis.
Polyacrylamide gel electrophoresis of fluorophore‐labeled hyaluronan and chondroitin sulfate disaccharides: Application to the analysis in cells and tissues
Authors: Karousou Evgenia G. , Militsopoulou Maria , Porta Giovanni , De Luca Giancarlo , Hascall Vincent C. , Passi Alberto 
Published: August 2004

AbstractThis report describes a new formulation of polyacrylamide gel electrophoresis of fluorophore‐labeled saccharides (PAGEFS) for the analysis of hyaluronan (HA) and chondroitin sulfate (CS) Δ‐disaccharides. PAGEFS relies on derivatization of reducing ends of HA‐ and the variously sulfated CS‐derived Δ‐disaccharides with 2‐aminoacridone (AMAC), followed by electrophoresis under optimized buffer conditions (Tris‐borate and Tris‐HCl) and on polyacrylamide gels (25% T/3.75% C). The method was applied to the analysis of glycosaminoglycans (GAGs) from the human umbilical cord tissue and GAGs isolated from human aortic smooth muscle cell cultures. The obtained results were in agreement with those obtained after an analysis with high‐performance liquid chromatography (HPLC). On the basis of these results, PAGEFS is a rapid and sensitive method for the analysis of the total amount of HA‐ and CS‐derived disaccharides, as it allows analyzing 20 samples in minigels in one run and provides quantitation with relatively high sensitivity (less than 25 pmol per disaccharide). In addition, PAGEFS overcomes the lack of commercial gels described previously for the separation of AMAC‐labeled disaccharides. Therefore, the method proposed here is an economic and useful tool for a fast screening of GAGs in biological samples, particularly when a high number of samples should be analyzed.