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Size, electrophoretic mobility, and ion dissociation of vesicles prepared with synthetic amphiphiles

Cuccovia, Iolanda Midea; Feitosa, Eloi; Chaimovich, Hernan; Sepulveda, Luis; Reed, Wayne
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 3722-3725
ENG
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Vesicles prepared with synthetic amphiphiles (dioctadecyldimethylammonium bromide and chloride, dihexadecyl phosphate and its sodium salt) were obtained by sonication, ethanol injections, and chloroform injections. The hydrodynamic diameter of vesicles (Dh), estimated from the diffusivity measured by quasielastic light scattering, ranged from 230 to 3000 Å. The electrophoretic mobility (Um) was measured by free-flow electrophoresis. The zeta potential (ζ) and the degree of counterion dissociation (α) of the vesicles were calculated from Um and conductivity data, α decreased with increasing Dh of the vesicles, probably due to the decreasing headgroup area and the increasing counterion association needed to relax the surface electrostatic potential. The electrophoretic mobility was also calculated (Uc) according to an impenetrable, nonconducting sphere model with a spherically symmetric charge distribution approximation. Within the limits of the experimental error(s) of the (different) methods employed and the assumptions made in the calculations, the fact that the Um/Uc ratio ranged from 1.3 to 7.5 was considered to be a good agreement between the calculated and the experimental values. © 1990 American Chemical Society.

Effects of UV radiation exposure on DNA and DNA repair enzymes

Camacho, Inês Sofia Cortes Eusébio
Fonte: Faculdade de Ciências e Tecnologia Publicador: Faculdade de Ciências e Tecnologia
Tipo: Dissertação de Mestrado
Publicado em //2012 ENG
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Dissertação para obtenção do Grau de Mestre em Biotecnologia; DNA integrity in the cell is under constant threat from damaging agents of endogenous or exogenous origin, such as UV light, ionizing radiation and oxidative stress. Although the effects of these carcinogens on DNA have been extensively studied, very little is known about their effect on DNA repair enzymes. The aim of the present work was the study of the effect of UV radiation on E. coli Endonuclease III, a DNA glycosylase belonging to base excision repair system. This enzyme was homologously overexpressed and then purified with a Fe/protein ratio of 3.88 ± 0.63 (fully‐loaded form). Endonuclease III exposure to UV radiation for 45 min (19.77 kJ dose) may lead to the destruction of the Fe‐S bonds of the [4Fe‐4S] cluster or to the conversion of this center into a different redox state. Electrophoretic mobility shift assays with protein‐DNA complex showed that Endonuclease III binding to plasmid DNA promotes a retardation of the free supercoiled DNA band, indicative of Endonuclease III‐DNA complex(es) formation. These assays also showed that Endonuclease III is able to bind both linear and supercoiled plasmid DNA, although with higher affinity for the linear form. Electrophoretic mobility shift assays performed after 45 min of UV irradiation (19.77 kJ) revealed that although shift occurred...

Solubilization of Minerals by Bacteria: Electrophoretic Mobility of Thiobacillus ferrooxidans in the Presence of Iron, Pyrite, and Sulfur

Blake, Robert C.; Shute, Elizabeth A.; Howard, Gary T.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1994 EN
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Thiobacillus ferroxidans is an obligate acidophile that respires aerobically on pyrite, elemental sulfur, or soluble ferrous ions. The electrophoretic mobility of the bacterium was determined by laser Doppler velocimetry under physiological conditions. When grown on pyrite or ferrous ions, washed cells were negatively charged at pH 2.0. The density of the negative charge depended on whether the conjugate base was sulfate, perchlorate, chloride, or nitrate. The addition of ferric ions shifted the net charge on the surface asymptotically to a positive value. When grown on elemental sulfur, washed cells were close to their isoelectric point at pH 2.0. Both pyrite and colloidal sulfur were negatively charged under the same conditions. The electrical double layer around the bacterial cells under physiological conditions exerted minimal electrostatic repulsion in possible interactions between the cell and either of its charged insoluble substrates. When Thiobacillus ferrooxidans was mixed with either pyrite or colloidal sulfur at pH 2.0, the mobility spectra of the free components disappeared with time to be replaced with a new colloidal particle whose electrophoretic properties were intermediate between those of the starting components. This new particle had the charge and size properties anticipated for a complex between the bacterium and its insoluble substrates. The utility of such measurements for the study of the interactions of chemolithotrophic bacteria with their insoluble substrates is discussed.

The rate with which spontaneous mutation alters the electrophoretic mobility of polypeptides.

Neel, J V; Satoh, C; Goriki, K; Fujita, M; Takahashi, N; Asakawa, J; Hazama, R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1986 EN
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Studies of a Japanese population, involving a total of 539,170 locus tests distributed over 36 polypeptides, yielded three presumptive spontaneous mutations altering the electrophoretic mobility of the polypeptide. This corresponds to a mutation rate of 0.6 X 10(-5) per locus per generation. The a priori probability that undetected discrepancies between legal and biological parentage might in our test system result in an apparent electrophoretic mutation in this population is calculated to be only 0.3 X 10(-7) per locus per generation. Since electrophoresis only detects about half of the amino acid substitutions due to mutations of nucleotides, the corrected rate for mutations causing amino acid substitutions in polypeptides is 1.2 X 10(-5) per locus per generation. With allowance for synonymous mutations and those resulting in stop codons, the total mutation rate for nucleotide changes in the exons encoding a polypeptide becomes approximately equal to 1.8 X 10(-5) per locus per generation. When the present observations are combined with all of the other available data concerning mutation resulting in electrophoretic variants, the electrophoretic rate drops to 0.3 X 10(-5) per locus per generation, the total locus rate drops to roughly 1.0 X 10(-5)...

Nucleotide sequences associated with differences in electrophoretic mobility of envelope glycoprotein gp70 and with GIX antigen phenotype of certain murine leukemia viruses

Donis-Keller, Helen; Rommelaere, Jean; Ellis, Ronald W.; Hopkins, Nancy
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1980 EN
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Previous genetic and biochemical studies led to the identification of two large RNase T1-resistant oligonucleotides, designated the GIX+ and GIX- oligonucleotides, whose presence in the genomes of closely related murine leukemia viruses is mutually exclusive and predictive of two properties of the viral envelope glycoprotein gp70. Viruses harboring the GIX+ oligonucleotide induce expression of the gp70-associated antigen GIX and possess gp70s with more rapid electrophoretic mobility on sodium dodecyl sulfate/polyacrylamide gels than viruses that possess the GIX- oligonucleotide. The latter viruses fail to induce GIX on infected fibroblasts. The GIX+ and GIX- oligonucleotides lie in corresponding positions in the 3′ third of the oligonucleotide maps of their respective viruses. We have determined the nucleotide sequences of the GIX+ and GIX- oligonucleotides. The sequence of the GIX- oligonucleotide is U-A-U-C-U-C-A-A-C-C-A-C-C-A-U-A-C-U-U-A-A-C-C-U-C-A-C-C-A-C-[unk]-G, and the sequence of the GIX+ oligonucleotide is U-A-U-C-U-C-A-A-C-C-A-C-C-A-U-A-C-U-U-G. Thus, a single base change could result in the interconversion of the two oligonucleotides. Consideration of the amino acids specified by the two oligonucleotides suggests that this single base difference may result in the presence of an additional oligosaccharide chain in the gp70s of the GIX- viruses. Evidence supporting this prediction has been obtained by M. R. Rosner...

Mutation in Shigella flexneri 2a Resulting in Increased Electrophoretic Mobility

Corwin, Laurence M.; Talevi, Leonard A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1972 EN
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An avirulent mutant of Shigella flexneri 2a, when grown in broth containing calcium ions, was found to have a three- to fourfold increase in electrophoretic mobility toward the anode when compared to its virulent parent. Under these same growth conditions, it was found that the avirulent strain was made more electronegative in the presence of ethylenediaminetetraacetic acid (EDTA). EDTA had no effect on the electrophoretic mobility of the virulent strain. In the absence of added calcium in the growth medium, no differences between the strains were observed. The alteration which has resulted in an increased negative charge has also stabilized the avirulent strain against loss of lipopolysaccharide from EDTA treatment.

A Model Allowing Continuous Variation in Electrophoretic Mobility of Neutral Alleles

Cobbs, Gary
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1979 EN
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The infinite-sites model with no recombination is extended to include mutations that affect electrophoretic mobility. The model allows the effect of a single-site mutation to have a continuous effect on mobility. Formulae are obtained for the variance of electrophoretic mobility of alleles after an arbitrary length of time. A special case of the general model is the case of stepwise production of neutral alleles with an arbitrary number of steps.

Effect of oxidized glutathione on some enzymes of erythrocytes and its relation to erythrocytic enzyme activity and electrophoretic mobility

Walter, Harry; Caccam, Juanita F.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1966 EN
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1. Oxidized glutathione reacts or interacts with some erythrocytic enzymes (glucose 6-phosphate dehydrogenase, EC 1.1.1.49, aspartate aminotransferase, EC 2.6.1.10) but not with some others (lactate dehydrogenase, EC 1.1.1.27). 2. GSSG does not diminish the activity of any of these enzymes and is therefore not responsible for the decreased enzyme activities associated with older erythrocytes. 3. It may be that the reaction of aspartate aminotransferase with GSSG is the cause for the more rapid anodic electrophoretic mobility of this enzyme derived from human erythrocytes when compared with the mobility of the same enzyme from other human tissues. 4. A reinterpretation of some related, previously published, data with regard to the electrophoretic mobility of the above-mentioned enzymes from young and old erythrocytes is presented.

Calculation of the Electrophoretic Mobility of a Particle Bearing Bound Polyelectrolyte Using the Nonlinear Poisson-Boltzmann Equation

Sharp, Kim A.; Brooks, Donald E.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1985 EN
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A numerical method for determining the electrophoretic mobility of a polyelectrolyte-coated particle is presented. The particle surface is modeled as having a permeable layer of polyelectrolyte molecules anchored to its surface. Fluid flow within the polyelectrolyte layer is subject to Stokes drag arising from the polyelectrolyte segments. The method allows arbitrary distribution of polymer segments and charge density normal to the surface to be used. The hydrodynamic plane of shear may also be varied. The potential profile is determined by a numerical solution to the nonlinearized Poisson-Boltzmann equation. The potential profile is then used in a numerical solution to the Navier-Stokes equation to give the required mobility. The use of the nonlinearized Poisson-Boltzmann equation extends the results to higher charge density/lower ionic strength conditions than previous treatments. The surface potentials and mobilities for three limiting charge distributions are compared for both the linear and nonlinear treatments to delimit the range of validity of the linear treatment. The utility of the numerical, nonlinear treatment is demonstrated by an improved fit to the electrophoretic mobility of human erythrocytes as a function of ionic strength in the range 10 to 150 mM.

The macrophage electrophoretic mobility (MEM) test for malignant disease. Further clinical investigations and studies on macrophage slowing factors

Preece, A. W.; Light, P. Ann
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1974 EN
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This paper describes further confirmation of the successful application of the macrophage electrophoretic mobility (MEM) test to the detection of malignant disease. Results from fifty-one patients and eighteen controls are presented. The apparatus and technique used for the measurement of macrophage electrophoretic mobility differed from those previously used in other investigations. Further information is presented on the nature and behaviour of macrophage slowing factor(s), and experiments concerning the possible use of cells other than guinea-pig macrophages as indicators of slowing factor production are also described.

Cell electrophoresis for diagnostic purposes. I. Diagnostic value of the electrophoretic mobility test (EMT) for the detection of gynaecological malignancies.

Hoffmann, W.; Werner, W.; Steiner, R.; Kaufmann, R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1981 EN
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Lymphocytes from 278 gynaecological patients (100 controls and 178 patients with a malignant condition) have been investigated for their response to encephalitogenic factor, cancer basic protein, and KCl extract of adenocarcinoma of the body of the uterus as "antigens", using tanned sheep erythrocytes ETS as indicator particles in the electrophoretic mobility test (EMT). Electrophoretic mobility was measured with a Zeiss cytopherometer. The study was split into three test series producing in the cancer group 66% correct positive test results (34% false negatives) and in the control group 83% correct negative results (17% false positives). Consequently, with the instrumentation used, EMT is, at least in our hands, not sufficiently reliable for the diagnosis of cancer.

Impact of Chemical and Structural Anisotropy on the Electrophoretic Mobility of Spherical Soft Multilayer Particles: The Case of Bacteriophage MS2

Langlet, Jérémie; Gaboriaud, Fabien; Gantzer, Christophe; Duval, Jérôme F. L.
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: Artigo de Revista Científica
EN
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We report a theoretical investigation of the electrohydrodynamic properties of spherical soft particles composed of permeable concentric layers that differ in thickness, soft material density, chemical composition, and flow penetration degree. Starting from a recent numerical scheme developed for the computation of the direct-current electrophoretic mobility (μ) of diffuse soft bioparticles, the dependence of μ on the electrolyte concentration and solution pH is evaluated taking the known three-layered structure of bacteriophage MS2 as a supporting model system (bulk RNA, RNA-protein bound layer, and coat protein). The electrokinetic results are discussed for various layer thicknesses, hydrodynamic flow penetration degrees, and chemical compositions, and are discussed on the basis of the equilibrium electrostatic potential and hydrodynamic flow field profiles that develop within and around the structured particle. This study allows for identifying the cases where the electrophoretic mobility is a function of the inner structural and chemical specificity of the particle and not only of its outer surface properties. Along these lines, we demonstrate the general inapplicability of the notions of zeta potential (ζ) and surface charge for quantitatively interpreting electrokinetic data collected for such systems. We further shed some light on the physical meaning of the isoelectric point. In particular...

Fast Determination of Mitochondria Electrophoretic Mobility Using Micro-Free Flow Electrophoresis

Kostal, Vratislav; Fonslow, Bryan R.; Arriaga, Edgar A.; Bowser, Michael T.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/11/2009 EN
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Fast, continuous separation of mitochondria from rat myoblasts using micro free-flow electrophoresis (μFFE) with online laser induced fluorescence detection (LIF) is reported. Mitochondrial electrophoretic profiles were acquired in less than 30s. Compared to macroscale FFE instruments, μFFE devices consumed approximately 100-fold less sample, used 10-fold less buffer and required a 15-fold lower electric field. Mitochondrial electrophoretic mobility distributions measured using μFFE were compared to those measured with a capillary electrophoresis instrument with laser induced fluorescence detection (CE-LIF). There was high similarity between the two distributions with CE-LIF distribution being offset by 1.8×10−4 cm2V−1s−1 with respect to the μFFE distribution. We hypothesize that this offset results from the differences in electric field strength used in the techniques. Compared to CE-LIF, analysis of mitochondria using μFFE greatly decreased separation time and required less separation voltage, while maintaining low sample (125 nL) and buffer (250 μL) volumes. These features together with the potential for collecting separated organelle fractions for further characterization make μFFE a very attractive tool for the high-throughput analysis of organelle subpopulations as well as investigating the fundamentals of the electrophoretic mobility of biological particles.

Direct visualization of electrophoretic mobility shift assays using nanoparticle-aptamer conjugates

Wang, Min S.; Reed, Scott M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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Here we demonstrate that aptamers tethered to gold nanoparticles enable direct visualization of protein-oligonucleotide interactions during gel electrophoresis. This technique is used to confirm that an aptamer previously identified as binding to C-reactive protein (CRP) only binds to the monomeric form of CRP. While native, pentameric CRP (pCRP) is used in clinical assays to predict cardiovascular disease (CVD) risk, it is the monomeric isoform that is more strongly associated with pro-inflammatory and pro-atherogenic effects. To visualize this selectivity, the CRP-aptamer was conjugated to streptavidin-coated gold nanoparticles and the mobility of the free oligonucleotide-nanoparticle conjugate (ON-NP) and the protein/ON-NP complex bands were visualized and recorded during electrophoresis using a simple digital camera. At a concentration of 6 mg/mL, monomeric CRP showed a significant decrease in the observed ON-NP mobility, whereas no change in mobility was observed with pCRP up to 18 mg/mL. Advantages of this nanoparticle-based electrophoretic mobility shift assay (NP-EMSA) over traditional EMSA include real-time detection of protein-oligonucleotide interactions, the avoidance of harmful radioisotopes, and elimination of the need for expensive gel imagers. The availability of both the NP-EMSA technique and an mCRP specific probe will allow for improved clinical diagnostic to more accurately predict future CVD risk.

Immunological activity of blood lymphocyte fractions: a study by the macrophage electrophoretic mobility method

Minderhoud, J. M.; Smith, J. K.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1972 EN
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Samples of human blood-lymphocytes from patients with cancer, multiple sclerosis or cerebral haemorrhage were separated on a discontinuous Ficoll gradient. Six to eight fractions were obtained in which one or two fractions contained a large number of small lymphocytes. The fractions were incubated with encephalitogenic factor or tumour antigen and guinea-pig macrophages, after which the macrophages' electrophoretic mobility (EMR) was measured. The fractions with a density between 1·075 and 1·090, and containing a high percentage of small lymphocytes, produced a significant EMR in these patients and it was concluded that the cells responsible for cellular immunological activity were also responsible for reductions in the electrophoretic mobility.

Determination of the molecular weight of DNA-bound protein(s) responsible for gel electrophoretic mobility shift of linear DNA fragments examplified with purified viral myb protein.

Bading, H
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 24/06/1988 EN
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A protein-DNA complex has less gel electrophoretic mobility than the free DNA fragment. One parameter for the degree of retardation of a linear DNA fragment in a protein-DNA complex is the molecular weight of the bound protein(s). The quotient of the migration distances of free DNA (m) and protein-DNA complex (m') is a function of the molecular weight (MW) of the bound protein(s). Based on the evaluation of the lac repressor induced mobility shift of a 203 bp DNA fragment containing the lac operator in a 5% non-denaturating polyacrylamide gel a direct proportionality could be shown between (m/m'-1) and MW with the proportionality factor K = 215 kDa. The factor K depends on the acrylamide concentration in the gel, getting lower values with increasing acrylamide concentrations. A calculation is given to determine the molecular weight of DNA-binding factors responsible for the decreased electrophoretic mobility of a linear DNA fragment. As an example this calculation was used in order to analyse DNA-binding of the isolated viral myb protein. It could be demonstrated that the viral myb protein binds to DNA as a monomer and as a dimer.

Label-Free Determination of the Number of Biomolecules Attached to Cells by Measurement of the Cell's Electrophoretic Mobility in a Microchannel

Aki, Atsushi; Nair, Baiju G.; Morimoto, Hisao; Kumar, D. Sakthi; Maekawa, Toru
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 29/12/2010 EN
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We developed a label-free method for a determination of the number of biomolecules attached to individual cells by measuring the electrophoretic mobility of the cells in a microchannel. The surface of a biological cell, which is dispersed in aqueous solution, is normally electrically charged and the charge quantity at the cell's surface is slightly changed once antibody molecules are attached to the cell, based on which we detect the attachment of antibody molecules to the surface of individual red blood cells by electrophoretic mobility measurement. We also analyzed the number of antibody molecules attached to the cell's surface using a flow cytometer. We found that there is a clear correlation between the number of antibody molecules attached to the individual cells and the electophoretic mobility of the cells. The present technique may well be utilized not only in the field of cell biology but also in the medical and pharmaceutical industries.

Computation of the electrophoretic mobility of proteins.

Chae, K S; Lenhoff, A M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1995 EN
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A scheme is presented for computing the electrophoretic mobility of proteins in free solution, accounting for the details of the protein shape and charge distribution. The method of Teubner is implemented using a boundary integral formulation within which the velocity distribution, the equilibrium electrical potential around the molecule, and the potential distribution due to the applied field are solved for numerically using the boundary element method. Good agreement of the numerical result is obtained for spheres with the corresponding semi-analytical specialization of Henry's analysis. For protein systems, the method is applied to lysozyme and ribonuclease A. In both cases, the predicted mobility tensors are fairly isotropic, with the resulting scalar mobilities being significantly smaller than for spheres of equal volume and net charge. Comparisons with previously published experimental results for ribonuclease show agreement to be excellent in the presence of a net charge, but poorer at the point of zero charge. The approach may be useful for evaluating approximate methods for estimating protein electrophoretic mobilities and for using electrophoretic measurements to obtain insight into charge distributions on proteins.

The relation between Escherichia coli surface functional groups' composition and their physicochemical properties

Hamadi,Fatima; Latrache,Hassan; Zahir,Hafida; Elghmari,Abderrahmene; Timinouni,Mohamed; Ellouali,Mostapha
Fonte: Sociedade Brasileira de Microbiologia Publicador: Sociedade Brasileira de Microbiologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/03/2008 EN
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Escherichia coli surface characteristics including hydrophobicity, electrophoretic mobility and surface functional groups' composition were investigated. These characteristics were determined respectively by water contact angle measurements, microelectrophoresis and x-ray photoelectron spectroscopy (XPS). The relation between the physicochemical properties and functional groups' composition was also examined. The electrophoretic mobility at pH 7 appeared to be governed on the cell surface by the (O=C) functional groups. The cell surface's hydrophilicity was associated with high levels of (C-(O.N)) and (OH-(C-O-C)) functional groups, whereas the cell surface's hydrophobicity was associated with (C-(C,H)) functional groups.

Electrophoretic Mobility of Polyelectrolytes within a Confining Well

Shendruk, Tyler N.; Bertrand, Martin; Slater, Gary W.
Fonte: Universidade Cornell Publicador: Universidade Cornell
Tipo: Artigo de Revista Científica
Publicado em 11/04/2015
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We present a numerical study of polyelectrolytes electrophoresing in free solution while squeezed by an axisymmetric confinement force transverse to their net displacement. Hybrid multi-particle collision dynamics and molecular dynamics simulations with mean-field finite Debye layers show that even though the polyelectrolyte chains remain "free-draining", their electrophoretic mobility increases with confinement in nanoconfining potential wells. The primary mechanism leading to the increase in mobility above the free-solution value, despite long-range hydrodynamic screening by counterion layers, is the orientation of polymer segments within Debye layers. The observed length-dependence of the electrophoretic mobility arises due to secondary effects of counterion condensation related to confinement compactification.