Capillary electrophoresis of affinity complexes between subviral 80S particles of human rhinovirus and monoclonal antibody 2G2

Human rhinoviruses (HRVs), the main etiologic agents of the common cold, transform into subviral B- or 80S particles (they sediment at 80S upon sucrose density gradient centrifugation) during infection and, in vitro, upon exposure to a temperature between 50 and 56 degrees C. With respect to the native virion they lack the genomic RNA and the viral capsid protein VP4. 80S particles are unstable and easily disintegrate into their components, VP1, VP2, and VP3 in buffers containing SDS. However, this detergent was found to be a necessary constituent of the BGE for the analysis of these viruses and their complexes with receptors and antibodies by CE. We here demonstrate that dodecylpoly(ethyleneglycol ether) (D-PEG) a nonionic detergent, is suitable for analysis of subviral particles as it preserves their integrity, in contrast to SDS. Electrophoresis of the 80S particles in borate buffer (pH 8.3, 100 mM) containing 10 mM D-PEG resulted in a well-defined electrophoretic peak. The identity of the peak was confirmed, among other means, by complexation with mAb 2G2, which recognizes a structural epitope exclusively present on subviral particles but not on native virus. Upon incubation of the 80S particles with mAb 2G2 the peak disappeared, but a new peak, attributed to the antibody complex emerged. The separation system allowed following the time course of the transformation of intact HRV serotype 2 into 80S particles upon incubation at temperatures between 40 and 65 degrees C. We also demonstrate that subviral particles derived from HRV2 labeled with the fluorescence dyes FITC or Cy3.5 were stable in the separation system containing D-PEG. Dye-modified particles were still recognized by mAb 2G2, suggesting that the exposed lysines that are derivatized by the reagent do not form part of the epitope of the antibody.     

Kinetics of thermal denaturation of human rhinoviruses in the presence of anti-viral capsid binders analyzed by capillary electrophoresis

In vivo, the icosahedral capsid of human rhinoviruses undergoes well-defined transitions during the infection pathway. Native virus, sedimenting at 150S, is converted to subviral particles with a sedimentation coefficient of 135S, which have lost the innermost capsid protein VP4. Upon release of the genomic RNA empty 80S capsids remain. Similar structural modifications are observed in vitro upon exposure to low pH and/or elevated temperature. Virions are stabilized against these transitions by various antiviral compounds, which bind to a hydrophobic pocket in the capsid protein VP1. Using capillary electrophoresis the kinetics of viral decay in the presence of such hydrophobic drugs was investigated. Assuming first-order kinetics, the increase of the time constant reflects the extent of stabilization. Exposure of the virions to 55 degrees C after presaturation with the antivirals increased the time constants (as compared to native virus) by a factor of 8-30, from a few minutes to several ten minutes. Denaturation of the stabilized capsid gave rise to heterogeneous material rather than to defined subviral particles. This was confirmed by electron microscopy and indicates that the structural modification of the virus follows a kinetically well-defined pathway which is disturbed by the drugs resulting in disorganized disruption of the virion.     

Influence of detergent additives on mobility of native and subviral rhinovirus particles in capillary electrophoresis

The electrophoretic properties of two human rhinovirus (HRV) serotypes, HRV2 and HRV14, their subviral particles, and their capsid proteins were investigated by CE using borate buffer, pH 8.3, as BGE and three different detergents as additives. In addition, the influence of modification of the capsid with an amine reactive fluorescent dye, Cy3.5, on migration in the electric field was assessed. We found that the reproducibility of the electrophoretic results was decisively dependent on the presence of the detergents above their respective CMC. As compared to the strong ionic detergent SDS, the nonionic, mild detergent dodecylpoly(ethyleneglycol ether) (D-PEG) efficiently and reproducibly resolved both, native viruses as well as subviral particles. Most of the analytes behaved as expected except native HRV2; this serotype showed a dramatically higher anionic mobility in SDS than in D-PEG. Additionally, its mobility decreased when each positive charge contributed from a lysine at the capsid surface was substituted by four negative charges upon derivatization with Cy3.5. We discuss the possibility that this effect is caused by differences in number and in arrangement of exposed lysines in the two serotypes leading to differences in the amount of bound SDS micelles.     

Identification of poliovirions and subviral particles by capillary electrophoresis

Poliovirions, purified from infected cell extracts with anion-exchange chromatography, can be analyzed and identified by CE in untreated fused silica capillaries using UV detection. Other subviral particles can be eluted as well from the same infected cell extract using a higher salt concentration buffer on the ion-exchange chromatography. Virions can be identified because of their conversion into empty capsids upon heating at 56°C. As a result of heating, the viral genome is released from the capsid. Here, we show that during this incubation some intermediate particles were found. The latter were identified by enzymatic peak shift analysis. The high salt concentration eluate subviral particles were analyzed with preincubation affinity CE together with their sensitivity for RNase and proteinase K treatment. Electropherograms of the higher salt concentration eluate display a mixture of at least four different subviral particles. One particle proved to have an [N1, H] antigenicity and was resistant to RNase and proteinase K digestion. The remaining particles were all sensitive to proteinase K treatment. This CE method proved to be valuable in the detection, identification and analysis of poliovirions and poliovirus particles offering an alternative powerful, cheap, fast and easy analysis method.     

Capillary electrophoresis of viruses, subviral particles and virus complexes

CZE and CIEF were so far applied to the analysis of tobacco mosaic virus, Semliki forest virus, human rhinovirus, adenovirus, norovirus and the bacteriophages T5 and MS2. The concentration of viral or subviral particles, of capsid proteins and viral genomes were determined, their electrophoretic mobilities and pI values were measured and bioaffinity reactions between viruses and antibodies, antibody fragments and receptor fragments were assessed. The role of detergents added to the BGE to obtain reproducible electrophoretic conditions was elucidated. The analytes were detected via their UV-absorbance or via fluorescence after derivatization of the viral capsid, the nucleic acid, or both. A new dimension to the detection is added by the possibility of making use of the viral infectivity. At least in theory, this allows for the unequivocal identification of a single infectious virus particle after collection at the capillary outlet. This review summarizes the 25 papers so far published on this topic.     

Measuring the electrophoretic mobility of concentrated suspensions in nonaqueous media

We present a new method of measuring the electrophoretic mobility of a particle in a concentrated suspension. The method is used to measure the electrophoretic mobility of PMMA particles (diameter 10 microm) suspended in a mixture of liquid hydrocarbons. The particle volume fraction of the suspension is varied from 0 up to 0.30 and the resulting variation of the electrophoretic mobility is discussed. The suspending liquid is such that its refractive index is very close to that of the particles. Thus the suspension is almost transparent and it is possible to follow through a microscope the motion of one particle. The suspension is subjected to a low-frequency electric field (0.5 Hz). The cell containing the suspension is mounted on a piezoelectric crystal. The displacement that compensates for the particle motion (when the particle image is steady) is determined.     

Electrophoretic mobility of a colloidal particle with constant surface charge density

When the electrophoretic mobility of a particle in an electrolyte solution is measured, the obtained electrophoretic mobility values are usually converted to the particle zeta potential with the help of a proper relationship between the electrophoretic mobility and the zeta potential. For a particle with constant surface charge density, however, the surface charge density should be a more characteristic quantity than the zeta potential because for such particles the zeta potential is not a constant quantity but depends on the electrolyte concentration. In this article, a systematic method that does not require numerical computer calculation is proposed to determine the surface charge density of a spherical colloidal particle on the basis of the particle electrophoretic mobility data. This method is based on two analytical equations, that is, the relationship between the electrophoretic mobility and zeta potential of the particle and the relationship between the zeta potential and surface charge density of the particle. The measured mobility values are analyzed with these two equations. As an example, the present method is applied to electrophoretic mobility data on gold nanoparticles (Agnihotri, S. M.; Ohshima, H.; Terada, H.; Tomoda, K.; Makino, K. Langmuir 2009, 25, 4804).     

Approximate analytic expressions for the electrophoretic mobility of spheroidal particles

Approximate analytic expressions are derived for the electrophoretic mobility of spheroidal particles (prolate and oblate) carrying low zeta potential in an electrolyte solution under an applied tangential or transverse electric field. The present approximation method, which is based on the observation that the electrophoretic mobility of a particle is determined mainly by the distortion of the applied electric field by the presence of the particle. The exact expression for the equilibrium electric potential distribution around the particle, which can be expressed as an infinite sum of spheroidal wave functions, is not needed in the present approximation. The electrophoretic mobility values calculated with these approximate expressions for spheroidal particles with constant surface potential or constant surface charge density are in excellent agreement with the exact numerical results of previous reports with the relative errors less than about 4%.     

Electrothermal flow effects in insulating (electrodeless) dielectrophoresis systems

We simulate electrothermally induced flow in polymeric, insulator-based dielectrophoresis (iDEP) systems with DC-offset, AC electric fields at finite thermal Péclet number, and we identify key regimes where electrothermal (ET) effects enhance particle deflection and trapping. We study a single, two-dimensional constriction in channel depth with parametric variations in electric field, channel geometry, fluid conductivity, particle electrophoretic (EP) mobility, and channel electroosmotic (EO) mobility. We report the effects of increasing particle EP mobility, channel EO mobility, and AC and DC field magnitudes on the mean constriction temperature and particle behavior. Specifically, we quantify particle deflection and trapping, referring to the deviation of particles from their pathlines due to dielectrophoresis as they pass a constriction and the stagnation of particles due to negative dielectrophoresis near a constriction, respectively. This work includes the coupling between fluid, heat, and electromagnetic phenomena via temperature-dependent physical parameters. Results indicate that the temperature distribution depends strongly on the fluid conductivity and electric field magnitude, and particle deflection and trapping depend strongly on the channel geometry. Electrothermal (ET) effects perturb the EO flow field, creating vorticity near the channel constriction and enhancing the deflection and trapping effects. ET effects alter particle deflection and trapping responses in insulator-based dielectrophoresis devices, especially at intermediate device aspect ratios (2 ≤ r ≤ 7) in solutions of higher conductivity (σ m ≥ 1 × 10(-3)S/m). The impact of ET effects on particle deflection and trapping are diminished when particle EP mobility or channel EO mobility is high. In almost all cases, ET effects enhance negative dielectrophoretic particle deflection and trapping phenomena.     

Colloid Vibration Potential in a Concentrated Suspension of Spherical Colloidal Particles

A relation between the dynamic electrophoretic mobility of spherical colloidal particles in a concentrated suspension and the colloid vibration potential (CVP) generated in the suspension by a sound wave is obtained from the analogy with the corresponding Onsager relation between electrophoretic mobility and sedimentation potential in concentrated suspensions previously derived on the basis of Kuwabara's cell model. The obtained expression for CVP is applicable to the case where the particle zeta potential is low, the particle relative permittivity is very small, and the overlapping of the electrical double layers of adjacent particles is negligible. It is found that CVP shows much stronger dependence on the particle volume fraction φ than predicted from the φ dependence of the dynamic electrophoretic mobility. It is also suggested that the same relation holds between the electrokinetic sonic amplitude of a concentrated suspension of spherical colloidal particles and the dynamic electrophoretic mobility. Copyright 1999 Academic Press.     

On the limiting electrophoretic mobility of a highly charged colloidal particle in an electrolyte solution

The electrophoretic mobility of a spherical charged colloidal particle in an electrolyte solution with large kappaa (where kappa= Debye-Hückel parameter and a= particle radius) tends to a nonzero constant value in the limit of high zeta potential. It is demonstrated that this is caused by the fact that counterions condensed near the highly charged particle surface do not contribute to the electrophoretic mobility and only co-ions govern the mobility. A simple method to derive the limiting electrophoretic mobility expression is given. The present method is also applied to cylindrical particles, showing that the leading term of the limiting electrophoretic mobility of a cylindrical particle in a transverse field with large kappaa is the same as that of a spherical particle. The electrophoretic mobility of a cylindrical particle in a tangential field, on the other hand, is proportional to the particle zeta potential and does not exhibit a constant limiting value for high zeta potentials.     

How does the nature of the steric stabilizer affect the pickering emulsifier performance of lightly cross-linked, acid-swellable poly(2-vinylpyridine) latexes?

A near-monodisperse styrene-functionalized poly[2-(dimethylamino)ethyl methacrylate] (PDMA) macromonomer was evaluated as a reactive steric stabilizer for the preparation of poly(2-vinylpyridine (P2VP) latexes via emulsion polymerization. The solution pH was shown to be a critical parameter for successful syntheses: stable latexes with minimal coagulum were only obtained at (or above) neutral pH. The presence of the grafted PDMA stabilizer in a near-monodisperse P2VP latex of 280 nm diameter was indicated by FT-IR spectroscopy and quantified at 6.0 wt % using 1H NMR spectroscopy. XPS studies confirmed that this stabilizer was located at the latex surface, as expected. Combined DLS and electrophoretic data indicated that these PDMA-P2VP particles exist in three states depending on the solution pH: swollen cationic microgels were obtained below pH 4.1, nonsolvated latex particles with a cationic stabilizer layer were obtained at intermediate pH, and flocculated latex particles with neutral PDMA stabilizer chains were obtained at around pH 8.5. Finally, this PDMA-P2VP latex was shown to be a superior Pickering emulsifier for stabilizing water-in-1-undecanol emulsions than either a poly(ethylene glycol)-stabilized P2VP latex or a charge-stabilized P2VP latex. This serves to illustrate the important role played by the steric stabilizer in determining particle wettability.     

Dynamic electrophoretic mobility of spherical colloidal particles in salt-free concentrated suspensions

In this contribution, the dynamic electrophoretic mobility of spherical colloidal particles in a salt-free concentrated suspension subjected to an oscillating electric field is studied theoretically using a cell model approach. Previous calculations focusing the analysis on cases of very low or very high particle surface charge are analyzed and extended to arbitrary conditions regarding particle surface charge, particle radius, volume fraction, counterion properties, and frequency of the applied electric field (sub-GHz range). Because no limit is imposed on the volume fractions of solids considered, the overlap of double layers of adjacent particles is accounted for. Our results display not only the so-called counterion condensation effect for high particle charge, previously described in the literature, but also its relative influence on the dynamic electrophoretic mobility throughout the whole frequency spectrum. Furthermore, we observe a competition between different relaxation processes related to the complex electric dipole moment induced on the particles by the field, as well as the influence of particle inertia at the high-frequency range. In addition, the influences of volume fraction, particle charge, particle radius, and ionic drag coefficient on the dynamic electrophoretic mobility as a function of frequency are extensively analyzed.     

Electrophoresis of soft particles: analytic approximations

An approximate analytic expression is derived for the electrophoretic mobility of a weakly charged spherical soft particle (i.e., a hard particle covered with a weakly charged polyelectrolyte layer) on the basis of the general mobility expression for soft particles (Ohshima, H., J. Colloid Interface Sci. 2000, 228, 190-193). The obtained mobility expression, which reproduces various approximate results so far derived and gives some new mobility formulas, covers all types of weakly charged soft particles with arbitrary values of the thickness of polymer layer, the radius of the particle core, the electrophoretic softness, and the Debye length, including spherical polyelectrolytes with no particle core as well as spherical hard particles with no polyelectrolyte layer.     

Effect of pressure on electrophoretic mobility of polystyrene latex particles

A capillary electrophoresis system that can apply arbitrary helium gas pressures at both inlet and outlet reservoirs was constructed. The system was used to investigate the effect of pressure on electrophoretic behavior of polystyrene latex particles. The electrophoretic mobility of latex particles was increased with the application of pressure (< 3.0 kgf/cm2). The shrinkage of particle diameter under pressurization was observed using a microscope, however, the magnitude of shrinkage was not enough to explain the increase in electrophoretic mobility. Therefore, the application of pressure might increase the electric charge of the latex particle. Since methanol inhibited the enhancement in the electrophoretic mobility of the latex particles, water might play an important role in increasing mobility.     

A method for determining electrophoretic and electroosmotic mobilities using AC and DC electric field particle displacements

We have developed a method for measuring the electrophoretic mobility of submicrometer, fluorescently labeled particles and the electroosmotic mobility of a microchannel. We derive explicit expressions for the unknown electrophoretic and the electroosmotic mobilities as a function of particle displacements resulting from alternating current (AC) and direct current (DC) applied electric fields. Images of particle displacements are captured using an epifluorescent microscope and a CCD camera. A custom image-processing code was developed to determine image streak lengths associated with AC measurements, and a custom particle tracking velocimetry (PTV) code was devised to determine DC particle displacements. Statistical analysis was applied to relate mobility estimates to measured particle displacement distributions.     

Effects of dielectric gradients-mediated ions partitioning on the electrophoresis of composite soft particles: An analytical theory

In this work, we report original analytical expressions defining the electrophoretic mobility of composite soft particles comprising an inner core and a surrounding polymer shell with differentiated permeabilities to ions from aqueous background electrolyte and to fluid flow developed under applied DC field conditions. The existence of dielectric permittivity gradients operational at the core/shell and shell/solution interfaces is accounted for within the Debye–Hückel approximation and flat plate configuration valid in the thin double layer regime. The proposed electrophoretic mobility expressions, applicable to weakly to moderately charged particles with size well exceeding the Debye layer thickness, involve the relevant parameters describing the particle core/shell structure and the electrohydrodynamic features of the core and shell particle components. It is shown that the analytical expressions reported so far in literature for the mobility of hard (impermeable) or porous particles correspond to asymptotic limits of the more generic results detailed here. The impacts of dielectric-mediated effects of ions partitioning between bulk solution and particle body on the electrophoretic response are further discussed. The obtained expressions pave the way for a refined quantitative, analytical interpretation of electrophoretic mobility data collected on soft (nano)particles (e.g., functionalized dendrimers and multilayered polyelectrolytic particles) or biological cells (e.g., viruses) for which the classical hard core-soft shell representation is not appropriate.     

A new approach to the studies of submicron particle suspensions based on the effect of pressure in capillary zone electrophoresis

A new approach based on the effect of pressure in CZE is suggested for acceleration of particle migration in electrophoretic runs resulting in reduction of the analysis time. It provides conditions for studying fast processes in suspensions. The effect of pressure on the migration of silica spheres with average diameters of 100, 150, and 390 nm was studied by CZE at an applied voltage of 25 kV. The particle hydrodynamic behavior was also investigated under the same capillary dimensions and BGE composition. The total particle mobility (excluding the average flow rate) was found to increase with increasing the pressure applied and particle size. The particle migration mechanism explaining the effect of pressure on particle velocity was shown to be almost the same as in wide‐bore hydrodynamic chromatography. It is based on changing radial distribution of particle concentration along the capillary cross section depending on particle diffusivity. On the basis of this mechanism appearance of a zone of negatively charged particles in electropherograms ahead of the marker peak can be explained.     

Influence of polyelectrolyte multilayer adsorption on the temperature sensitivity of poly(<Emphasis Type="Italic">N</Emphasis>-isopropylacrylamide) (PNiPAM) microgels

The electrophoretic mobility and temperature-dependent particle size of poly(N-isopropylacrylamide) (PNiPAM) microgels after alternating adsorption of poly(diallyldimethylammonium chloride) (PDADMAC) and poly(sodium 4-styrenesulfonate) (PSS) have been determined. First a PNiPAM-co-acrylic acid (AAc) shell was added to the PNiPAM microgel, then PDADMAC and PSS were adsorbed alternately. The studies of the electrophoretic mobility revealed charge reversal when a polyelectrolyte (PE) layer was adsorbed. Particle size measurements revealed a strong influence of polyelectrolyte adsorption on the temperature-dependent particle swelling. The strong influence of the adsorbed polyelectrolyte on the particle size is in contrast to polyelectrolyte multilayer adsorption on rigid particles.