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.     

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.     

Separation of cationic polymer particles and characterization of avidin-immobilized particles by capillary electrophoresis

Cationic polymer microparticles have received much attention especially in the field of biotechnology, such that their analysis and separation have become important. So far, the separation of cationic polymer particles with different size using CE has not been achieved and the cationic particles migrated as if they are negatively charged, probably due to electrostatic interaction between capillary wall and cationic polymer particles. In this paper, the separation of cationic polymer microparticles by CE was investigated in detail. The separation of cationic particles with different size was achieved in CE by taking into account the interaction between sample particles and the inner surface of capillaries. By employing a poly(vinyl alcohol)-coated capillary, a better size separation of amine-modified latex particles was obtained compared to a Polybrene-coated capillary. It was elucidated that the composition, concentration, and pH of the background solution were also important factors in the separation of colloidal particles to avoid the surface adsorption and the characteristic aggregation of polymer particles. Furthermore, the CE analysis was applied to the characterization of cationic protein-immobilized particles.     

Characterization of rhinovirus subviral A particles via capillary electrophoresis, electron microscopy and gas-phase electrophoretic mobility molecular analysis: Part I

During infection, enteroviruses, such as human rhinoviruses (HRVs), convert from the native, infective form with a sedimentation coefficient of 150S to empty subviral particles sedimenting at 80S (B particles). B particles lack viral capsid protein 4 (VP4) and the single-stranded RNA genome. On the way to this end stage, a metastable intermediate particle is observed in the cell early after infection. This subviral A particle still contains the RNA but lacks VP4 and sediments at 135S. Native (150S) HRV serotype 2 (HRV2) as well as its empty (80S) capsid have been well characterized by capillary electrophoresis. In the present paper, we demonstrate separation of at least two forms of subviral A particles on the midway between native virions and empty 80S capsids by CE. For one of these intermediates, we established a reproducible way for its preparation and characterized this particle in terms of its electrophoretic mobility and its appearance in transmission electron microscopy (TEM). Furthermore, the conversion of this intermediate to 80S particles was investigated. Gas-phase electrophoretic mobility molecular analysis (GEMMA) yielded additional insights into sample composition. More data on particle characterization including its protein composition and RNA content (for unambiguous identification of the detected intermediate as subviral A particle) will be presented in the second part of the publication.     

Separation and characterization of sub-microm- and microm-sized particles by capillary zone electrophoresis

The analytical separation and characterization of particles in the size range of sub-microm and microm diameters by capillary zone electrophoresis (CZE) has been reviewed. The theoretical basis, on which the mobility can be interpreted to provide information regarding characteristics of particle surface, has shortly been presented. Particular emphasis was put on the model dependence of that interpretation and the need in most applications to forego the classical idealized model of spherical particles with "smooth" surfaces and to apply more realistic models, which take the "hairy" surface of real particles into account. Some highlights of the literature on the CZE of polystyrene latex microspheres, organic and inorganic colloids, lipoprotein particles, viruses, liposomes, biological membrane vesicles, and biological cells have been discussed. Also summarized are the reports on the particle size dependence of mobility and peak broadening in CZE and on electrophoretic behavior of rodlike particles and particle aggregates. Finally, the effects of neutral polymers in the background electrolyte on particle mobility and peak width are reviewed.     

Recent advances in the analysis of biological particles by capillary electrophoresis

This review covers research papers published in the years 2005-2007 that describe the application of capillary electrophoresis to the analysis of biological particles such as whole cells, subcellular organelles, viruses and microorganisms.     

Highly efficient approach for characterizing nanometer-sized gold particles by capillary electrophoresis

This paper demonstrates that capillary electrophoresis (CE) can be employed for characterizing the sizes of nanometer-scale gold particles. We characterized the gold nanoparticles by effecting CE separation using a buffer of SDS (70 mM) and 3-cyclohexylamino-1-propanesulfonic acid (CAPS; 10 mM) at pH 11.0 and an applied voltage of 18 kV and obtained a linear relationship (R² > 0.99) between electrophoretic mobilities and size for nanoparticles whose diameters fall in the regime from 5.0 ± 0.5 to 41.2 ± 3.3 nm; the relative standard deviations of these electrophoretic mobilities are <0.8%. We evaluated the feasibility of employing these separation conditions for the size characterization by of gold nanoparticle samples that were synthesized by a rapid microwave heating method. We confirmed that this CE method is a valid one for size characterization by comparing the results obtained by CE with those provided by scanning electron microscopy (SEM); a good correlation exists between these two techniques. Our results demonstrate that CE can be employed to accelerate the analysis of the sizes of nanomaterials.     

Separation and detection of individual submicron particles by capillary electrophoresis with laser-light-scattering detection

Separation and detection of individual submicron polystyrene spheres using capillary electrophoresis with laser-light-scattering detection has been demonstrated. Electrophoretically separated particles were passed through a focused laser beam and light scattered from individual particles was collected at 90 degrees. Each diameter of polystyrene spheres injected (from 110 to 992 nm) resulted in the observation of a well-defined migration window containing multiple peaks, each arising from the light scattered by an individual particle. The migration time window for individual particles of a particular size corresponded well to the migration time of a peak from a population of particles of the same size detected using a UV absorbance detector. The electrophoretic mobility and scattered light intensity were determined for each particle detected. The average scattered light intensity for each particle size was consistent with Mie scattering theory. Particles as small as 110 nm in diameter were detected individually using this method, but particles with a diameter of 57 nm could not be individually detected. The number of single particle scattering events was counted and compared to the theoretical number of particles injected electrokinetically, and the detection efficiency determined ranged from 38 to 57% for polystyrene spheres of different sizes. The laser-light-scattering detection method was directly compared to laser-induced fluorescence detection using fluorescent polystyrene microspheres. The number of particles detected individually by each method was in agreement.     

Identification and quantification of natural isoxazolinone compounds by capillary zone electrophoresis.

A capillary zone electrophoresis (CZE) method that is specific, simple, rapid and also cheap was developed to analyse some natural UV-absorbing isoxazolinone compounds with toxic potential present in legume seedlings. The six most common natural isoxazolinone compounds were separated within 10 min with 25 mM potassium phosphate (pH 7.5) containing 8% 1-propanol as running buffer. A 60 cm coated fused-silica capillary (52.6 cm effective length x 75 microm I.D.), with an electric field of 375 V/cm at 30 degrees C was used. The limit of detection ranged from 0.01 mM (3.0 microg/ml) to 0.03 mM (7.7 microg/ml). Linearity between peak areas and concentrations ranging from 0.05 mM to 1.75 mM were determined for each isoxazolinone. The correlation coefficient was 0.9954 or greater. Both relative migration time and peak area were reproducible. The RSD of relative migration time is between 0.44 and 1.94% and RSD of peak area is between 1.26 and 6.86%. The concentrations of isoxazolinones in Lathyrus odoratus and L. sativus seedlings obtained by CZE were in agreement with the previous results from HPLC.     

Charge density profiling of circulating human low-density lipoprotein particles by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) has been utilized to profile the low-density (LDL) particles in human blood serum in this study. A 5 mM sodium phosphate buffer, pH 7.40, was chosen as the most suitable CE buffer and an extensive ultrafiltration (UF) procedure was applied to purify the LDL sample. Two LDL particle species, LDL with lower mobility and LDL- with higher mobility were observed. The electropherograms were highly reproducible with good precision of effective mobilities, corrected peak areas (CPAs) and CPA ratio of LDL-/LDL. LDL particles shown on the electropherogram were also characterized by several procedures. The applications of Sigma HDL cholesterol reagent and CE on-line 2-propanol precipitation indicated that the two particle species shown in the electropherogram belong to LDL. The LDL particles were found to associate with the buoyant LDL fraction and the LDL- particles associate with the dense LDL fraction. This study utilizes CZE for the profiling of LDL isoforms and provides a new analytical method for the resolution of LDL subspecies. It demonstrates a high-mobility LDL particle which circulates in healthy subjects and diminishes in atherosclerotic patients. Diminution of the high-mobility LDL subspecies may be linked to minimal formation of arterial plaque in atherosclerotic patients.     

Haplotyping by capillary electrophoresis

The investigation of the genetic background and phenotype structures of complex diseases, such as cardiovascular or psychiatric disorders and tumors, is one of the most scrutinized fields of the post genomic era. Besides the multiplex analysis of genetic markers and polymorphisms throughout the whole genome, more and more attention is focused on the interaction between the etiological factors of these traits. Haplotype determination, rather than multiplex genotyping seems to be one of the first building blocks of this endeavor. This review focuses on the importance and theoretical background of haplotyping, and summarizes the recent examples of novel and emerging haplotyping techniques by capillary gel electrophoresis based DNA fragment analysis, a powerful tool for the examination of the inheritance of complex traits.     

Identification and determination of effective components in Euphrasia regelii by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) method has been developed for simultaneous determination of eukovoside, cinnamic acid and ferulic acid in Euphrasia regelii for the first time. The electrophoresis buffer was 20 mmol/L sodium borate containing 10% (v/v) methanol (pH 8.50). The effects of concentration of borate and electrolyte pH on electrophoretic behavior and separation were studied. Regression equations revealed linear relationships (correlation coefficients 0.9995-0.9998) between the peak area of each analyte and the concentration. The levels of analytes in the different parts of Euphrasia regelii were easily determined with recoveries ranging from 95.5 to 104.2%.     

Study of intact virus‐like particles of human papillomavirus by capillary electrophoresis

Virus‐like particles of human papillomavirus (HPV‐VLP), resulting from the self‐assembly of the capsid proteins (L1 or L1 and L2), have been widely used to study HPV as they are similar to the native virion. Moreover, two prophylactic vaccines, Gardasil^® and Cervarix^®, are based on HPV‐VLP L1. Analytical techniques currently used to characterize HPV‐VLP, such as SDS‐PAGE, Western blot, ELISA, are time‐consuming and semiquantitative. In this study, CE was evaluated for the analysis of intact HPV16‐VLP. The usefulness of capillary inner wall coating as well as various BGEs, pH, and detergent additives were investigated. Reproducible HPV‐VLP analysis in CE was achieved using poly(ethylene oxide)‐coated capillary and a BGE containing high salt concentration and low SDS concentration. The developed method enables HPV‐VLP detection in less than 10 min (migration times RSD: 1.6%). The identity of HPV‐VLP peak was confirmed by comparison with a sample obtained from a wild‐type baculovirus and with VLP‐based vaccine, Gardasil^®, after adjuvant dissolution. Finally, we applied the developed methodology to VLP‐based vaccines, demonstrating that CE could be successfully used for vaccine quality control.     

Identification and determination of aesculin and aesculetin in ash barks by capillary zone electrophoresis

Summary A capillary zone electrophoresis method for identification and determination of aesculin and aesculetin has been established using borate-phosphate buffer containing 30% ethanol with on-column UV detection. A detailed investigation of the influence of changes in borate concentration, pH, applied voltage, temperature and organic modifier was then carried out. For both aesculin and aesculetin, a linear plot of migration time (MT) against borate concentration was obtained, and ln[measured peak area (MA)] and lnMT both gave linear plots against ln(applied voltage) with correlation coefficient r>0.999, which also resulted in a linear correlation between MA and MT (r≥0.9998) under varied voltage. Ethanol as organic modifier to the background electrolytes helped in separating aesculin and aesculetin from other components in ash barks. The reproducibility with relative standard deviation in MT and in normalized peak area(NA) and linearity based on NA against concentration were evaluated. Finally, the method was successfully applied to monitor the quality of different ash barks and to compare the effect of sample preparation on content of bioactive components in ash bark. Results indicate that CZE promises to be applicable to quality control of traditional Chinese medicines containing aesculin and aesculetin.     

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.     

Identification of individual DNA molecule of Mycobacterium tuberculosis by nested PCR-RFLP and capillary electrophoresis

The improvement of sensitivity and differentiation in rapidly identifying a small amount of mycobacteria in sputum has significant implications for reducing tuberculosis transmission. We previously applied the conventional PCR and capillary electrophoresis (CE) to establish the restriction fragment length polymorphism (RFLP) pattern of mycobacterial 65-kDa heat shock protein (hsp65) gene from colony specimens. However, the previous analysis did not provide enough sensitivity for sputum specimens in which the limitation of analysis might be hindered by PCR inhibitors and primer-dimers formation during amplification. In the current study, nested PCR (nPCR) had been redesigned for PCR-RFLP analysis (PRA) of mycobacterial hsp65 gene using CE. The results show both Mycobacterium tuberculosis complex and mycobacteria other than tuberculosis could be identified in the presence of PCR inhibitors. The interference due to primer-dimers was also minimized. Based on the Poisson distribution, the repeatability of single DNA molecule detection was greatly affected by sampling probability and might be improved significantly by increasing the sample loading. The PRA using nPCR and CE is not only able to detect the individual mycobacterial DNA molecule but also potentially differentiate the species.     

Application of capillary electrophoresis to the characterization of some colloidal particles

The advantages of the capillary electrophoresis method, known to be highly selective, have been tested on some standard latex colloids and nanoparticles of thorium phosphate, investigated separately or in mixtures. The results have been compared to those obtained by laser Doppler velocimetry. Both methods appear as complementary: capillary electrophoresis is more efficient to point out very fine particles among others, but is more restricting about the supporting medium.     

Pesticide analysis by capillary electrophoresis

In this work, a critical and updated revision of the current situation of the analysis of pesticides by Capillary Electrophoresis (CE) is presented. The review has been written in two main sections. The first one presents a thorough revision of the various offline and on-line sample preconcentration procedures that have been used in conjunction with CE to analyze these compounds. The second part reviews the various detection strategies (i.e., UV, LIF, MS, and electrochemical) and CE modes that have been applied to the analysis of pesticides. Future trends that can be expected from this hot research area are also discussed.