Determination of the electrophoretic mobility of bacteria and their separation by capillary zone electrophoresis

Conditions for the determination of electrophoretic mobilities of bacteria by capillary electrophoresis (CE) were explored. Most precise values are obtained using fused silica capillaries of 1–3 m length (0.25 mm inner diameter), a background buffer with an ionic strength of 0.0015 mol/L and a pH value of 7–10 at a field strength of 120 V/cm. Capillary electrophoretic separation of three different bacteria populations on the basis of their mobility differences could be realized. Electrophoretic band widths of all bacteria populations investigated are relatively large compared to molecule bands. It finds its explanation in the different distribution of surface charge density to cross-sectional area of each single cell of a population. Received: 30 January 1997 / Revised: 15 May 1997 / Accepted: 22 May 1997     

Adhesion of waste water bacteria to reverse osmosis membranes

A stirred cell was used to study initial adhesion of three sewage bacteria belonging to the genus Pseudomonas to the three reverse osmosis (RO) membranes BW30, PVD and CAB2, and the nanofiltration membrane NF45. Membranes were immersed in suspensions containing 10⁸ bacteria/ml for 10 min. All three strains were capable of rapidly colonising the four membranes, but to different extents. It was found that bacteria would sometimes aggregate upon adhering to particular RO membranes. The effects of solution ionic strength and pH, and conditioning of membranes (by prior exposure to filtrates of treated and untreated sewage) on the number of adherent bacteria were investigated. Minimal bacterial attachment occurred in a very low ionic strength milieu (deionised water). Salt concentrations corresponding to waste water and to twice that concentration resulted in significantly higher but statistically similar numbers of attached microbes. Adhesion of the three isolates was not affected by pH in the range of 4–8. The number of bacteria attaching to the membranes could be increased or reduced by conditioning films of sewage origin, conditioning films could also trigger or inhibit aggregation of adherent cells. Some surface properties of the membranes (roughness, hydrophobicity) and bacterial cells (electrophoretic mobility, functional groups by affinity chromatography) were also investigated.     

Use of electrophoretic techniques and MALDI–TOF MS for rapid and reliable characterization of bacteria: analysis of intact cells, cell lysates, and “washed pellets”

In this study electrophoretic and mass spectrometric analysis of three types of bacterial sample (intact cells, cell lysates, and “washed pellets”) were used to develop an effective procedure for the characterization of bacteria. The samples were prepared from specific bacterial strains. Five strains representing different species of the family Rhizobiaceae were selected as model microorganisms: Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viciae, R. galegae, R. loti, and Sinorhizobium meliloti. Samples of bacteria were subjected to analysis by four techniques: capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), gel IEF, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS). These methods are potential alternatives to DNA-based methods for rapid and reliable characterization of bacteria. Capillary electrophoretic (CZE and CIEF) analysis of intact cells was suitable for characterization of different bacterial species. CIEF fingerprints of “washed pellets” and gel IEF of cell lysates helped to distinguish between closely related bacterial species that were not sufficiently differentiated by capillary electrophoretic analysis of intact cells. MALDI–TOF MS of “washed pellets” enabled more reliable characterization of bacteria than analysis of intact cells or cell lysates. Electrophoretic techniques and MALDI–TOF MS can both be successfully used to complement standard methods for rapid characterization of bacteria.     

The effect of aggregation on the separation performance of bacteria in capillary electrophoresis

During the last 15 years, methods for the capillary electrophoretic separation of different bacteria species have been developed, which exploit their characteristic cell surface‐charge to volume ratio. A special variant, the polymer‐based CE of bacteria, includes a focusing step, which forces the bacteria cells to form aggregates at the beginning of the electrophoretic process, resulting in very high apparent efficiencies. Our experiments presented in this article reveal that the migration time of bacteria species in polymer‐based CE increases with a growing amount of injected cells. Thus, the electrophoretic mobilities are not characteristic for the single cells of one species, but for the aggregates of the bacteria species, which are formed during the focusing process. Electrophoretic mobility (EM) data are obviously inapplicable for the identification of bacteria if the concentration of the bacteria sample solution is not constant. Fractions taken during the electrophoretic separation of different bacteria species were cultivated and tested for species purity. Interestingly, the electrophoretic bands were never pure, as all of them contained different mixtures of the injected species. We attribute this to the formation of stable mixed‐species aggregates during polymer‐based focusing. The mixed clusters migrate in the electric field with consistent velocity as a whole and are not separated electrophoretically.     

Effect of monascus pigment derivatives on the electrophoretic mobility of bacteria, and the cell adsorption and antibacterial activities of pigments

Various amino acid derivatives of monascus pigments were synthesized. The effects of pigment derivatives on the pigment adsorption ratio, electrophoretic mobility (EPM) of bacterial cells, and antibacterial activity were investigated under varying conditions of pigment type, pigment concentration, pH, and ionic strength. Two hydrophobic and two hydrophilic derivatives were selected as model pigments. There was a close relationship between the antimicrobial activity and the pigment adsorption ratio. Against Escherichia coli, the hydrophobic l-Tyr and l-Phe derivatives (log P = 3.18 and 3.57) exhibited high antimicrobial activities (MIC = 8 and 16 mg/L) and high cellular adsorption ratios (9.6 and 10.9 mg/L). The hydrophilic l-Glu and l-Asn derivatives (log P = 1.40 and 0.47) exhibited low activities (MIC = 64 and 128 mg/L) and low adsorption ratios (4.7 and 4.0 mg/L). The electrophoretic mobility of 11 different bacteria varied between −1.93 × 10⁻⁸ and −1.19 × 10⁻⁸ m² V⁻¹ s⁻¹ regardless of Gram⁺ or Gram⁻. The l-Phe derivative showed low MIC values (high antimicrobial activities) against bacteria with a high electrophoretic mobility. A positive linearity between the pigment adsorption ratio and the electrophoretic mobility was established. When the four pigment derivatives were added to E. coli solutions, the electrophoretic mobility of cells in all cases sharply increased with an increasing pigment concentration. The mobility value was high for hydrophobic pigment derivatives in descending order of l-Phe (0.8 × 10⁻⁸ m² V⁻¹ s⁻¹), l-Tyr (0.68 × 10⁻⁸ m² V⁻¹ s⁻¹), l-Glu (0.46 × 10⁻⁸ m² V⁻¹ s⁻¹), and l-Asn (0.44 × 10⁻⁸ m² V⁻¹ s⁻¹). Additional adsorption of the hydrophobic derivatives probably occurred due to a hydrophobic interaction between the pigment and the pigment-coated cells. The electrophoretic mobility decreased gradually with an increasing pH and/or ionic strength with both addition and no addition of the pigment derivatives. The pattern of change of the pigment adsorption ratio under varying pH and/or ionic strength values was similar to the pattern for electrophoretic mobility.     

Characterization of carboxylated nanolatexes by capillary electrophoresis

Poly(styrene-co-acrylic acid) (St/AA) and poly(styrene-co-methacrylic acid) (St/MA) nanolatexes with different acid contents were prepared by emulsion copolymerization and were analyzed by capillary electrophoresis (CE) and by laser doppler velocimetry (LDV). Due to the intrinsic differences in the methodologies, CE (separative technique) and LDV (zetametry, nonseparative technique) lead to very different electrophoretic mobility distributions. Beyond these differences, the variation of the electrophoretic mobility is a complex and nonlinear function of the hydrodynamic radius, the ionic strength, and the zeta potential. To gain better insight on the influence of the ionic strength and the acid content on the electrophoretic behavior of the nanolatexes, the electrophoretic mobility data were changed into surface charge densities using the O'Brien, White, and Ohshima modeling. This approach leads to the conclusion that the surface charge density is mainly controlled at high ionic strength (～50 mM) by the adsorption of anionic surfactants coming from the sample. On the contrary, at low ionic strength, and/or in the presence of neutral surfactant in the electrolyte, the acid content was the main parameter controlling the surface charge density of the nanolatexes.     

Electrokinetic characterization of superparamagnetic nanoparticle–aptamer conjugates: design of new highly specific probes for miniaturized molecular diagnostics

With the view of designing new nanoparticle (NP)–aptamer conjugates and proving their suitability as biorecognition tools for miniaturized molecular diagnostics, new maghemite–silica core–shell NP–aptamer conjugates were characterized for the first time in terms of grafting rate and colloidal stability under electrophoretic conditions using capillary electrophoresis. After the grafting rate (on the order of six to 50) of the lysozyme-binding aptamer had been estimated, the electrophoretic stability and peak dispersion of the resulting oligonucleotide–NP conjugates were estimated so as to determine the optimal separation conditions in terms of buffer pH, ionic strength and nature, as well as temperature and electric field strength. The effective surface charge density of the NPs was close to zero for pH lower than 5, which led to some aggregation. The NPs were stable in the pH range from 5 to 9, and an increase in electrophoretic mobility was evidenced with increasing pH. Colloidal stability was preserved at physiological pH for both non-grafted NPs and grafted NPs in the 10–100 mM ionic strength range and in the 15–60 °C temperature range. A strong influence of the nature of the buffer counterion on NP electrophoretic mobility and peak dispersion was evidenced, thus indicating some interactions between buffer components and NP–aptamer conjugates. Whereas an electric field effect (50–900 V cm^?1) on NP electrophoretic mobility was evidenced, probably linked to counterion dissociation, temperature seems to have an appreciable effect on the zeta potential and aptamer configuration as well. This information is crucial for estimating the potentialities of such biorecognition tools in electrophoretic systems.     

Reliable electrophoretic mobilities free from Joule heating effects using CE

Ionic electrophoretic mobilities determined by means of CE experiments are sometimes different when compared to generally accepted values based on limiting ionic conductance measurements. While the effect of ionic strength on electrophoretic mobility has been long understood, the increase in the mobility that results from Joule heating (the resistive heating that occurs when a current passes through an electrolyte) has been largely overlooked. In this work, a simple method for obtaining reliable and reproducible values of electrophoretic mobility is described. The electrophoretic mobility is measured over a range of driving powers and the extrapolation to zero power dissipation is employed to eliminate the effect of Joule heating. These extrapolated values of electrophoretic mobility can then be used to calculate limiting ionic mobilities by making a correction for ionic strength; this somewhat complicated calculation is conveniently performed by using the freeware program PeakMaster 5. These straightforward procedures improve the agreement between experimentally determined and literature values of limiting ionic mobility by at least one order of magnitude. Using Tris-chromate BGE with a value of conductivity 0.34 S/m and ionic strength 59 mM at a modest dissipated power per unit length of 2.0 W/m, values of mobility for inorganic anions were increased by an average of 12.6% relative to their values free from the effects of Joule heating. These increases were accompanied by a reduction in mobilities due to the ionic strength effect, which was 11% for univalent and 28% for divalent inorganic ions compared to their limiting ionic mobilities. Additionally, it was possible to determine the limiting ionic mobility for a number of aromatic anions by using PeakMaster 5 to perform an ionic strength correction. A major significance of this work is in being able to use CE to obtain reliable and accurate values of electrophoretic mobilities with all its benefits, including understanding and interpretation of physicochemical phenomena and the ability to model and simulate such phenomena accurately.     

Size-based characterization of nanometric cationic maghemite particles using capillary zone electrophoresis

Size-sorted maghemite (gamma-Fe(2)O(3)) particle populations of number mean solid diameters ranging from 6 to 10 nm were suspended and directly characterized in their stabilizing acidic, citrated or basic aqueous media using CZE coupled with UV detection. Analytical conditions were optimized in order to ensure reliable mobility measurements of these ferrofluids in their anionic and cationic forms. Particular interest has been paid to the investigation of the positively charged ferrofluids since cationic colloids have received little attention so far. A strategy for capillary wall modification was chosen in order to prevent particle adsorption while preserving high analytical performances. The influence of experimental conditions such as particle volume fraction, injection volume, electric field strength and electrolyte nature on electrophoretic profiles and measured electrophoretic mobilities was evaluated. A size-dependent electrophoretic mobility was demonstrated and discussed in terms of the ratio of the particle radius to Debye length with reference to existing models (Henry, etc.). Although these nanometric particle distributions lie in a very narrow size range, partial separation was obtained with selectivity varying as a function of electrolyte ionic strength.     

Increased adhesion of Enterococcus faecalis strains with bimodal electrophoretic mobility distributions

Initial adhesion is a determinant in the development of microbial biofilms. It is influenced, amongst others, by the surface hydrophobicity and the electrostatic characteristics of the substratum and adhering organisms. Enterococcus faecalis strains, grown in pure cultures, generally display subpopulations with different electrokinetic features, reflected in a bimodal electrophoretic mobility distribution. Here, the initial adhesion kinetics of five heterogeneous and five homogeneous E. faecalis strains were followed in a parallel-plate flow chamber. After 4h of flow, heterogeneous strains adhered in significantly higher numbers than homogeneous strains (7.3 x 10(6) and 1.9 x 10(6)cm(-2), respectively), but the initial deposition rates were not significantly influenced (740 and 600 cm(-2)s(-1), respectively). Apparently, initial deposition of bacteria is mainly governed by attractive Lifshitz-Van der Waals forces that overwhelm the electrostatic repulsion energy barrier, thus resulting in similar initial deposition rates for the various bacterial populations investigated. In contrast, during later stages of adhesion, bacteria in heterogeneous cultures likely experience a lower electrostatic repulsion from already adhering bacteria than bacteria in homogeneous cultures, thus allowing a closer proximity of the bacteria with respect to each other, which ultimately leads to increased adhesion after 4 h.     

Examination of the electrophoretic behavior of liposomes

The electromigration of liposomes is a complex process resulting in many unexpected behaviors that are difficult to address with existing theories. In this study, the electrophoretic behaviors of liposome populations under various conditions were examined through the use of capillary electrophoresis and the results compared to classical electrokinetic, colloid, and spheroid theories. To elucidate the possible effects of applied field strength, bilayer rigidity, and surface charge on these behaviors, the electrophoretic mobilities of liposome populations were monitored while varying the applied potential, ionic strength of the medium, and the surface charge and cholesterol content of the liposomes. On the basis of comparisons made to the theoretical predictions, our results suggest that liposomal deformation and field-induced polarization may occur during electrophoresis and these mechanisms help to describe many of the observed behaviors.     

Binding constant determination of uranyl–citrate complex by ACE using a multi‐injection method

The binding constant determination of uranyl with small‐molecule ligands such as citric acid could provide fundamental knowledge for a better understanding of the study of uranyl complexation, which is of considerable importance for multiple purposes. In this work, the binding constant of uranyl–citrate complex was determined by ACE. Besides the common single‐injection method, a multi‐injection method to measure the electrophoretic mobility was also applied. The BGEs used contained HClO₄ and NaClO₄, with a pH of 1.98 ± 0.02 and ionic strength of 0.050 mol/L, then citric acid was added to reach different concentrations. The electrophoretic mobilities of the uranyl–citrate complex measured by both of the two methods were consistent, and then the binding constant was calculated by nonlinear fitting assuming that the reaction had a 1:1 stoichiometry and the complex was [(UO₂)(Cit)]^?. The binding constant obtained by the multi‐injection method was log K = 9.68 ± 0.07, and that obtained by the single‐injection method was log K = 9.73 ± 0.02. The results provided additional knowledge of the uranyl–citrate system, and they demonstrated that compared with other methods, ACE using the multi‐injection method could be an efficient, fast, and simple way to determine electrophoretic mobilities and to calculate binding constants.     

Electrophoretic mobility of the polymer-like micelles of tetradecyldimethylamine oxide hemihydrochloride

Effects of the surfactant concentration C_d and the NaCl concentration C_s on the electrophoretic mobilities U of the well-characterized polymer-like micelles have been investigated by the electrophoretic light scattering, using tetradecyldimethylamine oxide hemihydrochloride (C14DMAO·1/2HCl). At the high ionic strength of 0.1 mol kg⁻¹ NaCl, the electrophoretic mobilities were independent of C_d (5 mM < C_d < 100 mM), despite the concentration-dependent micelle growth of the polymer-like micelles. This suggests that the electrophoretic mobility of the polymer-like micelle at high ionic strengths is independent of the contour length (i.e., the molecular weight), as found on linear polyelectrolytes. Somewhat surprisingly, the entanglements of the polymer-like micelles gave small effect on the electrophoretic mobilities in the examined range of the surfactant concentration above an overlap concentration. The mobilities of the polymer-like micelle decreased with √C_s in a single exponential manner in the range of C_s from 0.02 to 0.3 mol kg⁻¹. It is suggested that the cylinder model can be applied to the electrophoretic mobilities of the polymer-like micelles at high ionic strengths (i.e. a free-draining behavior), since the persistence length of the polymer-like micelle (20 nm) is much larger than the Debye length at high ionic strength.     

Continuous microfluidic DNA and protein trapping and concentration by balancing transverse electrokinetic forces

Sample pre-concentration can be a critical element to improve sensitivity of integrated microchip assays. In this work a converging Y-inlet microfluidic channel with integrated coplanar electrodes was used to investigate transverse DNA and protein migration under uniform direct current (DC) electric fields to assess the ability to concentrate a sample prior to other enzymatic modifications or capillary electrophoretic separations. Employing a pressure-driven flow to perfuse the microchannel, negatively charged samples diluted in low and high ionic strength buffers were co-infused with a receiving buffer of the same ionic strength into a main daughter channel. Experimental results demonstrated that, depending of the buffer selection, different DNA migration and accumulation dynamics were seen. Charged analytes could traverse the channel width and accumulate at the positive bias electrode in a low electroosmotic mobility, high electrophoretic mobility, high ionic strength buffer or migrated towards an equilibrium position within the channel in a high electroosmotic mobility, high electrophoretic mobility, low ionic strength buffer. The various migration behaviours are the result of a balance between the electrophoretic force and a drag force induced by a recirculating electroosmotic flow generated across the channel width due to the bounding walls. Under continuous flow conditions, DNA samples were concentrated several-fold by balancing these transverse electrokinetic forces. The electrokinetic trapping technique presented here is a simple technique which could be expanded to concentrate or separate other analytes as a preconditioning step for downstream processes.     

Influence of buffer composition on the capillary electrophoretic separation of carbon nanoparticles

Carbon nanoparticles obtained from the flame of an oil lamp were examined by means of capillary electrophoresis. The influence of buffer composition on the separation of the mixture of negatively charged carbon nanoparticles was studied by varying buffer selection, pH, and concentration. The electrophoretic pattern was affected by both the co- and counter-ion in the buffer solution, influencing selectivity and peak shape. The capillary electrophoretic separations at different pH revealed species with large electrophoretic mobilities under a wide range of pH. The mobility of selected species in the mixture of nanoparticles showed a strong dependence upon the solution ionic strength. The mobility of these nanoparticles as a function of ionic strength was compared to classical electrokinetic theory, suggesting that under the experimental conditions utilized, the species are small, highly charged particles with appreciable zeta potentials, even at low pH.     

Superfast electrophoresis of electron-type conducting particles

The features of concentration polarization caused by electric current through a unipolar conductive particle are considered. The peculiarities of the formation of an induced space charge near a particle with electron-type conductivity are analysed. It has been shown that the theoretical values of electrophoretic velocity for these particles are essentially smaller than those calculated for particles with ion-type conductivity.A new method to observe the superfast electrophoresis is developed. The electrophoretic velocity of graphite and activated carbon particles of different size (diameter, 200–500 μm) displaced in distilled water and electrolyte solutions in strong electric fields (100–500 V cm⁻¹) was measured. It is shown that, in contrast to classical electrophoresis, the electrophoretic mobility of such particles increases with the particle size and the external field strength. The experimental and theoretical results are compared. The discrepancy between theory and experiment is analysed.     

On the use of the activation energy concept to investigate analyte and network deformations in entangled polymer solution capillary electrophoresis of synthetic polyelectrolytes

The activation energy associated with the electrophoretic migration of an analyte under given electrolyte conditions can be accessed through the determination of the analyte electrophoretic mobility at various temperatures. In the case of the electrophoretic separation of polyelectrolytes in the presence of an entangled polymer network, activation energy can be regarded as the energy needed by the analyte to overcome the obstacles created by the separating network. Any deformation undergone by the analyte or the network is expected to induce a decrease in the activation energy. In this work, the electrophoretic mobilities of poly(styrenesulfonates) (PSSs) of various molecular weights (Mr 16 x 10(3) to 990 x 10(3)) were determined in entangled polyethylene oxide (PEO) solutions as a function of temperature (in the 17-60 degrees C range) and the PSS activation energies were calculated. The influences of the PSS molecular weight, blob sizes zetab of the separating network (related to the PEO concentration), ionic strength of the electrolyte and electric field strength (75-600 V/cm) were investigated. The results were interpreted in terms of analyte and network deformations and were confronted with those previously obtained for DNA migration in polymer solutions and chemical gels. For a radius of gyration Rgzetab, suggesting PSS and network deformations in the latter case. Increasing ionic strength resulted in an increase in the PSS activation energy, because of the decrease of their radii of gyration, which makes them less deformable. Finally, the activation energies of all the PSSs are a decreasing function of field strength and at high field strength tend to reach a constant value close to that for a small molecule.     

Nonlinear electrophoresis of colloidal particles

Advances over the past decade in nonlinear electrophoresis of charged, dielectric colloidal particles in aqueous electrolytes are reviewed. Here, the word nonlinear refers to the fact that the ratio of the electrophoretic speed of the particle to the magnitude of the applied electric field—the electrophoretic mobility—is not independent of field strength. This is in stark contrast to the vast majority of work on (linear) colloidal electrophoresis over the last century, where the mobility is assumed to be a material property dependent only on the particle–electrolyte combination. The present discussion is focused on: (i) experimental measurements of the field-dependent mobility; (ii) an asymptotic scheme to calculate the mobility in the common thin-Debye-layer limit; and (iii) computations of nonlinear electrophoresis from numerical solution of the electrokinetic equations. The article concludes with suggestions for future work in this evolving area of colloid science.     

Electrokinetic characterization of polystyrene–non-ionic surfactant complexes

An experimental study on the electrophoretic mobility (μ_e) of polystyrene particles after the adsorption of non-ionic surfactants with different chain lengths is described. Two sulphate latexes with relatively low surface charge densities (3.2 and 4.8 μC cm⁻²) were used as solid substrate for the adsorption of four non-ionic surfactants, Triton X-100, Triton X-165, Triton X-305 and Triton X-405, each one with 9–10, 16, 30 and 40 molecules of ethylene oxide (EO), respectively. The electrophoretic mobility of the polystyrene–non-ionic surfactant complexes was studied versus the amount of adsorbed surfactant (Γ). The presence of non-ionic surfactant onto particles surface seems to produce a slight shifting of the slipping plane because the mobilities of the different complexes display a very small decreasing. The increase in the number of EO chains in the surfactant molecule seems to operate as a steric impediment which decreases the number of adsorbed large surfactant molecules. The electrophoretic mobilities of the latex–surfactant complexes with maximum adsorption were measured versus the pH and ionic strength of the dispersion. While the different complexes showed a similar qualitative behaviour compared with that of the bare latex against the pH, the adsorption of the surfactant reduces the typical maximum in the μ_e−log[electrolyte].