Capillary electrophoresis of microbial aggregates

Uncontrolled aggregation of bacterial cells is a significant disadvantage of electrophoretic separations. Various aspects of the electrophoretic behavior of different strains of Gram‐positive Bacillus cereus, Bacillus subtilis, Sarcina lutea, Staphylococcus aureus(1), and Micrococcus luteus bacteria and Gram‐negative Escherichia coli bacteria were investigated in this study. Our findings indicate that bacteria can be rapidly analyzed by CZE with surface charge modification by calcium ions (Ca²⁺). Bound Ca²⁺ ions increase zeta potential to more than 2.0 mV and significantly reduce repulsive forces. Under the above conditions, bacterial cells create compact aggregates, and fewer high‐intensity signals are observed in electropherograms. The above can be attributed to the bridging effect of Ca²⁺ between bacterial cells. CE was performed to analyze bacterial aggregates in an isotachophoretic mode. A single peak was observed in the electropherogram.     

Numerical simulations of the second-order electrokinetic bias observed with the gated injection mode in chips

It is well known that sample introduction via electrokinetic mode leads to a bias in conventional CE, which is proportional to the difference of electrophoretic mobilities between species. In electrophoretic separation chips using the gated injection mode, flow distribution at the crossjunction, which is linked to the electric field strength distribution during the loading step, induces an additional contribution to species discrimination. This second-order bias has a similar effect on quantitation like usual electrokinetic bias: the higher the analyte's apparent mobility, the larger the amount injected into the separation channel. The present paper assesses by numerical simulations the influence of several parameters, namely the injected amount, the electric field distribution, and the analyte-apparent Peclet number on this second-order bias.     

Capillary electrophoresis for fast detection of heterogeneous population in colistin‐resistant Gram‐negative bacteria

It has been shown that diverse strains of bacteria can be separated according to their characteristic surface properties by means of CE. We employed here this analytical technique to the study of colistin‐resistance in Gram‐negative bacteria, which involves the selection of mutants with modified outer membrane composition resulting in changes of surface cell properties. In the same way as with molecular entities, we performed firstly the validation of an ITP‐based CE method for three common pathogenic Gram‐negative bacteria namely Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Secondly, we compared the electrophoretic profiles of bacterial samples from a colistin‐susceptible clinical isolate of K. pneumoniae and from the corresponding colistin‐resistant derivative. By a simple CE run taking a few minutes, the coexistence of several bacterial subpopulations in the colistin‐resistant derivative was clearly evidenced. This work encourages further research that would allow applications of CE in clinical laboratory for a daily monitoring of bacterial population in cared patients when “last‐chance” colistin treatment is initiated against multidrug‐resistant bacteria.     

Determination of the bacterial pathogen Edwardsiella tarda in fish species by capillary electrophoresis with blue light-emitting diode-induced fluorescence

High-performance capillary electrophoresis (HPCE) has been applied to the identification, separation, and quantitation of intact bacteria. We demonstrate that a pathogen (Edwardsiella tarda) which causes systemic infection in commercially important fish species can be rapidly identified and determined (< 10 min) after direct injection into fish fluid by CE blue light-emitting diode (LED)-induced fluorescence. SYTO 13 (488 nm/509 nm), a cell-permeable green nucleic acid stain, was used to stain the cells. Remarkably high efficiency (> 1,200,000 theoretical plates/m) was achieved with this rapid and efficient CE method. It was found that proper sample vortexing (90 s) would be beneficial to disperse aggregated cells and facilitate the focusing of intact cells during electrophoresis. Ionization of the surface constituents of Edwardsiella tarda cells provided efficient surface charges for the intact cells to be separated from the EOF and damaged or lysed cells when the separation was performed in running buffer (3.94 mM Tris, 0.56 mM borate, 0.013 mM EDTA) at pH 10.5. The limit of detection (LOD) and recovery were found to be 4.2 x 10(4) cells/mL and 70.0%, respectively. This proposed CE method could become an effective tool for diagnosis and tracking of certain diseases caused by bacteria in fish species as well as in human beings.     

In‐capillary reactant mixing for monitoring glycerol kinase kinetics by CE

CE was used for the first time to study the two‐substrate enzyme glycerol kinase. The capillary was used as a nanoreactor in which the enzyme and its two substrates glycerol and adenosine‐5′‐triphosphate were in‐capillary mixed to realize the enzymatic assay. For kinetic parameters determination, reactants were injected (50 mbar × 5 s) as follows: (i) incubation buffer; (ii) adenosine‐5′‐triphosphate; (iii) enzyme, and (iv) glycerol. Enzymatic reaction was then initiated by mixing the reactants using electrophoretically mediated microanalysis (+20 kV for 6 s) followed by a zero‐potential amplification step of 3 min. Finally, electrophoretic separation was performed; the product adenosine‐5′‐diphosphate was detected at 254 nm and quantified. For enzyme inhibition, an allosteric inhibitor fructose‐1,6‐bisphosphate plug was injected before the first substrate plug and +20 kV for 8 s was applied for reactant mixing. A simple, economic, and robust CE method was developed for monitoring glycerol kinase activity and inhibition. Only a few tens of nanoliters of reactants were used. The results compared well with those reported in literature. This study indicates, for the first time, that at least four reactant plugs can be in‐capillary mixed using an electrophoretically mediated microanalysis approach.     

Polymer monolayers with a photosensitive crown‐ether

The mixed monolayers of a photosensitive crown‐ether (CE) and poly(maleic acid hexadecyl monoamide‐alt‐propylene) (P12) have been prepared and studied. Fluorescence spectra of CE have a strong band at 530–535 nm in solution (monomer) or at 593–603 nm in transferred monolayers (aggregates). Due to the interaction with polymer the fluorescence maximum of CE is shifted to 530 nm in the mixed monolayers with P12.     

聚合物涂层在毛细管电泳分离蛋白中的研究进展

毛细管电泳具有分析时间短,分离效率高,样品消耗量少等优点,在生物样品分离,特别是蛋白质分析领域有重要应用。然而,毛细管内壁硅羟基的解离给分离结果带来诸多不良影响。聚合物涂层能够抑制蛋白质在毛细管内壁的吸附以及调控电渗流,故对毛细管内壁进行有效修饰能够提高其对蛋白质的分离效率及分离稳定性。该文主要综述了动态及静态聚合物涂层毛细管的最新研究进展,并概述了近些年基于多巴胺/聚多巴胺发展起来的涂层毛细管的研究进展,最后展望了聚合物涂层毛细管的发展趋势。     

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.     

Determination of pathogenic bacteria by CZE with surface-modified capillaries

The importance of electromigration techniques in molecular biology and medicine is increasing rapidly, especially in systematic studies on proteomes and metabolomes. Staphylococcus aureus and Escherichia coli are bacterial species most frequently encountered in human infections, and many serious illnesses can be observed in the hospital environment. In this contribution we proposed a CE method with different modification of internal capillary surface and with monolithic beds as a selective material for determination of bacteria in clinical samples. The electrophoretic separation depends on the differential mobility of bacteria in the capillary and selective interactions between bacterial cells and stationary phases (modified surface, monolithic beads). Proposed procedures could become an effective tool for diagnosis of certain diseases caused by S. aureus and E. coli as well as Proteus vulgaris.     

In-line liquid-phase microextraction for selective enrichment and direct electrophoretic analysis of acidic drugs

This work describes an efficient in-line extraction-preconcentration unit coupled to the electrophoretic capillary based on a liquid-phase microextraction (LPME) process, which can be directly assembled to the cartridge of the commercial CE equipment. The unit permits analyte extraction, preconcentration and electrophoretic separation to be automatically performed in the commercial CE equipment without the need for additional hardware or software. This new approach was usefully used for the separation and determination of nonsteroidal anti-inflammatory drugs in human urine permitting at least to analyze 30 consecutive real samples. The LODs were lower than 2 microg/L and the reproducibility, expressed as RSD, was 3.1% for the same unit and only 4.8% between different units.     

Recent advances in nanomaterial‐based capillary electrophoresis

This review gives a summary of applications of different nanomateials, such as gold nanoparticles (AuNPs), carbon‐based nanoparticles, magnetic nanoparticles (MNPs), and nano‐sized metal organic frameworks (MOFs), in electrophoretic separations. This review also emphasizes the recent works in which nanoparticles (NPs) are used as pseudostationary phase (PSP) or immobilized on the capillary surface for enhancement of separation in CE, CEC, and microchips electrophoresis.     

An air‐assisted flow‐gating interface for capillary electrophoresis

A new kind of flow gating interface (FGI) has been designed for online connection of CE with flow‐through analytical techniques. The sample is injected into the separation capillary from a space from which the BGE was forced out by compressed air. A drop of sample solution with a volume of 75 nL is formed between the outlet of the delivery capillary supplying the solution from the flow‐through apparatus and the entrance to the CE capillary; the sample is hydrodynamically injected into the CE capillary from this drop. The sample is not mixed with the surrounding BGE solution during injection. The functioning of the proposed FGI is fully automated and the individual steps of the injection process are controlled by a computer. The injection sequence lasts several seconds and thus permits performance of rapid sequential analyses of the collected sample. FGI was tested for the separation of equimolar 50 μM mixture of the inorganic cations K⁺, Ba²⁺, Na⁺, Mg²⁺, and Li⁺ in 50 mM acetic acid/20 mM Tris (pH 4.5) as BGE. The obtained RSD values for the migration times varied in the range 0.7–1.0% and the values for the peak area were 0.7–1.4%; RSD were determined for ten repeated measurements.     

Continuous cell introduction and rapid dynamic lysis for high-throughput single-cell analysis on microfludic chips with hydrodynamic focusing

A chip-based microfluidic system for high-throughput single-cell analysis is described. The system was integrated with continuous introduction of individual cells, rapid dynamic lysis, capillary electrophoretic (CE) separation and laser induced fluorescence (LIF) detection. A cross microfluidic chip with one sheath-flow channel located on each side of the sampling channel was designed. The labeled cells were hydrodynamically focused by sheath-flow streams and sequentially introduced into the cross section of the microchip under hydrostatic pressure generated by adjusting liquid levels in the reservoirs. Combined with the electric field applied on the separation channel, the aligned cells were driven into the separation channel and rapidly lysed within 33ms at the entry of the separation channel by Triton X-100 added in the sheath-flow solution. The maximum rate for introducing individual cells into the separation channel was about 150cells/min. The introduction of sheath-flow streams also significantly reduced the concentration of phosphate-buffered saline (PBS) injected into the separation channel along with single cells, thus reducing Joule heating during electrophoretic separation. The performance of this microfluidic system was evaluated by analysis of reduced glutathione (GSH) and reactive oxygen species (ROS) in single erythrocytes. A throughput of 38cells/min was obtained. The proposed method is simple and robust for high-throughput single-cell analysis, allowing for analysis of cell population with considerable size to generate results with statistical significance.     

Sieving properties of end group‐halogenated Pluronic polymer matrix in DNA separation under nondenaturing CE analysis

CE‐SSCP analysis is a well‐established DNA separation method that is based on variations in mobility caused by sequence‐induced differences in the conformation of single‐stranded DNA. The resolution of CE‐SSCP analysis was improved by using a Pluronic polymer matrix, and it has been successfully applied in various genetic analyses. Because the Pluronic polymer forms a micellar cubic structure in the capillary, it provides a stable internal structure for high‐resolution CE‐SSCP analysis. We hypothesized that formation of micellar cubic structure is influenced by the end hydroxyl group of the Pluronic polymer, which affords structural stability through hydrogen bonding. To test this hypothesis, the hydroxyl group was halogenated to eliminate the hydrogen bonding without disturbing the polarity of polymer matrix. CE‐SSCP resolution of two DNA fragments with a single base difference was significantly worse in the halogenated polymer matrices due to band broadening. The viscoelastic properties of control (which has hydroxyl group), chlorinated, and brominated F108 solution upon heating were also investigated by rheological experiments, and we found that gelation was significantly associated with resolution. In this series of experiments, the effect of the hydroxyl group in Pluronic polymer matrix on separation resolution of CE‐SSCP analysis was demonstrated.     

Counterflow gradient electrophoresis for focusing and elution

Counterflow gradient electrofocusing uses the bulk flow of a liquid solution to counterbalance the electrophoretic migration of an analyte. When either the bulk velocity or the electrophoretic velocity of the analyte is made to vary across the length of the channel, there exists a unique zero‐velocity point for the analyte. This focusing method enables simultaneous separation and concentration of different analytes. The high resolution and sensitivity achieved are similar to that of isoelectric focusing, which separates analytes based on their isoelectric points, but the key difference is that analytes will instead focus based on their electrophoretic mobility. Dynamically changing the applied voltage or the counterflow rate over time will shift the zero‐velocity point, and therefore allows the focused analytes to pass through a fixed detection point, or elute from the separation channel. Throughout the review, a number of different counterflow gradient techniques will be discussed, along with their recent advancements and potential applications.     

Recent developments in the separation of inorganic and small organic ions by capillary electrophoresis

Which method should I use for ion analysis, ion chromatography (IC) or capillary electrophoresis (CE)? In terms of actual theoretical plates CE has a clear-cut advantage. The separation ability of IC is adequate for many sample types, and many separation scientists feel that IC offers greater reliability and confidence than CE. However, IC is a more mature technique and there has been more time to solve problems such as peak tailing and to improve reproducibility. The two techniques should be viewed as complementary. A number of recent developments in ion analysis by CE are discussed. These include some simple ways to control electroosmotic flow and improve reproducibility, separation of isotopes, improved methods of indirect photometric detection, a new contactless conductivity detector, separation of ions at low pH, and in solutions of high salt content. Progress in a new technique called IC-CE will be described in which a soluble ion-exchange polymer is added to the capillary electrolyte to separate anions based on differences in both electrophoretic mobility and ion-exchange interactions.     

Capillary electrophoretic study of green fluorescent hollow carbon nanoparticles

CE coupled with laser‐induced fluorescence and UV absorption detections has been applied to study the complexity of as‐synthesized green fluorescent hollow carbon nanoparticles (HC‐NP) samples. The effects of pH, type, and concentration of the run buffer and SDS on the separation of HC‐NP are studied in detail. It is observed that phosphate run buffer is more effective in separating the HC‐NP and the optimal run buffer is found to be 30 mM phosphate and 10 mM SDS at pH 9.0. The CE separation of this HC‐NP is based on the difference in size and electrophoretic mobility of HC‐NP. Some selected HC‐NP fractions are collected and further characterized by UV‐visible absorption and photoluminescence (PL) spectroscopy, MS, and transmission electron microscopy. The fractionated HC‐NP show profound differences in absorption, emission characteristics, and PL quantum yield that would have been otherwise misled by studying the complex mixture alone. It is anticipated that our CE methodology will open a new initiative on extensive studies of individual HC‐NP species in the biomedical, catalysis, electronic, and optical device, energy storage, material, and sensing field.     

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.     

Strategies in two‐dimensional liquid chromatographic separation of complex polymer systems

Complex synthetic polymer systems as for example copolymers exhibit distributions in at least two of the three basic molecular characteristics which are molar mass, chemical structure/composition and molecular architecture. Size exclusion chromatography (SEC) separates macromolecules according to their size in solution which simultaneously depends on all molecular characteristics. Therefore, multi‐dimensional liquid chromatographic techniques are to be applied to independently assess all different distributions present in the sample. So far, two‐dimensional separations have been attempted. In the first dimension separation column, selected liquid chromatographic mechanisms are intentionally combined to suppress effects of all but one molecular characteristic. Consequently, polymer species are separated exclusively or at least predominantly according to one single parameter. In the second dimension separation column, macromolecules are separated according to another molecular characteristic. In this contribution the methods are briefly reviewed in which effect of polymer molar mass on polymer retention is suppressed. The resulting ”one parameter separation systems” can be on‐line or off‐line connected to another separation system such as SEC to provide more detailed characterization of complex polymers. Besides, selected procedures for the re‐concentration of diluted polymer solutions are concisely treated. These may be utilized for increasing the concentration of sample(s) leaving the first dimension separation column. Eventually, some arrangements for controlled sample re‐introduction into the second dimension separation column are outlined.     

Capillary electrophoresis using a surfactant-treated capillary coupled with offline matrix-assisted laser desorption ionization mass spectrometry for high efficiency and sensitivity detection of proteins.

A method of combining capillary electrophoresis (CE) using a surfactant-modified capillary with matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is described for protein analysis. The CE-MALDI-MS coupling is based on CE fraction collection of nanoliter volume samples in less than 5 microl of dilute acid. This offline coupling does not require any special instrumentation and can be readily performed with commercial instruments. Protein adsorption during CE separation is prevented by coating the capillary with the surfactant didodecyldimethylammonium bromide. This surfactant binds strongly with the capillary wall, hence it does not desorb significantly to interfere with subsequent MALDI-MS analysis. It is shown that the use of a dilute acid for CE fraction collection is advantageous in lowering the detection limit of MALDI-MS compared to using an electrophoretic buffer. The detection limit for proteins such as cytochrome c is 23 fmol injected for CE, or 1.2 fmol spotted for MALDI-MS. This sensitivity is comparable to alternative CE-MALDI-MS coupling techniques using direct CE sample deposition on the MALDI target. In addition, the fraction collection approach has the advantage of allowing multiple reactions to be carried out on the fractioned sample. These reactions are very important in protein identification and structure analysis.