Combined capillary electrophoresis and DNA-fluorescence in situ hybridization for rapid molecular identification of Salmonella Typhimurium in mixed culture

CE, long a staple in analytical chemistry for molecular separations, has recently been adapted for separating heterogeneous mixtures of microbial cells based on intrinsic differences in cell morphology and surface charge. In this application, CE enables effective separations of both relatively broad categories of cells, as well as of more similar cell types. As a phenotypic approach, CE may be less applicable to certain populations, including those comprised of pleiomorphic cells or chain-forming cells, where differences in cell size, shape, or chain length may lead to broad, "unfocusable" distributions in cell surface charge. At the other end of the spectrum, closely related species having similar surface charge profiles may not be separable via CE alone. Successful combination of microbial CE with a compatible method for generating cell-specific signals could address these limitations, increasing the diagnostic power of this approach. Fluorescence in situ hybridization (FISH) is a rapid molecular technique for fluorescence-based labeling of whole target cells. In this work, we combined a simple CE-based presence/absence test with FISH to develop a bacterial detection assay having an additional "layer" of molecular specificity. Using this approach, we were able to differentiate Salmonella Typhimurium from Escherichia coli in mixed populations via CE. Both hybridizations and CE run times were short (10-15 min), bacterial populations were highly focused ( approximately 2-3 s peak width) and there was no need for a posthybridization wash step. As few as three injected cells of S. Typhimurium were detected against a background of approximately 300 injected E. coli cells, suggesting the possibility for single-cell detection of pathogens using this technique. This proof of concept study highlights the potential of CE-FISH as a promising new tool for molecular detection of specific bacterial cells within mixtures of closely related, physiologically inseparable populations.     

Capillary electrophoresis with wavelength-resolved laser-induced fluorescence detection

Capillary electrophoresis (CE) enables rapid separations with high separation efficiency and compatibility with small sample volumes. Laser-induced fluorescence detection can result in extremely low limits of detection in CE. Single-channel fluorescence detection, however, furnishes little qualitative information about a species being detected, except for its CE migration time. Use of multidimensional information often enables unambiguous identification of analytes. Combination of CE with information-rich wavelength-resolved fluorescence detection is analogous with ultraviolet-visible diode-array detection and furnishes both qualitative and quantitative chemical information about target species. This review discusses recent advances in wavelength-resolved laser-induced fluorescence detection coupled with CE, with an emphasis on instrument design.     

Enzymatic assay of marine bacterial phosphatases by capillary electrophoresis with laser-induced fluorescence detection

Microbial ectoenzyme activities in aquatic environments are important determinants of polymer hydrolysis and indicators of the state of microbial carbon, nitrogen, and phosphorus nutrition. Marine ectoenzymes are found on the cell surface or in the periplasmic space of gram-negative heterotrophic bacteria. Phosphatases, which remove phosphate groups from substrates, are one example of an ectoenzyme. Enzyme assays based on-capillary electrophoresis (CE) take advantage of CE's high-efficiency separation, extremely low sample volume requirements, and its ability to electrophoretically mix and separate zones of enzymes, substrates, and products all in one experimental run. CE has better resolving power and, when utilized with laser-induced fluorescence (LIF) detection, it is more sensitive than chromatography. CE-LIF is a promising tool for determining different phosphatases within a single microbial strain as well as the functional diversity between strains. In this study, four bacterial strains were studied (Shewanella sp., TW7, BB2AT2, and Vibrio alginolyticus) with each yielding at least one phosphatase that was kinetically characterized. K(m) values were calculated and found to be in the range of 0.0725-3.35 microM, whereas V(max) values ranged from 1.02 x 10(-3) to 1.05 x 10(-2) microM/min. The large range of values demonstrates differences among the phosphatases, suggesting different roles for each phosphatase not only between the species but also within a single bacterial species. This can have the important implications for organic matter processing in the sea.     

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.     

对数期金黄色葡萄球菌的离子交换色谱行为及其表征

建立了高效离子交换色谱和激光光散射仪联用的分离-检测系统研究对数期金黄色葡萄球菌 (Staphylococcus aureus)的方法。实验采用TSKgel SuperQ-TOYOPEARL 650C色谱柱,以20 mmol/L的哌嗪-盐酸缓冲液(pH 6.0)为流动相,倍增系数为21的光散射系统检测,发现对数期的S.aureus表现出两个有规律变化的色谱峰。透射电镜观察显示前峰的S.aureus为圆形,后峰为椭圆形且有一条明显的横隔壁。采用3-（4,5）-双甲基-2-噻唑-（2,5）-苯基溴化四氮唑盐     

Development and validation of a capillary electrophoresis method for the measurement of short-chain organic acids in natural rubber latex

Short-chain organic acid contents in serum of natural latex are interesting to measure and capillary electrophoresis (CE) has proved to be a good tool for their study. In the present work a method has been developed to identify the short-chain organic acids present in sera of natural rubber latex (oxalic, formic, fumaric, aconitic, succinic, malic, glutaric, citric, acetic, glycollic, propionic and quinic acids), the separation was optimised and the quantification method validated. The separation was performed on a CE system with UV detection at 200 nm. The separation was carried out with an uncoated fused-silica capillary (57 cm x 50 microm I.D.) and was operated at -10 kV potential. The separation buffers were prepared with 0.5 M H3PO4, 0.5 mM cetyltrimethylammonium bromide and pH adjusted by adding NaOH to 6.25 except for propionic acid which was better measured at pH 7.00. Validation parameters are adequate and limits of detection range from 0.005 mM to 1.6 mM. Short-chain organic acids were measured with this method in sera of three different types of latex.     

High-performance liquid chromatographic,capillary electrophoretic and capillary electrophoretic-electrospray ionisation mass spectrometric analysis of selected alkaloid groups

Systems for efficient separation of selected alkaloid groups by high performance liquid chromatography (HPLC), capillary electrophoresis (CE) and capillary electrophoresis coupled with electrospray ionisation mass spectrometry (CE-ESI-MS) are described. The optimized HPLC system was applied for the separation of 23 standard indole alkaloids as well as for qualitative and quantitative analyses of crude alkaloid extracts of Rauvolfia serpentina X Rhazya stricta hybrid cell cultures. The developed conditions for CE analysis proved to be efficient for separation of mixtures of standard indole and beta-carboline alkaloids. The described buffer system is also applicable in the combination of CE with electrospray ionisation mass spectrometry. This analytical technique allowed the separation and identification of components of standard indole alkaloid mixture as well as crude extracts of R. serpentina roots, R. serpentina cell suspension cultures and cortex of Aspidosperma quebracho-blanco. The influence of buffer composition and analyte structures on separation is discussed.     

A new single‐step quantitative pathogen detection system: Template‐tagging followed by multiplex asymmetric PCR using common primers and CE‐SSCP

Rapid diagnosis of bacterial infection is important for patient management and appropriate therapy during the early phase of bacteria‐induced disease. Among the existing techniques for identifying microbial, CE‐SSCP combined with 16S ribosomal RNA gene‐specific PCR has the benefits of excellent sensitivity, resolution, and reproducibility. However, even though CE‐SSCP can separate PCR products with high‐resolution, multiplex detection and quantification are complicated by primer‐dimer formation and non‐specific amplification. Here, we describe a novel technique for multiplex detection and quantification of pathogens by template‐tagging followed by multiplex asymmetric PCR and subsequent CE‐SSCP. More specifically, we reverse transcribed 16S ribosomal RNAs from seven septicemia‐inducing pathogens, tagged the templates with common end sequences, and amplified them using common primers. The resulting amplicons could be successfully separated by CE‐SSCP and quantified by comparison to an internal standard. This method yielded results that illustrate the potential of this system for diagnosing infectious disease.     

Capillary electrophoresis coupled with in‐column fiber‐optic laser‐induced fluorescence detection for the rapid separation of neodymium

In this study, in‐column fiber‐optic (ICFO) laser‐induced fluorescence (LIF) detection technique is coupled with capillary electrophoresis (CE) for the rapid separation of neodymium for the first time. The effects of buffer concentration, buffer pH, and separation voltage on the CE behaviors, including electrophoretic efficiency and detection sensitivity, are investigated in detail. Under the optimal condition determined in this study (15 mM borate buffer, pH 10.50, separation voltage 24 kV), neodymium could be separated effectively from the neighboring lanthanides (praseodymium and samarium) within several minutes, and the limit of detection for neodymium is estimated to be at the ppt level. The ICFO‐LIF‐CE system assembled in this study exhibits unique performance characteristics such as low cost and flexibility. Meanwhile, the separation efficiency and detection sensitivity of the assembled CE system are comparable to or somewhat better than those obtained in the previous traditional CE systems, indicating the potential of the assembled CE system for practical applications in the fields of spent nuclear fuel analysis, nuclear waste disposal/treatment, and nuclear forensics.     

Determination of the absolute mobility and the equivalent ionic conductivity of NpO2+ at 25 degrees C and at infinite dilution by capillary electrophoresis-inductively coupled plasma-mass spectrometry

The absolute mobility of NpO2+ and its equivalent ionic conductivity were extrapolated at 25 degrees C and at infinite dilution using a set of experimental data obtained at various ionic strengths. The separation was carried out by capillary electrophoresis (CE) at various concentrations of creatinine at a pH of 5. The detection of NpO2+ was performed by inductively coupled plasma mass spectrometry coupled on-line with CE. The following values have been found: mu0NpO2+ (25 degrees C) = (2.94 +/- 0.07) x 10(-4) cm2 V(-1) s(-1) and lambda0NpO2+ (x 10(4), 25 degrees C) = 28.3 +/- 0.7 m2 S mol(-1).     

Selective fluorescence labeling and sensitive determination of Staphylococcus aureus by microchip electrophoresis with a multiple-concentration approach

In this paper we explored the use of fluorescently labelled vancomycin to specifically bind and detect Staphylococcus aureus based on an on-line multiple-concentration on microchip electrophoresis.     

Determination of catecholamines by CE with direct chemiluminescence detection

A rapid and sensitive method to detect three catecholamines, isoprenaline, epinephrine, and dopamine, by CE coupled with direct luminol-potassium periodate chemiluminescence (CL) detection is described. The conditions for CE separation and CL reaction were systematically optimized. Under the optimum conditions, the baseline separation of three catecholamines was achieved within 6.5 min. The LODs obtained in standard solution were 5.3 x 10(-8 )mol/L for isoprenaline, 4.7 x 10(-8 )mol/L for epinephrine, and 1.5 x 10(-7 )mol/L for dopamine. The RSD of the migration time and peak area were less than 1.8 and 3.6% (n = 5), respectively. The present method was applied to the determination of the dopamine in urine samples of cigarette smokers and nonsmokers. The results obtained indicate that there is a close relationship between the content of dopamine in human urine and the amount of cigarettes smoked daily; the level of dopamine in smokers is higher than in nonsmokers.     

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.     

Large-volume sample stacking with polarity switching for the analysis of bacteria by capillary electrophoresis with laser-induced fluorescence detection

Capillary electrophoresis has been utilized for the rapid analysis of bacteria under specific experimental conditions. In this work, a method of large-volume sample stacking with polarity switching was evaluated for the analysis of bacteria by capillary electrophoresis with laser-induced fluorescence detection in order to enhance the detection sensitivity. The results indicated that the proposed method is not only effective for the focusing of bacterial cells, but also for the separation of mixtures of bacteria. With the optimized conditions, an enhancement factor of around 60-fold was obtained when long sample plug (up to 39.6% of capillary volume) was injected. Moreover, with the help of such stacking method, single, sharp, intense peak with high efficiency was observed without multiple peaks attributable to irregular clusters and aggregates of bacterial cells. This simple stacking approach appears to be promising as a rapid sterility test in various fields of applications.     

Evaluation of a dry, rehydratable film method for rapid enumeration of Staphylococcus aureus

Results with the new 3M Petrifilm Rapid S. aureus Count (RSA) Plate method were compared with those of the classical Baird-Parker agar (BPA) method for detection and enumeration of Staphylococcus aureus. Studies on 219 bacterial strains demonstrated that the Petrifilm RSA plate is more sensitive than and as specific as the classical BPA method for confirmed identification of S. aureus. Counts of colonies from 71 pure cultures, 61 naturally contaminated food samples, and more than 750 artificially inoculated food samples showed that the Petrifilm RSA method was as effective as the classical BPA method for identification and enumeration of S. aureus. The Petrifilm RSA method gave results in one-third the time required for the classical method.     

Capillary electrophoresis mass spectrometry and its application to the analysis of biological mixtures

Capillary electrophoresis (CE) mass spectrometry (MS), with its ability to separate compounds present in extremely small volume samples rapidly, with high separation efficiency, and with compound identification capability based on molecular weight, is an extremely valuable analytical technique for the analysis of complex biological mixtures. The highest sensitivities and separation efficiencies are usually achieved by using narrow capillaries (5-50 micro m i.d.) and by using sheathless CE-to-MS interfaces. The difficulties in CE-to-MS interfacing and the limited loadability of these narrow columns, however, have prevented CE-MS from becoming a widely used analytical technique. To remedy these limitations, several CE-MS interfacing techniques have recently been introduced. While electrospray ionization is the most commonly used ionization technique for interfacing CE-to-MS, matrix assisted laser desorption ionization has also been used, using both on-line and off-line techniques. Moreover, the high concentration detection limit of CE has been addressed by development of several sample concentration and sample focusing methods. In addition, a wide variety of techniques such as capillary zone electrophoresis, capillary isoelectric focusing, and on-column transient isotachophoresis have now been interfaced to MS. These advances have resulted in a rapid increase in the use of CE-MS in the analysis of complex biological mixtures. CE-MS has now been successfully applied to the analysis of a wide variety of compounds including amino acids, protein digests, protein mixtures, single cells, oligonucleotides, and various small molecules relevant to the pharmaceutical industry.     

毛细管电泳-质谱联用技术及其在蛋白质组学中的应用

蛋白质组学研究在生物学、精准医学等方面发挥着重要的作用。然而研究面临的巨大挑战来自生物样品的复杂性,因此在质谱（MS）鉴定技术不断革新的同时,发展分离技术以降低样品复杂度尤为重要。毛细管电泳（CE）技术具有上样体积小、分离效率高、分离速度快等优势,其与质谱的联用在蛋白质组学研究中越来越受到关注。低流速鞘流液和无鞘流液接口的发展及商品化推动了CE-MS技术的发展。目前毛细管区带电泳（CZE）、毛细管等电聚焦（CIEF）、毛细管电色谱（CEC）等分离模式已与质谱联用,其中CZE-MS应用最广泛。目前被广泛采用的蛋白质组学研究策略主要是基于酶解肽段分离鉴定的"自下而上（bottom-up）"策略。首先,CE-MS技术对酶解肽段的检测灵敏度高达1 zmol,已成功应用于单细胞蛋白质组学;其次,毛细管电泳技术与反相液相色谱互补,为疏水性质相近的肽段（尤其是翻译后修饰肽段）的分离鉴定提供了新的途径。基于整体蛋白质分离鉴定的自上而下"top-down"策略可以直接获得更精准、更完整的蛋白质信息。CE技术在蛋白质大分子的分离方面具有分离效率高、回收率高的优势,其与质谱的联用提高了整体蛋白质的鉴定灵敏度和覆盖度。非变性质谱（native MS）是一种在近生理条件下从完整蛋白质复合物水平上进行分析的质谱技术。CE与非变性质谱联用已被尝试用于蛋白质复合体的分离鉴定。该文引用了与CE-MS和蛋白质组学应用相关的93篇文献,综述了以上介绍的CE-MS的研究进展以及在蛋白质组学分析中的应用优势,并总结和展望了其应用前景。     

CE coupling with end-column electrochemiluminescence detection for chiral separation of disopyramide

CE with electrochemiluminescence (ECL) detection technique was successfully applied for the chiral separation of a kind of class IA antiarrhythmic racemic drug. To the best of our knowledge, this is the first report of ECL detection used in chiral CE. To get better detection sensitivity and good enantioresolution at the same time, the conditions of capillary inlet and outlet buffer were systematically optimized. Unlike the traditional chiral separation method, the buffers we used in the capillary inlet and outlet differed from each other in terms of buffer pH, ionic strength, type of BGE as well as buffer composition. Under the optimum conditions, baseline enantioseparation and highly sensitive detection of the enantiomers were achieved. Wide linear relationship of each enantiomer was achieved in the range of 5 x 10(-7) to 2 x 10(-5) mol/L with relative coefficients of 0.996 and 0.997, respectively. The detection limits were estimated to be 8 x 10(-8) and 1.0 x 10(-7) mol/L (S/N = 3) for the enantiomers, respectively. In addition, a successful application of this new method to the chiral separation of the racemic drug in spiked plasma samples confirmed the validity and applicability of the chiral CE-ECL method.     

Fast speciation analysis of iodophenol compounds in river waters by capillary electrophoresis-inductively coupled plasma-mass spectrometry with off-line solid-phase microextraction

An analytical methodology for the fast separation and determination of iodophenol species in natural water samples was developed using capillary electrophoresis (CE) coupled to inductively coupled plasma-mass spectrometry (ICP-MS). Based on the element-specific and highly sensitive detection provided by ICP-MS, the methodology has been applied to the analysis of 2-iodophenol, 4-iodophenol, and 2,4,6-triiodophenol. The use of solid-phase microextraction (SPME), after proper optimization, improved the signal by a factor of 100 leading to detection limits in the sub microg.L(-1). Different desorption conditions of iodophenol compounds from the SPME microfiber were studied to achieve the optimum preconcentration factor and best analytical performance. Different CE conditions were studied to achieve complete baseline separation of iodophenols in short migration times. Three different CE buffer systems were evaluated using ICP-MS detection. A buffer solution containing 20 mmol.L(-1) 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) and an applied potential of +22 kV were finally selected leading to a maximum separation time of 6.6 min. A relative standard deviation (%RSD) of about 5.0% for ten consecutive determinations was obtained. Finally, the speciation methodology developed was utilized for the determination of iodophenol compounds in natural water samples.     

Selective enrichment of ochratoxin A using human serum albumin bound magnetic beads as the concentrating probes for capillary electrophoresis/electrospray ionization-mass spectrometric analysis

Ochratoxin A (OTA) is a toxicant commonly present in many food products. Conventionally, immuno-affinity analysis is applied to rapidly screen the presence of OTA in food. However, antibodies are expensive. In this study, we present a new approach for selectively enriching OTA from aqueous samples using human serum albumin (HSA) bound magnetic beads as the affinity probes, followed by the analysis of CE/ESI-MS. In addition to demonstrating the feasibility of using the affinity probes to concentrate OTA, we also propose a rapid concentration and elution method for extraction, that is, OTA are extracted from aqueous samples by pipetting the samples in and out of a sample vial for 1 min followed by elution with pipetting for another minute. On the basis of the magnetic property, the affinity magnetic probe-target species could be rapidly isolated from the solution during the process of extraction and elution by magnetic separation. CE/ESI-MS, coupled by the electrodeless/sheathless interface, is used for the analysis of the samples. As this method features speed and cost-effectiveness, it is suitable for the purpose of rapid screening. In fact, the lowest detection limit for OTA is approximately 4 x 10(-3) mg/L.