High-speed Capillary Electrophoresis Research Articles

Nucleic acid strand displacement for indirect determination of foodborne bacteria by capillary electrophoresis and its application in antagonism and bacteriostasis studies.

Foodborne bacteria threaten human's health. Capillary electrophoresis (CE) is a powerful separation means for the determination of bacteria. Direct separation of bacteria suffers from the shortages of low resolution, channel adsorption, and bacterial aggregation. In this work, a method of nucleic acid strand displacement was developed to indirect separate the bacteria by CE. DNA complexes, consisting of probes and aptamers, were mixed with the three bacteria Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The aptamers could specifically bond with bacteria and release the probes. Through the separation of the probes, the bacteria could be indirectly determined by CE. This method avoided the shortages of direct separation of bacteria. Under the optimized conditions, the three probes for the bacteria could be separated and detected within 2.5min by high-speed CE with laser-induced fluorescence detection. The limits of detection for the bacteria were in the range 4.20×106 to 1.75×107 CFU/mL. Finally, the developed method was applied on the study of antagonism of the coexistent bacteria to reveal the relationship between them. Furthermore, the efficiency of bacteriostasis of three traditional Chinese medicines, Coptis chinensis, Schisandra chinensis, and honeysuckle, was also studied by this method.

3D printed cartridge for high-speed capillary electrophoresis with sheath liquid thermostatting and contactless conductivity detection

The high-speed capillary electrophoresis (HSCE) method is a technique that utilizes a high electric field strength applied through a short capillary to reduce the time required for sample separation. However, the increased electric field strength may result in pronounced Joule heating effects. To address this, we describe a 3D-printed cartridge with integrated contactless conductivity detection (C4D) head and a sheath liquid channel. The C4D electrodes and Faraday shield layers are fabricated by casting Wood's metal in chambers inside the cartridge. Effective thermostatting of the short capillary is achieved by flowing Fluorinert liquid, which provides better heat dissipation compared to airflow. A HSCE device is created by using the cartridge and a modified slotted-vial array sample-introduction approach. Analytes are introduced through electrokinetic injection. With the help of sheath liquid thermostatting, background electrolyte concentration can be increased to several hundred mM, resulting in improved sample stacking and peak resolutions. Additionally, the baseline signal is flattened. Typical cations such as NH4+, K+, Na+, Mg2+, Li+, and Ca2+ can be separated within 22 s with an applied field strength of 1200 V/cm. The limit of detection ranges from 2.5 to 4.6 μM with a relative standard deviation of migration times of 1.1–1.2% (n = 17). The method has been applied to detect cations in drinking water and black tea leaching for drink safety testing, and to identify explosive anions in paper swabs. Samples can be directly injected without the need for dilution.

Dual detection high-speed capillary electrophoresis for simultaneous serum protein analysis and immunoassays

Serum protein electrophoresis (SPE) separates serum proteins into bands whose shape and amplitude can alert clinicians to a range of disorders. This is followed by more specific immunoassays to quantify important antigens and confirm a diagnosis. Here we develop a high-speed capillary electrophoresis (HSCE) platform capable of simultaneous SPE and immunoassay measurements. A single laser excitation source is focused into the detection zone of the capillary to measure both refractive index (SPE) and fluorescence signals (immunoassays). The refractive index signal measures characteristic SPE profiles for human serum separated in 100 mM boric acid (pH 10), 100 mM arginine (pH 11), and 20 mM CHES (pH 10). For the immunoassay, the fluorescence electropherograms reveal that CHES provides the optimal buffer for measuring the immunocomplex and separating it from the free antigen. Immunoassays in CHES yield a LOD of 23 nM and a LOQ of 70 nM for the detection of fluorescein. The high pH reduces protein adsorption but reduces antibody affinity. Preliminary studies carried out in 50 mM barbital at pH 8 show improved stability of the immunocomplex and better separation for immunoassay quantification. Further optimization will open new capabilities for measuring orthogonal diagnostic signals in seconds with HSCE.

High-throughput monitoring of biomass conversion reaction with automatic time-resolved analysis

Reactions of biomass conversions are of great importance in fine chemistry for substantial development. While numerous studies have been performed to search for functional materials to catalyze biomass conversions, a robust and high-throughput analytical method is rather limited, which may hamper further integration and automation of the reactions. Here we propose an automatic and sequential method for the investigation of glucose conversion. By combining sequential sample injection and high-speed capillary electrophoresis (HSCE) techniques, we can monitor the glucose conversion from the beginning toward the end with a good temporal resolution. The HSCE assays are performed using short capillaries (effective length of 10 cm, i.d./o.d. of 50 μm/365 μm), and the analytes are separated at an electric field of 467 V/cm and are detected by UV-absorption at 200 nm with mixed 0.2 mM CTAB, 10 mM borate, 20 mM sorbic acid (pH 12.2) as the background electrolyte. All compounds involved in the reaction, including all products (fructose, 5-hydroxymethylfurfural, formic acid and levulinic acid) and the remaining substrate glucose, are efficiently separated and simultaneously detected from just one analysis with a temporal resolution of one minute. The method exhibits high-resolution separation, a wide linear range with limit-of-detection down to μg/mL-level, as well as excellent repeatability in sequential analysis. It is indicated that the proposed method is of great value in the analysis of complicated biomass conversion and could be potentially applied in various catalytic chemical reactions.

Study of antagonism between some intestinal bacteria with high-speed micellar electrokinetic chromatography.

In this work, high-speed micellar electrokinetic chromatography with LIF detection was applied to study the antagonism between three intestinal bacteria, Escherichia coli (E. coli), Bacillus licheniformis (B. licheniformis) and Bacillus subtilis (B. subtilis). The fluorescent derivatization for the bacteria was performed by labeling the bacteria with FITC. In a high-speed capillary electrophoresis (HSCE) device, the three bacteria could be completely separated within 4min under the separation mode MEKC. The BGE was 1 × TBE containing 30mM SDS and 1.5×10-5 g/mL polyethylene oxide. The limits of detection for E. coli, B. licheniformis and B. subtilis were 2.80×106 CFU/mL, 1.60×106 CFU/mL and 1.90×106 CFU/mL respectively. Lastly, the method was applied to investigate the antagonism between the three bacteria. The bacteria were mixed and cultured for 7 days. The samples were separated and determined every day to study the interaction between bacteria. The results showed that B. licheniformis and B. subtilis could not inhibit each other, but they could effectively inhibit the reproduction of E. coli. The method developed in this work was quick, sensitive and convenient, and it had great potential in the application of antagonism study for bacteria.

Direct detection of inorganic ions and underivatized amino acids in seconds using high-speed capillary electrophoresis coupled with back-scatter interferometry.

High speed capillary electrophoresis (HSCE) combined with refractive index (RI) detection is developed for the rapid separation and detection of inorganic ions and amino acids. A mixture of three inorganic ions (K+, Na+, Li+) and eight amino acids (Lys, Arg, Ala, Gly, Val, Thr, Trp, Asp) are detected using back scatter interferometry (BSI), without the need for chemical modifications or contrast. A thin-walled separation capillary (50 μm i.d. by 80 μm o.d.) helps mitigate Joule heating at the high field strengths required for rapid separations. This, combined with a short 8 cm length-to-detector (10 cm total length), enables separations on the seconds time scale. Using a background electrolyte (BGE) of 4 M acetic acid (pH 1.6) and a field strength of 900 V cm-1, all 11 analytes are separated in less than 40 s. Moreover, peaks in the BSI signal arising from the sample injection and EOF, enable electrophoretic mobilities to readily be obtained from apparent mobilities. This leads to excellent repeatability, with analyte electrophoretic mobilities varying from 0.39 to 1.56 % RSD over eight consecutive separations. The universal detection of inorganic ions and amino acids without prior chemical modification or additives in the BGE is an advantage of refractive index detection. A disadvantage arises from modest detection limits. Here, however, we show that submicromolar detection is possible with careful thermostatting of the thin separation capillary. A series of electropherograms are used to quantify arginine concentrations from 700 nM to 500 μM, using 50 μM Li+ as an internal standard. The resulting calibration curve leads to a calculated LOD of 376 nM and a LOQ of 1.76 μM. Diagnostically relevant amino acid panels are also separated, illustrating the potential for future applications in neurodegenerative and metabolic disease diagnostics. HSCE combined with BSI detection, therefore, is shown to be a rapid, sensitive, and universal approach for analyzing sample mixtures.

Portable and automated analyzer for rapid and high precision in vitro dissolution of drugs

We developed a novel portable and automated dissolution test analyzer for rapid and high precision in vitro dissolution testing of drugs. The analyzer consists of a flow-through-cell drug dissolution system, an automated sequential sampling system, a high-speed capillary electrophoresis (HSCE) system, and a data acquisition system. Combining the high-temporal resolution flow-gating sampling approach with HSCE, which has outstanding advantages of efficient separation and resolution, the analyzer can achieve rapid analysis and exhibits the ability in miniaturization for on-site assessment of different active pharmaceutical ingredients. To integrate the flow-through-cell dissolution system with HSCE, a specially designed flow-gating-injection (FGI) interface was employed. The performance of the analyzer was investigated by analyzing the dissolution of immediate-release drugs including single dose (amoxicillin dispersible tablets) and fixed dose combination (amoxicillin and clavulanate potassium) drug tablets with the high-temporal resolutions of 12 s and 20 s, respectively. The dissolution profiles of different active pharmaceutical ingredients could be simultaneously and automatically monitored with high repeatability and accuracy. The analyzer was successfully utilized for the pharmaceutical quality control and bio-relevant dissolution testing, as well as in vivo-in vitro correlation analysis. Our portable analyzer is miniaturized, convenient and of low-cost, and will provide a valuable tool for dissolution testing in pharmaceutical research and development.

High-Speed Capillary Electrophoresis Using a Thin-Wall Fused-Silica Capillary Combined with Backscatter Interferometry.

High-speed capillary electrophoresis (HSCE) is implemented using a 10 cm total length fused-silica capillary (50 μm i.d., 80 μm o.d.) combined with refractive index (RI) detection using backscatter interferometry (BSI). The short capillary length reduces analysis time while the ultrathin wall (15 μm) efficiently dissipates heat from the separation channel, mitigating the deleterious effects of Joule heating. The separation capillary is mounted on a temperature-controlled heat sink that stabilizes the temperature to ±0.004 °C. This temperature stabilization improves separation efficiency and enhances RI detection. Ohm's Law plots confirm the superior heat dissipation of the HSCE capillary compared to a similarly prepared conventional CE capillary (50 μm i.d., 363 μm o.d.). The speed and efficiency of HSCE combined with universal RI detection is illustrated through the separation of K+, Ba2+, Mg2+, Na+, Li+, and Tris+ in approximately 30 s, with efficiencies greater than 500 000 plates/m. Run-to-run repeatability is explored using nine consecutive electrokinetic injections of a K+, Na+, and Li+ mixture. The average migration times and %RSD for K+, Na+, and Li+ were measured to be 22.04 s (1.59%), 26.81 s (1.38%), and 29.80 s (2.21%), respectively. Finally, we show that the BSI signal is sensitive to the separation voltage through the Kerr mechanism. This leads to peaks in the electropherogram from the injection process that are useful for precisely defining the start of each separation and quantifying the amount of sample injected onto the capillary.

Automatic Dissolution Testing with High-Temporal Resolution for Both Immediate-Release and Fixed-Combination Drug Tablets

Dissolution testing plays many important roles throughout the pharmaceutical industry, from the research and development of drug products to the control and evaluation of drug quality. However, it is a challenging task to perform both high-efficient separation and high-temporal detection to achieve accurate dissolution profile of each active ingredient dissolved from a drug tablet. In our study, we report a novel non-manual-operation method for performing the automatic dissolution testing of drug tablets, by combining a program-controlled sequential analysis and high-speed capillary electrophoresis for efficient separation of active ingredients. The feasibility of the method for dissolution testing of real drug tablets as well as the performance of the proposed system has been demonstrated. The accuracy of drug dissolution testing is ensured by the excellent repeatability of the sequential analysis, as well as the similarity of the evaluation of dissolution testing. Our study show that the proposed method is capable to achieve simultaneous dissolution testing of multiple ingredients, and the matrix interferences can be avoided. Therefore it is of potential valuable applications in various fields of pharmaceutical research and drug regulation.

Simultaneous Analysis for the Multiple Periodontal Pathogens by High-Speed Capillary Electrophoresis

In the present study, one important branch of high-speed capillary electrophoresis (HSCE), short-end CE, was employed for the highly efficient analysis of Porphyromonas gingivalis (Pg), Treponema denticola (Td), and Tannerella forsythia (Tf), which are closely related to the occurrence of periodontal disease. Aiming to realize rapid and simultaneous detection, a 100 bp DNA ladder marker ranging from 100 to 1500 bp was employed as the standard for the optimization of polymer concentration and electric field strength in developed protocol. The results showed that the PCR (polymerase chain reaction) products of Pg (197 bp), Td (311 bp), and Tf (641 bp) were simultaneously identified and quantitated in approximately 90 s using electrophoretic conditions that included 0.4% hydroxyethylcellulose (HEC, 1300 K) and a 200 V/cm electric field with an effective length of separation equal to 2.5 cm. The detected concentrations of the above target samples, determined by a time-modified internal standard route, were 1.08, 1.60, and 2.46 ng/µl, respectively. The present short-end CE strategy featured favorable properties for reproducibility, accuracy and linearity, whose evaluation included the values of relative standard deviation and the correlation coefficient. The current work may provide an effective avenue for the clinical detection of periodontal disease.

Informatics analysis of capillary electropherograms of autologously doped and undoped blood

Detection of autologous blood doping using capillary electrophoresis and inverse least squares.

Separation and determination of microRNAs by high-speed capillary sieving electrophoresis.

In this work, high-speed capillary sieving electrophoresis with laser-induced fluorescence detection was applied to simultaneously determine three microRNAs. A developed manual sample introduction device for the high-speed capillary electrophoresis system was applied to perform sample injection. Strategies, including field-amplified sample injection and electrokinetic injection, were studied to improve the detection sensitivity. Under the optimal conditions, the limit of detection for DNA-159 could be lowered to 5.10×10-12 mol/L. In order to achieve enough separation resolution, two DNA probes were designed to have extra sequences that acted as the drag tails. Under the optimized conditions, the three DNA probes and the complexes of microRNA-156, microRNA-159, and microRNA-166 could be completely separated within 3.2min in backgroundelectrolyte (pH 8.7) containing 2.0% m/m polyvinyl pyrrolidone and 0.4%m/m hydroxyethyl cellulose. The limits of detection for the three microRNAs were 0.051, 0.11, and 0.25nmol/L, respectively. Then the method was applied to analyze the microRNAs spiked in the samples extracted from banana leaves. The recoveries ranged from 114.3 to 121.1% (n=3). The results showed that the method developed in this work was an effective means for microRNA assay.

A Low-Cost Palmtop High-Speed Capillary Electrophoresis Bioanalyzer with Laser Induced Fluorescence Detection

In this work, we developed a miniaturized palmtop high-speed capillary electrophoresis (CE) system integrating whole modules, including picoliter-scale sample injection, short capillary-based fast CE, high-voltage power supply, orthogonal laser induced fluorescence (LIF) detection, battery, system control, on-line data acquisition, processing, storage, and display modules. A strategy of minimalist miniaturization combining minimal system design and low-cost system construction was adopted to achieve the instrument miniaturization with extremely low cost, which is differing from the current microfabrication strategy used in most reported miniaturized CE systems. With such a strategy, the total size of the bioanalyzer was minimized to 90 × 75 × 77 mm (length × width × height) and the instrument cost was reduced to ca. $500, which demonstrated the smallest and lowest-cost CE instrument with LIF detection in so far reported systems. The present bioanalyzer also exhibited comparable analytical performances to previously-reported high-speed CE systems. A limit of detection of 1.02 nM sodium fluorescein was obtained. Fast separations were achieved for multiple types of samples as amino acids, amino acid enantiomers, DNA fragments, and proteins with high efficiency. We applied this instrument in colorectal cancer diagnosis for detecting KRAS mutation status by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method.

Rapid identification and quantitation for oral bacteria based on short-end capillary electrophoresis

High-speed capillary electrophoresis (HSCE) is a promising technology applied in ultra-rapid and high-performance analysis of biomolecules (such as nucleic acids, protein). In present study, the short-end capillary electrophoresis coupled with one novel space domain internal standard method (SDIS) was employed for the rapid and simultaneous analysis of specific genes from three oral bacteria (Porphyromonas gingivalis (P.g), Treponema denticola (T.d) and Tannerela forsythia (T.f)). The reliability, reproducibility and accuracy properties of above mentioned SDIS method were investigated in detail. The results showed the target gene fragments of P.g, T.d and T.f could be precisely, fast identified and quantitated within 95s via present short-end CE system. The analyte concentration and the ratio of space domain signals (between target sample and internal standard sample) featured a well linear relationship calculated via SDIS method. And the correlation coefficients R2 and detection limits for P.g, T.d, T.f genes were 0.9855, 0.9896, 0.9969 and 0.077, 0.114 and 0.098ng/μl, respectively.

Automatic Combination of Microfluidic Nanoliter-Scale Droplet Array with High-Speed Capillary Electrophoresis.

In this paper, we developed a novel approach for interfacing a microfluidic two-dimensional droplet array to a high-speed capillary electrophoresis (HSCE) system. Picoliter-scale sample injection (ca. 200 pL) from a nanoliter-scale droplet array covered by nonvolatile oil was automatically achieved using the spontaneous injection mode, without the interference from the cover oil and the need of special droplet extraction interface as in previously reported systems. The system was applied in consecutive separations of 25 different samples of amino acids with a whole separation time less than 15 min, as well as on-line monitoring of in-droplet derivatizing reaction of amino acids by fluorescein isothiocyanate (FITC) over 3 hours. High separation speed (up to 100 samples per hour) and high separation efficiency (up to 9.22 × 105 N/m) were achieved.

Combination of poly(diallyldimethylammonium chloride) and hydroxypropyl-γ-cyclodextrin for high-speed enantioseparation of phenothiazines by capillary electrophoresis

High-speed capillary electrophoresis (CE) enables the simple, rapid, and inexpensive analysis of large sets of chiral samples in the pharmaceutical industry. Hence, we developed a novel method for separating enantiomers of d,l-phenothiazines simply and rapidly, based on using poly(diallyldimethylammonium chloride) (PDDAC) as an additive and hydroxypropyl-γ-cyclodextrin (Hp-γ-CD) as a chiral selector in capillary electrophoresis. Adding 0.9% PDDAC to the background electrolyte generated a stable, high, and reversed electroosmotic flow (EOF). Hp-γ-CD not only worked as a complexing agent to increase the chiral resolution between d,l-phenothiazines but also decreased the effective electrophoretic mobility of these drugs. Combining PDDAC and Hp-γ-CD as buffer additives enabled CE to achieve a high-speed enantioseparation of five pairs of d,l-phenothiazines. A decrease in capillary length and an increase in the intensity of the electric field further shortened the separation time. When the background electrolyte contained 0.9% PDDAC, 5mM Hp-γ-CD, and 75mM formic acid (pH 3.0), enantioseparation of the d,l-phenothiazines was attained within 230s by applying a capillary length of 32.5cm and an electric field of 292Vcm−1. The limit of detection (LOD) of the d,l-phenothiazines at a signal-to-noise ratio of 3 ranged from 2 to 8μM. We demonstrated the feasibility of this method by detecting the five pairs of d,l-phenothiazines in urine samples.

Monitoring Neurochemical Release from Astrocytes Using in Vitro Microdialysis Coupled with High-Speed Capillary Electrophoresis

We have developed a novel in vitro approach for monitoring fast neurochemical dynamics in model cell systems using microdialysis sampling coupled with high-speed capillary electrophoresis (CE). Cells from an immortalized astrocyte line (C8-D1A) were cultured in direct contact with the porous membrane of a microdialysis probe. Confocal microscopy was used to confirm cell viability and confluency over the microdialysis sampling region. Small molecules released from the astrocytes were efficiently sampled by the probe due to the direct contact with the membrane. Microdialysis sampling was coupled with online, high-speed CE allowing changes in the dialysate concentration of small molecule amine neurochemicals to be monitored with 20 s temporal resolution. Basal release of a number of important analytes was detected including glycine, taurine, D-serine, and glutamate. The ability of the in vitro microdialysis-CE instrument to monitor dynamic changes in analyte concentration was assessed by transferring a probe cultured with astrocytes from a solution containing artificial cerebrospinal fluid (aCSF) to a high K(+) solution (100 mM K(+)-aCSF). Upon stimulation, the observed concentration of a number of key neurochemicals increased dramatically including glycine (700%), taurine (185%), and serine (215%). Amino acids such as phenylalanine and valine, which are not known to respond to cellular swelling mechanisms, were unaffected by the K(+) stimulation.

Progress of Short Capillary-based High Speed Capillary Electrophoresis Systems

An overview is presented on the development of short capillary-based high speed capillary electrophoresis systems.The progress and application of various sample injection approaches suitable for high-speed capillary electrophoresis are mainly introduced,including optical-gating injection,flow-gating injection,electrokinetic injection,spontaneous injection,hydrodynamic injection and diffusion injection.

Microfluidic Picoliter-Scale Translational Spontaneous Sample Introduction for High-Speed Capillary Electrophoresis

A novel microfluidic picoliter-scale sample introduction approach was developed by combining the spontaneous injection approach with a capillary electrophoresis (CE) system based on a short capillary and slotted-vial array. A droplet splitting phenomenon at the capillary inlet end during the spontaneous sample introduction process was observed for the first time. On the basis of this phenomenon, a translational spontaneous injection approach was established to reduce sample injection volumes to the sub-100 pL range. A versatile high-speed capillary electrophoresis (HSCE) system was built on the basis of this sample injection approach with separation performance comparable to or even better than those reported in microfluidic chip-based CE systems. The HSCE system was composed of a short fused-silica capillary and an automated sample introduction system with slotted sample and buffer reservoirs and a computer-programmed translational stage. The translational spontaneous sample injection was performed by linearly moving the stage, allowing the capillary inlet first to enter the sample solution and then removing it. A droplet was left at the tip end and spontaneously drawn into the capillary by surface tension effect to achieve sample injection. The stage was continuously moved to allow the capillary inlet to be immersed into the buffer solution, and CE separation was performed by applying a high voltage between the buffer and waste reservoirs. With the use of the novel system, high-speed and efficient capillary zone electrophoresis (CZE) separation of a mixture of five fluorescein isothiocyanate (FITC) labeled amino acids was achieved within 5.4 s in a short capillary with a separation length of 15 mm, reaching separation efficiencies up to 0.40 microm plate height. Outstanding peak height precisions ranging from 1.2% to 3.7% RSD were achieved in 51 consecutive separations. By extension of the separation length to 50 mm, both high-speed and high-resolution CZE separation of eight FITC-labeled amino acids could be obtained in less than 21 s with theoretical plates ranging from 163,000 to 251,000 (corresponding to 0.31-0.20 microm plate heights). The present HSCE system also allowed fast chiral separations of FITC-labeled amino acids under micellar electrokinetic chromatography (MEKC) mode within 6.5 s.

Multiplexed Detection of Protein−Peptide Interaction and Inhibition Using Capillary Electrophoresis

High-speed capillary electrophoresis (CE) was employed to detect binding and inhibition of SH2 domain proteins using fluorescently labeled phosphopeptides as affinity probes. Single SH2 protein-phosphopeptide complexes were detected and confirmed by competition and fluorescence anisotropy. The assay was then extended to a multiplexed system involving separation of three SH2 domain proteins: Src, SH2-Bbeta, and Fyn. The selectivity of the separation was improved by altering the charge of the peptide binding partners used, thus demonstrating a convenient way to control resolution for the multiplexed assay. The separation was completed within 6 s, allowing rapidly dissociating complexes to be detected. Two low molecular weight inhibitors were tested for inhibition selectivity and efficacy. One inhibitor interrupted binding interaction of all three proteins, while the other selectively inhibited Src only leaving SH2-Bbeta and Fyn complex barely affected. IC(50) of both selective and nonselective inhibitors were determined and compared for different proteins. The IC(50) of the nonselective inhibitor was 49 +/- 9, 323 +/- 42, and 228 +/- 19 microM (n = 3) for Src, SH2-Bbeta, and Fyn, respectively, indicating different efficacy of the nonselective inhibitor for different SH2 domain protein. It is concluded that high-speed CE has the potential for multiplexed screening of drugs that disrupt protein-protein interactions.