Analysis of angiotension-converting enzyme by capillary electrophoresis.

A method is described for determination of serum angiotension-converting enzyme by capillary electrophoresis (CE) based on incubation of the substrate, a synthetic peptide, with the serum outside the capillary and cleaving hippuric acid and a dipeptide. The reaction is stopped by the addition of acetonitrile, followed by injection of the supernatant on the capillary. The acetonitrile allows injection of a large volume of sample on the capillary. Both the substrate and the reaction product (hippuric acid) can be monitored at the same time. The CE step is rapid and can be performed in about 6 min. The CE method compared well to a kinetic assay method (= 0.98).     

Selective genotyping of individual cells by capillary polymerase chain reaction

On-line capillary polymerase chain reaction (PCR) coupled with laser-induced fluorescence detection was successfully demonstrated for individual human cells. A single 50 num inner diameter (ID) fused-silica capillary served both as the reaction vessel and for isolating single cells. SYBR Green I dye was added into the reaction mixture for dynamic fluorescence labeling. Because of the small ID of the capillary, PCR-amplified DNA fragments from single cells were localized in the capillary, providing discrete product zones with concentrations at readily detectable levels. With selective primer design, only cells containing the DNA of interest were amplified. By counting the number of peaks in the capillary via electromigration past a detection window, the number of targeted cell templates could be determined. Identification of the 295 bp fragment beta-actin gene from individual human lymphoblast cell was demonstrated. Independent on-column cell counting provided positive correlation between the starting cell templates and the final PCR products. This opens up the possibility of highly selective and sensitive disease diagnosis at an early stage, when only a few cells in the population are defective.     

Miniaturized movable contactless conductivity detection cell for capillary electrophoresis

A miniaturized capacitively coupled contactless conductivity detector (mini-C(4)D) cell has been designed which is small enough to allow it to slide along the effective capillary length inside the capillary cassette of an Agilent capiillary electrophoresis system (CE) (or other CE brand of similar construction), including the possibility of positioning it close to the point of optical detection (4 cm), or even putting two such detector cells in one cassette. The cell was tested and the performance characteristics (noise, sensitivity, and peak width) were compared with those obtained with the previously used large C(4)D cell. No significant differences were observed. The mini-C(4)D was used in simultaneous separations of common cations and anions where its advantage over a larger C(4)D cell is the ability to vary the point of detection with the mini-C(4)D cell continuously at any point along the capillary length, so that the optimum apparent selectivity can be chosen. Other applications include providing a convenient second point of detection in addition to photometric detection, such as to measure accurately the linear velocity of a zone, or to allow placement of two mini-C(4)D cells in one capillary cassette simultaneously.     

High-throughput analysis of telomerase by capillary electrophoresis

The enzyme telomerase is expressed in (85-90)% of all human cancers, but not in normal, non-stem cell somatic tissues. Clinical assays for telomerase in easily obtained body fluids would have great utility as noninvasive, cost-effective methods for the early detection of cancer. The most commonly used method for the detection and quantification of telomerase enzyme activity is the polymerase chain reaction (PCR)-based assay known as the telomerase repeat amplification protocol or TRAP assay. Most of the TRAP assay systems use a slab-gel based electrophoresis system to size and quantify the PCR-amplified extension products. We are developing high-throughput capillary electrophoresis (CE) methods for the analysis of TRAP/PCR products. The TRAP assay was conducted on lysates of the human lung cancer cell line A-549 in reactions containing 5-100 cells. TRAP/PCR products were generated using a fluorescent 4,7,2'4'5'7',-hexachloro-6-carboxyfluorescein(HEX)-labeled TS primer and analyzed on the Applied Biosystems Model 310 CE system using POP4 polymer. After analysis with GeneScan and Genotyper software, the total peak areas of the TRAP ladder extension products were computed using Microsoft Excel. Results were compared with unlabeled TRAP/PCR products analyzed on the Bio-Rad BioFocus 3000 CE system using 6% high molecular weight polyvinylpyrrolidone (HMW PVP) polymer and SYBR Green I dye. Both CE systems were able to resolve the TRAP ladder products with high reproducibility and sensitivity (5-15 cells). With the appropriate robotic sample handling system, these CE methods would enable performing the telomerase TRAP assay with increased sensitivity, reproducibility and automation over slab-gel methods.     

Enzyme and inhibition assay of urease by continuous monitoring of the ammonium formation based on capillary electrophoresis

We present here an easy‐to‐operate and efficient method for enzyme and inhibition assays of urease, which is a widely distributed and important enzyme that catalyzes the hydrolysis of urea to ammonia and CO₂. The assay was achieved by integrating CE technique and rapid on‐line derivatization method, allowing us to continuously drive the sample to the capillary, thus to measure the amount of the product ammonia from the beginning to the end of the reaction. The method exhibits excellent repeatability with RSD as low as 2.5% for the initial reaction rate (n = 5), with the LOD of ammonia of 20 μM (S/N = 5). The enzyme activity as well as the inhibition of urease by Cu²⁺ were investigated using the present method. The results show that Cu²⁺ is a noncompetitive inhibitor on urease, in accordance with the result published in the literature. The enzyme activity and inhibition kinetic constants were obtained and were found to be consistent with the results of traditional off‐line enzyme assays. Our study indicates that the present approach is a reliable and convenient method for analysis of the urease activity and inhibition kinetics by continuous on‐line monitoring of the ammonium formation based on CE.     

Capillary electrophoretic study of the synergistic biological effects of alkaloids from Chelidonium majus L. in normal and cancer cells

In this study, the synergistic biological action of five celandine alkaloids in normal and cancer cells was investigated by capillary electrophoresis with light-emitting diode-induced native fluorescence detection. The specific capacity of each alkaloid to penetrate into the cells was estimated by monitoring alkaloid concentration decreases in the cell medium during incubation with murine fibroblast NIH/3T3, mouse melanoma B16F10, and human breast cancer MCF7 cell lines. Mixtures of isoquinoline alkaloids containing protopine, chelidonine, sanguinarine, allocryptopine, and stylopine were applied to cell cultures for 20 and 40 min, and the content of alkaloids in the cell media was measured by capillary electrophoresis (CE). CE separation of isoquinoline alkaloids was performed in 30 mM phosphate buffer (pH 2.5). As these alkaloids have native fluorescence, they were directly detected using the commercially available UV light-emitting diode without troublesome fluorescent derivatization. The results showed a differential ability of celandine alkaloids to penetrate into the normal and cancer cell interior, which was inversely proportional to their cytotoxic activity. While the most effective transport of celandine alkaloids from the cell medium to the cell interior was observed for normal murine fibroblast NIH/3T3 cells (about 55% of total content), cytotoxicity tests demonstrated selective and profound apoptotic effects of a five-alkaloid combination in the mouse melanoma B16F10 cell line.     

Electrophoretically mediated microanalysis for in-capillaryelectrical cell lysis and fast enzyme quantification by capillary electrophoresis

In this study, a novel capillary electrophoresis (CE)-based enzymatic assay was developed to evaluate enzymatic activity in whole cells. β-Galactosidase expression was used as an example, as it is a biomarker for assessing replicative senescence in mammalian cells. It catalyzes the hydrolysis of para-nitrophenyl-β-d-galactopyranoside (PNPG) into para-nitrophenol (PNP). The CE-based assay consisted of four main steps: (1) hydrodynamic injection of whole intact cells into the capillary, (2) in-capillary lysis of these cells by using pulses of electric field (electroporation), (3) in-capillary hydrolysis of PNPG by the β-galactosidase—released from the lysed cells—by the electrophoretically mediated microanalysis (EMMA) approach, and (4) on-line detection and quantification of the PNP formed. The developed method was applied to Escherichia coli as well as to human keratinocyte cells at different replicative stages. Results obtained by CE were in excellent agreement with those obtained from off-line cell lysates which proves the efficiency of the in-capillary approach developed. This work shows for the first time that cell membranes can be disrupted in-capillary by electroporation and that the released enzyme can be subsequently quantified in the same capillary. Enzyme quantification in cells after their in-capillary lysis has never been conducted by CE. The developed CE approach is automated, economic, eco-friendly, and simple to conduct. It has attractive applications in bacteria or human cells for early disease diagnostics or insights for development in biology.

Capillary electrophoresis system with flow injection sample introduction and chemiluminescence detection on a chip platform

A capillary electrophoresis (CE) system with chemiluminescence (CL) detection was combined with flow injection (FI) sample introduction on a chip platform. A falling-drop interface was applied to perform FI split-flow sample introduction while achieving electrical isolation from the CE high voltage. A tubular reservoir at the capillary outlet served as both the CL reaction and detection cell for the luminol-peroxide-metallic ion chemiluminescent reaction, with the luminol included in the separation buffer and CL reagent H2O2 continuously introduced into the outlet reservoir. An optical fiber was positioned within the outlet reservoir directly opposite, and 300 microns away from, the capillary outlet for collecting and transferring the generated CL to the PMT. The peak height signals and the separation efficiency were almost independent of the reagent flow-rate, making the system a robust one. The performance of the system was illustrated by the separation of Co(II) and Cu(II), achieving baseline separation in 60 s. Detection limits (3 sigma) were 1.25 x 10(-8) and 2.3 x 10(-6) mol dm-3 for Co(II) and Cu(II), respectively. Peak height precision was 1.9% RSD (n = 9) at the 10(-7) mol dm-3 Co level.     

Development of off-line and on-line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substrates

Fast and convenient CE assays were developed for the screening of adenosine kinase (AK) inhibitors and substrates. In the first method, the enzymatic reaction was performed in a test tube and the samples were subsequently injected into the capillary by pressure and detected by their UV absorbance at 260 nm. An MEKC method using borate buffer (pH 9.5) containing 100 mM SDS (method A) was suitable for separating alternative substrates (nucleosides). For the CE determination of AMP formed as a product of the AK reaction, a phosphate buffer (pH 7.5 or 8.5) was used and a constant current (95 microA) was applied (method B). The methods employing a fused-silica capillary and normal polarity mode provided good resolution of substrates and products of the enzymatic reaction and a short analysis time of less than 10 min. To further optimize and miniaturize the AK assays, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product employing electrophoretically mediated microanalysis (EMMA, method C). After hydrodynamic injection of a plug of reaction buffer (20 mM Tris-HCl, 0.2 mM MgCl2, pH 7.4), followed by a plug containing the enzyme, and subsequent injection of a plug of reaction buffer containing 1 mM ATP, 100 microM adenosine, and 20 microM UMP as an internal standard (I.S.), as well as various concentrations of an inhibitor, the reaction was initiated by the application of 5 kV separation voltage (negative polarity) for 0.20 min to let the plugs interpenetrate. The voltage was turned off for 5 min (zero-potential amplification) and again turned on at a constant current of -60 microA to elute the products within 7 min. The method employing a polyacrylamide-coated capillary of 20 cm effective length and reverse polarity mode provided good resolution of substrates and products. Dose-response curves and calculated K(i) values for standard antagonists obtained by CE were in excellent agreement with data obtained by the standard radioactive assay.     

Improving detection in capillary electrophoresis with laser induced fluorescence via a bubble cell capillary and laser power adjustment

Bubble cells have been frequently employed in capillary electrophoresis (CE) to increase the light path length with UV detection to provide an increase in the observed sensitivity of CE; however this approach has not been commonly used for laser-induced fluorescence detection (LIF) with CE. In this paper we study the influence of laser power on the sensitivity of detection in using conventional and enlarged fused silica capillaries for CE with LIF. When using the bubble cell capillary, the laser power must be decreased relative to use of the conventional capillary to reduce the effects of photodegradation of the species being illuminated by the laser. Even though the light intensity was decreased, an increase in sensitivity of detection was observed for most compounds when a bubble cell was used. This increase ranged from a factor of 8 for riboflavin (410 nm excitation) to 3.2 for most aromatic compounds (266 nm excitation), when using a 3x bubble cell compared with a conventional capillary. The bubble cell capillary was used for native detection of IgG by LIF at 266 nm. A limit of detection of 60 ng mL(-1) was obtained from a 20 pg injection, which was 40 times more sensitive than silver staining in conventional SDS/PAGE.     

Chitosan Membranes Filled with Cyclosporine A as Possible Devices for Local Administration of Drugs in the Treatment of Breast Cancer

The aim of this work is the design, preparation and characterization of membranes based on cyclosporine A (CsA) and chitosan carboxylate (CC) to be used as an implantable subcutaneous medical device for a prolonged therapeutic effect in the treatment of breast cancer. The choice to use CsA is due to literature data that have demonstrated its possible antitumor activity on different types of neoplastic cells. To this end, CsA was bound to CC through an amidation reaction to obtain a prodrug to be dispersed in a chitosan-based polymeric membrane. The reaction intermediates and the final product were characterized by Fourier transform infrared spectroscopy (FT-IR) and proton nuclear magnetic resonance (¹H-NMR). Membranes were analyzed by differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). The data obtained showed the effective formation of the amide bond between CsA and CC and the complete dispersion of CsA inside the polymeric membrane. Furthermore, preliminary tests, conducted on MDA-MB-231, a type of breast cancer cell line, have shown a high reduction in the proliferation of cancer cells. These results indicate the possibility of using the obtained membranes as an interesting strategy for the release of cyclosporin-A in breast cancer patients.     

Cell cycle-dependent characterization of single MCF-7 breast cancer cells by 2-D CE

The composition of single MCF-7 breast cancer cells is characterized using 2-D CE. Individual MCF-7 cells were aspirated into a 30 mum inner diameter fused-silica capillary and lysed by contact with an SDS-containing buffer. Proteins and other primary amines were fluorescently labeled on-column using the fluorogenic dye 3-(2-furoyl)quinoline-2-carboxaldehyde. Labeled components were separated first according to molecular weight using capillary sieving electrophoresis (CSE) and then by MEKC. Analytes were detected in a sheath-flow cuvette using LIF. The expression profiles for MCF-7 cellular homogenate and a single MCF-7 cell are compared. As a proof-of-principle investigation, variation in expression was also compared within and between G1 and G2/M cell cycle phases for MCF-7 cells. Following their treatment with the viable nuclear stain Hoechst 33342, MCF-7 cells were sorted by flow cytometry on the basis of their ploidy. Sorted cells were then analyzed by 2-D CE. The degree of variability was >2.5 times larger between cells of different phases than between cells of the same phase. In typical 1 h 2-D CE separations using MCF-7 cells, over 100 components are resolved.     

High temporal resolution monitoring of enzyme reaction and inhibition using optically gated vacancy capillary electrophoresis and immobilized enzyme

A novel method for monitoring of enzyme reaction and inhibition with high temporal resolution was developed by using optically gated vacancy capillary electrophoresis (OGVCE) with laser-induced fluorescence (LIF) detection and immobilized enzyme. Trypsin cleavage reaction and inhibition were investigated by the presented OGVCE-LIF assay, using carboxyfluorescein (FAM) end-labeled Angiotensin as the substrate and commercially available immobilized trypsin. The substrate and the product were continuously loaded into the capillary by the electroosmotic flow while the immobilized enzyme remained in the sample vial. Substrate consumption and product formation were monitored simultaneously at 5 s interval during the whole reaction time. The enzymatic reaction rates obtained from the substrate and the product were highly consistent. The enzyme activity and the Michaelis constants of trypsin cleavage reaction, as well as the inhibition constant (for reversible competitive inhibitor) and the inhibition fraction (for irreversible inhibitor), were obtained. It was showed that the reported OGVCE-LIF method can perform fast, accurate, sensitive and reproducible CE enzyme assay with high temporal resolution, thus has great potential in application of the enzyme-substrate systems with fast reaction rate and the fluorescent substrate and products.     

Sensitive detection of tumor marker CA15-3 in human serum by capillary electrophoretic immunoassay with chemiluminescence detection

A new and sensitive non-competitive immunoassay (IA) for tumor marker carbohydrate antigen 15-3 (CA15-3) by CE coupling with ECL detection has been developed. This method is based on luminol-H(2)O(2 )reaction catalyzed by horseradish peroxidase (HRP). The optimum CE separation and CL detection conditions were investigated. After the non-competitive immunoreaction, the free HRP-labeled CA15-3 antibody (Ab*) and the bound Ab*-antigen (Ab*-Ag) complex were separated in a separation capillary and then catalyzed the CL reaction of luminol and H(2)O(2 )in a reaction capillary following the separation capillary. The calibration curve based on the peak areas of Ab*-Ag complex plotted against the concentrations of CA15-3 is in the range of 0-250 U/mL with a correlation coefficient of 0.9983 and the detection limit is 0.035 U/mL (S/N = 3). The response for five consecutive injections of 125 U/mL CA15-3 resulted in RSDs of 0.83% and 3.1% for the migration time and the peak area, respectively. The method was successfully used for the quantification of CA15-3 in human sera obtained from healthy persons and from patients with breast cancer.     

Sequential capillary electrophoresis analysis using optically gated sample injection and UV/vis detection

We present sequential CE analysis of amino acids and l ‐asparaginase‐catalyzed enzyme reaction, by combing the on‐line derivatization, optically gated (OG) injection and commercial‐available UV‐Vis detection. Various experimental conditions for sequential OG‐UV/vis CE analysis were investigated and optimized by analyzing a standard mixture of amino acids. High reproducibility of the sequential CE analysis was demonstrated with RSD values (n = 20) of 2.23, 2.57, and 0.70% for peak heights, peak areas, and migration times, respectively, and the LOD of 5.0 μM (for asparagine) and 2.0 μM (for aspartic acid) were obtained. With the application of the OG‐UV/vis CE analysis, sequential online CE enzyme assay of l ‐asparaginase‐catalyzed enzyme reaction was carried out by automatically and continuously monitoring the substrate consumption and the product formation every 12 s from the beginning to the end of the reaction. The Michaelis constants for the reaction were obtained and were found to be in good agreement with the results of traditional off‐line enzyme assays. The study demonstrated the feasibility and reliability of integrating the OG injection with UV/vis detection for sequential online CE analysis, which could be of potential value for online monitoring various chemical reaction and bioprocesses.     

CE combined with rolling circle amplification for sensitive DNA detection

Here we describe an assay which combines CE with rolling circle amplification (RCA) for sensitive DNA detection and quantification. RCA is an isothermal DNA replication technique that generates a long ssDNA with tandem repeats. It requires simpler temperature control in reaction and offers higher sequence specificity and greater quantitation capability compared to other amplification technologies. In this study, RCA amplified the DNA target via a circular template, and the product was digested into monomers for CE analysis. Less than 2 fmol of the DNA target could easily be detected using this RCA-CE assay and the assay has a dynamic range of two orders of magnitudes. Moreover, simultaneous detection of both the target DNA and the internal standard was achieved by designing two padlock probes with different sizes, which could significantly improve the quantification accuracy. The RCA-CE assay is easy to perform, readily adaptable for detection of multiple targets because of the high resolution power of CE, and is compatible with other applications employing RCA as a signal amplification tool. Additionally, this assay can be used with a capillary array system to perform sensitive, high-throughput genetic screening.     

Colorimetric quantification of mRNA expression in rare tumour cells amplified by multiple ligation-dependent probe amplification

An enzyme-linked oligonucleotide assay (ELONA) for quantification of mRNA expression of five genes involved in breast cancer, extracted from isolated rare tumour cells and amplified by multiplex ligation-dependent probe amplification (MLPA) is presented. In MLPA, a multiplex oligonucleotide ligation assay is combined with a PCR reaction in which all ligation products are amplified by use of a single primer pair. Biotinylated probes complementary to each of the target sequences were immobilised on the surface of a streptavidin-coated microtitre plate and exposed to single-stranded MLPA products. A universal reporting probe sequence modified with horseradish peroxidase (URP–HRP) and complementary to a universal primer used during the MLPA step was further added to the surface-bound duplex as a reporter probe. Simultaneous addition of anchoring probe and target, followed by addition of reporter probe, rather than sequential addition, was achieved with no significant effect on sensitivity and limits of detection, but considerably reduced the required assay time. Detection limits as low as 20 pmol L⁻¹, with an overall assay time of 95 min could be achieved with negligible cross-reactivity between probes and non-specific targets present in the MLPA-PCR product. The same MLPA-PCR product was analysed using capillary electrophoresis, the technique typically used for analysis of MLPA products, and good correlation was observed. The assay presented is easy to carry out, relatively inexpensive, rapid, does not require sophisticated instrumentation, and enables quantitative analysis, making it very promising for the analysis of MLPA products.     

Sensitive determination of sulfonamides in environmental water by capillary electrophoresis coupled with both silvering detection window and in‐capillary optical fiber light‐emitting diode‐induced fluorescence detector

A new detector, silvering detection window and in‐capillary optical fiber light‐emitting diode‐induced fluorescence detector (SDW‐ICOF‐LED‐IFD), is introduced for capillary electrophoresis (CE). The strategy of the work was that half surface of the detection window was coated with silver mirror, which could reflect the undetected fluorescence to the photomultiplier tube to be detected, consequently enhancing the detection sensitivity. Sulfonamides (SAs) are important antibiotics that achieved great applications in many fields. However, they pose a serious threat on the environment and human health when they enter into the environment. The SDW‐ICOF‐LED‐IFD‐CE system was used to determine fluorescein isothiocyanate (FITC)‐labeled sulfadoxine (SDM), sulfaguanidine (SGD) and sulfamonomethoxine sodium (SMM‐Na) in environmental water. The detection results obtained by the SDW‐ICOF‐LED‐IFD‐CE system were compared to those acquired by the CE with in‐capillary optical fiber light‐emitting diode‐induced fluorescence detection (ICOF‐LED‐IFD‐CE). The limits of detection (LODs) of SDW‐ICOF‐LED‐IFD‐CE and ICOF‐LED‐IFD‐CE were 1.0–2.0 nM and 2.5–7.7 nM (S/N = 3), respectively. The intraday (n = 6) and interday (n = 6) precision of migration time and corresponding peak area for both types of CE were all less than 0.86% and 3.68%, respectively. The accuracy of the proposed method was judged by employing standard addition method, and recoveries obtained were in the range of 92.5–102.9%. The results indicated that the sensitivity of the SDW‐ICOF‐LED‐IFD‐CE system was improved, and that its reproducibility and accuracy were satisfactory. It was successfully applied to analyze SAs in environmental water.     

Application of a capillary‐assembled microfluidic system for separation of cephalosporins

This paper demonstrates a simple and easy setting up of a fused‐silica capillary‐assembled microfluidic system (μCE). This system incorporates a split‐flow pressure injection of the sample into a microfluidic system made from PDMS and a short (～20 cm) length of fused‐silica capillary as a separation unit. The on‐capillary detection was carried out by fiber optic spectrometry. A mixture of six cephalosporin antibiotics was separated in the μCE system and the obtained results were compared to those achievable by conventional CE. The six components could be separated within 8.5 min with the number of theoretical plates around 10 000.     

Combination of flow injection with capillary electrophoresis: 8. Miniaturized capillary electrophoresis system with flow injection sample introduction and electrogenerated chemiluminescence detection

The combined flow injection (FI)-capillary electrophoresis (CE) system was further exploited by coupling to an electrogenerated chemiluminescence (ECL) detection system. A low-cost miniaturized CE system was developed on a chip platform to provide easy interface both with FI sample introduction and with ECL detection. A falling-drop interface was employed to perform FI split-flow sample introduction while achieving electrical isolation from the CE high voltage. A plexiglas reservoir at the capillary outlet served as both the reaction and detection cell for the ECL reaction, with Ru(bpy)₃²⁺ reagent continuously flowing through the cell. An optical fiber was positioned within the reservoir close to the capillary outlet for transferring the ECL emission to the PMT. The relative positions of the capillary outlet, working electrode and optical fiber as well as reagent renewal flow-rate were optimized to achieve both good sensitivity and separation efficiency under non-interrupted sampling conditions, involving large numbers of samples. An on-column joint often used in other works for isolating the ECL detection system from the CE separation voltage was not found necessary. The performance of the system was illustrated by the baseline separation of proline, valine and phenylalanine with a high throughput of 50 h⁻¹ and plate height of 14 μm for proline under 147 V cm⁻¹ field strength. Detection limits (3σ) were 1.2, 50 and 25 μM and peak height precisions were 1.4, 5.4 and 4.3% R.S.D. (n=9) for proline, valine and phenylalanine, respectively.